Study of Tnp1, Tekt1, and Plzf Genes Expression During an in vitro Three-Dimensional Neonatal Male Mice Testis Culture

PubMed

Alrahel, Ahmad; Movahedin, Mansoureh; Mazaheri, Zohre; Amidi, Fardin

2018-07-01

In vitro spermatogenesis has a long research history beginning in the early 20th century. This organ culture method was therefore abandoned, and alternative cell culture methods were chosen by many researchers. Here, whether Tnp1, Tekt1, and Plzf, which play a crucial role in spermatogenesis, can be expressed during testis organ culture was assessed. Testes of 10 mouse pups were first removed, and the testis tissue was then separated into smaller pieces of seminiferous tubules. The size of the pieces was arbitrary; approximately 1 mg in weight or 1 mm3 in size when compacted. Afterwards, the testis tissue fragments (1–3) were transferred to the hexahedrons, incubated in a culture incubator and cultured for 12 weeks. Histological assessment and molecular evaluation were carried out at the end of the study. The results showed that the expression of Tekt1 as a mitotic gene in mouse pups decreased significantly (p ≤ 0.05) in comparison to adult mouse testis. Meanwhile, the expression of Tnp1 as a meiotic gene increased significantly (p ≤ 0.05) as compared to neonate mouse testis at the beginning of the culture. The expression of Plzf showed no significant difference during the 12 weeks of culture (p ≥ 0.05). Based on histological study, different types of spermatocytes and post-meiotic stages of germ cells could not be detected. This kind of three-dimensional culture can induce expression of post-meiotic gene, Tnp1, but only at the molecular level and not beyond meiosis.

Heterophilic binding of the adhesion molecules poliovirus receptor and immunoglobulin superfamily 4A in the interaction between mouse spermatogenic and Sertoli cells.

PubMed

Wakayama, Tomohiko; Sai, Yoshimichi; Ito, Akihiko; Kato, Yukio; Kurobo, Miho; Murakami, Yoshinori; Nakashima, Emi; Tsuji, Akira; Kitamura, Yukihiko; Iseki, Shoichi

2007-06-01

The cell adhesion protein immunoglobulin superfamily 4A (IGSF4A) is expressed on the surfaces of spermatogenic cells in the mouse testis. During spermatogenesis, IGSF4A is considered to bind to the surface of Sertoli cells in a heterophilic manner. To identify this unknown partner of IGSF4A, we generated rat monoclonal antibodies against the membrane proteins of mouse Sertoli cells grown in primary culture. Using these monoclonal antibodies, we isolated a clone that immunostained Sertoli cells and reacted with the product of immunoprecipitation of the homogenate of mouse testis with anti-IGSF4A antibody. Subsequently, to identify the Sertoli cell membrane protein that is recognized by this monoclonal antibody, we performed expression cloning of a cDNA library from the mouse testis. As a result, we identified poliovirus receptor (PVR), which is another IGSF-type cell adhesion molecule, as the binding partner of IGSF4A. The antibodies raised against PVR and IGSF4A immunoprecipitated both antigens in the homogenate of mouse testis. Immunoreactivity for PVR was present in Sertoli cells but not in spermatogenic cells at all stages of spermatogenesis. Overexpression of PVR in TM4, a mouse Sertoli cell line, increased more than three-fold its capacity to adhere to Tera-2, which is a human cell line that expresses IGSF4A. These findings suggest that the heterophilic binding of PVR to IGSF4A is responsible, at least in part, for the interaction between Sertoli and spermatogenic cells during mouse spermatogenesis.

Localization and expression of Orexin A and its receptor in mouse testis during different stages of postnatal development.

PubMed

Joshi, Deepanshu; Singh, Shio Kumar

2017-01-15

Orexin A (OXA), a hypothalamic neuropeptide, is involved in regulation of various biological functions and its actions are mediated through G-protein-coupled receptor, OX1R. This neuropeptide has emerged as a central neuroendocrine modulator of reproductive functions. Both OXA and OX1R have been shown to be expressed in peripheral organs such as gastrointestinal and genital tracts. In the present study, localization and expression of OXA and OX1R in mouse testis during different stages of postnatal development have been investigated. Immunohistochemical results demonstrated localization of OXA and OX1R in both the interstitial and the tubular compartments of the testis throughout the period of postnatal development. In testicular sections on 0day postpartum (dpp), gonocytes, Sertoli cells and foetal Leydig cells showed OXA and OX1R-immunopositive signals. At 10dpp, Sertoli cells, spermatogonia, early spermatocytes and Leydig cells showed immunopositive signals for both, the ligand and the receptor. On 30 and 90dpp, the spermatogonia, Sertoli cells, spermatocytes, spermatids and Leydig cells showed the OXA and OX1R-immunopositive signals. At 90dpp, strong OXA-positive signals were seen in Leydig cells, primary spermatocytes and spermatogonia, while OX1R-immunopositive intense signals were observed in Leydig cells and elongated spermatids. Further, semiquantitative RT-PCR and immunoblot analyses showed that OXA and OX1R were expressed in the testis both at transcript and protein levels during different stages of postnatal development. The expression of OXA and OX1R increased progressively from day of birth (0dpp) until adulthood (90dpp), with maximal expression at 90 dpp. The results suggest that OXA and OX1R are expressed in the testis and that they may help in proliferation and development of germ cells, Leydig cells and Sertoli cells, and in the spermatogenic process and steroidogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

ARX/Arx is expressed in germ cells during spermatogenesis in both marsupial and mouse.

PubMed

Yu, Hongshi; Pask, Andrew J; Hu, Yanqiu; Shaw, Geoff; Renfree, Marilyn B

2014-03-01

The X-linked aristaless gene, ARX, is essential for the development of the gonads, forebrain, olfactory bulb, pancreas, and skeletal muscle in mice and humans. Mutations cause neurological diseases, often accompanied by ambiguous genitalia. There are a disproportionately high number of testis and brain genes on the human and mouse X chromosomes. It is still unknown whether the X chromosome accrued these genes during its evolution or whether genes that find themselves on the X chromosome evolve such roles. ARX was originally autosomal in mammals and remains so in marsupials, whereas in eutherian mammals it translocated to the X chromosome. In this study, we examined autosomal ARX in tammars and compared it with the X-linked Arx in mice. We detected ARX mRNA in the neural cells of the forebrain, midbrain and hindbrain, and olfactory bulbs in developing tammars, consistent with the expression in mice. ARX was detected by RT-PCR and mRNA in situ hybridization in the developing tammar wallaby gonads of both sexes, suggestive of a role in sexual development as in mice. We also detected ARX/Arx mRNA in the adult testis in both tammars and mice, suggesting a potential novel role for ARX/Arx in spermiogenesis. ARX transcripts were predominantly observed in round spermatids. Arx mRNA localization distributions in the mouse adult testis suggest that it escaped meiotic sex chromosome inactivation during spermatogenesis. Our findings suggest that ARX in the therian mammal ancestor already played a role in male reproduction before it was recruited to the X chromosome in eutherians.

Identification and characterization of Rhox13, a novel X-linked mouse homeobox gene

PubMed Central

Geyer, Christopher B.; Eddy, Edward M.

2008-01-01

Homeobox genes encode transcription factors whose expression organizes programs of development. A number of homeobox genes expressed in reproductive tissues have been identified recently, including a colinear cluster on the X chromosome in mice. This has led to an increased interest in understanding the role(s) of homeobox genes in regulating development of reproductive tissues including the testis, ovary, and placenta. Here we report the identification and characterization of a novel homeobox gene of the paired-like class on the X chromosome distal to the reproductive homeobox (Rhox) cluster in mice. Transcripts are found in the testis and ovary as early as 13.5 days post-coitum (dpc). Transcription ceases in the ovary by 3 days post-partum (dpp), but continues in the testis through adulthood. The Rhox13 gene encodes a 25.3 kDa protein expressed in the adult testis in germ cells at the basal aspect of the seminiferous epithelium. PMID:18675325

Interaction between basigin and monocarboxylate transporter 2 in the mouse testes and spermatozoa

PubMed Central

Chen, Cheng; Maekawa, Mamiko; Yamatoya, Kenji; Nozaki, Masami; Ito, Chizuru; Iwanaga, Toshihiko; Toshimori, Kiyotaka

2016-01-01

Basigin is a member of the immunoglobulin superfamily and plays various important roles in biological events including spermatogenesis. To examine the basigin molecular variants during spermatogenesis and sperm maturation in the mouse, immunoprecipitated basigin samples from testis and epididymal spermatozoa were analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The results demonstrated that basigin molecules from the testis and spermatozoa were separable into two major bands and that the differences in the molecular sizes were possibly because of an endoproteolytic cleavage. Since basigin is known to be a chaperone for the monocarboxylate transporter 1 (MCT1), the localization of basigin, MCT1 and MCT2 was examined during postnatal testicular development. Immunohistochemical studies showed different expression patterns of MCT1 and MCT2. MCT1 was localized on the surface of spermatogonia, spermatocytes, and spermatids. In contrast, MCT2 appeared on the principal piece of spermatozoa in the testis, where basigin was also observed. In mature epididymal spermatozoa, MCT2 was located on the midpiece, where basigin co-localized with MCT2 but not with MCT1. Furthermore, MCT2 was immunoprecipitated with basigin in mouse testes and sperm. These results suggest that basigin has a functional role as a binding partner with MCT2 in testicular and epididymal spermatozoa. PMID:26208397

Activation of transcriptional activity of HSE by a novel mouse zinc finger protein ZNFD specifically expressed in testis.

PubMed

Xu, Fengqin; Wang, Weiping; Lei, Chen; Liu, Qingmei; Qiu, Hao; Muraleedharan, Vinaydhar; Zhou, Bin; Cheng, Hongxia; Huang, Zhongkai; Xu, Weian; Li, Bichun; Wang, Minghua

2012-04-01

Zinc finger proteins (ZFPs) that contain multiple cysteine and/or histidine residues perform important roles in various cellular functions, including transcriptional regulation, cell proliferation, differentiation, and apoptosis. The Cys-Cys-His-His (C(2)H(2)) type of ZFPs are the well-defined members of this super family and are the largest and most complex proteins in eukaryotic genomes. In this study, we identified a novel C(2)H(2) type of zinc finger gene ZNFD from mice which has a 1,002 bp open reading frame and encodes a protein with 333 amino acid residues. The predicted 37.4 kDa protein contains a C(2)H(2) zinc finger domain. ZNFD gene is located on chromosome 18qD1. RT-PCR analysis revealed that the ZNFD gene was specifically expressed in mouse testis but not in other tissues. Subcellular localization analysis demonstrated that ZNFD was localized in the nucleus. Reporter gene assays showed that overexpression of ZNFD in the COS7 cells activates the transcriptional activities of heat shock element (HSE). Overall, these results suggest that ZNFD is a member of the zinc finger transcription factor family and it participates in the transcriptional regulation of HSE. Many heat shock proteins regulated by HSE are involved in testicular development. Therefore, our results suggest that ZNFD may probably participate in the development of mouse testis and function as a transcription activator in HSE-mediated gene expression and signaling pathways.

Tzfp represses the androgen receptor in mouse testis.

PubMed

Furu, Kari; Klungland, Arne

2013-01-01

The testis zinc finger protein (Tzfp), also known as Repressor of GATA, belongs to the BTB/POZ zinc finger family of transcription factors and is thought to play a role in spermatogenesis due to its remarkably high expression in testis. Despite many attempts to find the in vivo role of the protein, the molecular function is still largely unknown. Here, we address this issue using a novel mouse model with a disrupted Tzfp gene. Homozygous Tzfp null mice are born at reduced frequency but appear viable and fertile. Sertoli cells in testes lacking Tzfp display an increase in Androgen Receptor (AR) signaling, and several genes in the testis, including Gata1, Aie1 and Fanc, show increased expression. Our results indicate that Tzfp function as a transcriptional regulator and that loss of the protein leads to alterations in AR signaling and reduced number of apoptotic cells in the testicular tubules.

Global transcriptome analysis of the C57BL/6J mouse testis by SAGE: evidence for nonrandom gene order.

PubMed

Divina, Petr; Vlcek, Cestmír; Strnad, Petr; Paces, Václav; Forejt, Jirí

2005-03-05

We generated the gene expression profile of the total testis from the adult C57BL/6J male mice using serial analysis of gene expression (SAGE). Two high-quality SAGE libraries containing a total of 76 854 tags were constructed. An extensive bioinformatic analysis and comparison of SAGE transcriptomes of the total testis, testicular somatic cells and other mouse tissues was performed and the theory of male-biased gene accumulation on the X chromosome was tested. We sorted out 829 genes predominantly expressed from the germinal part and 944 genes from the somatic part of the testis. The genes preferentially and specifically expressed in total testis and testicular somatic cells were identified by comparing the testis SAGE transcriptomes to the available transcriptomes of seven non-testis tissues. We uncovered chromosomal clusters of adjacent genes with preferential expression in total testis and testicular somatic cells by a genome-wide search and found that the clusters encompassed a significantly higher number of genes than expected by chance. We observed a significant 3.2-fold enrichment of the proportion of X-linked genes specific for testicular somatic cells, while the proportions of X-linked genes specific for total testis and for other tissues were comparable. In contrast to the tissue-specific genes, an under-representation of X-linked genes in the total testis transcriptome but not in the transcriptomes of testicular somatic cells and other tissues was detected. Our results provide new evidence in favor of the theory of male-biased genes accumulation on the X chromosome in testicular somatic cells and indicate the opposite action of the meiotic X-inactivation in testicular germ cells.

Global transcriptome analysis of the C57BL/6J mouse testis by SAGE: evidence for nonrandom gene order

PubMed Central

Divina, Petr; Vlček, Čestmír; Strnad, Petr; Pačes, Václav; Forejt, Jiří

2005-01-01

Background We generated the gene expression profile of the total testis from the adult C57BL/6J male mice using serial analysis of gene expression (SAGE). Two high-quality SAGE libraries containing a total of 76 854 tags were constructed. An extensive bioinformatic analysis and comparison of SAGE transcriptomes of the total testis, testicular somatic cells and other mouse tissues was performed and the theory of male-biased gene accumulation on the X chromosome was tested. Results We sorted out 829 genes predominantly expressed from the germinal part and 944 genes from the somatic part of the testis. The genes preferentially and specifically expressed in total testis and testicular somatic cells were identified by comparing the testis SAGE transcriptomes to the available transcriptomes of seven non-testis tissues. We uncovered chromosomal clusters of adjacent genes with preferential expression in total testis and testicular somatic cells by a genome-wide search and found that the clusters encompassed a significantly higher number of genes than expected by chance. We observed a significant 3.2-fold enrichment of the proportion of X-linked genes specific for testicular somatic cells, while the proportions of X-linked genes specific for total testis and for other tissues were comparable. In contrast to the tissue-specific genes, an under-representation of X-linked genes in the total testis transcriptome but not in the transcriptomes of testicular somatic cells and other tissues was detected. Conclusion Our results provide new evidence in favor of the theory of male-biased genes accumulation on the X chromosome in testicular somatic cells and indicate the opposite action of the meiotic X-inactivation in testicular germ cells. PMID:15748293

Xenogeneic spermatogenesis following transplantation of hamster germ cells to mouse testes.

PubMed

Ogawa, T; Dobrinski, I; Avarbock, M R; Brinster, R L

1999-02-01

It was recently demonstrated that rat spermatogenesis can occur in the seminiferous tubules of an immunodeficient recipient mouse after transplantation of testis cells from a donor rat. In the present study, hamster donor testis cells were transplanted to mice to determine whether xenogeneic spermatogenesis would result. The hamster diverged at least 16 million years ago from the mouse and produces spermatozoa that are larger than, and have a shape distinctly different from, those of the mouse. In four separate experiments with a total of 13 recipient mice, hamster spermatogenesis was identified in the testes of each mouse. Approximately 6% of the tubules examined demonstrated xenogeneic spermatogenesis. In addition, cryopreserved hamster testis cells generated spermatogenesis in recipients. However, abnormalities were noted in hamster spermatids and acrosomes in seminiferous tubules of recipient mice. Hamster spermatozoa were also found in the epididymis of recipient animals, but these spermatozoa generally lacked acrosomes, and heads and tails were separated. Thus, defects in spermiogenesis occur in hamster spermatogenesis in the mouse, which may reflect a limited ability of endogenous mouse Sertoli cells to support fully the larger and evolutionarily distant hamster germ cell. The generation of spermatogenesis from frozen hamster cells now adds this species to the mouse and rat, in which spermatogonial stem cells also can be cryopreserved. This finding has immediate application to valuable animals of many species, because the cells could be stored until suitable recipients are identified or culture techniques devised to expand the stem cell population.

Accumulation of the mitochondrial form of the sulphydryl oxidase Erv1p/Alrp during the early stages of spermatogenesis.

PubMed

Klissenbauer, Monika; Winters, Silke; Heinlein, Uwe A O; Lisowsky, Thomas

2002-07-01

In this study, we investigated the expression of the mammalian FAD-dependent sulphydryl oxidase Erv1p/Alrp in the rat and mouse and during mouse spermatogenesis. Up to three forms of Alrp were identified in protein extracts from different tissues and organs, but very little enzyme was present in blood samples. The three forms of Alrp represent the full-length protein of 23 kDa and fragments of 21 kDa and 15 kDa. All forms of Alrp were assembled into dimers in vivo. In contrast to samples from other organs, the protein analysis of mouse testis identified predominantly full-length 23 kDa Alrp. This finding prompted us to investigate in more detail the expression of Alrp during spermatogenesis. Testis samples of individual mice from postnatal days 13-29 were probed with an antibody specific for mammalian Alrp. In addition, cells from whole testis preparations were fractionated on a bovine serum albumin column gradient. Protein expression of mouse Alrp was compared with those of testis-specific cyritestin, the cytoskeleton marker actin and mitochondrial subunit Vb of cytochrome oxidase and cytochrome c. These studies demonstrated a specific accumulation of full-length mouse Alrp during the early stages of spermatogenesis. The highest levels of Alrp were found in spermatogonia and primary spermatocytes. Levels of expression of Alrp did not correlate with the synthesis of components of the respiratory chain, indicating that full-length Alrp in the mitochondria of spermatogonia and spermatocytes has another function in addition to its role in oxidative phosphorylation.

Gene discovery in the hamster: a comparative genomics approach for gene annotation by sequencing of hamster testis cDNAs

PubMed Central

Oduru, Sreedhar; Campbell, Janee L; Karri, SriTulasi; Hendry, William J; Khan, Shafiq A; Williams, Simon C

2003-01-01

Background Complete genome annotation will likely be achieved through a combination of computer-based analysis of available genome sequences combined with direct experimental characterization of expressed regions of individual genomes. We have utilized a comparative genomics approach involving the sequencing of randomly selected hamster testis cDNAs to begin to identify genes not previously annotated on the human, mouse, rat and Fugu (pufferfish) genomes. Results 735 distinct sequences were analyzed for their relatedness to known sequences in public databases. Eight of these sequences were derived from previously unidentified genes and expression of these genes in testis was confirmed by Northern blotting. The genomic locations of each sequence were mapped in human, mouse, rat and pufferfish, where applicable, and the structure of their cognate genes was derived using computer-based predictions, genomic comparisons and analysis of uncharacterized cDNA sequences from human and macaque. Conclusion The use of a comparative genomics approach resulted in the identification of eight cDNAs that correspond to previously uncharacterized genes in the human genome. The proteins encoded by these genes included a new member of the kinesin superfamily, a SET/MYND-domain protein, and six proteins for which no specific function could be predicted. Each gene was expressed primarily in testis, suggesting that they may play roles in the development and/or function of testicular cells. PMID:12783626

A new bioluminescent imaging technology for studying oxidative stress in the testis and its impacts on fertility.

PubMed

Ma, Qixiang; Shao, Haozhen; Feng, Yanyan; Zhang, Linpeng; Li, Pengshou; Hu, Xiaowei; Ma, Zhitao; Lou, Hua; Zeng, Xianwei; Luo, Guangbin

2018-05-24

Excessive oxidative stress (OS) leads to cellular dysfunctions and cell death and constitutes a major cause of male infertility. However, the etiologies of increased reactive oxygen species (ROS) in male infertility is not fully understood. One major limitation is the lack of an in vivo imaging system that can be used to effectively study the impact of excessive ROS in the testis. Recently, we discovered that the hepatocellular carcinoma reporter (HCR) mice previously generated in our laboratory also expressed luciferase in the spermatids of the testis. The goal of the current study is to use the HCR mice to detect OS in the testis and to investigate the potential use of this new system in studying OS-induced male infertility. Bioluminescence imaging (BLI) was performed in HCR mice that were treated with peroxy caged luciferin-1 (PCL-1), an OS reporter, to establish a new mouse model for in vivo monitoring of the OS status inside the male reproductive tract. Subsequently, the effect of acetaminophen (APAP) overdose on the OS inside the testis and male fertility were determined. Lastly, APAP was co-administered with glutathione, an antioxidant reagent, to test if the HCR mice can serve as a model for the effective and rapid assessment of the potency of individual agents in modifying the OS inside the mouse testis. The OS level in the testis in the HCR mice was readily detected by BLI. The use of this new model led to the discovery that APAP caused a sudden rise of OS in the testis and was a potent toxicant for the male reproductive system. Moreover, administration of glutathione was effective in preventing the APAP-induced elevation of OS and in ameliorating all of the OS-induced anomalies in the testis. The HCR mice represent an excellent model for monitoring OS change in the mouse testis by real time BLI. APAP is a potent male reproductive toxicant and APAP-treated mice represent a valid model for OS-induced male infertility. This model can be used to study OS-induced damage in male reproductive tract and in assessing the effects of therapeutic agents on the relative levels of OS and male fertility. Copyright © 2018 Elsevier Inc. All rights reserved.

Loss of p300 and CBP disrupts histone acetylation at the mouse Sry promoter and causes XY gonadal sex reversal

PubMed Central

Carré, Gwenn-Aël; Siggers, Pam; Xipolita, Marilena; Brindle, Paul; Lutz, Beat; Wells, Sara; Greenfield, Andy

2018-01-01

Abstract CREB-binding protein (CBP, CREBBP, KAT3A) and its closely related paralogue p300 (EP300, KAT3B), together termed p300/CBP, are histone/lysine acetyl-transferases that control gene expression by modifying chromatin-associated proteins. Here, we report roles for both of these chromatin-modifying enzymes in mouse sex determination, the process by which the embryonic gonad develops into a testis or an ovary. By targeting gene ablation to embryonic gonadal somatic cells using an inducible Cre line, we show that gonads lacking either gene exhibit major abnormalities of XY gonad development at 14.5 dpc, including partial sex reversal. Embryos lacking three out of four functional copies of p300/Cbp exhibit complete XY gonadal sex reversal and have greatly reduced expression of the key testis-determining genes Sry and Sox9. An analysis of histone acetylation at the Sry promoter in mutant gonads at 11.5 dpc shows a reduction in levels of the positive histone mark H3K27Ac. Our data suggest a role for CBP/p300 in testis determination mediated by control of histone acetylation at the Sry locus and reveal a novel element in the epigenetic control of Sry and mammalian sex determination. They also suggest possible novel causes of human disorders of sex development (DSD). PMID:29145650

Cell adhesion molecules expression pattern indicates that somatic cells arbitrate gonadal sex of differentiating bipotential fetal mouse gonad.

PubMed

Piprek, Rafal P; Kolasa, Michal; Podkowa, Dagmara; Kloc, Malgorzata; Kubiak, Jacek Z

2017-10-01

Unlike other organ anlagens, the primordial gonad is sexually bipotential in all animals. In mouse, the bipotential gonad differentiates into testis or ovary depending on the genetic sex (XY or XX) of the fetus. During gonad development cells segregate, depending on genetic sex, into distinct compartments: testis cords and interstitium form in XY gonad, and germ cell cysts and stroma in XX gonad. However, our knowledge of mechanisms governing gonadal sex differentiation remains very vague. Because it is known that adhesion molecules (CAMs) play a key role in organogenesis, we suspected that diversified expression of CAMs should also play a crucial role in gonad development. Using microarray analysis we identified 129 CAMs and factors regulating cell adhesion during sexual differentiation of mouse gonad. To identify genes expressed differentially in three cell lines in XY and XX gonads: i) supporting (Sertoli or follicular cells), ii) interstitial or stromal cells, and iii) germ cells, we used transgenic mice expressing EGFP reporter gene and FACS cell sorting. Although a large number of CAMs expressed ubiquitously, expression of certain genes was cell line- and genetic sex-specific. The sets of CAMs differentially expressed in supporting versus interstitial/stromal cells may be responsible for segregation of these two cell lines during gonadal development. There was also a significant difference in CAMs expression pattern between XY supporting (Sertoli) and XX supporting (follicular) cells but not between XY and XX germ cells. This indicates that differential CAMs expression pattern in the somatic cells but not in the germ line arbitrates structural organization of gonadal anlagen into testis or ovary. Copyright © 2017 Elsevier B.V. All rights reserved.

EXPRESSION OF THE SPERMATOGENIC CELL-SPECIFIC GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDS) IN RAT TESTIS

EPA Science Inventory

The spermatogenic cell-specific variant of glyceraldehyde 3-phosphate dehydrogenase (GAPDS) has been cloned from a rat testis cDNA library and its pattern of expression determined. A 1417 nucleotide cDNA has been found to encode an enzyme with substantial homology to mouse GAPDS...

A physiologically based pharmacokinetic model for ethylene oxide in mouse, rat, and human.

PubMed

Fennell, T R; Brown, C D

2001-06-15

Ethylene oxide (EO) is widely used as a gaseous sterilant and industrial intermediate and is a direct-acting mutagen and carcinogen. The objective of these studies was to develop physiologically based pharmacokinetic (PB-PK) models for EO to describe the exposure-tissue dose relationship in rodents and humans. We previously reported results describing in vitro and in vivo kinetics of EO metabolism in male and female F344 rats and B6C3F1 mice. These studies were extended by determining the kinetics of EO metabolism in human liver cytosol and microsomes. The results indicate enzymatically catalyzed GSH conjugation via cytosolic glutathione S-transferase (cGST) and hydrolysis via microsomal epoxide hydrolase (mEH) occur in both rodents and humans. The in vitro kinetic constants were scaled to account for cytosolic (cGST) and microsomal (mEH) protein content and incorporated into PB-PK descriptions for mouse, rat, and human. Flow-limited models adequately predicted blood and tissue EO levels, disposition, and elimination kinetics determined experimentally in rats and mice, with the exception of testis concentrations, which were overestimated. Incorporation of a diffusion-limited description for testis improved the ability of the model to describe testis concentrations. The model accounted for nonlinear increases in blood and tissue concentrations that occur in mice on exposure to EO concentrations greater than 200 ppm. Species differences are predicted in the metabolism and exposure-dose relationship, with a nonlinear relationship observed in the mouse as a result of GSH depletion. These models represent an essential step in developing a mechanistically based EO exposure-dose-response description for estimating human risk from exposure to EO. Copyright 2001 Academic Press.

Replacement of serum with ocular fluid for cryopreservation of immature testes.

PubMed

Pothana, Lavanya; Devi, Lalitha; Venna, Naresh Kumar; Pentakota, Niharika; Varma, Vivek Phani; Jose, Jedy; Goel, Sandeep

2016-12-01

Cryopreservation of immature testis is a feasible approach for germplasm preservation of male animals. Combinations of dimethyl sulfoxide (DMSO) and foetal bovine serum (FBS) are used for testis cryopreservation. However, an alternative to FBS is needed, because FBS is expensive. Buffalo ocular fluid (BuOF), a slaughter house by-product, could be an economical option. The objective of the present study was to assess whether BuOF can replace FBS for cryopreservation of immature mouse (Mus musculus), rat (Rattus norvegicus), and buffalo (Bubalus bubalis) testes. Results showed that rodent and buffalo testes frozen in DMSO (10% for rodents and 20% for buffalo) with 20% FBS or BuOF had similar numbers of viable and DNA-damaged cells (P > 0.05). The expression of cell proliferation- (PCNA) and apoptosis-specific proteins (Annexin V and BAX/BCL2 ratio) were also comparable in mouse and buffalo testes frozen in DMSO with FBS or BuOF (P > 0.05). Interestingly, rat testis frozen in DMSO with BuOF had lower expression of Annexin V protein than testis frozen in DMSO with FBS (P < 0.05). The percentage of meiotic germ cells (pachytene-stage spermatocytes) in xenografts from testis frozen either in DMSO with BuOF or FBS did not significantly differ in rats or buffalo (P > 0.05). These findings provide evidence that BuOF has potential to replace FBS for cryopreservation of immature rodent and buffalo testis. Further investigation is needed to explore whether BuOF can replace FBS for testis cryopreservation of other species. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression of Pkd2l2 in testis is implicated in spermatogenesis.

PubMed

Chen, Ye; Zhang, Zheng; Lv, Xiao-Yan; Wang, Yi-Dong; Hu, Zhong-Guo; Sun, Huan; Tan, Rui-Zhi; Liu, Yu-Hang; Bian, Guo-Hui; Xiao, Yan; Li, Qin-Wei; Yang, Qiu-Tan; Ai, Jian-Zhong; Feng, Lu; Yang, Yang; Wei, Yu-Quan; Zhou, Qin

2008-08-01

Pkd2l2 is a novel member of the polycystic kidney disease (PKD) gene family in mammals. Prominently expressed in testis, this gene is still poorly understood. In this study, reverse transcription polymerase chain reaction (RT-PCR) results showed a time-dependent expression pattern of Pkd2l2 in postnatal mouse testis. Immunohistochemical analysis revealed that Pkd2l2 encoded a protein, polycystin-L2, which was predominantly detectable in the plasma membrane of spermatocytes and round spermatids, as well as in the head and tail of elongating spermatids within seminiferous tubules in mouse testis tissue sections of postnatal day 14 and adult mice. A green fluorescent fusion protein of Pkd2l2 resided in the plasma membrane of HEK 293 and MDCK cells, suggesting that it functions as a plasma membrane protein. Overexpression of Pkd2l2 increased the intracellular calcium concentration of MDCK cells, as detected by flow cytometry. Collectively, these data indicated that Pkd2l2 may be involved in the mid-late stage of spermatogenesis through modulation of the intracellular calcium concentration.

Abnormal spermatogenesis following sodium fluoride exposure is associated with the downregulation of CREM and ACT in the mouse testis.

PubMed

Wang, Chong; Chen, Yan; Manthari, Ram Kumar; Wang, Jundong

2018-04-01

cAMP response element modulator (CREM) is involved in regulating gene expression in normal spermatogenesis. The transcriptional activity of CREM is partly regulated by activator of CREM in the testis (ACT). To investigate the effects of different concentrations of sodium fluoride (NaF) on the gene and protein expression of CREM and ACT in the mouse testis, sexually mature male Kunming mice were exposed to 50, 100, or 150 mg/L NaF in their drinking water for 90 days. NaF reduced the sperm count and viability and increased the percentage of malformed sperm in a dose-dependent manner. The mRNA expression of CREM and ACT was markedly downregulated in the NaF-treated groups. Furthermore, immunohistochemistry revealed that CREM and ACT proteins were decreased significantly in the 50, 100, and 150 mg/L NaF-treated groups compared to the control group. These findings indicate that the decreased gene and protein expression of CREM and ACT in the testis is associated with an impairment of reproductive functions by NaF.

Akt1 protects against germ cell apoptosis in the post natal mouse testis following lactational exposure to 6-N-propylthiouracil

EPA Science Inventory

Lactational exposure to 6-propyl-2-thio-uracil (PTU), a neonatal goitrogen, leads to increased testis size and sperm production in rodents. Aktl, a gene involved in cell survival and proliferation is also phosphorylated by thyroxine (T4). Therefore, we examined the requirement f...

Diallyl disulfide attenuated carbon ion irradiation-induced apoptosis in mouse testis through changing the ratio of Tap73/ΔNp73 via mitochondrial pathway

PubMed Central

Di, Cui-xia; Han, Lu; Zhang, Hong; Xu, Shuai; Mao, Ai-hong; Sun, Chao; Liu, Yang; Si, Jing; Li, Hong-yan; Zhou, Xin; Liu, Bing; Miao, Guo-ying

2015-01-01

Diallyl disulfide (DADS), a major organosulfur compound derived from garlic, has various biological properties, including anti-cancer effects. However, the protective mechanism of DADS against radiation-induced mouse testis cell apoptosis has not been elucidated. In this study, the magnitude of radiation effects evoked by carbon ion irradiation was marked by morphology changes, significant rise in apoptotic cells, activation expression of p53, up regulation the ratio of pro-apoptotic Tap73/anti-apoptotic ΔNp73, as well as alterations of crucial mediator of the mitochondrial pathway. Interestingly, pretreatment with DADS attenuated carbon ion irradiation-induced morphology damages and apoptotic cells. Additionally, DADS elevated radiation-induced p53 and p21 expression, suggesting that p53 might be involved in the inhibition of cell cycle progression through up regulation of p21. Furthermore, administration with DADS prevented radiation-induced Tap73/ΔNp73 expression and consequently down regulated Bax/Bcl-2 ratio, cytochrome c release and caspase-3 expression, indicating that the balance between Tap73 and ΔNp73 had potential to activate p53 responsive genes. Thus, our results showed that radio protection effect of DADS on mouse testis is mediated by blocking apoptosis through changing the ratio of Tap73/ΔNp73 via mitochondrial pathway, suggesting that DADS could be used as a potential radio protection agent for the testis against heavy-ion radiation. PMID:26526304

Identification of human candidate genes for male infertility by digital differential display.

PubMed

Olesen, C; Hansen, C; Bendsen, E; Byskov, A G; Schwinger, E; Lopez-Pajares, I; Jensen, P K; Kristoffersson, U; Schubert, R; Van Assche, E; Wahlstroem, J; Lespinasse, J; Tommerup, N

2001-01-01

Evidence for the importance of genetic factors in male fertility is accumulating. In the literature and the Mendelian Cytogenetics Network database, 265 cases of infertile males with balanced reciprocal translocations have been described. The candidacy for infertility of 14 testis-expressed transcripts (TETs) were examined by comparing their chromosomal mapping position to the position of balanced reciprocal translocation breakpoints found in the 265 infertile males. The 14 TETs were selected by using digital differential display (electronic subtraction) to search for apparently testis-specific transcripts in the TIGR database. The testis specificity of the 14 TETs was further examined by reverse transcription-polymerase chain reaction (RT-PCR) on adult and fetal tissues showing that four TETs (TET1 to TET4) were testis-expressed only, six TETs (TET5 to TET10) appeared to be differentially expressed and the remaining four TETs (TET11 to TET14) were ubiquitously expressed. Interestingly, the two tesis expressed-only transcripts, TET1 and TET2, mapped to chromosomal regions where seven and six translocation breakpoints have been reported in infertile males respectively. Furthermore, one ubiquitously, but predominantly testis-expressed, transcript, TET11, mapped to 1p32-33, where 13 translocation breakpoints have been found in infertile males. Interestingly, the mouse mutation, skeletal fusions with sterility, sks, maps to the syntenic region in the mouse genome. Another transcript, TET7, was the human homologue of rat Tpx-1, which functions in the specific interaction of spermatogenic cells with Sertoli cells. TPX-1 maps to 6p21 where three cases of chromosomal breakpoints in infertile males have been reported. Finally, TET8 was a novel transcript which in the fetal stage is testis-specific, but in the adult is expressed in multiple tissues, including testis. We named this novel transcript fetal and adult testis-expressed transcript (FATE).

Generation and characterization of Lhx9 – GFPCreERT2 knock-in mouse line

PubMed Central

Xie, Xiaoling; Deng, Min; Gan, Lin

2014-01-01

Summary LHX9 is a LIM-homeodomain transcription factor essential for the development of gonads, spinal cord interneurons, and thalamic neurons to name a few. We recently reported the expression of LHX9 in retinal amacrine cells during development. In this study, we generated an Lhx9 - GFPCreERT2 (GCE) knock-in mouse line by knocking-in a GCE cassette at the Lhx9 locus, thus inactivating endogenous Lhx9. Lhx9GCE/+ mice were viable, fertile, and displayed no overt phenotypical characteristics. Lhx9GCE/GCE mice were all phenotypically female, smaller in size, viable, but infertile. The specificity and efficacy of the Lhx9-GCE mouse line was verified by crossing it to a Rosa26 - tdTomato reporter mouse line, which reveals the Cre recombinase activities in retinal amacrine cells, developing limbs, testis, hippocampal neurons, thalamic neurons, and cerebellar neurons. Taken together, the Lhx9-GCE mouse line could serve as a beneficial tool for lineage tracing and gene manipulation experiments. PMID:25112520

[Effects of infrasound on ultrastructure of testis cell in mice].

PubMed

Wei, Ya-Ning; Liu, Jing; Shu, Qing; Huang, Xiao-Feng; Chen, Jing-Zao

2002-01-01

To investigate the effects of infrasound on ultrastructure of testis in mouse. Twelve male BALB/C mice were randomly divided into three groups according to exposed duration on 1, 7 and 14 day. The mice were separately exposed to infrasound environment under 8 Hz/90 dB, 8 Hz/130 dB, 16 Hz/90 dB, 16 Hz/130 dB 2 hours per day. There was another control group which had three mice were separated into module with no infrasound. All the mice were killed on schedule. Then all the sections of testis were observed under electronic microscope. The alterations of structure and the chromatin were observed. Some acute alteration in one day group was found in testis cell, such as cellular denaturation and necrosis, intercellular edema, mitochondria swelling, liposome hyperplasia. When the infrasound was up to 8 Hz/130 dB, the damage showed seriously. In 7 and 14 day group, the acute alteration was gradually decreased. A plenty of abnormal sperm were found. And other alteration was chromatin condense. The effect of variational frequency was important in ultrastructure. The infrasound markedly effected to testicular cell morphology and secreting function. Infrasound will lead to the alteration of procreation in mouse.

Design, modelling and preliminary characterisation of microneedle-based electrodes for tissue electroporation in vivo

NASA Astrophysics Data System (ADS)

O'Mahony, Conor; Houlihan, Ruth; Grygoryev, Konstantin; Ning, Zhenfei; Williams, John; Moore, Tom

2016-10-01

We analysed the use of microneedle-based electrodes to enhance electroporation of mouse testis with DNA vectors for production of transgenic mice. Different microneedle formats were developed and tested, and we ultimately used electrodes based on arrays of 500 μm tall microneedles. In a series of experiments involving injection of a DNA vector expressing Green Fluorescent Protein (GFP) and electroporation using microneedle electrodes and a commercially available voltage supply, we compared the performance of flat and microneedle electrodes by measuring GFP expression at various timepoints after electroporation. Our main finding, supported by both experimental and simulated data, is that needles significantly enhanced electroporation of testis.

Gonadal Identity in the Absence of Pro-Testis Factor SOX9 and Pro-Ovary Factor Beta-Catenin in Mice1

PubMed Central

Nicol, Barbara; Yao, Humphrey H.-C.

2015-01-01

Sex-reversal cases in humans and genetic models in mice have revealed that the fate of the bipotential gonad hinges upon the balance between pro-testis SOX9 and pro-ovary beta-catenin pathways. Our central query was: if SOX9 and beta-catenin define the gonad's identity, then what do the gonads become when both factors are absent? To answer this question, we developed mouse models that lack either Sox9, beta-catenin, or both in the somatic cells of the fetal gonads and examined the morphological outcomes and transcriptome profiles. In the absence of Sox9 and beta-catenin, both XX and XY gonads progressively lean toward the testis fate, indicating that expression of certain pro-testis genes requires the repression of the beta-catenin pathway, rather than a direct activation by SOX9. We also observed that XY double knockout gonads were more masculinized than their XX counterpart. To identify the genes responsible for the initial events of masculinization and to determine how the genetic context (XX vs. XY) affects this process, we compared the transcriptomes of Sox9/beta-catenin mutant gonads and found that early molecular changes underlying the XY-specific masculinization involve the expression of Sry and 21 SRY direct target genes, such as Sox8 and Cyp26b1. These results imply that when both Sox9 and beta-catenin are absent, Sry is capable of activating other pro-testis genes and drive testis differentiation. Our findings not only provide insight into the mechanism of sex determination, but also identify candidate genes that are potentially involved in disorders of sex development. PMID:26108792

Imaging of Chromosome Dynamics in Mouse Testis Tissue by Immuno-FISH.

PubMed

Scherthan, Harry

2017-01-01

The mouse (Mus musculus) represents the central mammalian genetic model system for biomedical and developmental research. Mutant mouse models have provided important insights into chromosome dynamics during the complex meiotic differentiation program that compensates for the genome doubling at fertilization. Homologous chromosomes (homologues) undergo dynamic pairing and recombine during first meiotic prophase before they become partitioned into four haploid sets by two consecutive meiotic divisions that lack an intervening S-phase. Fluorescence in situ hybridization (FISH) has been instrumental in the visualization and imaging of the dynamic reshaping of chromosome territories and mobility during prophase I, in which meiotic telomeres were found to act as pacemakers for the chromosome pairing dance. FISH combined with immunofluorescence (IF) co-staining of nuclear proteins has been instrumental for the visualization and imaging of mammalian meiotic chromosome behavior. This chapter describes FISH and IF methods for the analysis of chromosome dynamics in nuclei of paraffin-embedded mouse testes. The techniques have proven useful for fresh and archived paraffin testis material of several mammalian species.

Prepubertal mouse testis growth and maturation and androgen production are acutely sensitive to di-n-butyl phthalate.

PubMed

Moody, Sarah; Goh, Hoey; Bielanowicz, Amanda; Rippon, Paul; Loveland, Kate L; Itman, Catherine

2013-09-01

Phthalates are plasticizers with widespread industrial, domestic, and medical applications. Epidemiological data indicating increased incidence of testicular dysgenesis in boys exposed to phthalates in utero are reinforced by studies demonstrating that phthalates impair fetal rodent testis development. Because humans are exposed to phthalates continuously from gestation through adulthood, it is imperative to understand what threat phthalates pose at other life stages. To determine the impact during prepuberty, we assessed the consequences of oral administration of 1 to 500 mg di-n-butyl phthalate (DBP)/kg/d in corn oil to wild-type (C57BL/6J) male mice from 4 to 14 days of age. Dose-dependent effects on testis growth correlated with reduced Sertoli cell proliferation. Histological and immunohistochemical analyses identified delayed spermatogenesis and impaired Sertoli cell maturation after exposure to 10 to 500 mg DBP/kg/d. Interference with the hypothalamic-pituitary-gonadal axis was indicated in mice fed 500 mg DBP/kg/d, which had elevated circulating inhibin but no change in serum FSH. Increased immunohistochemical staining for inhibin-α was apparent at doses of 10 to 500 mg DBP/kg/d. Serum testosterone and testicular androgen activity were lower in the 500 mg DBP/kg/d group; however, reduced anogenital distance in all DBP-treated mice suggested impaired androgen action at earlier time points. Long-term effects were evident, with smaller anogenital distance and indications of disrupted spermatogenesis in adult mice exposed prepubertally to doses from 1 mg DBP/kg/d. These data demonstrate the acute sensitivity of the prepubertal mouse testis to DBP at doses 50- to 500-fold lower than those used in rat and identify the upregulation of inhibin as a potential mechanism of DBP action.

Expression of the GM2 activator protein in mouse testis.

PubMed

Li, Yu-Teh; Li, Su-Chen; Chen, I-Li

2017-12-01

The GM2-activator protein (GM2-AP), revealed by Li et al. in 1973 in human liver, was initially identified as a protein cofactor that stimulated β-hexosaminidase A to hydrolyze N-acetylgalactosamine from GM2 ganglioside. This cofactor was found to be missing in human variant AB Tay-Sachs disease. Over the years, the GM2-AP has also been shown to be involved in kidney vesicular transport, lipid presentation by CD1 molecule to T-cells, and interaction of human sperm with zona pellucida. Since the expression of the GM2-AP via mRNA detection in mouse tissues was found to be the highest in testis, we became interested in the localization of the GM2-AP at cellular level in mouse testis during spermatogenesis. Using immunohistochemical analysis and electron microscopy, we found that the GM2-AP was predominantly localized in the basal cytoplasm and the attenuated processes of Sertoli cells. The stained structure appeared to be lysosomes. The most interesting finding was the association of the GM2-AP with the acrosomal apparatus in early spermatids. A modest to intense staining was observed in some acrosomal granules and acrosomal caps. The GM2-AP seemed to disappear from acrosomal caps in the later stage of spermatids, in which the nucleus became elongated and condensed. These results suggest that the GM2-AP may be involved in the normal functions of Sertoli cells and play important roles during the development of acrosomal caps in the early spermatids. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Comparative Analyses of H3K4 and H3K27 Trimethylations Between the Mouse Cerebrum and Testis

PubMed Central

Cui, Peng; Liu, Wanfei; Zhao, Yuhui; Lin, Qiang; Zhang, Daoyong; Ding, Feng; Xin, Chengqi; Zhang, Zhang; Song, Shuhui; Sun, Fanglin; Yu, Jun; Hu, Songnian

2012-01-01

The global features of H3K4 and H3K27 trimethylations (H3K4me3 and H3K27me3) have been well studied in recent years, but most of these studies were performed in mammalian cell lines. In this work, we generated the genome-wide maps of H3K4me3 and H3K27me3 of mouse cerebrum and testis using ChIP-seq and their high-coverage transcriptomes using ribominus RNA-seq with SOLiD technology. We examined the global patterns of H3K4me3 and H3K27me3 in both tissues and found that modifications are closely-associated with tissue-specific expression, function and development. Moreover, we revealed that H3K4me3 and H3K27me3 rarely occur in silent genes, which contradicts the findings in previous studies. Finally, we observed that bivalent domains, with both H3K4me3 and H3K27me3, existed ubiquitously in both tissues and demonstrated an invariable preference for the regulation of developmentally-related genes. However, the bivalent domains tend towards a “winner-takes-all” approach to regulate the expression of associated genes. We also verified the above results in mouse ES cells. As expected, the results in ES cells are consistent with those in cerebrum and testis. In conclusion, we present two very important findings. One is that H3K4me3 and H3K27me3 rarely occur in silent genes. The other is that bivalent domains may adopt a “winner-takes-all” principle to regulate gene expression. PMID:22768982

Selective ablation of Ppp1cc gene in testicular germ cells causes oligo-teratozoospermia and infertility in mice.

PubMed

Sinha, Nilam; Puri, Pawan; Nairn, Angus C; Vijayaraghavan, Srinivasan

2013-11-01

The four isoforms of serine/threonine phosphoprotein phosphatase 1 (PP1), derived from three genes, are among the most conserved proteins known. The Ppp1cc gene encodes two alternatively spliced variants, PP1 gamma1 (PPP1CC1) and PP1 gamma2 (PPP1CC2). Global deletion of the Ppp1cc gene, which causes loss of both isoforms, results in male infertility due to impaired spermatogenesis. This phenotype was assumed to be due to the loss of PPP1CC2, which is abundant in testis. While PPP1CC2 is predominant, other PP1 isoforms are also expressed in testis. Given the significant homology between the four PP1 isoforms, the lack of compensation by the other PP1 isoforms for loss of one, only in testis, is surprising. Here we document, for the first time, expression patterns of the PP1 isoforms in postnatal developing and adult mouse testis. The timing and sites of testis expression of PPP1CC1 and PPP1CC2 in testis are nonoverlapping. PPP1CC2 is the only one of the four PP1 isoforms not detected in sertoli cells and spermatogonia. Conversely, PPP1CC2 may be the only PP1 isoform expressed in postmeiotic germ cells. Deletion of the Ppp1cc gene in germ cells at the differentiated spermatogonia stage of development and beyond in Stra8 promoter-driven Cre transgenic mice results in oligo-terato-asthenozoospermia and male infertility, thus phenocopying global Ppp1cc null (-/-) mice. Taken together, these results confirm that spermatogenic defects observed in the global Ppp1cc knockout mice and in mice expressing low levels of PPP1CC2 in testis are due to compromised functions of PPP1CC2 in meiotic and postmeiotic germ cells.

Effect of mono-(2-ethylhexyl) phthalate on human and mouse fetal testis: In vitro and in vivo approaches

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muczynski, V.; CEA, DSV, iRCM, SCSR, LDRG, 92265 Fontenay-aux-Roses; INSERM, Unité 967, F-92265, Fontenay aux Roses

The present study was conducted to determine whether exposure to the mono-(2-ethylhexyl) phthalate (MEHP) represents a genuine threat to male human reproductive function. To this aim, we investigated the effects on human male fetal germ cells of a 10{sup −5} M exposure. This dose is slightly above the mean concentrations found in human fetal cord blood samples by biomonitoring studies. The in vitro experimental approach was further validated for phthalate toxicity assessment by comparing the effects of in vitro and in vivo exposure in mouse testes. Human fetal testes were recovered during the first trimester (7–12 weeks) of gestation andmore » cultured in the presence or not of 10{sup −5} M MEHP for three days. Apoptosis was quantified by measuring the percentage of Caspase-3 positive germ cells. The concentration of phthalate reaching the fetal gonads was determined by radioactivity measurements, after incubations with {sup 14}C-MEHP. A 10{sup −5} M exposure significantly increased the rate of apoptosis in human male fetal germ cells. The intratesticular MEHP concentration measured corresponded to the concentration added in vitro to the culture medium. Furthermore, a comparable effect on germ cell apoptosis in mouse fetal testes was induced both in vitro and in vivo. This study suggests that this 10{sup −5} M exposure is sufficient to induce changes to the in vivo development of the human fetal male germ cells. -- Highlights: ► 10{sup −5} M of MEHP impairs germ cell development in the human fetal testis. ► Organotypic culture is a suitable approach to investigate phthalate effects in human. ► MEHP is not metabolized in the human fetal testis. ► In mice, MEHP triggers similar effects both in vivo and in vitro.« less

Identification of a mouse B-type cyclin which exhibits developmentally regulated expression in the germ line

NASA Technical Reports Server (NTRS)

Chapman, D. L.; Wolgemuth, D. J.

1992-01-01

To begin to examine the function of cyclins in mammalian germ cells, we have screened an adult mouse testis cDNA library for the presence of B-type cyclins. We have isolated cDNAs that encode a murine B-type cyclin, which has been designated cycB1. cycB1 was shown to be expressed in several adult tissues and in the midgestation mouse embryo. In the adult tissues, the highest levels of cycB1 transcripts were seen in the testis and ovary, which contain germ cells at various stages of differentiation. The major transcripts corresponding to cycB1 are 1.7 and 2.5 kb, with the 1.7 kb species being the predominant testicular transcript and the 2.5 kb species more abundant in the ovary. Examination of cDNAs corresponding to the 2.5 kb and 1.7 kb mRNAs revealed that these transcripts encode identical proteins, differing only in the polyadenylation signal used and therefore in the length of their 3' untranslated regions. Northern blot and in situ hybridization analyses revealed that the predominant sites of cycB1 expression in the testis and ovary were in the germinal compartment, particularly in early round spermatids in the testis and growing oocytes in the ovary. Thus cycB1 is expressed in both meiotic and postmeiotic cells. This pattern of cycB1 expression further suggests that cycB1 may have different functions in the two cell types, only one of which correlates with progression of the cell cycle.

Identification of Hedgehog signaling outcomes in mouse testis development using a hanging drop-culture system.

PubMed

Szczepny, Anette; Hogarth, Cathryn A; Young, Julia; Loveland, Kate L

2009-02-01

The Hedgehog (Hh) signaling pathway affects fetal testis growth. Recently, we described the dynamic cellular production of Hh signaling pathway components in juvenile and adult rodent testes. The Hh signaling is understood to regulate cord formation in the fetal testis, but minimal knowledge exists regarding how Hh signaling impacts the postnatal testis. To investigate this, we employed hanging drop cultures, which are used routinely in embryoid body formation. This approach has the advantage of using small media volume, and we examined its suitability for short-term culture of both murine embryonic gonads and adult testis tubules. The effects of cyclopamine, a specific Hh signaling inhibitor, were examined following culture of Embryonic Day 11.5 urogenital ridges (as control) and adult seminiferous tubule fragments for 24-48 h using histological, cell proliferation, and gene expression analyses. Cultured embryonic testes displayed generally normal cord structure, anti-Müllerian hormone (Amh) expression, and cell proliferation; known Hh target gene expression (Gli1, osteopontin, official symbol Spp1, and Amh) was altered in response to cyclopamine. Cultured adult tubules exhibited some loss of seminiferous epithelium organization over 48 h. Spermatogonia continued to proliferate, however, and no significant loss of viability was noted overall. Addition of cyclopamine significantly affected levels of Gli1, Igfbp6, Ccnd2 (cyclin D2), Ccnb1 (cyclin B1), Spp1, Kit, and Amh mRNAs; these genes have been shown previously to be expressed in Sertoli and germ cells. These novel results identify Hh target genes in the testis and demonstrate this signaling pathway likely affects cell survival and differentiation in the context of normal adult testis.

Identification of Hedgehog Signaling Outcomes in Mouse Testis Development Using a Hanging Drop-Culture System1

PubMed Central

Szczepny, Anette; Hogarth, Cathryn A.; Young, Julia; Loveland, Kate L.

2008-01-01

The Hedgehog (Hh) signaling pathway affects fetal testis growth. Recently, we described the dynamic cellular production of Hh signaling pathway components in juvenile and adult rodent testes. The Hh signaling is understood to regulate cord formation in the fetal testis, but minimal knowledge exists regarding how Hh signaling impacts the postnatal testis. To investigate this, we employed hanging drop cultures, which are used routinely in embryoid body formation. This approach has the advantage of using small media volume, and we examined its suitability for short-term culture of both murine embryonic gonads and adult testis tubules. The effects of cyclopamine, a specific Hh signaling inhibitor, were examined following culture of Embryonic Day 11.5 urogenital ridges (as control) and adult seminiferous tubule fragments for 24–48 h using histological, cell proliferation, and gene expression analyses. Cultured embryonic testes displayed generally normal cord structure, anti-Müllerian hormone (Amh) expression, and cell proliferation; known Hh target gene expression (Gli1, osteopontin, official symbol Spp1, and Amh) was altered in response to cyclopamine. Cultured adult tubules exhibited some loss of seminiferous epithelium organization over 48 h. Spermatogonia continued to proliferate, however, and no significant loss of viability was noted overall. Addition of cyclopamine significantly affected levels of Gli1, Igfbp6, Ccnd2 (cyclin D2), Ccnb1 (cyclin B1), Spp1, Kit, and Amh mRNAs; these genes have been shown previously to be expressed in Sertoli and germ cells. These novel results identify Hh target genes in the testis and demonstrate this signaling pathway likely affects cell survival and differentiation in the context of normal adult testis. PMID:18843087

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells

PubMed Central

Singh, Satyendra; Klarmann, Kimberly D.; Coppola, Vincenzo; Keller, Jonathan R.; Tessarollo, Lino

2016-01-01

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies. PMID:27835883

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells.

PubMed

Puverel, Sandrine; Kiris, Erkan; Singh, Satyendra; Klarmann, Kimberly D; Coppola, Vincenzo; Keller, Jonathan R; Tessarollo, Lino

2016-12-20

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies.

Functional characterization of a mouse testicular olfactory receptor and its role in chemosensing and in regulation of sperm motility.

PubMed

Fukuda, Nanaho; Yomogida, Kentaro; Okabe, Masaru; Touhara, Kazushige

2004-11-15

Although a subset of the olfactory receptor (OR) gene family is expressed in testis, neither their developmental profile nor their physiological functions have been fully characterized. Here, we show that MOR23 (a mouse OR expressed in the olfactory epithelium and testis) functions as a chemosensing receptor in mouse germ cells. In situ hybridization showed that MOR23 was expressed in round spermatids during stages VI-VIII of spermatogenesis. Lyral, a cognate ligand of MOR23, caused an increase in intracellular Ca2+ in a fraction of spermatogenic cells and spermatozoa. We also generated transgenic mice that express high levels of MOR23 in the testis and examined the response of their germ cells to lyral. The results provided evidence that lyral-induced Ca2+ increases were indeed mediated by MOR23. In a sperm accumulation assay, spermatozoa migrated towards an increasing gradient of lyral. Tracking and sperm flagellar analyses suggest that Ca2+ increases caused by MOR23 activation lead to modulation of flagellar configuration, resulting in chemotaxis. By contrast, a gradient of a cAMP analog or K8.6 solution, which elicit Ca2+ influx in spermatozoa, did not cause sperm accumulation, indicating that chemosensing and regulation of sperm motility was due to an OR-mediated local Ca2+ increase. The present studies indicate that mouse testicular ORs might play a role in chemoreception during sperm-egg communication and thereby regulate fertilization.

Expression dynamics of self-renewal factors for spermatogonial stem cells in the mouse testis.

PubMed

Sakai, Mizuki; Masaki, Kaito; Aiba, Shota; Tone, Masaaki; Takashima, Seiji

2018-04-16

Glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells (SSCs). Although GDNF is indispensable for the maintenance of SSCs, the role of FGF2 in the testis remains to be elucidated. To clarify this, the expression dynamics and regulatory mechanisms of Fgf2 and Gdnf in the mouse testes were analyzed. It is well known that Sertoli cells express Gdnf, and its receptor is expressed in a subset of undifferentiated spermatogonia, including SSCs. However, we found that Fgf2 was mainly expressed in the germ cells and its receptors were expressed not only in the cultured spermatogonial cell line, but also in testicular somatic cells. Aging, hypophysectomy, retinoic acid treatment, and testicular injury induced distinct Fgf2 and Gdnf expression dynamics, suggesting a difference in the expression mechanism of Fgf2 and Gdnf in the testis. Such differences might cause a dynamic fluctuation of Gdnf/Fgf2 ratio depending on the intrinsic/extrinsic cues. Considering that FGF2-cultured spermatogonia exhibit more differentiated phenotype than those cultured with GDNF, FGF2 might play a role distinct from that of GDNF in the testis, despite the fact that both factors are self-renewal factor for SSC in vitro.

A new chapter in the bisphenol A story: bisphenol S and bisphenol F are not safe alternatives to this compound.

PubMed

Eladak, Soria; Grisin, Tiphany; Moison, Delphine; Guerquin, Marie-Justine; N'Tumba-Byn, Thierry; Pozzi-Gaudin, Stéphanie; Benachi, Alexandra; Livera, Gabriel; Rouiller-Fabre, Virginie; Habert, René

2015-01-01

Bisphenol A (BPA) is a widely studied typical endocrine-disrupting chemical, and one of the major new issues is the safe replacement of this commonly used compound. Bisphenol S (BPS) and bisphenol F (BPF) are already or are planned to be used as BPA alternatives. With the use of a culture system that we developed (fetal testis assay [FeTA]), we previously showed that 10 nmol/L BPA reduces basal testosterone secretion of human fetal testis explants and that the susceptibility to BPA is at least 100-fold lower in rat and mouse fetal testes. Here, we show that addition of LH in the FeTA system considerably enhances BPA minimum effective concentration in mouse and human but not in rat fetal testes. Then, using the FeTA system without LH (the experimental conditions in which mouse and human fetal testes are most sensitive to BPA), we found that, as for BPA, 10 nmol/L BPS or BPF is sufficient to decrease basal testosterone secretion by human fetal testes with often nonmonotonic dose-response curves. In fetal mouse testes, the dose-response curves were mostly monotonic and the minimum effective concentrations were 1,000 nmol/L for BPA and BPF and 100 nmol/L for BPS. Finally, 10,000 nmol/L BPA, BPS, or BPF reduced Insl3 expression in cultured mouse fetal testes. This is the first report describing BPS and BPF adverse effects on a physiologic function in humans and rodents. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

CRISPR/Cas9-mediated Dax1 knockout in the monkey recapitulates human AHC-HH.

PubMed

Kang, Yu; Zheng, Bo; Shen, Bin; Chen, Yongchang; Wang, Lei; Wang, Jianying; Niu, Yuyu; Cui, Yiqiang; Zhou, Jiankui; Wang, Hong; Guo, Xuejiang; Hu, Bian; Zhou, Qi; Sha, Jiahao; Ji, Weizhi; Huang, Xingxu

2015-12-20

Mutations in the DAX1 locus cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH), which manifest with primary adrenal insufficiency and incomplete or absent sexual maturation, respectively. The associated defects in spermatogenesis can range from spermatogenic arrest to Sertoli cell only syndrome. Conclusions from Dax1 knockout mouse models provide only limited insight into AHC/HH disease mechanisms, because mouse models exhibit more extensive abnormalities in testicular development, including disorganized and incompletely formed testis cords with decreased number of peritubular myoid cells and male-to-female sex reversal. We previously reported successful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome targeting in cynomolgus monkeys. Here, we describe a male fetal monkey in which targeted genome editing using CRISPR/Cas9 produced Dax1-null mutations in most somatic tissues and in the gonads. This DAX1-deficient monkey displayed defects in adrenal gland development and abnormal testis architecture with small cords, expanded blood vessels and extensive fibrosis. Sertoli cell formation was not affected. This phenotype strongly resembles findings in human patients with AHC-HH caused by mutations in DAX1. We further detected upregulation of Wnt/β-catenin-VEGF signaling in the fetal Dax1-deficient testis, suggesting abnormal activation of signaling pathways in the absence of DAX1 as one mechanism of AHC-HH. Our study reveals novel insight into the role of DAX1 in HH and provides proof-of-principle for the generation of monkey models of human disease via CRISPR/Cas9-mediated gene targeting. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Rat organic solute carrier protein 1 (rOscp1) mediated the transport of organic solutes in Xenopus laevis oocytes: isolation and pharmacological characterization of rOscp1.

PubMed

Izuno, Hisanori; Kobayashi, Yasuna; Sanada, Yutaka; Nihei, Daisuke; Suzuki, Masako; Kohyama, Noriko; Ohbayashi, Masayuki; Yamamoto, Toshinori

2007-09-22

Rat organic solute carrier protein 1 (rOscp1) was isolated from a rat testis cDNA library. Isolated rOscp1 cDNA consisted of 1089 base pairs that encoded a 363-amino acid protein, and the amino acid sequence was 88% and 93% identical to that of human OSCP1 (hOSCP1) and mouse Oscp1 (mOscp1), respectively. The message for rOscp1 is highly detected in rat testis. When expressed in X. oocytes, rOscp1 mediated the high affinity transport of p-aminohippurate (PAH) with a Km value of 15.7+/-1.9 microM, and rOscp1-mediated organic solutes were exhibited in time- and Na+-independent manners. rOscp1 also transported various structurally heterogenous compounds such as testosterone, dehydroepiandrosterone sulfate (DHEA-S), and taurocholate with some differences in substrate specificity compared with hOSCP1. Immunohistochemical analysis revealed that the rOscp1 protein is localized in the basal membrane side of Sertoli cells as observed in mouse testis [Kobayashi et al., 2007; Kobayashi, Y., Tsuchiya, A., Hayashi, T., Kohyama, N., Ohbayashi, M., Yamamoto, T., 2007. Isolation and characterization of polyspecific mouse organic solute carrier protein 1 (mOscp1). Drug Metabolism and Disposition 35 (7), 1239-1245]. Thus, the present results indicate that a newly isolated cDNA clone, rOscp1, is a polyspecific organic solute carrier protein with some differences in substrate specificity compared with human and mouse OSCP1.

A Novel Testis-Specific Gene, Ccdc136, Is Required for Acrosome Formation and Fertilization in Mice.

PubMed

Geng, Qiang; Ni, Liwei; Ouyang, Bin; Hu, Yanhua; Zhao, Yu; Guo, Jun

2016-10-01

Testis-specific genes are essential for the spermatogenesis in mammalian male reproduction. In this study, we have identified a novel testis-specific gene, Ccdc136 (coiled-coil domain containing 136), from the results of high-throughput gene expression profiling in the developmental stage of mouse testes. Ccdc136 was conserved across species in evolution. Quantitative real-time polymerase chain reaction and Western blot analyses showed that Ccdc136 messenger RNA and protein were extraordinarily expressed in mouse testes, which was first presented at postnatal 3 week and increased in an age-dependent manner before adulthood. Immunofluorescence staining revealed that CCDC136 protein was most abundantly located in the acrosome of round spermatids and elongating spermatids within seminiferous tubules of the adult mouse testes. To investigate the function of Ccdc136 in mouse testes, we generated the Ccdc136-knockout mice using Cas9/RNA-mediated gene targeting technology. Interestingly, we found Ccdc136(-/-) males were infertile, due to severe defect of disrupting acrosome formation. The expression levels of proteins (SPACA1 and PICK1) involved in acrosome formation were significantly downregulated in the testes of Ccdc136(-/-) mice than wide-type mice. Moreover, in vitro fertilization assay revealed that anti-CCDC136 antibody could remarkably inhibit fertilization, suggesting CCDC136 also plays an important role in fertilization. All of these demonstrated the essential role of CCDC136-mediated acrosome formation in spermatogenesis and fertilization, which might also provide new insight into the genetic causes of human infertility. © The Author(s) 2016.

Epitopes of human testis-specific lactate dehydrogenase deduced from a cDNA sequence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Millan, J.L.; Driscoll, C.E.; LeVan, K.M.

The sequence and structure of human testis-specific L-lactate dehydrogenase (LDHC/sub 4/, LDHX; (L)-lactate:NAD/sup +/ oxidoreductase, EC 1.1.1.27) has been derived from analysis of a complementary DNA (cDNA) clone comprising the complete protein coding region of the enzyme. From the deduced amino acid sequence, human LDHC/sub 4/ is as different from rodent LDHC/sub 4/ (73% homology) as it is from human LDHA/sub 4/ (76% homology) and porcine LDHB/sub 4/ (68% homology). Subunit homologies are consistent with the conclusion that the LDHC gene arose by at least two independent duplication events. Furthermore, the lower degree of homology between mouse and human LDHC/submore » 4/ and the appearance of this isozyme late in evolution suggests a higher rate of mutation in the mammalian LDHC genes than in the LDHA and -B genes. Comparison of exposed amino acid residues of discrete anti-genic determinants of mouse and human LDHC/sub 4/ reveals significant differences. Knowledge of the human LDHC/sub 4/ sequence will help design human-specific peptides useful in the development of a contraceptive vaccine.« less

Backdoor pathway for dihydrotestosterone biosynthesis: implications for normal and abnormal human sex development.

PubMed

Fukami, Maki; Homma, Keiko; Hasegawa, Tomonobu; Ogata, Tsutomu

2013-04-01

We review the current knowledge about the "backdoor" pathway for the biosynthesis of dihydrotestosterone (DHT). While DHT is produced from cholesterol through the conventional "frontdoor" pathway via testosterone, recent studies have provided compelling evidence for the presence of an alternative "backdoor" pathway to DHT without testosterone intermediacy. This backdoor pathway is known to exist in the tammar wallaby pouch young testis and the immature mouse testis, and has been suggested to be present in the human as well. Indeed, molecular analysis has identified pathologic mutations of genes involved in the backdoor pathway in genetic male patients with undermasculinized external genitalia, and urine steroid profile analysis has argued for the relevance of the activated backdoor pathway to abnormal virilization in genetic females with cytochrome P450 oxidoreductase deficiency and 21-hydroxylase deficiency. It is likely that the backdoor pathway is primarily operating in the fetal testis in a physiological condition to produce a sufficient amount of DHT for male sex development, and that the backdoor pathway is driven with a possible interaction between fetal and permanent adrenals in pathologic conditions with increased 17-hydroxyprogesterone levels. These findings provide novel insights into androgen biosynthesis in both physiological and pathological conditions. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

Genome-wide mapping in a house mouse hybrid zone reveals hybrid sterility loci and Dobzhansky-Muller interactions.

PubMed

Turner, Leslie M; Harr, Bettina

2014-12-09

Mapping hybrid defects in contact zones between incipient species can identify genomic regions contributing to reproductive isolation and reveal genetic mechanisms of speciation. The house mouse features a rare combination of sophisticated genetic tools and natural hybrid zones between subspecies. Male hybrids often show reduced fertility, a common reproductive barrier between incipient species. Laboratory crosses have identified sterility loci, but each encompasses hundreds of genes. We map genetic determinants of testis weight and testis gene expression using offspring of mice captured in a hybrid zone between M. musculus musculus and M. m. domesticus. Many generations of admixture enables high-resolution mapping of loci contributing to these sterility-related phenotypes. We identify complex interactions among sterility loci, suggesting multiple, non-independent genetic incompatibilities contribute to barriers to gene flow in the hybrid zone.

Utilization of DR1 as true RARE in regulating the Ssm, a novel retinoic acid-target gene in the mouse testis.

PubMed

Han, Kyuyong; Song, Haengseok; Moon, Irene; Augustin, Robert; Moley, Kelle; Rogers, Melissa; Lim, Hyunjung

2007-03-01

Various nuclear receptors form dimers to activate target genes via specific response elements located within promoters or enhancers. Retinoid X receptor (RXR) serves as a dimerization partner for many nuclear receptors including retinoic acid receptor (RAR) and peroxisome proliferator-activated receptor (PPAR). Dimers show differential preference towards directly repeated response elements with 1-5 nucleotide spacing, and direct repeat 1 (DR1) is a promiscuous element which recruits RAR/RXR, RXR/RXR, and PPAR/RXR in vitro. In the present investigation, we report identification of a novel RAR/RXR target gene which is regulated by DR1s in the promoter region. This gene, namely spermatocyte-specific marker (Ssm), recruits all the three combinations of nuclear receptors in vitro, but in vivo regulation is observed by trans-retinoic acid-activated RAR/RXR dimer. Indeed, chromatin immunoprecipitation experiment demonstrates binding of RARbeta and RXRalpha in the promoter region of the Ssm. Interestingly, expression of Ssm is almost exclusively observed in spermatocytes in the adult mouse testis, where RA signaling is known to regulate developmental program of male germ cells. The results show that Ssm is a RAR/RXR target gene uniquely using DR1 and exhibits stage-specific expression in the mouse testis with potential function in later stages of spermatogenesis. This finding exemplifies usage of DR1s as retinoic acid response element (RARE) under a specific in vivo context.

Fidgetin-Like1 Is a Strong Candidate for a Dynamic Impairment of Male Meiosis Leading to Reduced Testis Weight in Mice

PubMed Central

L'Hôte, David; Vatin, Magalie; Auer, Jana; Castille, Johan; Passet, Bruno; Montagutelli, Xavier

2011-01-01

Background In a previous work, using an interspecific recombinant congenic mouse model, we reported a genomic region of 23 Mb on mouse chromosome 11 implicated in testis weight decrease and moderate teratozoospermia (∼20–30%), a Quantitative Trait Locus (QTL) called Ltw1. The objective of the present study is to identify the gene underlying this phenotype. Results In the present study, we refined the QTL position to a 5 Mb fragment encompassing only 11 genes. We showed that the low testis weight phenotype was due to kinetic alterations occurring during the first wave of the spermatogenesis where we could point out to an abnormal lengthening of spermatocyte prophase. We identify Fidgetin-like 1 (Fignl1) as the gene underlying the phenotype, since if fulfilled both the physiological and molecular characteristics required. Indeed, amongst the 11 positional candidates it is the only gene that is expressed during meiosis at the spermatocyte stage, and that presents with non-synonymous coding variations differentiating the two mouse strains at the origin of the cross. Conclusions This work prompted us to propose Fignl1 as a novel actor in mammal's male meiosis dynamics which has fundamental interest. Besides, this gene is a new potential candidate for human infertilities caused by teratozoospermia and blockades of spermatogenesis. In addition this study demonstrates that interspecific models may be useful for understanding complex quantitative traits. PMID:22110678

Loss of Mitogen-Activated Protein Kinase Kinase Kinase 4 (MAP3K4) Reveals a Requirement for MAPK Signalling in Mouse Sex Determination

PubMed Central

Bogani, Debora; Siggers, Pam; Brixey, Rachel; Warr, Nick; Beddow, Sarah; Edwards, Jessica; Williams, Debbie; Wilhelm, Dagmar; Koopman, Peter; Flavell, Richard A.; Chi, Hongbo; Ostrer, Harry; Wells, Sara; Cheeseman, Michael; Greenfield, Andy

2009-01-01

Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY) gonad, sex-determining region of the Y (SRY) protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK) signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg) mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4), a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas). These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and create a novel entry point into the molecular and cellular mechanisms underlying sex determination in mice and disorders of sexual development in humans. PMID:19753101

A comparative study of sperm production in two species of Australian arid zone rodents (Pseudomys australis, Notomys alexis) with marked differences in testis size.

PubMed

Peirce, E J; Breed, W G

2001-02-01

The plains rat, Pseudomys australis, and the spinifex hopping mouse, Notomys alexis, show marked differences in the size of their testes and in the number of spermatozoa within the epididymides. In the present study, the dynamics of sperm production and the duration of sperm transit along the male excurrent ducts were compared between these two species. The durations of the cycle of the seminiferous epithelium, spermatogenesis and sperm transit were determined by tracking cells using autoradiography after [(3)H]thymidine incorporation. Daily sperm production was determined from counts of testicular spermatids after homogenization and further estimates of sperm transit were obtained by dividing sperm reserves within the various regions of the extratesticular ducts by the daily sperm production of the attached testis. In the plains rat, the mean duration of the cycle of the seminiferous epithelium was 11.2 days, the duration of spermatogenesis was 45 days, daily sperm production was 2.6 x 10(7) spermatozoa per gram of testis and epididymal transit of spermatozoa took approximately 9 days (caput 0.8 days; corpus 1.5 days; cauda 6.5 days). In contrast, in the hopping mouse, the mean duration of the cycle of the seminiferous epithelium was 14 days, the duration of spermatogenesis was 56 days and daily sperm production per gram of testis was < 1.0 x 10(7). Epididymal transit of spermatozoa was completed in about 4 days (caput + corpus < 1 day; cauda 3 days); however, spermatozoa may be stored for an additional 1.5-2.0 days in the vas deferens. These results indicate that, in addition to small testes, the hopping mouse shows a low efficiency of sperm production, a relatively long duration of spermatogenesis and rapid passage of spermatozoa through the epididymis, all of which contribute to low epididymal sperm counts. These data are considered in relation to interspecific differences in sperm competition.

The roles of TAM receptor tyrosine kinases in the mammalian testis and immunoprivileged sites.

PubMed

Deng, Tingting; Chen, Qiaoyuan; Han, Daishu

2016-01-01

Three members of a receptor tyrosine kinase family, including Tyro3, Axl, and Mer, are collectively called as TAM receptors. TAM receptors have two common ligands, namely, growth arrest specific gene 6 (Gas6) and protein S (ProS). The TAM-Gas6/ProS system is essential for phagocytic removal of apoptotic cells, and plays critical roles in regulating immune response. Genetic studies have shown that TAM receptors are essential regulators of the tissue homeostasis in immunoprivileged sites, including the testis, retina and brain. The mechanisms by which the TAM-Gas6/ProS system regulates the tissue homeostasis in immunoprivileged sites are emerging. The roles of the TAM-Gas6/ProS system in regulating the immune privilege were intensively investigated in the mouse testis, and several studies were performed in the eye and brain. This review summarizes our current understanding of TAM signaling in the testis and other immunoprivileged tissues, as well as highlights topics that are worthy of further investigation.

Genome-wide mapping in a house mouse hybrid zone reveals hybrid sterility loci and Dobzhansky-Muller interactions

PubMed Central

Turner, Leslie M; Harr, Bettina

2014-01-01

Mapping hybrid defects in contact zones between incipient species can identify genomic regions contributing to reproductive isolation and reveal genetic mechanisms of speciation. The house mouse features a rare combination of sophisticated genetic tools and natural hybrid zones between subspecies. Male hybrids often show reduced fertility, a common reproductive barrier between incipient species. Laboratory crosses have identified sterility loci, but each encompasses hundreds of genes. We map genetic determinants of testis weight and testis gene expression using offspring of mice captured in a hybrid zone between M. musculus musculus and M. m. domesticus. Many generations of admixture enables high-resolution mapping of loci contributing to these sterility-related phenotypes. We identify complex interactions among sterility loci, suggesting multiple, non-independent genetic incompatibilities contribute to barriers to gene flow in the hybrid zone. DOI: http://dx.doi.org/10.7554/eLife.02504.001 PMID:25487987

Sex Reversal in C57BL/6J XY Mice Caused by Increased Expression of Ovarian Genes and Insufficient Activation of the Testis Determining Pathway

PubMed Central

Correa, Stephanie M.; Washburn, Linda L.; Kahlon, Ravi S.; Musson, Michelle C.; Bouma, Gerrit J.; Eicher, Eva M.; Albrecht, Kenneth H.

2012-01-01

Sex reversal can occur in XY humans with only a single functional WT1 or SF1 allele or a duplication of the chromosome region containing WNT4. In contrast, XY mice with only a single functional Wt1, Sf1, or Wnt4 allele, or mice that over-express Wnt4 from a transgene, reportedly are not sex-reversed. Because genetic background plays a critical role in testis differentiation, particularly in C57BL/6J (B6) mice, we tested the hypothesis that Wt1, Sf1, and Wnt4 are dosage sensitive in B6 XY mice. We found that reduced Wt1 or Sf1 dosage in B6 XYB6 mice impaired testis differentiation, but no ovarian tissue developed. If, however, a YAKR chromosome replaced the YB6 chromosome, these otherwise genetically identical B6 XY mice developed ovarian tissue. In contrast, reduced Wnt4 dosage increased the amount of testicular tissue present in Sf1+/− B6 XYAKR, Wt1+/− B6 XYAKR, B6 XYPOS, and B6 XYAKR fetuses. We propose that Wt1B6 and Sf1B6 are hypomorphic alleles of testis-determining pathway genes and that Wnt4B6 is a hypermorphic allele of an ovary-determining pathway gene. The latter hypothesis is supported by the finding that expression of Wnt4 and four other genes in the ovary-determining pathway are elevated in normal B6 XX E12.5 ovaries. We propose that B6 mice are sensitive to XY sex reversal, at least in part, because they carry Wt1B6 and/or Sf1B6 alleles that compromise testis differentiation and a Wnt4B6 allele that promotes ovary differentiation and thereby antagonizes testis differentiation. Addition of a “weak” Sry allele, such as the one on the YPOS chromosome, to the sensitized B6 background results in inappropriate development of ovarian tissue. We conclude that Wt1, Sf1, and Wnt4 are dosage-sensitive in mice, this dosage-sensitivity is genetic background-dependant, and the mouse strains described here are good models for the investigation of human dosage-sensitive XY sex reversal. PMID:22496664

Identification of tumor-restricted antigens NY-BR-1, SCP-1, and a new cancer/testis-like antigen NW-BR-3 by serological screening of a testicular library with breast cancer serum.

PubMed

Jäger, Dirk; Unkelbach, Marc; Frei, Claudia; Bert, Florian; Scanlan, Matthew J; Jäger, Elke; Old, Lloyd J; Chen, Yao-Tseng; Knuth, Alexander

2002-06-28

Serological analysis of recombinant cDNA expression libraries (SEREX) has led to the identification of several categories of new tumor antigens. We analyzed a testicular cDNA expression library with serum obtained from a breast cancer patient and isolated 13 genes designated NW-BR-1 through NW-BR-13. Of these, 3 showed tumor-restricted expression (NW-BR-1, -2 and -3), the others being expressed ubiquitously. NW-BR-3, representing 9 of 24 primary clones, showed tissue-restricted mRNA expression, being expressed in normal testis but not in 15 other normal tissues tested by Northern blotting. RT-PCR analysis showed strong NW-BR-3 expression in normal testis, weak expression in brain, kidney, trachea, uterus and normal prostate, and was negative in liver, heart, lung, colon, small intestine, bone marrow, breast, thymus, muscle, spleen, and stomach. NW-BR-3 mRNA expression was found in different tumor tissues and tumor cell lines by RT-PCR, thus showing a 'cancer/testis' (CT)-like mRNA expression pattern. NW-BR-3 shares 71% nucleotide and amino acid homology to a mouse gene cloned from mouse testicular tissue. Based on the mRNA expression pattern, NW-BR-3 represents a new candidate target gene for cancer immunotherapy. NW-BR-1 and NW-BR-2 also showed tumor-restricted mRNA expression. NW-BR-1 is a partial clone of the breast differentiation antigen NY-BR-1 previously identified by SEREX. NY-BR-1 is expressed in normal breast, testis and 80% of breast cancers. NW-BR-2 is identical to the CT antigen SCP-1, initially isolated by SEREX analysis of renal cancer. This study provides further evidence that SEREX is a powerful tool to identify new tumor antigens potentially relevant for immunotherapy approaches.

Wt1 dictates the fate of fetal and adult Leydig cells during development in the mouse testis.

PubMed

Wen, Qing; Zheng, Qiao-Song; Li, Xi-Xia; Hu, Zhao-Yuan; Gao, Fei; Cheng, C Yan; Liu, Yi-Xun

2014-12-15

Wilms' tumor 1 (Wt1) is a tumor suppressor gene encoding ∼24 zinc finger transcription factors. In the mammalian testis, Wt1 is expressed mostly by Sertoli cells (SCs) involved in testis development, spermatogenesis, and adult Leydig cell (ALC) steroidogenesis. Global knockout (KO) of Wt1 is lethal in mice due to defects in embryogenesis. Herein, we showed that Wt1 is involved in regulating fetal Leydig cell (FLC) degeneration and ALC differentiation during testicular development. Using Wt1(-/flox);Amh-Cre mice that specifically deleted Wt1 in the SC vs. age-matched wild-type (WT) controls, FLC-like-clusters were found in Wt1-deficient testes that remained mitotically active from postnatal day 1 (P1) to P56, and no ALC was detected at these ages. Leydig cells in mutant adult testes displayed morphological features of FLC. Also, FLC-like cells in adult mutant testes had reduced expression in ALC-associated genes Ptgds, Sult1e1, Vcam1, Hsd11b1, Hsd3b6, and Hsd17b3 but high expression of FLC-associated genes Thbs2 and Hsd3b1. Whereas serum LH and testosterone level in mutant mice were not different from controls, intratesticular testosterone level was significantly reduced. Deletion of Wt1 gene also perturbed the expression of steroidogenic enzymes Star, P450c17, Hsd3b6, Hsd3b1, Hsd17b1, and Hsd17b3. FLCs in adult mutant testes failed to convert androstenedione to testosterone due to a lack of Hsd17b3, and this defect was rescued by coculturing with fetal SCs. In summary, FLC-like cells in mutant testes are putative FLCs that remain mitotically active in adult mice, illustrating that Wt1 dictates the fate of FLC and ALC during postnatal testis development. Copyright © 2014 the American Physiological Society.

The fragmented testis method: development and its advantages of a new quantitative evaluation technique for detection of testis-ova in male fish.

PubMed

Lin, Bin-Le; Hagino, Satoshi; Kagoshima, Michio; Iwamatsu, Takashi

2009-02-01

A new quantitative evaluation technique, termed the fragmented testis method, has been developed for the detection of testis-ova in genotypic male fish using the medaka (Oryzias latipes). The routine traditional histological method for detection of testis-ova in male fish exposed to estrogens or suspected endocrine-disrupting chemicals has several disadvantages, including possible oversight of testis-ova due to limited sampling of selected tissue sections. The method we have developed here allows for the accurate determination of the developmental stages and the number and the size of testis-ova in a whole testis. Each testis was removed from the fish specimen, fixed with 10% buffered formalin solution, and then divided into small fragments on a glass slide with a dissecting needle or scalpel and aciform forceps in glycerin solution containing a small amount of methylene blue or toluidine blue. If present, all developing testis-ova of various sizes in fragmented testicular tissues were clearly stained and were observable under a dissecting microscope. Testis-ova occurred in controls were ascertained, while spermatozoa were also distinguishable using this method. This proved to be a convenient and cost-effective method for quantitatively evaluating testis-ova appearance in fish, and it may help to clarify the mechanism of testis-ova formation and the biological significance of testis-ova in future studies of endocrine disruption.

Cloning and sequence analysis of a cDNA encoding the alpha-subunit of mouse beta-N-acetylhexosaminidase and comparison with the human enzyme.

PubMed Central

Beccari, T; Hoade, J; Orlacchio, A; Stirling, J L

1992-01-01

cDNAs encoding the mouse beta-N-acetylhexosaminidase alpha-subunit were isolated from a mouse testis library. The longest of these (1.7 kb) was sequenced and showed 83% similarity with the human alpha-subunit cDNA sequence. The 5' end of the coding sequence was obtained from a genomic DNA clone. Alignment of the human and mouse sequences showed that all three putative N-glycosylation sites are conserved, but that the mouse alpha-subunit has an additional site towards the C-terminus. All eight cysteines in the human sequence are conserved in the mouse. There are an additional two cysteines in the mouse alpha-subunit signal peptide. All amino acids affected in Tay-Sachs-disease mutations are conserved in the mouse. Images Fig. 1. PMID:1379046

Cloning of apg-2 encoding a novel member of heat shock protein 110 family.

PubMed

Kaneko, Y; Kimura, T; Kishishita, M; Noda, Y; Fujita, J

1997-04-11

Chinese hamster heat shock protein 110-encoding gene (hsp110), mouse apg-1 and human hsp70RY are structurally related genes, with the first two encoding about 110-kDa HSPs [Yoon et al. (1995) J. Biol. Chem. 270, 15725-15733; Kaneko et al. (1997) J. Biol. Chem., in press; Fathallah et al. (1993) J. Immunol. 151, 810-813]. Using apg-1 cDNA as a probe, we isolated a novel cDNA, apg-2 from a mouse testis cDNA library, which was highly homologous to human hsp70RY. However, the predicted amino acid (aa) sequence of APG-2 was longer (841 aa) than that of HSP70RY (701 aa) and comparable to those of HSP110 and APG-1. Northern blot analysis revealed that the expression of apg-2 transcripts was ubiquitous in various mouse tissues, and most abundant in the testis and ovary. While induction of hsp70 transcripts was observed in mouse TAMA26 Sertoli cells and NIH/3T3 fibroblasts on temperature shift from 37 degrees C to 42 degrees C (traditional heat shock) or from 32 degrees C to 39 degrees C, apg-2 transcripts were not induced under either condition. These results suggest that apg-2 is an isoform of mouse homolog of hsp70RY, but that it belongs to the hsp110 family instead of hsp70 family, and that it plays a role under non-stress conditions.

Expression Profile of NOTCH3 in Mouse Spermatogonia.

PubMed

Okada, Ryu; Fujimagari, Megumi; Koya, Eri; Hirose, Yoshikazu; Sato, Tomomi; Nishina, Yukio

2017-01-01

Stable and sustainable spermatogenesis is supported by the strict regulation of self-renewal and differentiation of spermatogonial stem cells (SSC), which are a rare population of undifferentiated spermatogonia. It has been revealed that some signaling factors regulate the self-renewal of SSC; however, the molecular mechanism of SSC maintenance is still not completely understood. Notch signaling is an evolutionarily conserved juxtacrine signaling that plays important roles in the cell fate determination of various tissue stem cells. Recently, analyses of loss- and gain-of-function suggested that Notch signaling was necessary for normal spermatogenesis. However, the expression of Notch signal components in spermatogonia is still unclear. Here, we analyzed the distribution of NOTCH3-expressing spermatogonia and the target genes. Double immunostaining with differentiation markers revealed that NOTCH3 was expressed in some undifferentiated and differentiated spermatogonia in mouse testes. To define the target gene of Notch3 signaling in spermatogonia, we analyzed the mRNA expression pattern of Hes and Hey family genes during testis development. Hes1 abundance was decreased during testis development, suggesting that spermatogonia may express Hes1. Immunohistochemical analysis showed that HES1 was expressed in prepubertal spermatogonia, whereas it was expressed predominantly in adult Sertoli cells and weakly in adult spermatogonia. Furthermore, NOTCH3-HES1 double-positive spermatogonia were in pup and adult testes. These results suggest that Notch3 signaling in spermatogonia could promote Hes1 expression. © 2017 S. Karger AG, Basel.

Insulin Rescues Impaired Spermatogenesis via the Hypothalamic-Pituitary-Gonadal Axis in Akita Diabetic Mice and Restores Male Fertility

PubMed Central

Schoeller, Erica L.; Albanna, Gabriella; Frolova, Antonina I.; Moley, Kelle H.

2012-01-01

The mechanism responsible for poor reproductive outcomes in type 1 diabetic males is not well understood. In light of new evidence that the Sertoli cells of the testis secrete insulin, it is currently unclear whether diabetic subfertility is the result of deficiency of pancreatic insulin, testicular insulin, or both. In this study, the Akita mouse diabetic model, which expresses a mutant, nonfunctional form of ins2 in testes and pancreas, was used to distinguish between systemic and local effects of insulin deficiency on the process of spermatogenesis and fertility. We determined that Akita homozygous male mice are infertile and have reduced testis size and abnormal morphology. Spermatogonial germ cells are still present but are unable to mature into spermatocytes and spermatids. Exogenous insulin treatment regenerates testes and restores fertility, but this plasma insulin cannot pass through the blood-testis barrier. We conclude that insulin does not rescue fertility through direct interaction with the testis; instead, it restores function of the hypothalamic-pituitary-gonadal axis and, thus, normalizes hormone levels of luteinizing hormone and testosterone. Although we show that the Sertoli cells of the testis secrete insulin protein, this insulin does not appear to be critical for fertility. PMID:22522616

Gene knockout of Zmym3 in mice arrests spermatogenesis at meiotic metaphase with defects in spindle assembly checkpoint.

PubMed

Hu, Xiangjing; Shen, Bin; Liao, Shangying; Ning, Yan; Ma, Longfei; Chen, Jian; Lin, Xiwen; Zhang, Daoqin; Li, Zhen; Zheng, Chunwei; Feng, Yanmin; Huang, Xingxu; Han, Chunsheng

2017-06-29

ZMYM3, a member of the MYM-type zinc finger protein family and a component of a LSD1-containing transcription repressor complex, is predominantly expressed in the mouse brain and testis. Here, we show that ZMYM3 in the mouse testis is expressed in somatic cells and germ cells until pachytene spermatocytes. Knockout (KO) of Zmym3 in mice using the CRISPR-Cas9 system resulted in adult male infertility. Spermatogenesis of the KO mice was arrested at the metaphase of the first meiotic division (MI). ZMYM3 co-immunoprecipitated with LSD1 in spermatogonial stem cells, but its KO did not change the levels of LSD1 or H3K4me1/2 or H3K9me2. However, Zmym3 KO resulted in elevated numbers of apoptotic germ cells and of MI spermatocytes that are positive for BUB3, which is a key player in spindle assembly checkpoint. Zmym3 KO also resulted in up-regulated expression of meiotic genes in spermatogonia. These results show that ZMYM3 has an essential role in metaphase to anaphase transition during mouse spermatogenesis by regulating the expression of diverse families of genes.

The C-X-C signalling system in the rodent vs primate testis: impact on germ cell niche interaction.

PubMed

Heckmann, Laura; Pock, Tim; Tröndle, Ina; Neuhaus, Nina

2018-05-01

In zebrafish, action of the chemokine Cxcl12 is mediated through its G-protein-coupled seven-transmembrane domain receptor Cxcr4 and the atypical receptor Cxcr7. Employing this animal model, it was revealed that this Cxcl12 signalling system plays a crucial role for directed migration of primordial germ cells (PGC) during early testicular development. Importantly, subsequent studies indicated that this regulatory mechanism is evolutionarily conserved also in mice. What is more, the functional role of the CXCL12 system does not seem to be limited to early phases of testicular development. Data from mouse studies rather demonstrate that CXCL12 and its receptors are also involved in the homing process of gonocytes into their niches at the basal membrane of the seminiferous tubules. Intriguingly, even the spermatogonial stem cells (SSCs) present in the adult mouse testis appear to maintain the ability to migrate towards a CXCL12 gradient as demonstrated by functional in vitro migration assays and in vivo germ cell transplantation assays. These findings not only indicate a role of the CXCL12 system throughout male germ cell development in mice but also suggest that this system may be evolutionarily conserved. In this review, we take into account the available literature focusing on the localization patterns of the CXCL12 system not only in rodents but also in primates, including the human. Based on these data, we discuss whether the CXCL12 system is also conserved between rodents and primates and discuss the known and potential functional consequences. © 2018 Society for Reproduction and Fertility.

Age-specific absolute and relative organ weight distributions for B6C3F1 mice.

PubMed

Marino, Dale J

2012-01-01

The B6C3F1 mouse is the standard mouse strain used in toxicology studies conducted by the National Cancer Institute (NCI) and the National Toxicology Program (NTP). While numerous reports have been published on growth, survival, and tumor incidence, no overall compilation of organ weight data is available. Importantly, organ weight change is an endpoint used by regulatory agencies to develop toxicity reference values (TRVs) for use in human health risk assessments. Furthermore, physiologically based pharmacokinetic (PBPK) models, which utilize relative organ weights, are increasingly being used to develop TRVs. Therefore, all available absolute and relative organ weight data for untreated control B6C3F1 mice were collected from NCI/NTP studies in order to develop age-specific distributions. Results show that organ weights were collected more frequently in NCI/NTP studies at 2-wk (60 studies), 3-mo (147 studies), and 15-mo (40 studies) intervals than at other intervals, and more frequently from feeding and inhalation than drinking water studies. Liver, right kidney, lung, heart, thymus, and brain weights were most frequently collected. From the collected data, the mean and standard deviation for absolute and relative organ weights were calculated. Results show age-related increases in absolute liver, right kidney, lung, and heart weights and relatively stable brain and right testis weights. The results suggest a general variability trend in absolute organ weights of brain < right testis < right kidney < heart < liver < lung < spleen < thymus. This report describes the results of this effort.

Mouse RC/BTB2, a Member of the RCC1 Superfamily, Localizes to Spermatid Acrosomal Vesicles

PubMed Central

Shen, Xuening; Nagarkatti-Gude, David R.; Hess, Rex A.; Henderson, Scott C.; Strauss, Jerome F.; Zhang, Zhibing

2012-01-01

Mouse RC/BTB2 is an unstudied protein of the RCC1 (Regulator of Chromosome Condensation) superfamily. Because of the significant remodeling of chromatin that occurs during spermiogenesis, we characterized the expression and localization of mouse RC/BTB2 in the testis and male germ cells. The Rc/btb2 gene yields two major transcripts: 2.3 kb Rc/btb2-s, present in most somatic tissues examined; and 2.5 kb Rc/btb2-t, which contains a unique non-translated exon in its 5′-UTR that is only detected in the testis. During the first wave of spermatogenesis, Rc/btb2-t mRNA is expressed from day 8 after birth, reaching highest levels of expression at day 30 after birth. The full-length protein contains three RCC1 domains in the N-terminus, and a BTB domain in the C-terminus. In the testis, the protein is detectable from day 12, but is progressively up-regulated to day 30 and day 42 after birth. In spermatids, some of the protein co-localizes with acrosomal markers sp56 and peanut lectin, indicating that it is an acrosomal protein. A GFP-tagged RCC1 domain is present throughout the cytoplasm of transfected CHO cells. However, both GFP-tagged, full-length RC/BTB2 and a GFP-tagged BTB domain localize to vesicles in close proximity to the nuclear membrane, suggesting that the BTB domain might play a role in mediating full-length RC/BTB2 localization. Since RCC1 domains associate with Ran, a small GTPase that regulates molecular trafficking, it is possible that RC/BTB2 plays a role in transporting proteins during acrosome formation. PMID:22768142

Separation of mouse testis cells on a Celsep (TM) apparatus and their usefulness as a source of high molecular weight DNA or RNA

NASA Technical Reports Server (NTRS)

Wolgemuth, D. J.; Gizang-Ginsberg, E.; Engelmyer, E.; Gavin, B. J.; Ponzetto, C.

1985-01-01

The use of a self-contained unit-gravity cell separation apparatus for separation of populations of mouse testicular cells is described. The apparatus, a Celsep (TM), maximizes the unit area over which sedimentation occurs, reduces the amount of separation medium employed, and is quite reproducible. Cells thus isolated have been good sources for isolation of DNA, and notably, high molecular weight RNA.

Promoter mapping of the mouse Tcp-10bt gene in transgenic mice identifies essential male germ cell regulatory sequences.

PubMed

Ewulonu, U K; Snyder, L; Silver, L M; Schimenti, J C

1996-03-01

Transgenic mice were generated to localize essential promoter elements in the mouse testis-expressed Tcp-10 genes. These genes are expressed exclusively in male germ cells, and exhibit a diffuse range of transcriptional start sites, possibly due to the absence of a TATA box. A series of transgene constructs containing different amounts of 5' flanking DNA revealed that all sequences necessary for appropriate temporal and tissue-specific transcription of Tcp-10 reside between positions -1 to -973. All transgenic animals containing these sequences expressed a chimeric transgene at high levels, in a pattern that paralleled the endogenous genes. These experiments further defined a 227 bp fragment from -746 to -973 that was absolutely essential for expression. In a gel-shift assay, this 227-bp fragment bound nuclear protein from testis, but not other tissues, to yield two retarded bands. Sequence analysis of this fragment revealed a half-site for the AP-2 transcription factor recognition sequence. Gel shift assays using native or mutant oligonucleotides demonstrated that the putative AP-2 recognition sequence was essential for generating the retarded bands. Since the binding activity is testis-specific, but AP-2 expression is not exclusive to male germ cells, it is possible that transcription of Tcp-10 requires interaction between AP-2 and a germ cell-specific transcription factor.

Enriched expression of the ciliopathy gene Ick in cell proliferating regions of adult mice.

PubMed

Tsutsumi, Ryotaro; Chaya, Taro; Furukawa, Takahisa

2018-04-07

Cilia are essential for sensory and motile functions across species. In humans, ciliary dysfunction causes "ciliopathies", which show severe developmental abnormalities in various tissues. Several missense mutations in intestinal cell kinase (ICK) gene lead to endocrine-cerebro-osteodysplasia syndrome or short rib-polydactyly syndrome, lethal recessive developmental ciliopathies. We and others previously reported that Ick-deficient mice exhibit neonatal lethality with developmental defects. Mechanistically, Ick regulates intraflagellar transport and cilia length at ciliary tips. Although Ick plays important roles during mammalian development, roles of Ick at the adult stage are poorly understood. In the current study, we investigated the Ick gene expression in adult mouse tissues. RT-PCR analysis showed that Ick is ubiquitously expressed, with enrichment in the retina, brain, lung, intestine, and reproductive system. In the adult brain, we found that Ick expression is enriched in the walls of the lateral ventricle, in the rostral migratory stream of the olfactory bulb, and in the subgranular zone of the hippocampal dentate gyrus by in situ hybridization analysis. We also observed that Ick staining pattern is similar to pachytene spermatocyte to spermatid markers in the mature testis and to an intestinal stem cell marker in the adult small intestine. These results suggest that Ick is expressed in proliferating regions in the adult mouse brain, testis, and intestine. Copyright © 2018 Elsevier B.V. All rights reserved.

Pattern and density of vascularization in mammalian testes, ovaries, and ovotestes.

PubMed

Lupiáñez, Darío G; Real, Francisca M; Dadhich, Rajesh K; Carmona, Francisco D; Burgos, Miguel; Barrionuevo, Francisco J; Jiménez, Rafael

2012-05-01

According to the classical paradigm, the vasculature of the embryonic testis is more dense and complex than that of the ovary, but recent studies based on whole-mount detection of Caveolin-1 (CAV1) as an endothelial cell marker, have suggested that the level of ovarian vascularization is higher than previously assumed. However, this new hypothesis has been neither tested using alternative methodology nor investigated in other mammalian species. In this paper, we have studied the vascularization process in the gonads of males and females of two mammalian species, the mouse (Mus musculus) and the Iberian mole (Talpa occidentalis). Our results show that the pattern of testis vascularization is very well conserved among mammals, including both pre- and postnatal stages of development and, at least in the mole, it is conserved irrespectively of whether the testicular tissue is XY or XX. We have shown that CAV1 is present not only in endothelial cells but also in prefollicular oocytes and in an ovarian population of somatic cortical cells. These data clearly establish that: (1) according to the classical hypothesis, the degree of vascularization of the developing ovary is lower than that of the testis, (2) ovarian vascularization is also evolutionarily conserved as it occurs similarly both in moles and in mice, and (3) that the degree of vascular development of the mammalian ovary is age-dependent increasing significatively at puberty. The expression of CAV1 in the ovary of most animal taxa, from nematodes to mammals, strongly suggests a role for this gene in the female meiosis. © 2012 WILEY PERIODICALS, INC.

Tissue distribution and developmental expression of type XVI collagen in the mouse.

PubMed

Lai, C H; Chu, M L

1996-04-01

The expression of a recently identified collagen, alpha 1 (XVI), in adult mouse tissue and developing mouse embryo was examined by immunohistochemistry and in situ hybridization. A polyclonal antiserum was raised against a recombinant fusion protein, which contained a segment of 161 amino acids in the N-terminal noncollagenous domain of the human alpha 1 (XVI) collagen. Immunoprecipitation of metabolically labelled human or mouse fibroblast cell lysates with this antibody revealed a major, bacterial collagenase sensitive polypeptide of approximately 210 kDa. The size agrees with the prediction from the full-length cDNA. Immunofluorescence examination of adult mouse tissues using the affinity purified antibody revealed a rather broad distribution of the protein. The heart, kidney, intestine, ovary, testis, eye, arterial walls and smooth muscles all exhibited significant levels of expression, while the skeletal muscle, lung and brain showed very restricted and low signals. During development, no significant expression of the mRNA or protein was observed in embryo of day 8 of gestation, but strong signals was detected in placental trophoblasts. Expression in embryos was detectable first after day 11 of gestation with weak positive signals appearing in the heart. In later stages of development, stronger RNA hybridizations were observed in a variety of tissues, particularly in atrial and ventricular walls of the developing heart, spinal root neural fibers and skin. These data demonstrate that type XVI collagen represents another collagenous component widely distributed in the extracellular matrix and may contribute to the structural integrity of various tissues.

Validation of an automated counting procedure for phthalate-induced testicular multinucleated germ cells

EPA Science Inventory

In utero exposure to certain phthalate esters results in testicular toxicity, characterized at the tissue level by induction of multinucleated germ cells (MNGs) in rat, mouse, and human fetal testis. Phthalate exposures also result in a decrease in testicular testosterone in rats...

Sex-biased miRNAs in gonad and their potential roles for testis development in yellow catfish.

PubMed

Jing, Jing; Wu, Junjie; Liu, Wei; Xiong, Shuting; Ma, Wenge; Zhang, Jin; Wang, Weimin; Gui, Jian-Fang; Mei, Jie

2014-01-01

Recently, YY super-male yellow catfish had been created by hormonal-induced sex reversal and sex-linked markers, which provides a promising research model for fish sex differentiation and gonad development, especially for testis development. MicroRNAs (miRNAs) have been revealed to play crucial roles in the gene regulation and gonad development in vertebrates. In this study, three small RNA libraries constructed from gonad tissues of XX female, XY male and YY super-male yellow catfish were sequenced. The sequencing data generated a total of 384 conserved miRNAs and 113 potential novel miRNAs, among which 23, 30 and 14 miRNAs were specifically detected in XX ovary, XY testis, and YY testis, respectively. We observed relative lower expression of several miR-200 family members, including miR-141 and miR-429 in YY testis compared with XY testis. Histological analysis indicated a higher degree of testis maturity in YY super-males compared with XY males, as shown by larger spermatogenic cyst, more spermatids and fewer spermatocytes in the spermatogenic cyst. Moreover, five miR-200 family members were significantly up-regulated in testis when treated by 17α-ethinylestradiol (EE2), high dose of which will impair testis development and cell proliferation. The down-regulation of miR-141 and 429 coincides with the progression of testis development in both yellow catfish and human. At last, the expression pattern of nine arbitrarily selected miRNAs detected by quantitative RT-PCR was consistent with the Solexa sequencing results. Our study provides a comprehensive miRNA transcriptome analysis for gonad of yellow catfish with different sex genotypes, and identifies a number of sex-biased miRNAs, some of that are potentially involved in testis development and spermatogenesis.

Expression of Apg-1, a member of the Hsp110 family, in the human testis and sperm.

PubMed

Nonoguchi, K; Tokuchi, H; Okuno, H; Watanabe, H; Egawa, H; Saito, K; Ogawa, O; Fujita, J

2001-06-01

Apg-1 encodes a heat shock protein belonging to the Hsp110 family and is inducible by a 32 degrees C to 39 degrees C heat shock in somatic cells. In mouse testicular germ cells Apg-1 mRNA is constitutively expressed depending on the developmental stage. As human Apg-1 has recently been identified, the expression of Apg-1 in the human testis and sperm was investigated. Expression and heat-inducibility of Apg-1 in the human testicular germ cell tumor cell line, NEC8, was analyzed. Using an antimouse Apg-1 antibody, expression of Apg-1 in the human testis and sperm was examined by western blotting after confirmation of the specificity of the antibody. The cells expressing Apg-1 in the testis were further determined by immunohistochemistry. Slight induction of Apg-1 mRNA was detected in NEC8 cells after 32 degrees C to 39 degrees C temperature shift. In the human testis, the antibody specifically recognized Apg-1, which was absent in the testis without germ cells (Sertoli-cell-only syndrome) or arrested at spermatogonia. Spermatocytes and spermatids, but not testicular somatic cells, were positively stained with the anti-Apg-1 antibody. By western blot analysis, Apg-1 was detected in the preparation enriched for sperm from normal volunteers and infertile patients, but not from azoospermia patients. Apg-1 is developmentally expressed in human testicular germ cells and sperm, suggesting its role in spermatogenesis and fertilization. Identification of substrates for Apg-1 chaperone activity will help elucidate its function.

Proteolytic degradation of heat shock protein A2 occurs in response to oxidative stress in male germ cells of the mouse.

PubMed

Bromfield, Elizabeth G; Aitken, R John; McLaughlin, Eileen A; Nixon, Brett

2017-02-10

Does oxidative stress compromise the protein expression of heat shock protein A2 (HSPA2) in the developing germ cells of the mouse testis? Oxidative stress leads to the modification of HSPA2 by the lipid aldehyde 4-hydroxynonenal (4HNE) and initiates its degradation via the ubiquitin-proteasome system. Previous work has revealed a deficiency in HSPA2 protein expression within the spermatozoa of infertile men that have failed fertilization in a clinical setting. While the biological basis of this reduction in HSPA2 remains to be established, we have recently shown that the HSPA2 expressed in the spermatozoa of normozoospermic individuals is highly susceptible to adduction, a form of post-translational modification, by the lipid aldehyde 4HNE that has been causally linked to the degradation of its substrates. This modification of HSPA2 by 4HNE adduction dramatically reduced human sperm-egg interaction in vitro. Moreover, studies in a mouse model offer compelling evidence that the co-chaperone BCL2-associated athanogene 6 (BAG6) plays a key role in regulating the stability of HSPA2 in the testis, by preventing its ubiquitination and subsequent proteolytic degradation. Dose-dependent studies were used to establish a 4HNE-treatment regime for primary culture(s) of male mouse germ cells. The influence of 4HNE on HSPA2 protein stability was subsequently assessed in treated germ cells. Additionally, sperm lysates from infertile patients with established zona pellucida recognition defects were examined for the presence of 4HNE and ubiquitin adducts. A minimum of three biological replicates were performed to test statistical significance. Oxidative stress was induced in pachytene spermatocytes and round spermatids isolated from the mouse testis, as well as a GC-2 cell line, using 50-200 µM 4HNE or hydrogen peroxide (H2O2), and the expression of HSPA2 was monitored via immunocytochemistry and immunoblotting approaches. Using the GC-2 cell line as a model, the ubiquitination and degradation of HSPA2 was assessed using immunoprecipitation techniques and pharmacological inhibition of proteasomal and lysosomal degradation pathways. Finally, the interaction between BAG6 and HSPA2 was examined in response to 4HNE exposure via proximity ligation assays. HSPA2 protein levels were significantly reduced compared with controls after 4HNE treatment of round spermatids (P < 0.01) and GC-2 cells (P < 0.001) but not pachytene spermatocytes. Using GC-2 cells as a model, HSPA2 was shown to be both adducted by 4HNE and targeted for ubiquitination in response to cellular oxidative stress. Inhibition of the proteasome with MG132 prevented HSPA2 degradation after 4HNE treatment indicating that the degradation of HSPA2 is likely to occur via a proteasomal pathway. Moreover, our assessment of proteasome activity provided evidence that 4HNE treatment can significantly increase the proteasome activity of GC-2 cells (P < 0.05 versus control). Finally, 4HNE exposure to GC-2 cells resulted in the dissociation of HSPA2 from its regulatory co-chaperone BAG6, a key mediator of HSPA2 stability in male germ cells. While these experiments were performed using a mouse germ cell-model system, our analyses of patient sperm lysate imply that these mechanisms are conserved between mouse and human germ cells. This study suggests a causative link between non-enzymatic post-translational modifications and the relative levels of HSPA2 in the spermatozoa of a specific sub-class of infertile males. In doing so, this work enhances our understanding of failed sperm-egg recognition and may assist in the development of targeted antioxidant-based approaches for ameliorating the production of cytotoxic lipid aldehydes in the testis in an attempt to prevent this form of infertility. Not applicable. This work was supported by the National Health and Medical Research Council of Australia (APP1101953). The authors have no competing interests to declare. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Cloning and characterization of murine fanconi anemia group A gene: Fanca protein is expressed in lymphoid tissues, testis, and ovary.

PubMed

van de Vrugt, H J; Cheng, N C; de Vries, Y; Rooimans, M A; de Groot, J; Scheper, R J; Zhi, Y; Hoatlin, M E; Joenje, H; Arwert, F

2000-04-01

Fanconi anemia (FA) is an autosomal recessive disorder in humans characterized by bone marrow failure, cancer predisposition, and cellular hypersensitivity to cross-linking agents such as mitomycin C and diepoxybutane. FA genes display a caretaker function essential for maintenance of genomic integrity. We have cloned the murine homolog of FANCA, the gene mutated in the major FA complementation group (FA-A). The full-length mouse Fanca cDNA consists of 4503 bp and encodes a protein with a predicted molecular weight of 161 kDa. The deduced Fanca mouse protein shares 81% amino acid sequence similarity and 66% identity with the human protein. The nuclear localization signal and partial leucine zipper consensus motifs found in the human FANCA protein were also present in the murine homolog. In spite of the species difference, the murine Fanca cDNA was capable of correcting the cross-linker sensitive phenotype of human FA-A cells, suggesting functional conservation. Based on Northern as well as Western blots, Fanca was mainly expressed in lymphoid tissues, testis, and ovary. This expression pattern correlates with some of the clinical symptoms observed in FA patients. The availability of the murine Fanca cDNA now allows the gene to be studied in experimental mouse models.

Testis tumor associated to microlithiasis.

PubMed

Jesus, Lisieux Eyer de; Maciel, Felipe; Monnerat, Andrea Lima C; Fernandes, Marcia Antunes; Dekermache, Samuel

2013-12-01

To discuss the relationship between testicular microlithiasis and testis tumors in children and to consider the chances of testis preserving surgery in specific cases. Pre-adolescent presenting testicular microlithiasis and a larger left testis, corresponding to a cystic testicular tumor. The tumor was excised, with ipsilateral testis preservation. Histology diagnosed a testis dermoid tumor. The relationship between testis tumors and testicular microlithiasis is ill defined in children. Pediatric urologists need to develop specific follow-up protocols for pre-pubertal children.

High levels of the extracellular matrix proteoglycan decorin are associated with inhibition of testicular function

PubMed Central

Adam, Marion; Urbanski, Henryk F.; Garyfallou, Vasilios T.; Welsch, Ulrich; Köhn, Frank M.; Schwarzer, J. Ullrich; Strauss, Leena; Poutanen, Matti; Mayerhofer, Artur

2011-01-01

Decorin (DCN), a component of the extracellular matrix of the peritubular wall and the interstitial areas of the human testis, can interact with growth factor (GF) signaling, thereby blocking downstream actions of GFs. In the present study the expression and regulation of DCN using both human testes and two experimental animal models, namely the rhesus monkey and mouse, were examined. DCN protein was present in peritubular and interstitial areas of adult human and monkey testes, while it was almost undetectable in adult wild-type mice. Interestingly, the levels and sites of testicular DCN expression in the monkeys were inversely correlated with testicular maturation markers. A strong DCN expression associated with the abundant connective tissue of the interstitial areas in the postnatal through prepubertal phases was observed. In adult and old monkeys the DCN pattern was similar to the one in normal human testes, presenting strong expression at the peritubular region. In the testes of both infertile men and in a mouse model of inflammation associated infertility (aromatase-overexpressing transgenic mice), the fibrotic changes and increased numbers of Tumor necrosis factor (TNF)-α-producing immune cells were shown to be associated with increased production of DCN. Furthermore, studies with human testicular peritubular cells isolated from fibrotic testis indicated that TNF-α significantly increased DCN production. The data, thus, show that an increased DCN level is associated with impaired testicular function, supporting our hypothesis that DCN interferes with paracrine signaling of the testis in health and disease. PMID:22413766

Identification of mediator complex 26 (Crsp7) gametologs on platypus X1 and Y5 sex chromosomes: a candidate testis-determining gene in monotremes?

PubMed

Tsend-Ayush, Enkhjargal; Kortschak, R Daniel; Bernard, Pascal; Lim, Shu Ly; Ryan, Janelle; Rosenkranz, Ruben; Borodina, Tatiana; Dohm, Juliane C; Himmelbauer, Heinz; Harley, Vincent R; Grützner, Frank

2012-01-01

The basal lineage of monotremes features an extraordinarily complex sex chromosome system which has provided novel insights into the evolution of mammalian sex chromosomes. Recently, sequence information from autosomes, X chromosomes, and XY-shared pseudoautosomal regions has become available. However, no gene has so far been described on any of the Y chromosome-specific regions. We analyzed sequences derived from Y-specific BAC clones to identify genes with potentially male-specific function. Here, we report the identification and characterization of the mediator complex protein gametologs on platypus Y5 (Crspy). We also identified the X-chromosomal copy which unexpectedly maps to X1 (Crspx). Sequence comparison shows extensive divergence between the X and Y copy, but we found no significant positive selection on either gametolog. Expression analysis shows widespread expression of Crspx. Crspy is expressed exclusively in males with particularly strong expression in testis and kidney. Reporter gene assays to investigate whether Crspx/y can act on the recently discovered mouse Sox9 testis-specific enhancer element did reveal a modest effect together with mouse Sox9 + Sf1, but showed overall no significant upregulation of the reporter gene. This is the first report of a differentiated functional male-specific gene on platypus Y chromosomes, providing new insights into sex chromosome evolution and a candidate gene for male-specific function in monotremes.

The Non-Peptide Vasopressin V1b Receptor Antagonist, SSR149415, Ameliorates Spermatogenesis Function in a Mouse Model of Chronic Social Defeat Stress.

PubMed

Wang, Bin; Zhou, Jian; Zhuang, Yan-Yan; Wang, Liang-Liang; Pu, Jin-Xian; Huang, Yu-Hua; Xia, Fei; Lv, Jin-Xing

2017-11-01

To determine the effects of SSR149415 on testis and spermatogenesis in male mice subjected to chronic social defeat stress, C57BL/6 male mice were divided into two groups: Control and Stress. Then Stress group was subdivided into four subgroups administered water, SSR149415 (1 mg/kg/day), SSR149415 (10 mg/kg/day), SSR149415 (30 mg/kg/day), respectively. The behavioral alterations revealed by social interaction test and open field test were measured. The physical indices, including body weight and gonad weight (testis and epididymis) as well as testis/body weight and cauda epididymis/body weight were detected. Serum hormones, including testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were determined. Sperm count and abnormality as well as testicular histology structure were assessed. The germ cells apoptosis were also evaluated. Chronic social defeat stress-induced behavioral abnormality, as well as gonad atrophy (testis and epididymis) was significantly alleviated in stressed male mice exposed to SSR149415. Regressed serum testosterone levels and elevated serum FSH and LH levels exhibited by stressed male mice were observably reversed following SSR149415 administration. Chronic social defeat stress-induced damage in testicular histology structure and semen quality were also improved after SSR149415 administration. In addition, SSR149415 significantly reversed chronic social defeat stress-induced germ cells apoptosis. Overall, we provide clear evidence indicating the amelioration of chronic social defeat stress-induced behavioral abnormality and testicular dysfunction via SSR149415, promoting the development of drug-directed therapy against this disease. J. Cell. Biochem. 118: 3891-3898, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Testis tumor associated to microlithiasis

PubMed Central

de Jesus, Lisieux Eyer; Maciel, Felipe; Monnerat, Andrea Lima C.; Fernandes, Marcia Antunes; Dekermache, Samuel

2013-01-01

OBJECTIVE: To discuss the relationship between testicular microlithiasis and testis tumors in children and to consider the chances of testis preserving surgery in specific cases. CASE DESCRIPTION: Pre-adolescent presenting testicular microlithiasis and a larger left testis, corresponding to a cystic testicular tumor. The tumor was excised, with ipsilateral testis preservation. Histology diagnosed a testis dermoid tumor. COMMENTS: The relationship between testis tumors and testicular microlithiasis is ill defined in children. Pediatric urologists need to develop specific follow-up protocols for pre-pubertal children. PMID:24473964

Effect of the anti-androgenic endocrine disruptor vinclozolin on embryonic testis cord formation and postnatal testis development and function.

PubMed

Uzumcu, Mehmet; Suzuki, Hiroetsu; Skinner, Michael K

2004-01-01

Vinclozolin is a systemic dicarboximide fungicide that is used on fruits, vegetables, ornamental plants, and turf grass. Vinclozolin and its metabolites are known to be endocrine disruptors and act as androgen receptor antagonists. The hypothesis tested in the current study is that transient embryonic exposure to an anti-androgenic endocrine disruptor at the time of testis determination alters testis development and subsequently influences adult spermatogenic capacity and male reproduction. The effects of vinclozolin on embryonic testicular cord formation in vitro were examined, as well as the effects of transient in utero vinclozolin exposure on postnatal testis development and function. Embryonic day 13 (E13, sperm-positive vaginal smear day = E0) gonads were cultured in the absence or presence of vinclozolin (50-500microM). Vinclozolin treated gonads had significantly fewer cords (P < 0.05) and the histology of the cords that formed were abnormal as compared to vehicle-treated organs. Pregnant rats were exposed to vinclozolin (100 mg/kg/day) between embryonic days 8 and 14 (E8-E14) of development. Testis morphology and function were analyzed from postnatal day (P) 0, pubertal P20, and adult P60. No significant effect of vinclozolin on testis histology or germ cell viability was observed in P0 testis. The pubertal P20 testis from vinclozolin exposed animals had significantly higher numbers of apoptotic germ cells (P < 0.01), but testis weight was not affected. The adult P60 sperm motility was significantly lower in vinclozolin exposed males (P < 0.01). In addition, apoptotic germ cell number in testis of vinclozolin exposed animals was higher in adult P60 animals. Observations demonstrate that vinclozolin can effect embryonic testicular cord formation in vitro and that transient in utero exposure to vinclozolin increases apoptotic germ cell numbers in the testis of pubertal and adult animals. This correlated to reduced sperm motility in the adult. In conclusion, transient exposure to vinclozolin during the time of testis differentiation (i.e. cord formation) alters testis development and function. Observations indicate that transient exposure to an anti-androgenic endocrine disruptor during embryonic development causes delayed effects later in adult life on spermatogenic capacity.

Roles of steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF) 2 in androgen receptor activity in mice

PubMed Central

Ye, Xiangcang; Han, Sang Jun; Tsai, Sophia Y.; DeMayo, Francesco J.; Xu, Jianming; Tsai, Ming-Jer; O'Malley, Bert W.

2005-01-01

Genetic disruption of the steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF)2/SRC-2 in mouse resulted in distinctive mutant phenotypes. To quantify their roles in the function of androgen receptor (AR) transcriptional activity in vivo, we generated a unique transgenic AR-reporter mouse and analyzed the cell-specific contributions of SRC-1 and TIF2 to the activity of AR in mouse testis. Transgenic AR-luciferase and transgenic AR-lacZ mice harbor a recombinant mouse AR gene, ARGAL4DBD, which is functionally coupled with a upstream activation sequence-mediated reporter gene (AR activity indicator). After characterization of these mice in terms of AR function, we further derived bigenic mice by crossing AR activity indicator mice with the SRC-1-/- or TIF2+/- mutant mice. Analyses of the resultant bigenic mice by in vivo imaging and luciferase assays showed that testicular AR activity was decreased significantly in those with the TIF2+/- mutation but not in the SRC-1+/- background, suggesting that TIF2 serves as the preferential coactivator for AR in testis. Immunohistological analysis confirmed that AR and TIF2 coexist in mouse testicular Sertoli cell nuclei under normal conditions. Although SRC-1 concentrates in Sertoli cell nuclei in the absence of TIF2, nuclear SRC-1 is not able to rescue AR activity in the TIF2 mutant background. Interestingly, SRC-1 appears to negatively influence AR activity, thereby counterbalancing the TIF2-stimulated AR activity. Our results provide unique in vivo insights to the multidimensional cell-type-specific interactions between AR and coregulators. PMID:15983373

Roles of steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF) 2 in androgen receptor activity in mice.

PubMed

Ye, Xiangcang; Han, Sang Jun; Tsai, Sophia Y; DeMayo, Francesco J; Xu, Jianming; Tsai, Ming-Jer; O'Malley, Bert W

2005-07-05

Genetic disruption of the steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF)2/SRC-2 in mouse resulted in distinctive mutant phenotypes. To quantify their roles in the function of androgen receptor (AR) transcriptional activity in vivo, we generated a unique transgenic AR-reporter mouse and analyzed the cell-specific contributions of SRC-1 and TIF2 to the activity of AR in mouse testis. Transgenic AR-luciferase and transgenic AR-lacZ mice harbor a recombinant mouse AR gene, AR(GAL4DBD), which is functionally coupled with a upstream activation sequence-mediated reporter gene (AR activity indicator). After characterization of these mice in terms of AR function, we further derived bigenic mice by crossing AR activity indicator mice with the SRC-1-/- or TIF2+/- mutant mice. Analyses of the resultant bigenic mice by in vivo imaging and luciferase assays showed that testicular AR activity was decreased significantly in those with the TIF2+/- mutation but not in the SRC-1+/- background, suggesting that TIF2 serves as the preferential coactivator for AR in testis. Immunohistological analysis confirmed that AR and TIF2 coexist in mouse testicular Sertoli cell nuclei under normal conditions. Although SRC-1 concentrates in Sertoli cell nuclei in the absence of TIF2, nuclear SRC-1 is not able to rescue AR activity in the TIF2 mutant background. Interestingly, SRC-1 appears to negatively influence AR activity, thereby counterbalancing the TIF2-stimulated AR activity. Our results provide unique in vivo insights to the multidimensional cell-type-specific interactions between AR and coregulators.

Improvement of In Situ PCR by Optimization of PCR Cycle Number and Proteinase K Concentration: Localization of X Chromosome-Linked Phosphoglycerate Kinase-1 Gene in Mouse Reproductive Organs

PubMed Central

Hishikawa, Yoshitaka; An, Shucai; Yamamoto-Fukuda, Tomomi; Shibata, Yasuaki; Koji, Takehiko

2009-01-01

In situ polymerase chain reaction (in situ PCR), which can detect a few copies of genes within a cell by amplifying the target gene, was developed to better understand the biological functions of tissues. In this study, we optimized the protocol conditions for the detection of X chromosome-linked phosphoglycerate kinase-1 (pgk-1) gene in paraffin-embedded sections of mouse reproductive organs. The effects of various concentrations of proteinase K (PK) and PCR cycle numbers were examined. To label the amplified DNA, we used digoxigenin-dUTP (Dig), Cy-3-dUTP (Cy-3), or FluorX-dCTP (FluorX). The optimal concentration of PK was 50 µg/ml for the ovary and 10 µg/ml for the testis. Ten PCR cycles were optimal for Dig and 25 cycles were optimal for FluorX and Cy-3 in the ovary and testis. The signal-to-noise ratio of FluorX and Cy-3 for ovarian tissue was better than that of Dig. Using the above conditions, we detected 1–4 and 1–2 spots of pgk-1 in the nuclei of granulosa and germ cells, respectively. Our results indicate that in situ PCR is useful for detecting a specific gene in paraffin-embedded sections under optimized conditions of both PCR cycle number and PK concentration. PMID:19492023

FGF9, activin and TGFβ promote testicular characteristics in an XX gonad organ culture model.

PubMed

Gustin, Sonja E; Stringer, Jessica M; Hogg, Kirsten; Sinclair, Andrew H; Western, Patrick S

2016-11-01

Testis development is dependent on the key sex-determining factors SRY and SOX9, which activate the essential ligand FGF9. Although FGF9 plays a central role in testis development, it is unable to induce testis formation on its own. However, other growth factors, including activins and TGFβs, also present testis during testis formation. In this study, we investigated the potential of FGF9 combined with activin and TGFβ to induce testis development in cultured XX gonads. Our data demonstrated differing individual and combined abilities of FGF9, activin and TGFβ to promote supporting cell proliferation, Sertoli cell development and male germ line differentiation in cultured XX gonads. FGF9 promoted proliferation of supporting cells in XX foetal gonads at rates similar to those observed in vivo during testis cord formation in XY gonads but was insufficient to initiate testis development. However, when FGF9, activin and TGFβ were combined, aspects of testicular development were induced, including the expression of Sox9, morphological reorganisation of the gonad and deposition of laminin around germ cells. Enhancing β-catenin activity diminished the testis-promoting activities of the combined growth factors. The male promoting activity of FGF9 and the combined growth factors directly or indirectly extended to the germ line, in which a mixed phenotype was observed. FGF9 and the combined growth factors promoted male germ line development, including mitotic arrest, but expression of pluripotency genes was maintained, rather than being repressed. Together, our data provide evidence that combined signalling by FGF9, activin and TGFβ can induce testicular characteristics in XX gonads. © 2016 Society for Reproduction and Fertility.

Genetics of Gonadal Stem Cell Renewal

PubMed Central

Greenspan, Leah Joy; de Cuevas, Margaret

2015-01-01

Stem cells are necessary for the maintenance of many adult tissues. Signals within the stem cell microenvironment, or niche, regulate the self-renewal and differentiation capability of these cells. Misregulation of these signals through mutation or damage can lead to overgrowth or depletion of different stem cell pools. In this review, we focus on the Drosophila testis and ovary, both of which contain well-defined niches, as well as the mouse testis, which has become a more approachable stem cell system with recent technical advances. We discuss the signals that regulate gonadal stem cells in their niches, how these signals mediate self-renewal and differentiation under homeostatic conditions, and how stress, whether from mutations or damage, can cause changes in cell fate and drive stem cell competition. PMID:26355592

Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone.

PubMed

Gonen, Nitzan; Quinn, Alexander; O'Neill, Helen C; Koopman, Peter; Lovell-Badge, Robin

2017-01-01

During mouse sex determination, transient expression of the Y-linked gene Sry up-regulates its direct target gene Sox9, via a 3.2 kb testis specific enhancer of Sox9 (TES), which includes a core 1.4 kb element, TESCO. SOX9 activity leads to differentiation of Sertoli cells, rather than granulosa cells from the bipotential supporting cell precursor lineage. Here, we present functional analysis of TES/TESCO, using CRISPR/Cas9 genome editing in mice. Deletion of TESCO or TES reduced Sox9 expression levels in XY fetal gonads to 60 or 45% respectively relative to wild type gonads, and reduced expression of the SOX9 target Amh. Although human patients heterozygous for null mutations in SOX9, which are assumed to have 50% of normal expression, often show XY female sex reversal, mice deleted for one copy of Sox9 do not. Consistent with this, we did not observe sex reversal in either TESCO-/- or TES-/- XY embryos or adult mice. However, embryos carrying both a conditional Sox9 null allele and the TES deletion developed ovotestes. Quantitative analysis of these revealed levels of 23% expression of Sox9 compared to wild type, and a significant increase in the expression of the granulosa cell marker Foxl2. This indicates that the threshold in mice where sex reversal begins to be seen is about half that of the ~50% levels predicted in humans. Our results demonstrate that TES/TESCO is a crucial enhancer regulating Sox9 expression in the gonad, but point to the existence of additional enhancers that act redundantly.

The Regulation of Sox9 Gene Expression by the GATA4/FOG2 Transcriptional Complex in Dominant XX Sex Reversal Mouse Models.

PubMed Central

Manuylov, Nikolay L.; Fujiwara, Yuko; Adameyko, Igor I.; Poulat, Francis

2007-01-01

We have previously established an in vivo requirement for GATA4 and FOG2 transcription factors in sexual differentiation. Fog2 null mouse fetuses or fetuses homozygous for a targeted mutation in Gata4 (Gata4ki), which cripples the GATA4-FOG2 interaction, exhibit a profound and early block in testis differentiation in both sexes. Others have shown that XX mice with the Ods transgenic insertion or the Wt1-Sox9 YAC transgene overexpress the testis differentiation gene, Sox9. Thus, these XX animals undergo dominant sex-reversal by developing into phenotypically normal, but sterile, males. Now we have determined that Fog2 haploinsufficiency prevents (suppresses) this dominant sex-reversal and Fog2+/− Wt1-Sox9 or Ods XX animals develop normally - as fertile females. The suppression of sex-reversal in Fog2 heterozygous females results from approximately 50% downregulation of the expression from the transgene-associated allele of Sox9. The GATA4/FOG2-dependent sex reversal observed in the transgenic XX gonads has to rely on gene targets other than the Y chromosome-linked Sry gene. Importantly, Fog2 null or Gata4ki/ki embryos (either XX or XY) fail to express detectable levels of Sox9 despite carrying the Ods mutation or Wt1-Sox9 transgene. Fog2 haploinsufficiency leads to a decreased amount of SOX9-positive cells in XY gonads. We conclude that FOG2 is a limiting factor in the formation of a functional GATA4/FOG2 transcription complex that is required for Sox9 expression during gonadogenesis. PMID:17540364

Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone

PubMed Central

O’Neill, Helen C.; Koopman, Peter; Lovell-Badge, Robin

2017-01-01

During mouse sex determination, transient expression of the Y-linked gene Sry up-regulates its direct target gene Sox9, via a 3.2 kb testis specific enhancer of Sox9 (TES), which includes a core 1.4 kb element, TESCO. SOX9 activity leads to differentiation of Sertoli cells, rather than granulosa cells from the bipotential supporting cell precursor lineage. Here, we present functional analysis of TES/TESCO, using CRISPR/Cas9 genome editing in mice. Deletion of TESCO or TES reduced Sox9 expression levels in XY fetal gonads to 60 or 45% respectively relative to wild type gonads, and reduced expression of the SOX9 target Amh. Although human patients heterozygous for null mutations in SOX9, which are assumed to have 50% of normal expression, often show XY female sex reversal, mice deleted for one copy of Sox9 do not. Consistent with this, we did not observe sex reversal in either TESCO-/- or TES-/- XY embryos or adult mice. However, embryos carrying both a conditional Sox9 null allele and the TES deletion developed ovotestes. Quantitative analysis of these revealed levels of 23% expression of Sox9 compared to wild type, and a significant increase in the expression of the granulosa cell marker Foxl2. This indicates that the threshold in mice where sex reversal begins to be seen is about half that of the ~50% levels predicted in humans. Our results demonstrate that TES/TESCO is a crucial enhancer regulating Sox9 expression in the gonad, but point to the existence of additional enhancers that act redundantly. PMID:28045957

Male sex determination: insights into molecular mechanisms

PubMed Central

McClelland, Kathryn; Bowles, Josephine; Koopman, Peter

2012-01-01

Disorders of sex development often arise from anomalies in the molecular or cellular networks that guide the differentiation of the embryonic gonad into either a testis or an ovary, two functionally distinct organs. The activation of the Y-linked gene Sry (sex-determining region Y) and its downstream target Sox9 (Sry box-containing gene 9) triggers testis differentiation by stimulating the differentiation of Sertoli cells, which then direct testis morphogenesis. Once engaged, a genetic pathway promotes the testis development while actively suppressing genes involved in ovarian development. This review focuses on the events of testis determination and the struggle to maintain male fate in the face of antagonistic pressure from the underlying female programme. PMID:22179516

Evaluation of the testicular toxicity of prenatal exposure to bisphenol A based on microarray analysis combined with MeSH annotation.

PubMed

Tainaka, Hitoshi; Takahashi, Hikari; Umezawa, Masakazu; Tanaka, Hiromitsu; Nishimune, Yoshitake; Oshio, Shigeru; Takeda, Ken

2012-01-01

Bisphenol A (BPA) is known to be an endocrine disruptor that affects the development of reproductive system. The aim of the present study was to investigate a group of testicular genes dysregulated by prenatal exposure to BPA. Pregnant ICR mice were treated with BPA by subcutaneous administration on days 7 and 14 of pregnancy. Tissue and blood samples were collected from 6-week-old male offspring. Testes were subjected to gene expression analysis using a testis-specific microarray (Testis2), consisting of 2,482 mouse cDNA clones annotated with Medical Subject Headings (MeSH) terms indicative of testicular components and functions. To interpret the microarray data, we used the MeSH terms significantly associated with the altered genes. As a result, MeSH terms related to androgens and Sertoli cells were extracted in BPA-treated groups. Among the genes related to Sertoli cells, downregulation of Msi1h, Ncoa1, Nid1, Hspb2, and Gata6 were detected in the testis of mice treated with BPA (twice administered 50 mg/kg). The MeSH terms associated with this group of genes may provide useful means to interpret the testicular toxicity of BPA. This article concludes that prenatal BPA exposure downregulates expression of genes associated with Sertoli cell function and affects the reproductive function of male offspring. Additionally, a method using MeSH to extract a group of genes was useful for predicting the testicular and reproductive toxicity of prenatal BPA exposure.

Comparative Sperm Proteomics in Mouse Species with Divergent Mating Systems

PubMed Central

Vicens, Alberto; Borziak, Kirill; Karr, Timothy L.; Roldan, Eduardo R.S.

2017-01-01

Abstract Sexual selection is the pervasive force underlying the dramatic divergence of sperm form and function. Although it has been demonstrated that testis gene expression evolves rapidly, exploration of the proteomic basis of sperm diversity is in its infancy. We have employed a whole-cell proteomics approach to characterize sperm divergence among closely related Mus species that experience different sperm competition regimes and exhibit pronounced variation in sperm energetics, motility and fertilization capacity. Interspecific comparisons revealed significant abundance differences amongst proteins involved in fertilization capacity, including those that govern sperm-zona pellucida interactions, axoneme components and metabolic proteins. Ancestral reconstruction of relative testis size suggests that the reduction of zona pellucida binding proteins and heavy-chain dyneins was associated with a relaxation in sperm competition in the M. musculus lineage. Additionally, the decreased reliance on ATP derived from glycolysis in high sperm competition species was reflected in abundance decreases in glycolytic proteins of the principle piece in M. spretus and M. spicilegus. Comparison of protein abundance and stage-specific testis expression revealed a significant correlation during spermatid development when dynamic morphological changes occur. Proteins underlying sperm diversification were also more likely to be subject to translational repression, suggesting that sperm composition is influenced by the evolution of translation control mechanisms. The identification of functionally coherent classes of proteins relating to sperm competition highlights the utility of evolutionary proteomic analyses and reveals that both intensified and relaxed sperm competition can have a pronounced impact on the molecular composition of the male gamete. PMID:28333336

Critical Role of Hepatic Cyp450s in the Testis-Specific Toxicity of (5R)-5-Hydroxytriptolide in C57BL/6 Mice

PubMed Central

Yu, Cunzhi; Li, Yu; Liu, Mingxia; Gao, Man; Li, Chenggang; Yan, Hong; Li, Chunzhu; Sun, Lihan; Mo, Liying; Wu, Chunyong; Qi, Xinming; Ren, Jin

2017-01-01

Low solubility, tissue accumulation, and toxicity are chief obstacles to developing triptolide derivatives, so a better understanding of the pharmacokinetics and toxicity of triptolide derivatives will help with these limitations. To address this, we studied pharmacokinetics and toxicity of (5R)-5-hydroxytriptolide (LLDT-8), a novel triptolide derivative immunosuppressant in a conditional knockout (KO) mouse model with liver-specific deletion of CYP450 reductase. Compared to wild type (WT) mice, after LLDT-8 treatment, KO mice suffered severe testicular toxicity (decreased testicular weight, spermatocytes apoptosis) unlike WT mice. Moreover, KO mice had greater LLDT-8 exposure as confirmed with elevated AUC and Cmax, increased drug half-life, and greater tissue distribution. γ-H2AX, a marker of meiosis process, its localization and protein level in testis showed a distinct meiosis block induced by LLDT-8. RNA polymerase II (Pol II), an essential factor for RNA storage and synapsis in spermatogenesis, decreased in testes of KO mice after LLDT-8 treatment. Germ-cell line based assays confirmed that LLDT-8 selectively inhibited Pol II in spermatocyte-like cells. Importantly, the analysis of androgen receptor (AR) related genes showed that LLDT-8 did not change AR-related signaling in testes. Thus, hepatic CYP450s were responsible for in vivo metabolism and clearance of LLDT-8 and aggravated testicular injury may be due to increased LLDT-8 exposure in testis and subsequent Pol II reduction. PMID:29209210

Exposure to 2,4-dichlorophenoxyacetic acid induces oxidative stress and apoptosis in mouse testis.

PubMed

Zhang, Dalei; Wu, Yaling; Yuan, Yangyang; Liu, Wenwen; Kuang, Haibin; Yang, Jianhua; Yang, Bei; Wu, Lei; Zou, Weiying; Xu, Changshui

2017-09-01

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is used worldwide. It has been associated with a variety of toxicities in rodents. In this study, male mice were orally administered 2,4-D at 50, 100 or 200mg/kg/day to investigate testicular toxicity and the possible mechanisms of action. Exposure to 2,4-D at high concentrations (100 and 200mg/kg/day) for 14 consecutive days caused spermatogenic disruption and seminiferous epithelial destruction. Furthermore, 2,4-D administration (100 and 200mg/kg/day) increased the formation of the lipid peroxidation product malondialdehyde and decreased activities of the antioxidant enzymes superoxide dismutase and catalase in the testis. Moreover, 2,4-D exposure up-regulated the expression of p53 and Bax protein and down-regulated the expression of Bcl-2 protein in the testis. These results demonstrate that oxidative stress and apoptosis may be involved in testicular toxicity induced by high concentrations of 2,4-D in mice. Copyright © 2016 Elsevier Inc. All rights reserved.

Genetic regulation of mammalian gonad development.

PubMed

Eggers, Stefanie; Ohnesorg, Thomas; Sinclair, Andrew

2014-11-01

Sex-specific gonadal development starts with formation of the bipotential gonad, which then differentiates into either a mature testis or an ovary. This process is dependent on activation of either the testis-specific or the ovary-specific pathway while the opposite pathway is continuously repressed. A network of transcription factors tightly regulates initiation and maintenance of these distinct pathways; disruption of these networks can lead to disorders of sex development in humans and male-to-female or female-to-male sex reversal in mice. Sry is the Y-linked master switch that is both required and sufficient to drive the testis-determining pathway. Another key component of the testis pathway is Sox9, which acts immediately downstream of Sry. In contrast to the testis pathway, no single sex-determining factor has been identified in the ovary pathway; however, multiple genes, such as Foxl2, Rspo1, Ctnnb1, and Wnt4, seem to work synergistically and in parallel to ensure proper ovary development. Our understanding of the regulatory networks that underpin testis and ovary development has grown substantially over the past two decades.

Thyroid Hormone Role and Economy in the Developing Testis.

PubMed

Hernandez, Arturo

2018-01-01

Thyroid hormones (TH) exhibit pleiotropic regulatory effects on growth, development, and metabolism, and it is becoming increasingly apparent that the developing testis is an important target for them. Testicular development is highly dependent on TH status. Both hypo- and hyperthyroidism affect testis size and the proliferation and differentiation of Sertoli, Leydig, and germ cells, with consequences for steroidogenesis, spermatogenesis, and male fertility. These observations suggest that an appropriate content of TH and by implication TH action in the testis, whether the result of systemic hormonal levels or regulatory mechanisms at the local level, is critical for normal testicular and reproductive function. The available evidence indicates the presence in the developing testis of a number of transporters, deiodinases and receptors that could play a role in the timely delivery of TH action on testicular cells. These include the thyroid hormone receptor alpha (THRA), the MCT8 transporter, the TH-activating deiodinase DIO2, and the TH-inactivating deiodinase DIO3, all of which appear to modulate testicular TH economy and testis outcomes. © 2018 Elsevier Inc. All rights reserved.

FOXL2 impairment in human disease.

PubMed

Verdin, Hannah; De Baere, Elfride

2012-01-01

FOXL2 encodes a forkhead transcription factor that plays important roles in the ovary during development and in post-natal, adult life. Here, we focus on the clinical consequences of FOXL2 impairment in human disease. In line with other forkhead transcription factors, its constitutional genetic defects and a somatic mutation lead to developmental disease and cancer, respectively. More than 100 unique constitutional mutations and regulatory defects have been found in blepharophimosis syndrome (BPES), a complex eyelid malformation associated (type I) or not (type II) with premature ovarian failure (POF). In agreement with the BPES phenotype, FOXL2 is expressed in the developing eyelids and in fetal and adult ovaries. Two knock-out mice and at least one natural animal model, the Polled Intersex Syndrome goat, are known. They recapitulate the BPES phenotype and have provided many insights into the ovarian pathology. Only a few constitutional mutations have been described in nonsyndromic POF. Moreover, a recurrent somatic mutation p.C134W was found to be specific for adult ovarian granulo-sa cell tumors. Functional studies investigating the consequences of FOXL2 mutations or regulatory defects have shed light on the molecular pathogenesis of the aforementioned conditions, and contributed considerably to genotype-phenotype correlations. Recently, a conditional knock-out of Foxl2 in the mouse induced somatic transdifferentiation of ovary into testis in adult mice, suggesting that Foxl2 has an anti-testis function in the adult ovary. This changed our view on the ovary and testis as terminally differentiated organs in adult mammals. Finally, this might have potential implications for the understanding and treatment of frequent conditions such as POF and polycystic ovary syndrome. Copyright © 2012 S. Karger AG, Basel.

Characterization of two distinct dual specificity phosphatases encoded in alternative open reading frames of a single gene located on human chromosome 10q22.2.

PubMed

Chen, Hsu-Hsin; Luche, Ralf; Wei, Bo; Tonks, Nicholas K

2004-10-01

Dual specificity phosphatases (DSPs) are members of the protein-tyrosine phosphatase superfamily that dephosphorylate both phosphotyrosine and phosphoserine/threonine residues in vitro. Many DSPs have been found to play important roles in various aspects of cellular function and to be involved in human disease. We have identified a gene located on human chromosome 10q22.2, which utilizes alternative open reading frames (ORFs) to encode the following two distinct DSPs: the previously described testis and skeletal muscle-specific dual specificity phosphatase (TMDP) and a novel DSP, muscle-restricted dual specificity phosphatase (MDSP). Use of alternative ORFs encoding distinct proteins from a single gene is extremely rare in eukaryotes, and in all previously reported cases the two proteins produced from one gene are unrelated. To our knowledge this is the first example of a gene from which two distinct proteins of the same family are expressed using alternative ORFs. Here we provide evidence that both MDSP and TMDP proteins are expressed in vivo and are restricted to specific tissues, skeletal muscle and testis, respectively. Most interestingly, the protein expression profiles of both MDSP and TMDP during mouse postnatal development are strikingly similar. MDSP is expressed at very low levels in myotubes and early postnatal muscle. TMDP is not detectable in testis lysate in the first 3 weeks of life. The expression of both MDSP and TMDP proteins was markedly increased at approximately the 3rd week after birth and continued to increase gradually into adulthood, implying that the physiological functions of both DSPs are specific to the mature/late-developing organs. The conserved gene structure and the similarity in postnatal expression profile of these two proteins suggest biological significance of the unusual gene arrangement.

Differential investment into testes and sperm production in alternative male reproductive tactics of the African striped mouse (Rhabdomys pumilio).

PubMed

Schradin, Carsten; Eder, Susanne; Müller, Karin

2012-05-01

Males that follow alternative reproductive tactics might differ in their investment into testis development and sperm production. The resource-allocation hypothesis predicts that males following a sneaker tactic should invest more into sperm production than dominant territorial males which should invest more into mate guarding. This hypothesis is supported by studies in species where individual males cannot switch between tactics (fixed tactics). Here we present the first data for a species where males can switch between tactics (plastic tactics). We studied African striped mice (Rhabdomys pumilio) in captivity, mimicking three tactics observed in the field: philopatric group-living males, singly-housed males representing roaming males, and group-living breeding males. We measured quantitative and qualitative reproductive traits, as well as serum and testis hormone concentrations. We found no support for the resource-allocation hypothesis, since breeding and singly-housed males invested similarly in testes and sperm. However, philopatric males had significantly smaller testes and epididymides, lower sperm counts, lower testosterone and higher corticosterone levels than males of the two other tactics. Philopatric males did not reach a larger body mass than singly-housed males with well developed reproductive traits, indicating that they did not trade investment in sperm production against growth. Interestingly, testis testosterone concentrations of philopatric males did not differ from those of other males. Our data suggest that philopatric males are reproductively suppressed by the breeding male, but might be ready to increase their serum testosterone levels when social and environmental conditions allow for this physiological switch accompanying the behavioral switch between tactics. Copyright © 2012 Elsevier Inc. All rights reserved.

Hybrid male sterility is caused by mitochondrial DNA deletion.

PubMed

Hayashida, Kenji; Kohno, Shigeru

2009-07-01

Although it is known that the hybrid male mouse is sterile just like any other animal's heterogametic sex, the reason why only the male germ cells are impaired has yet to be discovered. TdT-mediated dUTP nick end labeling assay using a confocal fluorescence microscope and DNA fragmentation assay of hybrid testis indicated destruction of the mitochondrial DNA (mtDNA) rather than the nuclear DNA. Previously we reported that maternal mtDNA inheritance is through selective sperm mtDNA elimination based on the sperm factor and two egg factors, and expression of these three factors was recognized in the hybrid testis. It was thereby assumed that mtDNA destruction caused by the expression of maternal mtDNA inheritance system in male germ cells is implicated in the hybrid male sterility of mice.

Genome-wide analysis of long non-coding RNAs and their role in postnatal porcine testis development.

PubMed

Weng, Bo; Ran, Maoliang; Chen, Bin; He, Changqing; Dong, Lianhua; Peng, Fuzhi

2017-10-01

A comprehensive and systematic understanding of the roles of lncRNAs in the postnatal development of the pig testis has still not been achieved. In the present study, we obtained more than one billion clean reads and identified 15,528 lncRNA transcripts; these transcripts included 5032 known and 10,496 novel porcine lncRNA transcripts and corresponded to 10,041 lncRNA genes. Pairwise comparisons identified 449 known and 324 novel lncRNAs that showed differential expression patterns. GO and KEGG pathway enrichment analyses revealed that the targeted genes were involved in metabolic pathways regulating testis development and spermatogenesis, such as the TGF-beta pathway, the PI3K-Akt pathway, the Wnt/β-catenin pathway, and the AMPK pathway. Using this information, we predicted some lncRNAs and coding gene pairs were predicted that may function in testis development and spermatogenesis; these are listed in detail. This study has provided the most comprehensive catalog to date of lncRNAs in the postnatal pig testis and will aid our understanding of their functional roles in testis development and spermatogenesis. Copyright © 2017. Published by Elsevier Inc.

Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval.

PubMed

Smith, Ryan P; Lowe, Greg J; Kavoussi, Parviz K; Steers, William D; Costabile, Raymond A; Herr, John C; Shetty, Jagathpala; Lysiak, Jeffrey J

2012-05-01

Microdissection testicular sperm extraction markedly improves the sperm retrieval rates in men with nonobstructive azoospermia. However, localizing sperm foci can be time-consuming and it is not always successful. Fiberoptic confocal fluorescent microscopy offers the advantage of rapid in vivo detection of fluorescently labeled sperm in the seminiferous tubules. After establishing the feasibility of fiberoptic confocal fluorescent microscopy to identify antibody labeled sperm in vivo C57/B6 mice underwent intraperitoneal injection of busulfan to induce azoospermia. During spermatogenesis reestablishment at approximately 16 weeks the mice were anesthetized and the testes were delivered through a low midline incision. Fluorescein isothiocyanate labeled antibody to intra-acrosomal protein Hs-14 was injected retrograde into a single murine rete testis. The testes were imaged in vivo with fiberoptic confocal fluorescent microscopy and sperm foci were detected. The respective seminiferous tubules were excised and squash prepared for immunofluorescence microscopy. Sperm foci were identified in the testis injected with fluorescently tagged antibody by in vivo fiberoptic confocal fluorescence microscopy. The contralateral control testis of each mouse showed no specific signal. Immunofluorescence microscopy of the excised tubules provided morphological confirmation of the presence of labeled sperm with an absence in controls. Findings were consistent in the feasibility portion of the study and in the busulfan model of nonobstructive azoospermia. Fiberoptic confocal fluorescent microscopy was feasible during microdissection testicular sperm extraction in an azoospermic mouse model to identify fluorescently labeled sperm in vivo. Translation to the clinical setting could decrease operative time and improve the sperm harvest rate. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Low-dose carbon ion irradiation effects on DNA damage and oxidative stress in the mouse testis

NASA Astrophysics Data System (ADS)

Liu, Yang; Long, Jing; Zhang, Luwei; Zhang, Hong; Liu, Bin; Zhao, Weiping; Wu, Zhehua

2011-01-01

To investigate the effects of low-dose carbon ion irradiation on reproductive system of mice, the testes of outbred Kunming strain mice were whole-body irradiated with 0, 0.05, 0.1, 0.5 and 1 Gy, respectively. We measured DNA double-strand breaks (DNA DSBs) and oxidative stress parameters including malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and testis weight and sperm count at 12 h, 21 d and 35 d after irradiation in mouse testis. At 12 h postirradiation, a significant increase in DNA DSB level but no pronounced alterations in MDA content or SOD activity were observed in 0.5 and 1 Gy groups compared with the control group. At 21 d postirradiation, there was a significant reduction in sperm count and distinct enhancements of DSB level and MDA content in 0.5 and 1 Gy groups in comparison with control. At 35 d postirradiation, the levels of DNA DSBs and MDA, and SOD activity returned to the baseline except for the MDA content in 1 Gy (P < 0.05), while extreme falls of sperm count were still observed in 0.5 (P < 0.01) and 1 Gy (P < 0.01) groups. For the 0.05 or 0.1 Gy group, no differences were found in DNA DSB level and MDA content between control and at 12 h, 21 d and 35 d after irradiation, indicating that lower doses of carbon ion irradiation have no significant influence on spermatogenesis processes. In this study, male germ cells irradiated with over 0.5 Gy of carbon ions are difficult to repair completely marked by the sperm count. Furthermore, these data suggest that the deleterious effects may be chronic or delayed in reproductive system after whole-body exposure to acute high-dose carbon ions.

The Fate of Spermatogonial Stem Cells in the Cryptorchid Testes of RXFP2 Deficient Mice

PubMed Central

Ferguson, Lydia; How, Javier J.; Agoulnik, Alexander I.

2013-01-01

The environmental niche of the spermatogonial stem cell pool is critical to ensure the continued generation of the germ cell population. To study the consequences of an aberrant testicular environment in cryptorchidism we used a mouse model with a deletion of Rxfp2 gene resulting in a high intra-abdominal testicular position. Mutant males were infertile with the gross morphology of the cryptorchid testis progressively deteriorating with age. Few spermatogonia were identifiable in 12 month old cryptorchid testes. Gene expression analysis showed no difference between mutant and control testes at postnatal day 10. In three month old males a decrease in expression of spermatogonial stem cell (SSC) markers Id4, Nanos2, and Ret was shown. The direct counting of ID4+ cells supported a significant decrease of SSCs. In contrast, the expression of Plzf, a marker for undifferentiated and differentiating spermatogonia was not reduced, and the number of PLZF+ cells in the cryptorchid testis was higher in three month old testes, but equal to control in six month old mutants. The PLZF+ cells did not show a higher rate of apoptosis in cryptorchid testis. The expression of the Sertoli cell FGF2 gene required for SSC maintenance was significantly reduced in mutant testis. Based on these findings we propose that the deregulation of somatic and germ cell genes in the cryptorchid testis, directs the SSCs towards the differentiation pathway. This leads to a depletion of the SSC pool and an increase in the number of PLZF+ spermatogonial cells, which too, eventually decreases with the exhaustion of the stem cell pool. Such a dynamic suggests that an early correction of cryptorchidism is critical for the retention of the SSC pool. PMID:24098584

Protective effects of nuclear factor erythroid 2-related factor 2 on whole body heat stress-induced oxidative damage in the mouse testis.

PubMed

Li, Yansen; Huang, Yi; Piao, Yuanguo; Nagaoka, Kentaro; Watanabe, Gen; Taya, Kazuyoshi; Li, ChunMei

2013-03-21

Whole body heat stress had detrimental effect on male reproductive function. It's known that the nuclear factor erythroid 2-related factor 2 (Nrf2) activates expression of cytoprotective genes to enable cell adaptation to protect against oxidative stress. However, it's still unclear about the exactly effects of Nrf2 on the testis. Here, we investigate the protective effect of Nrf2 on whole body heat stress-induced oxidative damage in mouse testis. Male mice were exposed to the elevated ambient temperature (42°C) daily for 2 h. During the period of twelve consecutive days, mice were sacrificed on days 1, 2, 4, 8 and 12 immediately following heat exposure. Testes weight, enzymatic antioxidant activities and concentrations of malondialdehyde (MDA) and glutathione (GSH) in the testes were determined and immunohistochemical detection of Nrf2 protein and mRNA expression of Nrf2-regulated genes were analyzed to assess the status of Nrf2-antioxidant system. Heat-exposed mice presented significant increases in rectal, scrotal surface and body surface temperature. The concentrations of cortisol and testosterone in serum fluctuated with the number of exposed days. There were significant decrease in testes weight and relative testes weight on day 12 compared with those on other days, but significant increases in catalase (CAT) activity on day 1 and GSH level on day 4 compared with control group. The activities of total superoxide dismutase (T-SOD) and copper-zinc SOD (CuZn-SOD) increased significantly on days 8 and 12. Moreover, prominent nuclear accumulation of Nrf2 protein was observed in Leydig cells on day 2, accompanying with up-regulated mRNA levels of Nrf2-regulated genes such as Nrf2, heme oxygenase 1 (HO-1), γ-Glutamylcysteine synthetase (GCLC) and NAD (P) H: quinone oxidoreductase 1 (NQO1)) in heat-treated groups. These results suggest that Nrf2 displayed nuclear accumulation and protective activity in the process of heat treated-induced oxidative stress in mouse testes, indicating that Nrf2 might be a potential target for new drugs designed to protect germ cell and Leydig cell from oxidative stress.

Heterogeneity in sexual bipotentiality and plasticity of granulosa cells in developing mouse ovaries.

PubMed

Harikae, Kyoko; Miura, Kento; Shinomura, Mai; Matoba, Shogo; Hiramatsu, Ryuji; Tsunekawa, Naoki; Kanai-Azuma, Masami; Kurohmaru, Masamichi; Morohashi, Ken-Ichirou; Kanai, Yoshiakira

2013-07-01

In mammalian sex determination, SRY directly upregulates the expression of SOX9, the master regulatory transcription factor in Sertoli cell differentiation, leading to testis formation. Without SRY action, the bipotential gonadal cells become pre-granulosa cells, which results in ovarian follicle development. When, where and how pre-granulosa cells are determined to differentiate into developing ovaries, however, remains unclear. By monitoring SRY-dependent SOX9 inducibility (SDSI) in an Sry-inducible mouse system, we were able to identify spatiotemporal changes in the sexual bipotentiality/plasticity of ovarian somatic cells throughout life. The early pre-granulosa cells maintain the SDSI until 11.5 d.p.c., after which most pre-granulosa cells rapidly lose this ability by 12.0 d.p.c. Unexpectedly, we found a subpopulation of the pre-granulosa cells near the mesonephric tissue that continuously retains SDSI throughout fetal and early postnatal stages. After birth, these SDSI-positive pre-granulosa cells contribute to the initial round of folliculogenesis by the secondary follicle stage. In experimental sex reversal of 13.5-d.p.c. ovaries grafted into adult male nude mice, the differentiated granulosa cells re-acquire the SDSI before other signs of masculinization. Our data provide direct evidence of an unexpectedly high sexual heterogeneity of granulosa cells in developing mouse ovaries in a stage- and region-specific manner. Discovery of such sexually bipotential granulosa cells provides a novel entry point to the understanding of masculinization in various cases of XX disorders of sexual development in mammalian ovaries.

Evaluate the Influence of Eupatorium adenophorum Extract with Mice Organ

NASA Astrophysics Data System (ADS)

Nong, Xiang; Yang, Can; Yang, Yaojun; Liang, Zi; Hu, Qiang; Zhang, Ting

2018-01-01

In order to study the influence of extract from Eupatorium adenophorum in mice organs, this experiment will be the basis of further study that make Eupatorium adenophorum become Phyto contraceptive, this experiment take the feeding respectively way after the completion of the 1D, 5D, 10d, 15d of Eupatorium adenophorum mice by intragastrical administration of levonorgestrel group and blank control group. After the same operation in different periods of small rat heart and kidney the uterus, testis, and other organs were observed. The results showed that after extraction of E. adenophorum changes in female mice uterus shape was perfused significantly, showed swelling larger. Data analysis of each viscera coefficient was found E. adenophorum had No obvious effect on the heart, kidneys and testicles of mice. but there are obvious differences date between the treatment group and the blank group. (5d: F=10. 800 P=0. 043 cases) from tissue sections we can see female mice uterus cell morphology changes significantly, there was a similar appearance change in the uterus of the female mice with the estradiol For a male mouse testis of E.adenophorum gavage had No obvious effect. And it is found that the heart, the treated mice kidney, testis, ovary and other organs were observed in each period of time the organization had No obvious change; only female mice uterus tissue sections of individual cells became larger, and the organization of the gap larger. This research shows that E.adenophorum extract has the potential to develop botanical contraceptives, we will conduct in-depth study.

Irinotecan metabolite SN38 results in germ cell loss in the testis but not in the ovary of prepubertal mice.

PubMed

Lopes, Federica; Smith, Rowena; Nash, Sophie; Mitchell, Rod T; Spears, Norah

2016-11-01

Does the Irinotecan metabolite 7-ethyl-10-hydroxycamptothecan (SN38) damage the gonads of male and female prepubertal mice? The Irinotecan metabolite SN38 reduces germ cell numbers within the seminiferous tubules of mouse testes at concentrations that are relevant to cancer patients, while in contrast it has little if any effect on the female germ cell population. Little is known about the role of the chemotherapeutic agent Irinotecan on female fertility, with only one article to date reporting menopausal symptoms in perimenopausal women treated with Irinotecan, while no data are available either on adult male fertility or on the impact of Irinotecan on the subsequent fertility of prepubertal cancer patients, female or male. Male and female gonads were obtained from postnatal day 5 C57BL/6 mice and exposed in vitro to a range of concentrations of the Irinotecan metabolite SN38: 0.002, 0.01, 0.05, 0.1 or 1 µg ml -1 for the testis and 0.1, 1, 2.5 or 5 µg ml -1 for the ovary, with treated gonads compared to control gonads not exposed to SN38. SN38 was dissolved in 0.5% dimethyl sulfoxide, with controls exposed to the same concentration of diluent. The number of testis fragments used for each analysis ranged between 3 and 9 per treatment group, while the number of ovaries used for each analysis ranged between 4 and 12 per treatment group. Neonatal mouse gonads were developed in vitro, with tissue analysed at the end of the 4-6 day culture period, following immunofluorescence or hematoxylin and eosin staining. Statistical analyses were performed using one-way ANOVA followed by Bonferroni post-hoc test for normally distributed data and Kruskal-Wallis test followed by Dunns post-test for non-parametric data. Abnormal testis morphology was observed when tissues were exposed to SN38, with a smaller seminiferous tubule diameter at the highest concentration of SN38 (1 µg ml -1 , p < 0.001 versus control) and increased number of Sertoli cell-only tubules at the two highest concentrations of SN38 (0.1 µg ml -1 , p < 0.001; 1 µg ml -1 , p < 0.0001, both versus control). Within seminiferous tubules, a dose response decrease was observed in both germ cell number (mouse vasa homologue (MVH)-positive cells) and in proliferating cell number (bromodeoxyuridine (BrdU)-positive cells), with significance reached at the two highest concentrations of SN38 (0.1 µg ml -1 , p < 0.01 for both; 1 µg ml -1 , p < 0.001-MVH, p < 0.01-BrdU; all versus control). No change was seen in protein expression of the apoptotic marker cleaved caspase 3. Double immunofluorescence showed that occasional proliferating germ cells were present in treated testes, even after exposure to the highest drug concentration. When prepubertal ovaries were treated with SN38, no effect was seen on germ cell number, apoptosis or cell proliferation, even after exposure to the highest drug concentrations. As with any study using in vitro experiments with an experimental animal model, caution is required when extrapolating the present findings to humans. Differences between human and mouse spermatogonial development also need to be considered when assessing the effect of chemotherapeutic exposure. However, the prepubertal testes and ovaries used in the present studies contain germ cell populations that are representative of those found in prepubertal patients, and experimental tissues were exposed to drug concentrations within the range found in patient plasma. Our findings demonstrate that the prepubertal mouse ovary is relatively insensitive to exposure to the Irinotecan metabolite SN38, while it induces a marked dose-dependent sensitivity in the testicular germ cell population. The study identifies the importance of further investigation to identify the risk of infertility in young male cancer patients treated with Irinotecan. None. Work supported by Medical Research Grant (MRC) grant G1002118 and Children with Cancer UK grant 15-198. The authors declare that there is no conflict of interest that could prejudice the impartiality of the present research. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

ACTIONS OF THE ENDOCRINE DISRUPTOR METHOXYCHLOR AND ITS ESTROGENIC METABOLITE ON IN VITRO EMBRYONIC RAT SEMINIFEROUS CORD FORMATION AND PERINATAL TESTIS GROWTH. (R827405)

EPA Science Inventory

Abstract

The current study examines the actions of methoxychlor and its estrogenic metabolite, 2, 2-bis-(p-hydroxyphenyl)-1, 1, 1-trichloroethane (HPTE), on seminiferous cord formation and growth of the developing rat testis. The developing testis in the embryonic and ...

Signaling through the TGF Beta-Activin Receptors ALK4/5/7 Regulates Testis Formation and Male Germ Cell Development

PubMed Central

Stringer, Jessica M.; van den Bergen, Jocelyn A.; Wilhelm, Dagmar; Sinclair, Andrew H.; Western, Patrick S.

2013-01-01

The developing testis provides an environment that nurtures germ cell development, ultimately ensuring spermatogenesis and fertility. Impacts on this environment are considered to underlie aberrant germ cell development and formation of germ cell tumour precursors. The signaling events involved in testis formation and male fetal germ cell development remain largely unknown. Analysis of knockout mice lacking single Tgfβ family members has indicated that Tgfβ's are not required for sex determination. However, due to functional redundancy, it is possible that additional functions for these ligands in gonad development remain to be discovered. Using FACS purified gonadal cells, in this study we show that the genes encoding Activin's, TGFβ's, Nodal and their respective receptors, are expressed in sex and cell type specific patterns suggesting particular roles in testis and germ cell development. Inhibition of signaling through the receptors ALK4, ALK5 and ALK7, and ALK5 alone, demonstrated that TGFβ signaling is required for testis cord formation during the critical testis-determining period. We also show that signaling through the Activin/NODAL receptors, ALK4 and ALK7 is required for promoting differentiation of male germ cells and their entry into mitotic arrest. Finally, our data demonstrate that Nodal is specifically expressed in male germ cells and expression of the key pluripotency gene, Nanog was significantly reduced when signaling through ALK4/5/7 was blocked. Our strategy of inhibiting multiple Activin/NODAL/TGFβ receptors reduces the functional redundancy between these signaling pathways, thereby revealing new and essential roles for TGFβ and Activin signaling during testis formation and male germ cell development. PMID:23342175

Germ Cells Are Not Required to Establish the Female Pathway in Mouse Fetal Gonads

PubMed Central

Maatouk, Danielle M.; Mork, Lindsey; Hinson, Ashley; Kobayashi, Akio; McMahon, Andrew P.; Capel, Blanche

2012-01-01

The fetal gonad is composed of a mixture of somatic cell lineages and germ cells. The fate of the gonad, male or female, is determined by a population of somatic cells that differentiate into Sertoli or granulosa cells and direct testis or ovary development. It is well established that germ cells are not required for the establishment or maintenance of Sertoli cells or testis cords in the male gonad. However, in the agametic ovary, follicles do not form suggesting that germ cells may influence granulosa cell development. Prior investigations of ovaries in which pre-meiotic germ cells were ablated during fetal life reported no histological changes during stages prior to birth. However, whether granulosa cells underwent normal molecular differentiation was not investigated. In cases where germ cell loss occurred secondary to other mutations, transdifferentiation of granulosa cells towards a Sertoli cell fate was observed, raising questions about whether germ cells play an active role in establishing or maintaining the fate of granulosa cells. We developed a group of molecular markers associated with ovarian development, and show here that the loss of pre-meiotic germ cells does not disrupt the somatic ovarian differentiation program during fetal life, or cause transdifferentiation as defined by expression of Sertoli markers. Since we do not find defects in the ovarian somatic program, the subsequent failure to form follicles at perinatal stages is likely attributable to the absence of germ cells rather than to defects in the somatic cells. PMID:23091613

Expression of peroxisome proliferator-activated receptor alpha messenger ribonucleic acid and protein in human and rat testis.

PubMed

Schultz, R; Yan, W; Toppari, J; Völkl, A; Gustafsson, J A; Pelto-Huikko, M

1999-07-01

Peroxisome proliferator-activated receptor a (PPARalpha), a member of the steroid hormone receptor superfamily, has been linked to lipid homeostasis and tumorigenesis in tissues with high expression of receptor protein. On the other hand, the role of PPARalpha in tissues with a lower expression is not well known. Here we demonstrate the localization of PPARalpha messenger RNA (mRNA) and protein in developing and adult rat testis. Additionally, we demonstrate the expression of PPARalpha protein in adult human testis. Our experiments with Northern analysis, in situ hybridization and immunocytochemistry reveal a complex distribution of PPARalpha in tubular and interstitial cells of both adult and developing rat testis. The overall expression is rather low but may be modified by exogenous or endogenous stimuli. An up-regulation of PPARalpha mRNA could be observed after stimulation with FSH. In the developing rat testis, a clear expression of PPARalpha mRNA was present from the first days after birth. Additionally, PPARalpha mRNA and protein increased toward adulthood. In adult human testis PPARalpha immunoreactivity (IR) was present in interstitial Leydig cells and tubular cells. In the seminiferous epithelium of adult human testis the expression of PPARalpha-IR could be seen in meiotic spermatocytes, spermatids and myoid peritubular cells. The findings of our study suggest that PPARalpha may be involved in the regulation of growth and differentiation of tubular and interstitial cells in rat and human testis.

Gene expression profiles in the testis associated with testis-ova in adult Japanese medaka (Oryziaslatipes) exposed to 17α-ethinylestradiol.

PubMed

Hirakawa, Ikumi; Miyagawa, Shinichi; Katsu, Yoshinao; Kagami, Yoshihiro; Tatarazako, Norihisa; Kobayashi, Tohru; Kusano, Teruhiko; Mizutani, Takeshi; Ogino, Yukiko; Takeuchi, Takashi; Ohta, Yasuhiko; Iguchi, Taisen

2012-05-01

The occurrence of oocytes in the testis (testis-ova) of several fish species is often associated with exposure of estrogenic chemicals. However, induction mechanisms of the testis-ova remain to be elucidated. To develop marker genes for detecting testis-ova in the testis, adult male medaka were exposed to nominal concentration of 100 ng L(-1) of 17α-ethinylestradiol (EE2) for 3-5 weeks, and 800 ng estradiol benzoate (EB) for 3 weeks (experiment I), and a measured concentration of 20 ng L(-1) EE2 for 1-6 weeks (experiment II). Histological analysis was performed for the testis, and microarray analyses were performed for the testis, liver and brain. Microarray analysis in the estrogen-exposed medaka liver showed vitellogenin and choriogenin as estrogen responsive genes. Testis-ova were induced in the testis after 4 weeks of exposure to 100 ng L(-1) EE2, 3 weeks of exposure to 800 ng EB, and 6 weeks of exposure to 20 ng L(-1) EE2. Microarray analysis of estrogen-exposed testes revealed up-regulation of genes related to zona pellucida (ZP) and the oocytes marker gene, 42Sp50. Using quantitative RT-PCR we confirmed that Zpc5 gene can be used as a marker for the detection of testis-ova in male medaka. Copyright © 2011 Elsevier Ltd. All rights reserved.

Role of Cytochrome c in Apoptosis: Increased Sensitivity to Tumor Necrosis Factor Alpha Is Associated with Respiratory Defects but Not with Lack of Cytochrome c Release▿

PubMed Central

Vempati, Uma D.; Diaz, Francisca; Barrientos, Antoni; Narisawa, Sonoko; Mian, Abdul M.; Millán, José Luis; Boise, Lawrence H.; Moraes, Carlos T.

2007-01-01

Although the role of cytochrome c in apoptosis is well established, details of its participation in signaling pathways in vivo are not completely understood. The knockout for the somatic isoform of cytochrome c caused embryonic lethality in mice, but derived embryonic fibroblasts were shown to be resistant to apoptosis induced by agents known to trigger the intrinsic apoptotic pathway. In contrast, these cells were reported to be hypersensitive to tumor necrosis factor alpha (TNF-α)-induced apoptosis, which signals through the extrinsic pathway. Surprisingly, we found that this cell line (CRL 2613) respired at close to normal levels because of an aberrant activation of a testis isoform of cytochrome c, which, albeit expressed at low levels, was able to replace the somatic isoform for respiration and apoptosis. To produce a bona fide cytochrome c knockout, we developed a mouse knockout for both the testis and somatic isoforms of cytochrome c. The mouse was made viable by the introduction of a ubiquitously expressed cytochrome c transgene flanked by loxP sites. Lung fibroblasts in which the transgene was deleted showed no cytochrome c expression, no respiration, and resistance to agents that activate the intrinsic and to a lesser but significant extent also the extrinsic pathways. Comparison of these cells with lines with a defective oxidative phosphorylation system showed that cells with defective respiration have increased sensitivity to TNF-α-induced apoptosis, but this process was still amplified by cytochrome c. These studies underscore the importance of oxidative phosphorylation and apoptosome function to both the intrinsic and extrinsic apoptotic pathways. PMID:17210651

Ermelin, an endoplasmic reticulum transmembrane protein, contains the novel HELP domain conserved in eukaryotes.

PubMed

Suzuki, Akiko; Endo, Takeshi

2002-02-06

We have cloned a cDNA encoding a novel protein referred to as ermelin from mouse C2 skeletal muscle cells. This protein contained six hydrophobic amino acid stretches corresponding to transmembrane domains, two histidine-rich sequences, and a sequence homologous to the fusion peptides of certain fusion proteins. Ermelin also contained a novel modular sequence, designated as HELP domain, which was highly conserved among eukaryotes, from yeast to higher plants and animals. All these HELP domain-containing proteins, including mouse KE4, Drosophila Catsup, and Arabidopsis IAR1, possessed multipass transmembrane domains and histidine-rich sequences. Ermelin was predominantly expressed in brain and testis, and induced during neuronal differentiation of N1E-115 neuroblastoma cells but downregulated during myogenic differentiation of C2 cells. The mRNA was accumulated in hippocampus and cerebellum of brain and central areas of seminiferous tubules in testis. Epitope-tagging experiments located ermelin and KE4 to a network structure throughout the cytoplasm. Staining with the fluorescent dye DiOC(6)(3) identified this structure as the endoplasmic reticulum. These results suggest that at least some, if not all, of the HELP domain-containing proteins are multipass endoplasmic reticulum membrane proteins with functions conserved among eukaryotes.

Distinct transcripts are recognized by sense and antisense riboprobes for a member of the murine HSP70 gene family, HSP70.2, in various reproductive tissues

NASA Technical Reports Server (NTRS)

Murashov, A. K.; Wolgemuth, D. J.

1996-01-01

The expression of hsp70.2, an hsp70 gene family member, originally characterized by its high levels of expression in germ cells in the adult mouse testis, was detected in several other reproductive tissues, including epididymis, prostate, and seminal vesicles, as well as in extraembryonic tissues of mid-gestation fetuses. In addition, hybridization with RNA probes transcribed in the sense orientation surprisingly indicated the presence of slightly larger "antisense" transcripts in several tissues. The levels of antisense transcripts varied among the tissues, with the highest signal detected in the prostate and no signal being detectable in the testis. Consistent with these results, in situ hybridization analysis clearly localized the sense-orientation transcripts to pachytene spermatocytes, while no antisense-orientation transcripts were observed in adjacent sections of the same tubules. Our findings have thus shown that although hsp70.2 was expressed abundantly and in a highly stage-specific manner in the male germ line, it was also expressed in other murine tissues. Furthermore, we have made the surprising observation of antisense transcription of the hsp70.2 gene in several mouse tissues, revealing another level of complexity in the regulation and function of heat shock proteins.

Testicular biodistribution of 450 nm fluorescent latex particles after intramuscular injection in mice.

PubMed

Klein, J-P; Boudard, D; Cadusseau, J; Palle, S; Forest, V; Pourchez, J; Cottier, M

2013-06-01

The significant expansion in the use of nanoparticles and submicron particles during the last 20 years has led to increasing concern about their potential toxicity to humans and particularly their impact on male fertility. Currently, an insufficient number of studies have focused on the testicular biodistribution of particles. The aim of our study was to assess the distribution of 450 nm fluorescent particles in mouse testes after intramuscular injection. To this end, testes were removed from 5 groups of 3 mice each at 1 h (H1), 4 days (D4), 21 days (D21), 45 days (D45) and 90 days (D90) after the injection of 7.28 × 10⁹ particles in the tibialis anterior muscles of each mouse. We examined histological sections from these samples by epifluorescence microscopy and confocal microscopy and identified testicular biodistribution of a small number of particles in groups H1, D4, D21, D45 and D90. Using CD11b immunostaining, we showed that particles were not carried into the testis by macrophages. The intratesticular repartition of particles mainly followed testicular vascularization. Finally, we found some particles in seminiferous tubules but could not determine if the blood-testis barrier was crossed.

Testicular biodistribution of 450 nm fluorescent latex particles after intramuscular injection in mice

PubMed Central

Klein, Jean-Philippe; Boudard, Delphine; Cadusseau, Josette; Palle, Sabine; Forest, Valérie; Pourchez, Jérémie; Cottier, Michèle

2013-01-01

The significant expansion in the use of nanoparticles and submicron particles during the last 20 years has led to increasing concern about their potential toxicity to humans and particularly their impact on male fertility. Currently, an insufficient number of studies have focused on the testicular biodistribution of particles. The aim of our study was to assess the distribution of 450 nm fluorescent particles in mouse testes after intramuscular injection. To this end, testes were removed from 5 groups of 3 mice each at 1 h (H1), 4 days (D4), 21 days (D21), 45 days (D45) and 90 days (D90) after the injection of 7.28 × 109 particles in the tibialis anterior muscles of each mouse. We examined histological sections from these samples by epifluorescence microscopy and confocal microscopy and identified testicular biodistribution of a small number of particles in groups H1, D4, D21, D45 and D90. Using CD11b immunostaining, we showed that particles were not carried into the testis by macrophages. The intratesticular repartition of particles mainly followed testicular vascularization. Finally, we found some particles in seminiferous tubules but could not determine if the blood–testis barrier was crossed. PMID:23329290

Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways

PubMed Central

Seigneurin-Berny, Daphné; Verdel, André; Curtet, Sandrine; Lemercier, Claudie; Garin, Jérôme; Rousseaux, Sophie; Khochbin, Saadi

2001-01-01

The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination. PMID:11689694

C-X-C motif chemokine ligand 10 produced by mouse Sertoli cells in response to mumps virus infection induces male germ cell apoptosis

PubMed Central

Jiang, Qian; Wang, Fei; Shi, Lili; Zhao, Xiang; Gong, Maolei; Liu, Weihua; Song, Chengyi; Li, Qihan; Chen, Yongmei; Wu, Han; Han, Daishu

2017-01-01

Mumps virus (MuV) infection usually results in germ cell degeneration in the testis, which is an etiological factor for male infertility. However, the mechanisms by which MuV infection damages male germ cells remain unclear. The present study showed that C-X-C motif chemokine ligand 10 (CXCL10) is produced by mouse Sertoli cells in response to MuV infection, which induces germ cell apoptosis through the activation of caspase-3. CXC chemokine receptor 3 (CXCR3), a functional receptor of CXCL10, is constitutively expressed in male germ cells. Neutralizing antibodies against CXCR3 and an inhibitor of caspase-3 activation significantly inhibited CXCL10-induced male germ cell apoptosis. Furthermore, the tumor necrosis factor-α (TNF-α) upregulated CXCL10 production in Sertoli cells after MuV infection. The knockout of either CXCL10 or TNF-α reduced germ cell apoptosis in the co-cultures of germ cells and Sertoli cells in response to MuV infection. Local injection of MuV into the testes of mice confirmed the involvement of CXCL10 in germ cell apoptosis in vivo. These results provide novel insights into MuV-induced germ cell apoptosis in the testis. PMID:29072682

Disruption of Testis Cords by Cyclopamine or Forskolin Reveals Independent Cellular Pathways in Testis Organogenesis

PubMed Central

Yao, Humphrey Hung-Chang; Capel, Blanche

2014-01-01

Most studies to date indicate that the formation of testis cords is critical for proper Sertoli cell differentiation, inhibition of germ cell meiosis, and regulation of Leydig cell differentiation. However, the connections between these events are poorly understood. The objective of this study was to dissect the molecular and cellular relationships between these events in testis formation. We took advantage of the different effects of two hedgehog signaling inhibitors, cyclopamine and forskolin, on gonad explant cultures. Both hedgehog inhibitors phenocopied the disruptive effect of Dhh−/− on formation of testis cords without influencing Sertoli cell differentiation. However, they exhibited different effects on other cellular events during testis development. Treatment with cyclopamine did not affect inhibition of germ cell meiosis and mesonephric cell migration but caused defects in Leydig cell differentiation. In contrast, forskolin treatment induced germ cell meiosis, inhibited mesonephric cell migration, and had no effect on Leydig cell differentiation. By carefully contrasting the different effects of these two hedgehog inhibitors, we demonstrate that although formation of testis cords and development of other cell types normally take place in a tightly regulated sequence, each of these events can occur independent of the others. PMID:12051821

Dynamics of testis-ova in a wild population of Japanese pond frogs, Rana nigromaculata.

PubMed

Kobayashi, Tohru; Kumakura, Masahiko; Yoshie, Sumio; Sugishima, Tomomi; Horie, Yoshifumi

2015-02-01

Although many studies have reported the occurrence of testis-ova in wild frog populations, the origin and trigger of testis-ova differentiation/development remain unclear. A high frequency of testis-ova has been previously reported for wild populations of the Japanese pond frog, Rana nigromaculata (cf. Iwasawa and Asai, '59). In the present study, we aimed to clarify the dynamics of testis-ova in this frog species, including the origin and artificial induction of testis-ova. Testis-ova were observed in both mature frogs and puberty-stage frogs (i.e., 0- and 1-year-old frogs). However, the early stages of testis-ova (~pachytene stage) were mostly observed in puberty-stage male frogs at the onset of spermatogenesis. The early stages of testis-ova were observed in the cysts of early secondary spermatogonia and the single cysts of the primary spermatogonium. This finding indicates that testis-ova differentiation occurs during spermatogonial proliferation and that it is correlated with the initiation of spermatogenesis. We also examined whether estrogen exposure induced testis-ova differentiation and how it is correlated with the progression of spermatogenesis. When 1-year-old frogs were exposed to estradiol-17β during spring (i.e., when spermatogenesis was initiated), testis-ova differentiation was induced in a dose-dependent manner. However, this phenomenon did not occur in 1-year-old frogs during summer, (i.e., when the transition from spermatogonia to spermatocytes mainly occurs). These results present the first evidence that testis-ova of the Japanese pond frog are derived from primary and early secondary spermatogonia, and that estrogen exposure induces testis-ova differentiation accompanied by the initiation of spermatogenesis. © 2015 Wiley Periodicals, Inc.

Desert hedgehog is a mammal-specific gene expressed during testicular and ovarian development in a marsupial.

PubMed

O'Hara, William A; Azar, Walid J; Behringer, Richard R; Renfree, Marilyn B; Pask, Andrew J

2011-12-01

Desert hedgehog (DHH) belongs to the hedgehog gene family that act as secreted intercellular signal transducers. DHH is an essential morphogen for normal testicular development and function in both mice and humans but is not present in the avian lineage. Like other hedgehog proteins, DHH signals through the patched (PTCH) receptors 1 and 2. Here we examine the expression and protein distribution of DHH, PTCH1 and PTCH2 in the developing testes of a marsupial mammal (the tammar wallaby) to determine whether DHH signalling is a conserved factor in gonadal development in all therian mammals. DHH, PTCH1 and PTCH2 were present in the marsupial genome and highly conserved with their eutherian orthologues. Phylogenetic analyses indicate that DHH has recently evolved and is a mammal-specific hedgehog orthologue. The marsupial PTCH2 receptor had an additional exon (exon 21a) not annotated in eutherian PTCH2 proteins. Interestingly we found evidence of this exon in humans and show that its translation would result in a truncated protein with functions similar to PTCH1. We also show that DHH expression was not restricted to the testes during gonadal development (as in mice), but was also expressed in the developing ovary. Expression of DHH, PTCH1 and PTCH2 in the adult tammar testis and ovary was consistent with findings in the adult mouse. These data suggest that there is a highly conserved role for DHH signalling in the differentiation and function of the mammalian testis and that DHH may be necessary for marsupial ovarian development. The receptors PTCH1 and PTCH2 are highly conserved mediators of hedgehog signalling in both the developing and adult marsupial gonads. Together these findings indicate DHH is an essential therian mammal-specific morphogen in gonadal development and gametogenesis.

Immunocytochemical localization of a histone H2A variant in the mammalian nucleolar chromatin.

PubMed

Bhatnagar, Y M; McCullar, M K; Chronister, R B

1984-11-01

The distribution of protein "A", a minor variant of H2A present in the mouse testis, was studied in the liver and brain nuclei using peroxidase-antiperoxidase technique. The data presented here suggest that nucleolar-associated chromatin is highly enriched in protein "A". Microspectrophotometric measurements corroborate the immunocytochemical data. The regional differentiation in the eukaryotic chromatin, therefore, may involve qualitative changes in the histone composition.

Cell junction dynamics in the testis: Sertoli-germ cell interactions and male contraceptive development.

PubMed

Cheng, C Yan; Mruk, Dolores D

2002-10-01

Spermatogenesis is an intriguing but complicated biological process. However, many studies since the 1960s have focused either on the hormonal events of the hypothalamus-pituitary-testicular axis or morphological events that take place in the seminiferous epithelium. Recent advances in biochemistry, cell biology, and molecular biology have shifted attention to understanding some of the key events that regulate spermatogenesis, such as germ cell apoptosis, cell cycle regulation, Sertoli-germ cell communication, and junction dynamics. In this review, we discuss the physiology and biology of junction dynamics in the testis, in particular how these events affect interactions of Sertoli and germ cells in the seminiferous epithelium behind the blood-testis barrier. We also discuss how these events regulate the opening and closing of the blood-testis barrier to permit the timely passage of preleptotene and leptotene spermatocytes across the blood-testis barrier. This is physiologically important since developing germ cells must translocate across the blood-testis barrier as well as traverse the seminiferous epithelium during their development. We also discuss several available in vitro and in vivo models that can be used to study Sertoli-germ cell anchoring junctions and Sertoli-Sertoli tight junctions. An in-depth survey in this subject has also identified several potential targets to be tackled to perturb spermatogenesis, which will likely lead to the development of novel male contraceptives.

Loss of Smad4 in Sertoli and Leydig Cells Leads to Testicular Dysgenesis and Hemorrhagic Tumor Formation in Mice1

PubMed Central

Archambeault, Denise R.; Yao, Humphrey Hung-Chang

2014-01-01

ABSTRACT As the central component of canonical TGFbeta superfamily signaling, SMAD4 is a critical regulator of organ development, patterning, tumorigenesis, and many other biological processes. Because numerous TGFbeta superfamily ligands are expressed in developing testes, there may exist specific requirements for SMAD4 in individual testicular cell types. Previously, we reported that expansion of the fetal testis cords requires expression of SMAD4 by the Sertoli cell lineage. To further uncover the role of Smad4 in murine testes, we produced conditional knockout mice lacking Smad4 in either Leydig cells or in both Sertoli and Leydig cells simultaneously. Loss of Smad4 concomitantly in Sertoli and Leydig cells led to underdevelopment of the testis cords during fetal life and mild testicular dysgenesis in young adulthood (decreased testis size, partially dysgenic seminiferous tubules, and low sperm production). When the Sertoli/Leydig cell Smad4 conditional knockout mice aged (56- to 62-wk old), the testis phenotypes became exacerbated with the appearance of hemorrhagic tumors, Leydig cell adenomas, and a complete loss of spermatogenesis. In contrast, loss of Smad4 in Leydig cells alone did not appreciably alter fetal and adult testis development. Our findings support a cell type-specific requirement of Smad4 in testis development and suppression of testicular tumors. PMID:24501173

X-y interactions underlie sperm head abnormality in hybrid male house mice.

PubMed

Campbell, Polly; Nachman, Michael W

2014-04-01

The genetic basis of hybrid male sterility in house mice is complex, highly polygenic, and strongly X linked. Previous work suggested that there might be interactions between the Mus musculus musculus X and the M. m. domesticus Y with a large negative effect on sperm head morphology in hybrid males with an F1 autosomal background. To test this, we introgressed the M. m. domesticus Y onto a M. m. musculus background and measured the change in sperm morphology, testis weight, and sperm count across early backcross generations and in 11th generation backcross males in which the opportunity for X-autosome incompatibilities is effectively eliminated. We found that abnormality in sperm morphology persists in M. m. domesticus Y introgression males, and that this phenotype is rescued by M. m. domesticus introgressions on the X chromosome. In contrast, the severe reductions in testis weight and sperm count that characterize F1 males were eliminated after one generation of backcrossing. These results indicate that X-Y incompatibilities contribute specifically to sperm morphology. In contrast, X-autosome incompatibilities contribute to low testis weight, low sperm count, and sperm morphology. Restoration of normal testis weight and sperm count in first generation backcross males suggests that a small number of complex incompatibilities between loci on the M. m. musculus X and the M. m. domesticus autosomes underlie F1 male sterility. Together, these results provide insight into the genetic architecture of F1 male sterility and help to explain genome-wide patterns of introgression across the house mouse hybrid zone.

Extraordinary Sequence Divergence at Tsga8, an X-linked Gene Involved in Mouse Spermiogenesis

PubMed Central

Good, Jeffrey M.; Vanderpool, Dan; Smith, Kimberly L.; Nachman, Michael W.

2011-01-01

The X chromosome plays an important role in both adaptive evolution and speciation. We used a molecular evolutionary screen of X-linked genes potentially involved in reproductive isolation in mice to identify putative targets of recurrent positive selection. We then sequenced five very rapidly evolving genes within and between several closely related species of mice in the genus Mus. All five genes were involved in male reproduction and four of the genes showed evidence of recurrent positive selection. The most remarkable evolutionary patterns were found at Testis-specific gene a8 (Tsga8), a spermatogenesis-specific gene expressed during postmeiotic chromatin condensation and nuclear transformation. Tsga8 was characterized by extremely high levels of insertion–deletion variation of an alanine-rich repetitive motif in natural populations of Mus domesticus and M. musculus, differing in length from the reference mouse genome by up to 89 amino acids (27% of the total protein length). This population-level variation was coupled with striking divergence in protein sequence and length between closely related mouse species. Although no clear orthologs had previously been described for Tsga8 in other mammalian species, we have identified a highly divergent hypothetical gene on the rat X chromosome that shares clear orthology with the 5′ and 3′ ends of Tsga8. Further inspection of this ortholog verified that it is expressed in rat testis and shares remarkable similarity with mouse Tsga8 across several general features of the protein sequence despite no conservation of nucleotide sequence across over 60% of the rat-coding domain. Overall, Tsga8 appears to be one of the most rapidly evolving genes to have been described in rodents. We discuss the potential evolutionary causes and functional implications of this extraordinary divergence and the possible contribution of Tsga8 and the other four genes we examined to reproductive isolation in mice. PMID:21186189

Testicular tissue cryopreservation and spermatogonial stem cell transplantation to restore fertility: from bench to bedside

PubMed Central

2014-01-01

Male infertility management has made significant progress during the past three decades, especially after the introduction of intracytoplasmic sperm injection in 1992. However, many boys and men still suffer from primary testicular failure due to acquired or genetic causes. New and novel treatments are needed to address these issues. Spermatogenesis originates from spermatogonial stem cells (SSCs) that reside in the testis. Many of these men lack SSCs or have lost SSCs over time as a result of specific medical conditions or toxic exposures. Loss of SSCs is critical in prepubertal boys who suffer from cancer and are going through gonadotoxic cancer treatments, as there is no option of sperm cryopresrvation due to sexual immaturity. The development of SSC transplantation in a mouse model to repopulate spermatozoa in depleted testes has opened new avenues of research in other animal models, including non-human primates. Recent advances in cryopreservation and in vitro propagation of human SSCs offer promise for human SSC autotransplantation in the near future. Ongoing research is focusing on safety and technical issues of human SSC autotransplantation. This is the time to counsel parents and boys at risk of infertility on the possibility of cryopreserving and banking a small amount of testis tissue for potential future use in SSC transplantation. PMID:25157677

Sox9 overexpression in uterine epithelia induces endometrial gland hyperplasia

PubMed Central

Gonzalez, Gabriel; Mehra, Shyamin; Wang, Ying; Akiyama, Haruhiko

2016-01-01

SOX9 is a high mobility group transcription factor that is required in many biological processes, including cartilage differentiation, endoderm progenitor maintenance, hair differentiation, and testis determination. SOX9 has also been linked to colorectal, prostate, and lung cancer. We found that SOX9 is expressed in the epithelium of the adult mouse and human uterus, predominantly marking the uterine glands. To determine if SOX9 plays a role in the development of endometrial cancer we overexpressed Sox9 in the uterine epithelium using a progesterone receptor-Cre mouse model. Sox9 overexpression in the uterine epithelium led to the formation of simple and complex cystic glandular structures in the endometrium of aged-females. Histological analysis revealed that these structures appeared morphologically similar to structures present in patients with endometrial hyperplastic lesions and endometrial polyps that are thought to be precursors of endometrial cancer. The molecular mechanisms that cause the glandular epithelium to become hyperplastic, leading to endometrial cancer are still poorly understood. These findings indicate that chronic overexpression of Sox9 in the uterine epithelium can induce the development of endometrial hyperplastic lesions. Thus, SOX9 expression may be a factor in the formation of endometrial cancer. PMID:27262401

Goat RSPO1 over-expression rescues sex-reversal in Rspo1-knockout XX mice but does not perturb testis differentiation in XY or sex-reversed XX mice.

PubMed

Buscara, Laurine; Montazer-Torbati, Fatemeh; Chadi, Sead; Auguste, Aurélie; Laubier, Johann; Chassot, Anne-Amandine; Renault, Lauriane; Passet, Bruno; Costa, José; Pannetier, Maëlle; Vilotte, Marthe; Chaboissier, Marie-Christine; Vilotte, Jean-Luc; Pailhoux, Eric; Le Provost, Fabienne

2009-08-01

RSPO1 is a newly discovered gene involved in sex differentiation. Two goat BAC clones encompassing the RSPO1 gene (gRSPO1) were injected into mouse oocytes and several transgenic lines derived. Both clones induced gRSPO1 over-expression in various tissues, including male and female gonads, with no obvious phenotype and normal sex-ratios. Introgression of the gRSPO1 transgene into a mouse RSPO1 knockout genotype resulted in the rescue of the fertility and the disappearance of the masculinized gonadic features of the females, demonstrating the functionality of the goat protein in a mouse context. On the contrary, over-expression of gRSPO1 within a mSRY or a gSRY-XX genotypes did not interfere with the SRY-induced male phenotype.

The molecular and cellular basis of gonadal sex reversal in mice and humans

PubMed Central

Warr, Nick; Greenfield, Andy

2012-01-01

The mammalian gonad is adapted for the production of germ cells and is an endocrine gland that controls sexual maturation and fertility. Gonadal sex reversal, namely, the development of ovaries in an XY individual or testes in an XX, has fascinated biologists for decades. The phenomenon suggests the existence of genetic suppressors of the male and female developmental pathways and molecular genetic studies, particularly in the mouse, have revealed controlled antagonism at the core of mammalian sex determination. Both testis and ovary determination represent design solutions to a number of problems: how to generate cells with the right properties to populate the organ primordium; how to produce distinct organs from an initially bipotential primordium; how to pattern an organ when the expression of key cell fate determinants is initiated only in a discrete region of the primordium and extends to other regions asynchronously; how to coordinate the interaction between distinct cell types in time and space and stabilize the resulting morphology; and how to maintain the differentiated state of the organ throughout the adult period. Some of these, and related problems, are common to organogenesis in general; some are distinctive to gonad development. In this review, we discuss recent studies of the molecular and cellular events underlying testis and ovary development, with an emphasis on the phenomenon of gonadal sex reversal and its causes in mice and humans. Finally, we discuss sex-determining loci and disorders of sex development in humans and the future of research in this important area. WIREs Dev Biol 2012, 1:559–577. doi: 10.1002/wdev.42 PMID:23801533

Sertoli Cells Are Susceptible to ZIKV Infection in Mouse Testis.

PubMed

Sheng, Zi-Yang; Gao, Na; Wang, Zhao-Yang; Cui, Xiao-Yun; Zhou, De-Shan; Fan, Dong-Ying; Chen, Hui; Wang, Pei-Gang; An, Jing

2017-01-01

Flaviviruses including Dengue virus (DENV), Yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) are global health problems that caused several serious diseases such as fever, hemorrhagic fever, and encephalitis in the past century. Recently, Zika virus (ZIKV) which spreads from Asia to American and causes millions of infections emerges as a new dangerous member of the genus of Flavivirus . Unlike other well-known flaviviruses, ZIKV can be transmitted sexually and infect testes in murine models. Its impacts on sperm functions, and the exact susceptible cells, however, are not entirely clear. To investigate these issues, we infected interferon α/β and γ receptors deficient AG6 mice with ZIKV and examined the outcomes of infection using an assortment of physiological, histopathological, immunological, and virological techniques. We found that infected mice displayed signs of reproductive system disorder, altered androgen levels in serum, and high viral load in semen and testes. Additionally, histopathological examinations revealed marked atrophy of seminiferous tubules and significant reduction in lumen size. Notably, these were accompanied by positive staining of ZIKV antigens on sertoli cells, detection of viral particles and vacuole changes within cytoplasm of sertoli cells. The susceptibility of sertoli cells to ZIKV was further validated in vitro study using cell lines. Importantly, the disruption of tight junctions within testis and altered sperm morphology were also observed in ZIKV infected mice. It is well-known that tight junctions formed by adjacent sertoli cells are major component of blood testis barrier, which plays important roles in maintenance of microenvironment for spermagenesis in testis. Taken together, these results demonstrate that sertoli cells are susceptible to ZIKV infection, which results in the disruption of tight junctions in testis and causes abnormal spermatogenesis in mice. These results also imply that long-term impact of ZIKV infection on human male reproductive system requires close monitoring.

Drug transporters, the blood–testis barrier, and spermatogenesis

PubMed Central

Su, Linlin; Mruk, Dolores D; Cheng, C Yan

2015-01-01

The blood–testis barrier (BTB), which is created by adjacent Sertoli cells near the basement membrane, serves as a ‘gatekeeper’ to prohibit harmful substances from reaching developing germ cells, most notably postmeiotic spermatids. The BTB also divides the seminiferous epithelium into the basal and adluminal (apical) compartment so that postmeiotic spermatid development, namely spermiogenesis, can take place in a specialized microenvironment in the apical compartment behind the BTB. The BTB also contributes, at least in part, to the immune privilege status of the testis, so that anti-sperm antibodies are not developed against antigens that are expressed transiently during spermatogenesis. Recent studies have shown that numerous drug transporters are expressed by Sertoli cells. However, many of these same drug transporters are also expressed by spermatogonia, spermatocytes, round spermatids, elongating spermatids, and elongated spermatids, suggesting that the developing germ cells are also able to selectively pump drugs ‘in’ and/or ‘out’ via influx or efflux pumps. We review herein the latest developments regarding the role of drug transporters in spermatogenesis. We also propose a model utilized by the testis to protect germ cell development from ‘harmful’ environmental toxicants and xenobiotics and/or from ‘therapeutic’ substances (e.g. anticancer drugs). We also discuss how drug transporters that are supposed to protect spermatogenesis can work against the testis in some instances. For example, when drugs (e.g. male contraceptives) that can perturb germ cell adhesion and/or maturation are actively pumped out of the testis or are prevented from entering the apical compartment, such as by efflux pumps. PMID:21134990

Protective effects of nuclear factor erythroid 2-related factor 2 on whole body heat stress-induced oxidative damage in the mouse testis

PubMed Central

2013-01-01

Background Whole body heat stress had detrimental effect on male reproductive function. It's known that the nuclear factor erythroid 2-related factor 2 (Nrf2) activates expression of cytoprotective genes to enable cell adaptation to protect against oxidative stress. However, it’s still unclear about the exactly effects of Nrf2 on the testis. Here, we investigate the protective effect of Nrf2 on whole body heat stress-induced oxidative damage in mouse testis. Methods Male mice were exposed to the elevated ambient temperature (42°C) daily for 2 h. During the period of twelve consecutive days, mice were sacrificed on days 1, 2, 4, 8 and 12 immediately following heat exposure. Testes weight, enzymatic antioxidant activities and concentrations of malondialdehyde (MDA) and glutathione (GSH) in the testes were determined and immunohistochemical detection of Nrf2 protein and mRNA expression of Nrf2-regulated genes were analyzed to assess the status of Nrf2-antioxidant system. Results Heat-exposed mice presented significant increases in rectal, scrotal surface and body surface temperature. The concentrations of cortisol and testosterone in serum fluctuated with the number of exposed days. There were significant decrease in testes weight and relative testes weight on day 12 compared with those on other days, but significant increases in catalase (CAT) activity on day 1 and GSH level on day 4 compared with control group. The activities of total superoxide dismutase (T-SOD) and copper-zinc SOD (CuZn-SOD) increased significantly on days 8 and 12. Moreover, prominent nuclear accumulation of Nrf2 protein was observed in Leydig cells on day 2, accompanying with up-regulated mRNA levels of Nrf2-regulated genes such as Nrf2, heme oxygenase 1 (HO-1), γ-Glutamylcysteine synthetase (GCLC) and NAD (P) H: quinone oxidoreductase 1 (NQO1)) in heat-treated groups. Conclusions These results suggest that Nrf2 displayed nuclear accumulation and protective activity in the process of heat treated-induced oxidative stress in mouse testes, indicating that Nrf2 might be a potential target for new drugs designed to protect germ cell and Leydig cell from oxidative stress. PMID:23514035

Ehd4 is required to attain normal pre-pubertal testis size but dispensable for fertility in male mice

PubMed Central

George, Manju; Rainey, Mark A.; Naramura, Mayumi; Ying, GuoGuang; Harms, Don W.; Vitaterna, Martha H.; Doglio, Lynn; Crawford, Susan E.; Hess, Rex A.; Band, Vimla; Band, Hamid

2010-01-01

The four highly homologous members of the C-terminal EH domain-containing (EHD) protein family (EHD1-4) regulates endocytic recycling. To delineate the role of EHD4 in normal physiology and development, mice with a conditional knockout of the Ehd4 gene were generated. PCR of genomic DNA and Western blotting of organ lysates from Ehd4−/− mice confirmed EHD4 deletion. Ehd4−/− mice were viable and born at expected Mendelian ratios; however, males showed a 50% reduction in testis weight, obvious from postnatal day 31. An early (day 10) increase in germ cell proliferation and apoptosis and a later increase in apoptosis (day 31) were seen in the Ehd4−/− testis. Other defects included a progressive reduction in seminiferous tubule diameter, dysregulation of seminiferous epithelium and head abnormalities in elongated spermatids. As a consequence, lower sperm counts and reduced fertility were observed in Ehd4−/− males. Interestingly, EHD protein expression was seen to be temporally regulated in the testis and levels peaked between days 10 and 15. In the adult testis, EHD4 was highly expressed in primary spermatocytes and EHD4 deletion altered the levels of other EHD proteins in an age-dependent manner. We conclude that high levels of EHD1in the adult Ehd4−/− testis functionally compensate for lack of EHD4 and prevents the development of severe fertility defects. Our results suggest a role for EHD4 in the proper development of post-mitotic and post-meiotic germ cells and implicate EHD protein-mediated endocytic recycling as an important process in germ cell development and testis function. PMID:20213691

Effects of maternal dietary selenium (Se-enriched yeast) on testis development, testosterone level and testicular steroidogenesis-related gene expression of their male kids in Taihang Black Goats.

PubMed

Shi, Lei; Song, Ruigao; Yao, Xiaolei; Duan, Yunli; Ren, Youshe; Zhang, Chunxiang; Yue, Wenbin; Lei, Fulin

2018-07-01

To investigate the effects of maternal dietary selenium (Se-enriched yeast) on testis development, testosterone level and steroidogenesis-related gene expression in testis of their male kids, selected pregnant Taihang Black Goats were randomly allotted to four treatment groups. They were fed the basal gestation and lactation diets supplemented with 0 (control), 0.5, 2.0 and 4.0 mg of Se/kg DM. Thirty days after weaning, testes were collected from the kids. After the morphological development status of testis was examined, tissue samples were collected for analyzing testosterone concentration and histological parameters. Testosterone synthesis-related genes were detected using real-time PCR. Localization and quantification of androgen receptor (AR) in testis of goats were determined by immunohistochemical and western blot analysis. The results show that Se supplementation in the diet of dams led to higher (p < 0.05) testicular weight, volume, length, width, transverse and vertical grith of their male kids. Excessive Se (4.0 mg/kg) can inhibit the development of testis by decreasing testicular weight and volume. The density of spermatogenic cells and Leydig cells in the Se treatment groups was significantly (p < 0.05) higher than that in the control. Maternal dietary Se did not affect the thickness of testes, thickness of germinal epithelium and diameter of seminiferous tubule. Se supplemented in the diet of dams improved the testosterone level in testis tissue and serum, and promote the expression of testosterone-related genes. The mRNA expression of StAR, 3β-HSD and CYP11A1 was decreased with the increasing dietary Se levels of dams. Maternal dietary Se can improve the AR protein abundance in testis of their offspring. AR immunopositive product was detected in Leydig cells, peritubular myoid cells, perivascular smooth muscle cells, primary spermatocytes and spermatids. The expression of AR in spermatogenetic cells is stage specific. This study suggests that maternal dietary Se can influence the testis development and spermatogenesis of their male kids by modulating testosterone synthesis in goats. More attention should be given to the potential role of maternal nutrition in improving reproductive performance of their offspring. Copyright © 2018 Elsevier Inc. All rights reserved.

Quantitative assessment of testicular germ cell production and kinematic and morphometric parameters of ejaculated spermatozoa in the grey mouse lemur, Microcebus murinus.

PubMed

Aslam, H; Schneiders, A; Perret, M; Weinbauer, G F; Hodges, J K

2002-02-01

Germ cell production and organization of the testicular epithelium in a prosimian species, the grey mouse lemur, Microcebus murinus, was investigated to extend knowledge of comparative primate spermatogenesis. In addition, semen samples collected from adult male lemurs (body weight 53-92 g; n = 16) by rectal probe electroejaculation were evaluated using computer-assisted morphometric and kinematic analysis of spermatozoa. Epididymidal spermatozoa were collected from six animals after hemicastration; the testes were weighed and prepared for stereological analysis and flow cytometry. The relative testis mass (as a percentage of body weight) ranged between 1.17 and 5.6%. Twelve stages of testicular seminiferous epithelium as described for macaques were applied and only a single stage was observed in most of the seminiferous tubule cross-sections. On average (mean SD), a single testis contained 1870 +/- 829 x 10(6) germ cells and 35 +/- 12 x 10(6) Sertoli cells. Germ cell ratios (preleptotene:type B spermatogonia = 2, round spermatid:pachytene = 3; elongated spermatid:round spermatids = 1) indicated high spermatogenic efficacy. Sperm head dimensions and tail lengths of the ejaculated and epididymidal spermatozoa were similar. Percentages of defects (neck/mid-piece and tail) were low ( 10%) and similar for ejaculated and epididymidal spermatozoa. Spermatozoa were highly motile, characterized by extensive lateral head displacement, but relatively low progressive motility. In conclusion, the grey mouse lemur has unusually large testes with a highly efficient spermatogenic process and large sperm output. These features, together with the high proportion of morphologically normal and highly motile spermatozoa in the ejaculates, indicate that Microcebus murinus is a species in which sperm competition after ejaculation is likely to occur. The predominantly single spermatogenic stage system seems to be an ancestral feature among primates.

Disruption of a Spermatogenic Cell-Specific Mouse Enolase 4 (Eno4) Gene Causes Sperm Structural Defects and Male Infertility1

PubMed Central

Nakamura, Noriko; Dai, Qunsheng; Williams, Jason; Goulding, Eugenia H.; Willis, William D.; Brown, Paula R.; Eddy, Edward M.

2013-01-01

ABSTRACT Sperm utilize glycolysis to generate ATP required for motility, and several spermatogenic cell-specific glycolytic isozymes are associated with the fibrous sheath (FS) in the principal piece of the sperm flagellum. We used proteomics and molecular biology approaches to confirm earlier reports that a novel enolase is present in mouse sperm. We then found that a pan-enolase antibody, but not antibodies to ENO2 and ENO3, recognized a protein in the principal piece of the mouse sperm flagellum. Database analyses identified two previously uncharacterized enolase family-like candidate genes, 64306537H0Rik and Gm5506. Northern analysis indicated that 64306537H0Rik (renamed Eno4) was transcribed in testes of mice by Postnatal Day 12. To determine the role of ENO4, we generated mice using embryonic stem cells in which an Eno4 allele was disrupted by a gene trap containing a beta galactosidase (beta-gal) reporter (Eno4+/Gt). Expression of beta-gal occurred in the testis, and male mice homozygous for the gene trap allele (Eno4Gt/Gt) were infertile. Epididymal sperm numbers were 2-fold lower and sperm motility was reduced substantially in Eno4Gt/Gt mice compared to wild-type mice. Sperm from Eno4Gt/Gt mice had a coiled flagellum and a disorganized FS. The Gm5506 gene encodes a protein identical to ENO1 and also is transcribed at a low level in testis. We conclude that ENO4 is required for normal assembly of the FS and provides most of the enolase activity in sperm and that Eno1 and/or Gm5506 may encode a minor portion of the enolase activity in sperm. PMID:23446454

Pathoproteomics of testicular tissue deficient in the GARP component VPS54: the wobbler mouse model of globozoospermia.

PubMed

Jockusch, Harald; Holland, Ashling; Staunton, Lisa; Schmitt-John, Thomas; Heimann, Peter; Dowling, Paul; Ohlendieck, Kay

2014-04-01

In human globozoospermia, round-headed spermatozoa lack an acrosome and therefore cannot properly interact with oocytes. In the wobbler (WR) mouse, an L967Q missense mutation in the vesicular protein-sorting factor VPS54 causes motor neuron degeneration and globozoospermia. Although electron microscopy of WR testis shows all major components of spermatogenesis, they appear in a deranged morphology that prevents the formation of the acrosome. In order to determine proteome-wide changes, affected testes were analysed by 2D-DIGE and MS. The concentration of 8 proteins was increased and that of 35 proteins decreased as compared to wild type. Mass spectrometric analysis identified proteins with an altered concentration to be associated with metabolite transport, fatty acid metabolism, cellular interactions, microtubule assembly and stress response (chaperones Hsp70-2 and Hsp90α). Minor changes were observed for proteins involved in cell redox homeostasis, cytoskeleton formation, PTMs, detoxification and metabolism. The most dramatically decreased protein in WR testis was identified as fatty acid binding protein FABP3, as confirmed by immunoblot analysis. We conclude that a missense mutation in VPS54, an essential component of the Golgi-associated retrograde protein complex, not only prevents the formation of an acrosome but also initiates a cascade of metabolic abnormalities and a stress reaction. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The nuclear import factor importin α4 can protect against oxidative stress.

PubMed

Young, Julia C; Ly-Huynh, Jennifer D; Lescesen, Helen; Miyamoto, Yoichi; Browne, Cate; Yoneda, Yoshihiro; Koopman, Peter; Loveland, Kate L; Jans, David A

2013-10-01

The importin (IMP) superfamily of nuclear transport proteins is essential to key developmental pathways, including in the murine testis where expression of the 6 distinct IMPα proteins is highly dynamic. Present predominantly from the spermatocyte stage onwards, IMPα4 is unique in showing a striking nuclear localization, a property we previously found to be linked to maintenance of pluripotency in embryonic stem cells and to the cellular stress response in cultured cells. Here we examine the role of IMPα4 in vivo for the first time using a novel transgenic mouse model in which we overexpress an IMPα4-EGFP fusion protein from the protamine 1 promoter to recapitulate endogenous testicular germ cell IMPα4 expression in spermatids. IMPα4 overexpression did not affect overall fertility, testis morphology/weight or spermatogenic progression under normal conditions, but conferred significantly (>30%) increased resistance to oxidative stress specifically in the spermatid subpopulation expressing the transgene. Consistent with a cell-specific role for IMPα4 in protecting against oxidative stress, haploid germ cells from IMPα4 null mice were significantly (c. 30%) less resistant to oxidative stress than wild type controls. These results from two unique and complementary mouse models demonstrate a novel protective role for IMPα4 in stress responses specifically within haploid male germline cells, with implications for male fertility and genetic integrity. Copyright © 2013 Elsevier B.V. All rights reserved.

Expression of uncharacterized male germ cell-specific genes and discovery of novel sperm-tail proteins in mice.

PubMed

Kwon, Jun Tae; Ham, Sera; Jeon, Suyeon; Kim, Youil; Oh, Seungmin; Cho, Chunghee

2017-01-01

The identification and characterization of germ cell-specific genes are essential if we hope to comprehensively understand the mechanisms of spermatogenesis and fertilization. Here, we searched the mouse UniGene databases and identified 13 novel genes as being putatively testis-specific or -predominant. Our in silico and in vitro analyses revealed that the expressions of these genes are testis- and germ cell-specific, and that they are regulated in a stage-specific manner during spermatogenesis. We generated antibodies against the proteins encoded by seven of the genes to facilitate their characterization in male germ cells. Immunoblotting and immunofluorescence analyses revealed that one of these proteins was expressed only in testicular germ cells, three were expressed in both testicular germ cells and testicular sperm, and the remaining three were expressed in sperm of the testicular stages and in mature sperm from the epididymis. Further analysis of the latter three proteins showed that they were all associated with cytoskeletal structures in the sperm flagellum. Among them, MORN5, which is predicted to contain three MORN motifs, is conserved between mouse and human sperm. In conclusion, we herein identify 13 authentic genes with male germ cell-specific expression, and provide comprehensive information about these genes and their encoded products. Our finding will facilitate future investigations into the functional roles of these novel genes in spermatogenesis and sperm functions.

Effective Condition for Whole Testis Cryopreservation of Endangered Miho Spine Loach (Cobitis choii) Through the Optimization of Mud Loach (Misgurnus mizolepis) Whole Testis Cryopreservation Condition.

PubMed

Kim, J J; Nam, Y K; Bang, I C; Gong, S P

　　BACKGROUND: Miho spine loach (Cobitis choii) is an endangered Korean endemic fish. Whole testis cryopreservation is a good way for species preservation, but needs to the sacrifice of a large number of fish to optimize the freezing condition. Considering this limitation, a surrogate fish species was used for the protocol development. This study was to establish the effective condition for Miho spine loach whole testis cryopreservation by optimizing the conditions for whole testis cryopreservation in an allied species, mud loach (Misgurnus mizolepis). The condition for whole testis cryopreservation was optimized in mud loach first, and then the optimal condition was applied to Miho spine loach testes. The optimal condition for mud loach testis cryopreservation consists of the freezing medium containing 1.3 M dimethyl sulfoxide, 6% fetal bovine serum and 0.3 M trehalose, -1 C/min cooling rate and 26 degree C thawing temperature, which also permits effective cryopreservation of Miho spine loach testes. An effective cryopreservation condition for whole testis of the endangered Miho spine loach has been established by using mud loach as a surrogate fish.

Mutation of Gonadal soma-derived factor induces medaka XY gonads to undergo ovarian development.

PubMed

Imai, Takuto; Saino, Kentaro; Matsuda, Masaru

2015-11-06

Gonochoristic species have a bipotential gonad that develops into a testis or an ovary. In species whose sex is determined by a genetic factor, the expression of a sex-determining gene is the first cue that directs the development of a bipotential gonad. Subsequent expression of downstream genes induces the gonad to develop into a testis or an ovary. The TGF-ß family member Gonadal soma-derived factor (Gsdf) is thought to be an important gene for gonadal development in teleost fish, and it is expressed at higher levels in the testis than in the ovary from early to mature stages. However, there is little functional information about the gene. In this study, we targeted the Gsdf coding region in the medaka fish Oryzias latipes using transcription activator-like effector nucleases (TALENs) and studied the phenotypes of the Gsdf mutant medaka. Although normal and heterozygous XY gonads developed into a testis, all XY gonads with a homozygous mutation in Gsdf developed into an ovary at early developmental stages. However, two-thirds of Gsdf mutant XY gonads developed into testes in the adult stages. These results demonstrate that although a gonad can develop into a complete testis in the absence of Gsdf, Gsdf function is critical for directing the bipotential gonad at early developmental stages. Therefore, Gsdf is an endogenous inducer of testicular development similar to a master sex-determining gene. Copyright © 2015 Elsevier Inc. All rights reserved.

Sperm calcineurin inhibition prevents mouse fertility with implications for male contraceptive.

PubMed

Miyata, Haruhiko; Satouh, Yuhkoh; Mashiko, Daisuke; Muto, Masanaga; Nozawa, Kaori; Shiba, Kogiku; Fujihara, Yoshitaka; Isotani, Ayako; Inaba, Kazuo; Ikawa, Masahito

2015-10-23

Calcineurin inhibitors, such as cyclosporine A and FK506, are used as immunosuppressant drugs, but their adverse effects on male reproductive function remain unclear. The testis expresses somatic calcineurin and a sperm-specific isoform that contains a catalytic subunit (PPP3CC) and a regulatory subunit (PPP3R2). We demonstrate herein that male mice lacking Ppp3cc or Ppp3r2 genes (knockout mice) are infertile, with reduced sperm motility owing to an inflexible midpiece. Treatment of mice with cyclosporine A or FK506 creates phenocopies of the sperm motility and morphological defects. These defects appear within 4 to 5 days of treatment, which indicates that sperm-specific calcineurin confers midpiece flexibility during epididymal transit. Male mouse fertility recovered a week after we discontinued treatment. Because human spermatozoa contain PPP3CC and PPP3R2 as a form of calcineurin, inhibition of this sperm-specific calcineurin may lead to the development of a reversible male contraceptive that would target spermatozoa in the epididymis. Copyright © 2015, American Association for the Advancement of Science.

Validation of an automated counting procedure for phthalate-induced testicular multinucleated germ cells.

PubMed

Spade, Daniel J; Bai, Cathy Yue; Lambright, Christy; Conley, Justin M; Boekelheide, Kim; Gray, L Earl

2018-06-15

In utero exposure to certain phthalate esters results in testicular toxicity, characterized at the tissue level by induction of multinucleated germ cells (MNGs) in rat, mouse, and human fetal testis. Phthalate exposures also result in a decrease in testicular testosterone in rats. The anti-androgenic effects of phthalates have been more thoroughly quantified than testicular pathology due to the significant time requirement associated with manual counting of MNGs on histological sections. An automated counting method was developed in ImageJ to quantify MNGs in digital images of hematoxylin-stained rat fetal testis tissue sections. Timed pregnant Sprague Dawley rats were exposed by daily oral gavage from gestation day 17 to 21 with one of eight phthalate test compounds or corn oil vehicle. Both the manual counting method and the automated image analysis method identified di-n-butyl phthalate, butyl benzyl phthalate, dipentyl phthalate, and di-(2-ethylhexyl) phthalate as positive for induction of MNGs. Dimethyl phthalate, diethyl phthalate, the brominated phthalate di-(2-ethylhexyl) tetrabromophthalate, and dioctyl terephthalate were negative. The correlation between automated and manual scoring metrics was high (r = 0.923). Results of MNG analysis were consistent with these compounds' anti-androgenic activities, which were confirmed in an ex vivo testosterone production assay. In conclusion, we have developed a reliable image analysis method that can be used to facilitate dose-response studies for the reproducible induction of MNGs by in utero phthalate exposure. Copyright © 2018 Elsevier B.V. All rights reserved.

Coexpression of Nuclear Receptors and Histone Methylation Modifying Genes in the Testis: Implications for Endocrine Disruptor Modes of Action

PubMed Central

Anderson, Alison M.; Carter, Kim W.; Anderson, Denise; Wise, Michael J.

2012-01-01

Background Endocrine disruptor chemicals elicit adverse health effects by perturbing nuclear receptor signalling systems. It has been speculated that these compounds may also perturb epigenetic mechanisms and thus contribute to the early origin of adult onset disease. We hypothesised that histone methylation may be a component of the epigenome that is susceptible to perturbation. We used coexpression analysis of publicly available data to investigate the combinatorial actions of nuclear receptors and genes involved in histone methylation in normal testis and when faced with endocrine disruptor compounds. Methodology/Principal Findings The expression patterns of a set of genes were profiled across testis tissue in human, rat and mouse, plus control and exposed samples from four toxicity experiments in the rat. Our results indicate that histone methylation events are a more general component of nuclear receptor mediated transcriptional regulation in the testis than previously appreciated. Coexpression patterns support the role of a gatekeeper mechanism involving the histone methylation modifiers Kdm1, Prdm2, and Ehmt1 and indicate that this mechanism is a common determinant of transcriptional integrity for genes critical to diverse physiological endpoints relevant to endocrine disruption. Coexpression patterns following exposure to vinclozolin and dibutyl phthalate suggest that coactivity of the demethylase Kdm1 in particular warrants further investigation in relation to endocrine disruptor mode of action. Conclusions/Significance This study provides proof of concept that a bioinformatics approach that profiles genes related to a specific hypothesis across multiple biological settings can provide powerful insight into coregulatory activity that would be difficult to discern at an individual experiment level or by traditional differential expression analysis methods. PMID:22496781

New TFII-I family target genes involved in embryonic development.

PubMed

Makeyev, Aleksandr V; Bayarsaihan, Dashzeveg

2009-09-04

Two members of the TFII-I family transcription factor genes, GTF2I and GTF2IRD1, are the prime candidates responsible for the craniofacial and cognitive abnormalities of Williams syndrome patients. We have previously generated mouse lines with targeted disruption of Gtf2i and Gtf2ird1. Microarray analysis revealed significant changes in the expression profile of mutant embryos. Here we described three unknown genes that were dramatically down-regulated in mutants. The 2410018M08Rik/Scand3 gene encodes a protein of unknown function with CHCH and hATC domains. Scand3 is down-regulated during mouse embryonic stem cell (ES) differentiation. 4933436H12Rik is a testis-specific gene, which encodes a protein with no known domains. It is expressed in mouse ES cells. 1110008P08Rik/Kbtbd7 encodes an adapter protein with BTB/POZ, BACK, and Kelch motifs, previously shown to recruit substrates to the enzymatic complexes of the histone modifying or E3 ubiquitin ligase activities. Based on its expression pattern Kbtbd7 may have a specific role in brain development and function. All three genes possess well-conserved TFII-I-binding consensus sites within proximal promoters. Therefore our analysis suggests that these genes can be direct targets of TFII-I proteins and their impaired expression, as a result of the GTF2I and GTF2IRD1 haploinsufficiency, could contribute to the etiology of Williams syndrome.

RNAi as a tool to control the sex ratio of mouse offspring by interrupting Zfx/Zfy genes in the testis.

PubMed

Zhang, YongSheng; Xi, JiFeng; Jia, Bin; Wang, XiangZu; Wang, XuHai; Li, ChaoCheng; Li, YaQiang; Zeng, XianCun; Ying, RuiWen; Li, Xin; Jiang, Song; Yuan, FangYuan

2017-04-01

The objective of this study was to explore a novel method to alter the sex-ratio balance of mouse offspring by silencing the paralogous genes Zfx/Zfy (Zinc finger X/Y-chromosomal transcription factor gene) during spermatogenesis. Four recombined vectors PRZ1, PRZ2, PRZ3, and PRZ4 (RNAi-Ready-pSIREN-RetroQ-ZsGreen) were constructed for interrupting the Zfx gene. Additionally, a recombined vector Psilencer/Zfy-shRNA was constructed for interrupting the Zfy gene. Male mice were randomly divided into 8 groups, with 20 animals per group. Five groups of mice were injected with PRZ1, PRZ2, PRZ3, PRZ4, and Psilencer/Zfy-shRNA vectors, respectively. The three control groups were injected with an equal volume of physiological saline, empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector, and empty Psilencer/Zfy-shRNA vector, respectively. All groups were injected every 7 days for a total of four injections. Fourteen days after the fourth injection, 10 male mice from each group were mated individually with 10 females. Testicular tissue of 10 male mice in each group was collected, and the expression level of Zfx/Zfy mRNA was determined by qRT-PCR. Results showed that, compared with the empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector and the physiological saline group, expression of Zfx mRNA decreased significantly after injection of PRZ1 (p < 0.01), PRZ3 (p < 0.01), and PRZ4 (p < 0.01), and 78.75 ± 7.50% of the offspring were male in PRZ4 group, significantly higher than the offspring derived from the empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector and physiological saline group (p < 0.01). In the PRZ1 group, the expression of Zfx mRNA was also significantly lower (p < 0.01), but the male rate of offspring was not different (p > 0.05). Conversely, the expression of Zfy mRNA decreased significantly after injection of Psilencer/Zfy-shRNA (p < 0.01) and 31.00 ± 11.00% of the offspring were male, significantly lower than in the physiological saline group (p < 0.01). In conclusion, our findings show that RNAi-mediated disruption of Zfx/Zfy in mouse testis affected X/Y spermatogenesis. Additionally, results suggest that the paralogous genes Zfx/Zfy play an important role in the process of X and Y sperm development. The individual interference of Zfx/Zfy may predict the outcome of X and Y haploid sperms. Presented herein is an advanced method developed to control mouse X/Y spermatogenesis and sex ratio of offspring.

[Molecular mechanisms in sex determination: from gene regulation to pathology].

PubMed

Ravel, C; Chantot-Bastaraud, S; Siffroi, J-P

2004-01-01

Testis determination is the complex process by which the bipotential gonad becomes a normal testis during embryo development. As a consequence, this process leads to sexual differentiation corresponding to the masculinization of both genital track and external genitalia. The whole phenomenon is under genetic control and is particularly driven by the presence of the Y chromosome and by the SRY gene, which acts as the key initiator of the early steps of testis determination. However, many other autosomal genes, present in both males and females, are expressed during testis formation in a gene activation pathway, which is far to be totally elucidated. All these genes act in a dosage-sensitive manner by which quantitative gene abnormalities, due to chromosomal deletions, duplications or mosaicism, may lead to testis determination failure and sex reversal.

[A preparative method for isolating the synaptonemal complexes from mammalian spermatocytes].

PubMed

Dadashev, S Ia; Bogdanov, Iu F; Gorach, G G; Kolomiets, O L; Karpova, O I

1993-01-01

A method of isolation of synaptonemal complexes (SC) from mouse, rat and Syrian hamster spermatocytes is described. A fraction of pachytene spermatocyte nuclei was obtained by centrifugation of the testis homogenate in stepwise sucrose gradient and then lysed. The resulting chromatine was hydrolysed with DNAse II, and a fraction of isolated SCs was obtained by ultracentrifugation of the hydrolysate. The method can be applied for obtaining the SC fraction from spermatocytes sufficient for cytological, biochemical and molecular biology studies.

De novo transcriptome analysis and differentially expressed genes in the ovary and testis of the Japanese mantis shrimp Oratosquilla oratoria by RNA-Seq.

PubMed

Yan, Hongwei; Cui, Xin; Shen, Xufang; Wang, Lianshun; Jiang, Linan; Liu, Haiying; Liu, Ying; Liu, Qi; Jiang, Chen

2018-06-01

The mantis shrimp Oratosquilla oratoria is a widely distributed, commercially important crustacean species. Although its conservation and the development of successful artificial breeding technologies have recently received considerable attention, there are currently no available data regarding the molecular mechanisms in controlling reproduction. In this study, we performed transcriptome sequencing of the testis, ovary, female and male eyestalks and the androgenic gland of O. oratoria, and compared the expression pattern of transcripts from the testis and ovary libraries to identify genes involved in gonadal development. A total of 147,130,937 clean reads were retrieved after removing the adapters in reads and filtering out low-quality data. All the reads were assembled into 94,990 unigenes (23,133 in testis and ovary) with an average length of 783 base pairs (bp) and N50 of 1502 bp. A search of all-unigenes against COG, GO, KEGG, KOG, Pfam, Swiss-Prot and Nr databases resulted in a total of 19,404 annotated unigenes. Comparison of the sequences in the ovary and testis libraries revealed that 1188 unigenes were up-regulated in the ovary and 2732 were up-regulated in the testis. Twenty ovary-up-regulated and 21 testis-up-regulated unigenes were confirmed by quantitative real-time PCR. Additionally, 13,437 simple sequence repeats (SSRs) and 275,799 putative single nucleotide polymorphisms (SNPs) were identified. The important functional genes and pathways identified here provide a valuable dataset for understanding the molecular mechanisms controlling gonad development in O. oratoria, and the numerous (13,437 SSRs and 275,799 SNPs) molecular markers obtained here will provide fundamental basis for functional genomic and population genetic studies of O. oratoria. Copyright © 2018 Elsevier Inc. All rights reserved.

[Pharmacological study on Tianxiong (tuber of Aconitum carmichaeli Debx.), a Chinese drug for reinforcing the kidney yang retail in Hong Kong market].

PubMed

Cao, H; Wang, S T; Wu, L Y; Wang, X T; Jiang, A P

2001-06-01

To explore the pharmacodynamic mechanism of Tianxiong (Aconitum carmichaeli) in tonifying the kidney and supporting Yang, so as to provide evidences for further development of new drugs treating Yang-eficiency of the kidney. Observing parameters such as visceral index, survival time of low-temperature swimming for hydrocortisone-induced Yang-deficiency model mouse and testis-removed kidney-deficiency model rat. The decoction of processed Tianxiong could strengthen the antifatigual ability and prolong the survival time of low-temperature swimming for mice, and promote immunization in rats. Tianxiong is able to reinforce the kidney Yang, which reconforms the conclusion of "replenishing the fire of vital gate and the Qi of kidney" recorded in Chinese historical literature and proved by overseas clinical practice.

Sex determination in mammalian germ cells

PubMed Central

Spiller, Cassy M; Bowles, Josephine

2015-01-01

Germ cells are the precursors of the sperm and oocytes and hence are critical for survival of the species. In mammals, they are specified during fetal life, migrate to the developing gonads and then undergo a critical period during which they are instructed, by the soma, to adopt the appropriate sexual fate. In a fetal ovary, germ cells enter meiosis and commit to oogenesis, whereas in a fetal testis, they avoid entry into meiosis and instead undergo mitotic arrest and mature toward spermatogenesis. Here, we discuss what we know so far about the regulation of sex-specific differentiation of germ cells, considering extrinsic molecular cues produced by somatic cells, as well as critical intrinsic changes within the germ cells. This review focuses almost exclusively on our understanding of these events in the mouse model. PMID:25791730

Identification and characterization of an SPO11 homolog in the mouse.

PubMed

Metzler-Guillemain, C; de Massy, B

2000-01-01

The SPO11/TOPVIA family includes proteins from archaebacteria and eukaryotes. The protein member from the archaebacterium Sulfulobus shibatae is the catalytic subunit of TopoVI DNA topoisomerase. In Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster, SPO11 is required for meiotic recombination, suggesting a conserved mechanism for the initiation step of this process. Indeed, S. cerevisiae SPO11 has been shown to be directly involved in the formation of meiotic DNA double-strand breaks that initiate meiotic recombination. Here, we report the identification of a Mus musculus Spo11 cDNA, which encodes a protein closely related to all members of the SPO11/TOPVIA family. cDNAs resulting from alternative splicing were detected, suggesting that there are potential variants of the mouse SPO11 protein. By RNA-blotting analysis, expression of the mouse Spo11 gene was detected only in the testis, in agreement with its predicted function in the initiation of meiotic recombination. We mapped the mouse Spo11 gene to chromosome 2, band H2-H4.

Morphological and histomorphological structures of testes and ovaries in early developmental stages of the silkworm, Bombyx mori.

PubMed

Sakai, Hiroki; Kirino, Yohei; Katsuma, Susumu; Aoki, Fugaku; Suzuki, Masataka G

2016-01-01

The gonad develops as a testis in male or an ovary in female. In the silkworm, B. mori , little is known about testis and ovary in the embryonic stages and early larval stages. In this study, we performed morphological and histomorphological observations of ovaries and testes from the late embryonic stage to the 1st instar larval stage. Results obtained with lack of accurate information on sex of examined individuals may be misleading, thus we performed phenotypic observations of gonads by utilizing sex-limited strain that enables us to easily discriminate female embryos from male ones based on those egg colors. In testis, four testicular follicles were clearly observed in the testis at the first instar larval stage, and boundary layers were formed between the testicular follicles. At the late embryonic stage, the testis consisted of four testicular follicles, while the boundary layers were still obscure. In ovary, four ovarioles were easily recognizable in the ovary at the first instar larval stage, and boundary layers were formed between the ovarioles. However, in the late embryonic stage, it was quite difficult to identify four ovarioles. Morphological characteristics were almost similar between testis and ovary in early developmental stages. Our present study demonstrates that the most reliable difference between testis and ovary in early developmental stages is the attaching point of the duct. Formation and development of the duct may be sensitive to the sex-determining signal and display sexual dimorphism in early embryonic stages.

Expression analysis of sox3 during testicular development, recrudescence, and after hCG induction in catfish, Clarias batrachus.

PubMed

Rajakumar, Anbazhagan; Senthilkumaran, Balasubramanian

2014-01-01

In teleosts, the expression of steroidogenic enzymes and related transcription factor genes occurs in a stage- and tissue-specific manner causing sexual development. The role of sox3, an evolutionary ancestor of SRY, has not been studied in detail. Therefore, the full-length cDNA of sox3 (1,197 kb) was cloned from catfish testis, and mRNA expression was analyzed during gonadal development, during the seasonal reproductive cycle, and after human chorionic gonadotropin (hCG) induction. Tissue distribution analysis showed that sox3 expression was higher in testis, ovary, and brain compared to other tissues analyzed. Developing and mature testis showed higher sox3 expression than ovary of corresponding stages, and more sox3 transcripts were found during the spawning phase of the seasonal reproductive cycle. Expression of sox3 was upregulated by hCG after in vivo and in vitro induction, suggesting that gonadotropins might stimulate it. In situ hybridization and immunohistochemistry showed the presence of sox3 mRNA and protein in somatic and interstitial cell layers of the testis. Sox3 could also be found in the zona radiata of developing and mature oocytes. Exposure of methyltestosterone (1 µg/l) and ethinylestradiol (1 µg/l) for 21 days during testicular development showed lower sox3 expression levels in the testis and brain, indicating a certain feedback intervention. These results suggest a possible role for Sox3 in the regulation of testicular development and function. © 2014 S. Karger AG, Basel.

Sphingomyelin Synthase 1 Is Essential for Male Fertility in Mice

PubMed Central

Scherthan, Harry; Horsch, Marion; Beckers, Johannes; Fuchs, Helmut; Gailus-Durner, Valerie; Hrabě de Angelis, Martin; Ford, Steven J.; Burton, Neal C.; Razansky, Daniel; Trümbach, Dietrich; Aichler, Michaela; Walch, Axel Karl; Calzada-Wack, Julia; Neff, Frauke; Wurst, Wolfgang; Hartmann, Tobias; Floss, Thomas

2016-01-01

Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1-/- mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility. PMID:27788151

A novel TRIM family member, Trim69, regulates zebrafish development through p53-mediated apoptosis.

PubMed

Han, Ruiqin; Zhao, Qing; Zong, Shudong; Miao, Shiying; Song, Wei; Wang, Linfang

2016-05-01

Trim69 contains the hallmark domains of a tripartite motif (TRIM) protein, including a Ring-finger domain, B-box domain, and coiled-coil domain. Trim69 is structurally and evolutionarily conserved in zebrafish, mouse, rat, human, and chimpanzee. The role of this protein is unclear, however, so we investigated its function in zebrafish development. Trim69 is extensively expressed in zebrafish adults and developing embryos-particularly in the testis, brain, ovary, and heart-and its expression decreases in a time- and stage-dependent manner. Loss of trim69 in zebrafish induces apoptosis and activates apoptosis-related processes; indeed, the tp53 pathway was up-regulated in response to the knockdown. Expression of human trim69 rescued the apoptotic phenotype, while overexpression of trim69 does not increase cellular apoptosis. Taken together, our results suggest that trim69 participates in tp53-mediated apoptosis during zebrafish development. Mol. Reprod. Dev. 83: 442-454, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Mitotic Arrest in Teratoma Susceptible Fetal Male Germ Cells

PubMed Central

Western, Patrick S.; Ralli, Rachael A.; Wakeling, Stephanie I.; Lo, Camden; van den Bergen, Jocelyn A.; Miles, Denise C.; Sinclair, Andrew H.

2011-01-01

Formation of germ cell derived teratomas occurs in mice of the 129/SvJ strain, but not in C57Bl/6 inbred or CD1 outbred mice. Despite this, there have been few comparative studies aimed at determining the similarities and differences between teratoma susceptible and non-susceptible mouse strains. This study examines the entry of fetal germ cells into the male pathway and mitotic arrest in 129T2/SvJ mice. We find that although the entry of fetal germ cells into mitotic arrest is similar between 129T2/SvJ, C57Bl/6 and CD1 mice, there were significant differences in the size and germ cell content of the testis cords in these strains. In 129T2/SvJ mice germ cell mitotic arrest involves upregulation of p27KIP1, p15INK4B, activation of RB, the expression of male germ cell differentiation markers NANOS2, DNMT3L and MILI and repression of the pluripotency network. The germ-line markers DPPA2 and DPPA4 show reciprocal repression and upregulation, respectively, while FGFR3 is substantially enriched in the nucleus of differentiating male germ cells. Further understanding of fetal male germ cell differentiation promises to provide insight into disorders of the testis and germ cell lineage, such as testis tumour formation and infertility. PMID:21674058

staggerer phenotype in retinoid-related orphan receptor α-deficient mice

PubMed Central

Steinmayr, Markus; André, Elisabeth; Conquet, François; Rondi-Reig, Laure; Delhaye-Bouchaud, Nicole; Auclair, Nathalie; Daniel, Hervé; Crépel, Francis; Mariani, Jean; Sotelo, Constantino; Becker-André, Michael

1998-01-01

Retinoid-related orphan receptor α (RORα) is a member of the nuclear receptor superfamily. To study its physiological role we generated null-mutant mice by targeted insertion of a lacZ reporter gene encoding the enzyme β-galactosidase. In heterozygous RORα+/− mice we found β-galactosidase activity, indicative of RORα protein expression, confined to the central nervous system, skin and testis. In the central nervous system, the RORα gene is expressed in cerebellar Purkinje cells, the thalamus, the suprachiasmatic nuclei, and retinal ganglion cells. In skin, RORα is strongly expressed in the hair follicle, the epidermis, and the sebaceous gland. Finally, the peritubular cells of the testis and the epithelial cells of the epididymis also strongly express RORα. Recently, it was reported that the ataxic mouse mutant staggerer (sg/sg) is caused by a deletion in the RORα gene. The analysis of the cerebellar and the behavioral phenotype of homozygous RORα−/− mice proves identity to sg/sg mice. Although the absence of RORα causes dramatic developmental effects in the cerebellum, it has no apparent morphological effect on thalamus, hypothalamus, and retina. Similarly, testis and skin of RORα−/− mice display a normal phenotype. However, the pelage hair of both sg/sg and RORα−/− is significantly less dense and when shaved shows reluctance to regrow. PMID:9520475

Positional cloning of the PIS mutation in goats and its impact on understanding mammalian sex-differentiation

PubMed Central

2005-01-01

In goats, the PIS (polled intersex syndrome) mutation is responsible for both the absence of horns in males and females and sex-reversal affecting exclusively XX individuals. The mode of inheritance is dominant for the polled trait and recessive for sex-reversal. In XX PIS-/- mutants, the expression of testis-specific genes is observed very precociously during gonad development. Nevertheless, a delay of 4–5 days is observed in comparison with normal testis differentiation in XY males. By positional cloning, we demonstrate that the PIS mutation is an 11.7-kb regulatory-deletion affecting the expression of two genes, PISRT1 and FOXL2 which could act synergistically to promote ovarian differentiation. The transcriptional extinction of these two genes leads, very early, to testis-formation in XX homozygous PIS-/- mutants. According to their expression profiles and bibliographic data, we propose that FOXL2 may be an ovary-differentiating gene, and the non-coding RNA PISRT1, an anti-testis factor repressing SOX9, a key regulator of testis differentiation. Under this hypothesis, SRY, the testis-determining factor would inhibit these two genes in the gonads of XY males, to ensure testis differentiation. PMID:15601595

Positional cloning of the PIS mutation in goats and its impact on understanding mammalian sex-differentiation.

PubMed

Pailhoux, Eric; Vigier, Bernard; Schibler, Laurent; Cribiu, Edmond P; Cotinot, Corinne; Vaiman, Daniel

2005-01-01

In goats, the PIS (polled intersex syndrome) mutation is responsible for both the absence of horns in males and females and sex-reversal affecting exclusively XX individuals. The mode of inheritance is dominant for the polled trait and recessive for sex-reversal. In XX PIS-/- mutants, the expression of testis-specific genes is observed very precociously during gonad development. Nevertheless, a delay of 4-5 days is observed in comparison with normal testis differentiation in XY males. By positional cloning, we demonstrate that the PIS mutation is an 11.7-kb regulatory-deletion affecting the expression of two genes, PISRT1 and FOXL2 which could act synergistically to promote ovarian differentiation. The transcriptional extinction of these two genes leads, very early, to testis-formation in XX homozygous PIS-/- mutants. According to their expression profiles and bibliographic data, we propose that FOXL2 may be an ovary-differentiating gene, and the non-coding RNA PISRT1, an anti-testis factor repressing SOX9, a key regulator of testis differentiation. Under this hypothesis, SRY, the testis-determining factor would inhibit these two genes in the gonads of XY males, to ensure testis differentiation.

Effects of simulated microgravity on mouse Sertoli cells in culture

NASA Astrophysics Data System (ADS)

Angela, Masini Maria; Prato, Paola; Linda, Scarabelli; Lanza, Cristina; Palmero, Silvio; Pointis, Georges; Ricci, Franco; Strollo, Felice

With the advent of space flights questions concerning the effects of microgravity (0xG) on hu-man reproduction physiology have got priority Spermatogenesis is a complex, highly ordered process of cell division and differentiation by which spermatogonial cells give rise to mature spermatozoa. Sertoli cells play a crucial role in the development of germ cells and the regulation of spermatogenesis. In this study the influence of 0xG on Sertoli cells was evaluated. A Sertoli cell line from mouse testis (42GPA9) was analyzed for cytoskeletal (using the 3D reconstruction generated from a stack of confocal images) and SHBG changes by immunohistochemistry, for antioxidant agents by RT-PCR and for culture medium lactate concentrations by wet chemistry. Cells were cultured for 6, 24 and 48 hrs on a three-dimensional Random Positioning Machine (3D-RPM); static controls (1xG) were positioned on the supporting frame. At the end of each experiment, cultured cells were either fixed in paraformaldehyde or RNA-extracted or used for culture medium lactate measurements as needed. At 0xG Sertoli cytoskeleton got disorganized, microtubules fragmented and SHBG undetectable already after 24 hrs, with alterations wors-ening further until 48 hrs; various antioxidant systems (SOD, GST, PARP, MTs) appreciably increased during the first 24 hrs but significantly decreased at 48 hrs. No changes occurred in 1xG samples. At least initially, 0xG seems to perturb antioxidant protection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate production at 0xG slightly decreased only after 24 hrs. Further experiments need to be carried out in space to investigate upon steroidogenesis and germ cell differentiation within the testis, to rule out eventually pending male infertility consequences, which would be a problem nowadays, when life expectancy increases and male fertility might become a social issue often extending into 60 years and over. (experiment funded by ASI, through a grant within the OSMA project).

A proton pump ATPase with testis-specific E1-subunit isoform required for acrosome acidification.

PubMed

Sun-Wada, Ge-Hong; Imai-Senga, Yoko; Yamamoto, Akitsugu; Murata, Yoshiko; Hirata, Tomoyuki; Wada, Yoh; Futai, Masamitsu

2002-05-17

The vacuolar-type H(+)-ATPases (V-ATPases) are a family of multimeric proton pumps involved in a wide variety of physiological processes. We have identified two novel mouse genes, Atp6e1 and Atp6e2, encoding testis-specific (E1) and ubiquitous (E2) V-ATPase subunit E isoforms, respectively. The E1 transcript appears about 3 weeks after birth, corresponding to the start of meiosis, and is expressed specifically in round spermatids in seminiferous tubules. Immunohistochemistry with isoform-specific antibodies revealed that the V-ATPase with E1 and a2 isoforms is located specifically in developing acrosomes of spermatids and acrosomes in mature sperm. In contrast, the E2 isoform was expressed in all tissues examined and present in the perinuclear compartments of spermatocytes. The E1 isoform exhibits 70% identity with the E2, and both isoforms functionally complemented a null mutation of the yeast counterpart VMA4, indicating that they are bona fide V-ATPase subunits. The chimeric enzymes showed slightly lower K(m)(ATP) than yeast V-ATPase. Consistent with the temperature-sensitive growth of Deltavma4-expressing E1 isoform, vacuolar membrane vesicles exhibited temperature-sensitive coupling between ATP hydrolysis and proton transport. These results suggest that E1 isoform is essential for energy coupling involved in acidification of acrosome.

Characterization of Nestin-positive stem Leydig cells as a potential source for the treatment of testicular Leydig cell dysfunction

PubMed Central

Jiang, Mei Hua; Cai, Bing; Tuo, Ying; Wang, Jiancheng; Zang, Zhi Jun; Tu, Xiang'an; Gao, Yong; Su, Zhijian; Li, Weiqiang; Li, Guilan; Zhang, Min; Jiao, Jianwei; Wan, Zi; Deng, Chunhua; Lahn, Bruce T; Xiang, Andy Peng

2014-01-01

The ability to identify and isolate lineage-specific stem cells from adult tissues could facilitate cell replacement therapy. Leydig cells (LCs) are the primary source of androgen in the mammalian testis, and the prospective identification of stem Leydig cells (SLCs) may offer new opportunities for treating testosterone deficiency. Here, in a transgenic mouse model expressing GFP driven by the Nestin (Nes) promoter, we observed Nes-GFP+ cells located in the testicular interstitial compartment where SLCs normally reside. We showed that these Nes-GFP+ cells expressed LIFR and PDGFR-α, but not LC lineage markers. We further observed that these cells were capable of clonogenic self-renewal and extensive proliferation in vitro and could differentiate into neural or mesenchymal cell lineages, as well as LCs, with the ability to produce testosterone, under defined conditions. Moreover, when transplanted into the testes of LC-disrupted or aging models, the Nes-GFP+ cells colonized the interstitium and partially increased testosterone production, and then accelerated meiotic and post-meiotic germ cell recovery. In addition, we further demonstrated that CD51 might be a putative cell surface marker for SLCs, similar with Nestin. Taken together, these results suggest that Nes-GFP+ cells from the testis have the characteristics of SLCs, and our study would shed new light on developing stem cell replacement therapy for testosterone deficiency. PMID:25418539

Molecular cloning and subcellular localization of Tektin2-binding protein 1 (Ccdc 172) in rat spermatozoa.

PubMed

Yamaguchi, Airi; Kaneko, Takane; Inai, Tetsuichiro; Iida, Hiroshi

2014-04-01

Tektins (TEKTs) are composed of a family of filament-forming proteins localized in cilia and flagella. Five types of mammalian TEKTs have been reported, all of which have been verified to be present in sperm flagella. TEKT2, which is indispensable for sperm structure, mobility, and fertilization, was present at the periphery of the outer dense fiber (ODF) in the sperm flagella. By yeast two-hybrid screening, we intended to isolate flagellar proteins that could interact with TEKT2, which resulted in the isolation of novel two genes from the mouse testis library, referred as a TEKT2-binding protein 1 (TEKT2BP1) and -protein 2 (TEKT2BP2). In this study, we characterized TEKT2BP1, which is registered as a coiled-coil domain-containing protein 172 (Ccdc172) in the latest database. RT-PCR analysis indicated that TEKT2BP1 was predominantly expressed in rat testis and that its expression was increased after 3 weeks of postnatal development. Immunocytochemical studies discovered that TEKT2BP1 localized in the middle piece of rat spermatozoa, predominantly concentrated at the mitochondria sheath of the flagella. We hypothesize that the TEKT2-TEKT2BP1 complex might be involved in the structural linkage between the ODF and mitochondria in the middle piece of the sperm flagella.

Sertoli cell-specific ablation of miR-17-92 cluster significantly alters whole testis transcriptome without apparent phenotypic effects.

PubMed

Hurtado, Alicia; Real, Francisca M; Palomino, Rogelio; Carmona, Francisco David; Burgos, Miguel; Jiménez, Rafael; Barrionuevo, Francisco J

2018-01-01

MicroRNAs are frequently organized into polycistronic clusters whose transcription is controlled by a single promoter. The miR-17-92 cluster is expressed in most embryonic and postnatal organs. It is a potent oncogene associated to several types of cancer and it is involved in several important developmental processes. In the testis, expression of the miR-17-92 cluster in the germ cells is necessary to maintain normal spermatogenesis. This cluster is also expressed in Sertoli cells (the somatic cells of the seminiferous tubules), which require miRNAs for correct cell development and survival. To study the possible role of miR-17-92 in Sertoli cell development and function and, in order to overcome the postnatal lethality of miR-17-92-/ mice, we conditionally deleted it in embryonic Sertoli cells shortly after the sex determination stage using an Amh-Cre allele. Mutant mice developed apparently normal testes and were fertile, but their testis transcriptomes contained hundreds of moderately deregulated genes, indicating that testis homeostasis is tightly controlled in mammals and that miR-17-92 expression in Sertoli cells contribute to maintain normal gene expression levels, but is unnecessary for testis development and function. Our results show that significant deregulation of hundreds of genes might have no functional consequences.

Characterizing Transcriptional Networks in Male Rainbow Darter (Etheostoma caeruleum) that Regulate Testis Development over a Complete Reproductive Cycle

PubMed Central

McMaster, Mark E.; Servos, Mark R.; Martyniuk, Christopher J.; Munkittrick, Kelly R.

2016-01-01

Intersex is a condition that has been associated with exposure to sewage effluents in male rainbow darter (Etheostoma caeruleum). To better understand changes in the transcriptome that are associated with intersex, we characterized annual changes in the testis transcriptome in wild, unexposed fish. Rainbow darter males were collected from the Grand River (Ontario, Canada) in May (spawning), August (post-spawning), October (recrudescence), January (developing) and March (pre-spawning). Histology was used to determine the proportion of spermatogenic cell types that were present during each period of testicular maturation. Regression analysis determined that the proportion of spermatozoa versus spermatocytes in all stages of development (R2 ≥ 0.58) were inversely related; however this was not the case when males were in the post-spawning period. Gene networks that were specific to the transition from developing to pre-spawning stages included nitric oxide biosynthesis, response to wounding, sperm cell function, and stem cell maintenance. The pre-spawning to spawning transition included gene networks related to amino acid import, glycogenesis, Sertoli cell proliferation, sperm capacitation, and sperm motility. The spawning to post-spawning transition included unique gene networks associated with chromosome condensation, ribosome biogenesis and assembly, and mitotic spindle assembly. Lastly, the transition from post-spawning to recrudescence included gene networks associated with egg activation, epithelial to mesenchymal transition, membrane fluidity, and sperm cell adhesion. Noteworthy was that there were a significant number of gene networks related to immune system function that were differentially expressed throughout reproduction, suggesting that immune network signalling has a prominent role in the male testis. Transcripts in the testis of post-spawning individuals showed patterns of expression that were most different for the majority of transcripts investigated when compared to the other stages. Interestingly, many transcripts associated with female sex differentiation (i.e. esr1, sox9, cdca8 and survivin) were significantly higher in the testis during the post-spawning season compared to other testis stages. At post-spawning, there were higher levels of estrogen and androgen receptors (esr1, esr2, ar) in the testis, while there was a decrease in the levels of sperm associated antigen 1 (spag1) and spermatogenesis associated 4 (spata4) mRNA. Cyp17a was more abundant in the testis of fish in the pre-spawning, spawning, and post-spawning seasons compared to those individuals that were recrudescent while aromatase (cyp19a) did not vary in expression over the year. This study identifies cell process related to testis development in a seasonally spawning species and improves our understanding regarding the molecular signaling events that underlie testicular growth. This is significant because, while there are a number of studies characterizing molecular pathways in the ovary, there are comparatively less describing transcriptomic patterns in the testis in wild fish. PMID:27861489

Characterization of immortalized dairy goat male germline stem cells (mGSCs).

PubMed

Zhu, Haijing; Ma, Jing; Du, Rui; Zheng, Liming; Wu, Jiang; Song, Wencong; Niu, Zhiwei; He, Xin; Du, Enqi; Zhao, Shanting; Hua, Jinlian

2014-09-01

Male germline stem cells (mGSCs), in charge for the fertility in male testis, are the only kind of adult stem cells that transmit genetic information to next generation, with promising prospects in germplasm resources preservation and optimization, and production of transgenic animals. Mouse male germline stem cell lines have been established and are valuable for studying the mechanisms of spermatogenesis. However, there is a lack of stable mGSC cell lines in livestock, which restricts the progress of transgenic research and related biotechnology. Here, we firstly established an immortalized dairy goat mGSC cell line to study the biological properties and the signaling pathways associated with mGSCs self-renewal and differentiation. The ectopic factors SV40 large T antigen and Bmi1 genes were transduced into dairy goat mGSCs, and the results showed that the proliferation of these cells that were named mGSCs-I-SB was improved significantly. They maintained the typical characteristics including the expression of mGSC markers, and the potential to differentiate into all three germ layers, sperm-like cells in vitro. Additionally, mGSCs-I-SB survived and differentiated into three germ layer cell types when they were transplanted into chicken embryos. Importantly, the cells also survived in mouse spermatogenesis deficiency model testis which seemed to be the golden standard to examine mGSCs. Conclusively, our results demonstrate that mGSCs-I-SB present the characteristics of mGSCs and may promote the future study on goat mGSCs. © 2014 Wiley Periodicals, Inc.

Diazepam administration prevents testosterone decrease and lipofuscin accumulation in testis of mouse exposed to chronic noise stress.

PubMed

Ruffoli, R; Carpi, A; Giambelluca, M A; Grasso, L; Scavuzzo, M C; Giannessi F, F

2006-10-01

Lipofuscin is an autofluorescent and undegradable material, which accumulates in tissues during ageing and under different types of stress. Among these, oxidative stress represents a major trigger for lipofuscin formation. However, prolonged noise exposure is also an effective stressful stimuli. Diazepam may inhibit lipofuscinogenesis in liver and prevent the noise-induced reduction of the steroidogenesis in the adrenal gland. The aim of the study was to ascertain whether chronic noise exposure causes lipofuscin accumulation in mouse testis, and to evaluate the effects of diazepam administration. Eight-week old mice were either exposed for 6 weeks (6 h day(-1)) to white-noise (group A), or received diazepam (3 mg kg(-1), i.p.) before noise exposures (group B), while a further group was used as control (group C). Light fluorescence and transmission electron microscopy revealed lipofuscin in large amounts in the Leydig cells in mice of group A, which concomitantly had low serum testosterone levels; pre-treatment with diazepam occluded both effects. The present study indicates that: (i) chronic noise exposure causes lipofuscin accumulation at the level of the Leydig cells and a decrease in testosterone; (ii) all these effects are suppressed by pre-treatment with diazepam. As the Leydig cells represent the only cellular type of the interstitial testicular tissue having peripheral benzodiazepine receptors, these results could be explained by the capacity of the peripheral benzodiazepine receptors to prevent reactive oxygen species damage and to increase the resistance of these cells to oxidative stress.

Effective Delivery of Male Contraceptives Behind the Blood-Testis Barrier (BTB) – Lesson from Adjudin

PubMed Central

Chen, Haiqi; Mruk, Dolores D.; Xia, Weiliang; Bonanomi, Michele; Silvestrini, Bruno; Cheng, Chuen-Yan

2016-01-01

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. It divides the seminiferous epithelium of the seminiferous tubule, the functional unit of the testis, where spermatogenesis takes place, into the basal and the adluminal (apical) compartments. Functionally, the BTB provides a unique microenvironment for meiosis I/II and post-meiotic spermatid development which take place exclusively in the apical compartment, away from the host immune system, and it contributes to the immune privilege status of testis. However, the BTB also poses major obstacles in developing male contraceptives (e.g., adjudin) that exert their effects on germ cells in the apical compartment, such as by disrupting spermatid adhesion to the Sertoli cell, causing germ cell exfoliation from the testis. Besides the tight junction (TJ) between adjacent Sertoli cells at the BTB that restricts the entry of contraceptives from the microvessels in the interstitium to the adluminal compartment, drug transporters, such as P-glycoprotein and multidrug resistance-associated protein 1 (MRP1), are also present that actively pump drugs out of the testis, limiting drug bioavailability. Recent advances in drug formulations, such as drug particle micronization (<50 μm) and co-grinding of drug particles with ß-cyclodextrin have improved bioavailability of contraceptives via considerable increase in solubility. Herein, we discuss development in drug formulations using adjudin as an example. We also put emphasis on the possible use of nanotechnology to deliver adjudin to the apical compartment with multidrug magnetic mesoporous silica nanoparticles. These advances in technology will significantly enhance our ability to develop effective non-hormonal male contraceptives for men. PMID:26758796

Effective Delivery of Male Contraceptives Behind the Blood-Testis Barrier (BTB) - Lesson from Adjudin.

PubMed

Chen, Haiqi; Mruk, Dolores D; Xia, Weiliang; Bonanomi, Michele; Silvestrini, Bruno; Cheng, Chuen-Yan

2016-01-01

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. It divides the seminiferous epithelium of the seminiferous tubule, the functional unit of the testis, where spermatogenesis takes place, into the basal and the adluminal (apical) compartments. Functionally, the BTB provides a unique microenvironment for meiosis I/II and post-meiotic spermatid development which take place exclusively in the apical compartment, away from the host immune system, and it contributes to the immune privilege status of testis. However, the BTB also poses major obstacles in developing male contraceptives (e.g., adjudin) that exert their effects on germ cells in the apical compartment, such as by disrupting spermatid adhesion to the Sertoli cell, causing germ cell exfoliation from the testis. Besides the tight junction (TJ) between adjacent Sertoli cells at the BTB that restricts the entry of contraceptives from the microvessels in the interstitium to the adluminal compartment, drug transporters, such as P-glycoprotein and multidrug resistance-associated protein 1 (MRP1), are also present that actively pump drugs out of the testis, limiting drug bioavailability. Recent advances in drug formulations, such as drug particle micronization (<50 μm) and co-grinding of drug particles with ß-cyclodextrin have improved bioavailability of contraceptives via considerable increase in solubility. Herein, we discuss development in drug formulations using adjudin as an example. We also put emphasis on the possible use of nanotechnology to deliver adjudin to the apical compartment with multidrug magnetic mesoporous silica nanoparticles. These advances in technology will significantly enhance our ability to develop effective non-hormonal male contraceptives for men.

The Drosophila ovarian and testis stem cell niches: similar somatic stem cells and signals.

PubMed

Decotto, Eva; Spradling, Allan C

2005-10-01

The stem cell niches at the apex of Drosophila ovaries and testes have been viewed as distinct in two major respects. While both contain germline stem cells, the testis niche also contains "cyst progenitor" stem cells, which divide to produce somatic cells that encase developing germ cells. Moreover, while both niches utilize BMP signaling, the testis niche requires a key JAK/STAT signal. We now show, by lineage marking, that the ovarian niche also contains a second type of stem cell. These "escort stem cells" morphologically resemble testis cyst progenitor cells and their daughters encase developing cysts before undergoing apoptosis at the time of follicle formation. In addition, we show that JAK/STAT signaling also plays a critical role in ovarian niche function, and acts within escort cells. These observations reveal striking similarities in the stem cell niches of male and female gonads, and suggest that they are largely governed by common mechanisms.

hCG-induced endoplasmic reticulum stress triggers apoptosis and reduces steroidogenic enzyme expression through activating transcription factor 6 in Leydig cells of the testis

PubMed Central

Park, Sun-Ji; Kim, Tae-Shin; Park, Choon-Keun; Lee, Sang-Hee; Kim, Jin-Man; Lee, Kyu-Sun; Lee, In-kyu; Park, Jeen-Woo; Lawson, Mark A; Lee, Dong-Seok

2014-01-01

Endoplasmic reticulum (ER) stress generally occurs in secretory cell types. It has been reported that Leydig cells, which produce testosterone in response to human chorionic gonadotropin (hCG), express key steroidogenic enzymes for the regulation of testosterone synthesis. In this study, we analyzed whether hCG induces ER stress via three unfolded protein response (UPR) pathways in mouse Leydig tumor (mLTC-1) cells and the testis. Treatment with hCG induced ER stress in mLTC-1 cells via the ATF6, IRE1a/XBP1, and eIF2α/GADD34/ATF4 UPR pathways, and transient expression of 50 kDa protein activating transcription factor 6 (p50ATF6) reduced the expression level of steroidogenic 3β-hydroxy-steroid dehydrogenase Δ5-Δ4-isomerase (3β-HSD) enzyme. In an in vivo model, high-level hCG treatment induced expression of p50ATF6 while that of steroidogenic enzymes, especially 3β-HSD, 17α-hydroxylase/C17–20 lyase (CYP17), and 17β-hydrozysteroid dehydrogenase (17β-HSD), was reduced. Expression levels of steroidogenic enzymes were restored by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Furthermore, lentivirus-mediated transient expression of p50ATF6 reduced the expression level of 3β-HSD in the testis. Protein expression levels of phospho-JNK, CHOP, and cleaved caspases-12 and -3 as markers of ER stress-mediated apoptosis markedly increased in response to high-level hCG treatment in mLTC-1 cells and the testis. Based on transmission electron microscopy and H&E staining of the testis, it was shown that abnormal ER morphology and destruction of testicular histology induced by high-level hCG treatment were reversed by the addition of TUDCA. These findings suggest that hCG-induced ER stress plays important roles in steroidogenic enzyme expression via modulation of the ATF6 pathway as well as ER stress-mediated apoptosis in Leydig cells. PMID:23256993

IL-6 mediates differentiation disorder during spermatogenesis in obesity-associated inflammation by affecting the expression of Zfp637 through the SOCS3/STAT3 pathway.

PubMed

Huang, Guizhen; Yuan, Miao; Zhang, Jie; Li, Jun; Gong, Di; Li, Yanyan; Zhang, Jie; Lin, Ping; Huang, Lugang

2016-06-22

Zfp637 is a recently identified zinc finger protein, and its functions remain largely unknown. Here, we innovatively demonstrate the effects of Zfp637 on the differentiation of mouse spermatogonia and on its downstream target gene SOX2 in vitro. Obesity has been recognized as a chronic inflammatory disease that leads to decreased sexual function and sexual development disorders. We observed higher levels of IL-6 in serum and testis homogenates from obese mice compared with control mice. We also demonstrated that high levels of IL-6 inhibited Zfp637 expression, and we elucidated the underlying mechanisms. SOCS3 overexpression and STAT3 phosphorylation inhibitor (AG490) were used to investigate the function of the SOCS3/STAT3 pathway during this process. Our results showed that exposure of mouse spermatogonial cells to high levels of IL-6 inhibited Zfp637 expression by increasing SOCS3 expression and inhibiting the phosphorylation of STAT3, further reducing cellular differentiation. Consistent with the in vitro results, we observed increasing expression levels of SOCS3 and SOX2, but a reduction of Zfp637 expression, in obese mouse testes. In conclusion, Zfp637 plays a crucial role in spermatogenesis by downregulating SOX2 expression, and IL-6 can decrease the expression of Zfp637 through the SOCS3/STAT3 signaling pathway.

SKAP, an outer kinetochore protein, is required for mouse germ cell development

PubMed Central

Grey, Corinne; Espeut, Julien; Ametsitsi, Rachel; Kumar, Rajeev; Luksza, Malgorzata; Brun, Christine; Verlhac, Marie-Hélene; Suja, José Angél; de Massy, Bernard

2016-01-01

In sexually reproducing organisms, accurate gametogenesis is crucial for the transmission of genetic material from one generation to the next. This requires the faithful segregation of chromosomes during mitotic and meiotic divisions. One of the main players in this process is the kinetochore, a large multi-protein complex that forms at the interface of centromeres and microtubules. Here, we analyzed the expression profile and function of small kinetochore-associated protein (SKAP) in the mouse. We found that two distinct SKAP isoforms are specifically expressed in the germline: a smaller isoform, which is detected in spermatogonia and spermatocytes and localized in the outer mitotic and meiotic kinetochores from metaphase to telophase, and a larger isoform, which is expressed in the cytoplasm of elongating spermatids. We generated SKAP-deficient mice and found that testis size and sperm production were severely reduced in mutant males. This phenotype was partially caused by defects during spermatogonia proliferation before entry into meiosis. We conclude that mouse SKAP, while being dispensable for somatic cell divisions, has an important role in the successful outcome of male gametogenesis. In germ cells, analogous to what has been suggested in studies using immortalized cells, SKAP most likely stabilizes the interaction between kinetochores and microtubules, where it might be needed as an extra safeguard to ensure the correct segregation of mitotic and meiotic chromosomes. PMID:26667018

Phenotyping male infertility in the mouse: how to get the most out of a 'non-performer'.

PubMed

Borg, Claire L; Wolski, Katja M; Gibbs, Gerard M; O'Bryan, Moira K

2010-01-01

Functional male gametes are produced through complex processes that take place within the testis, epididymis and female reproductive tract. A breakdown at any of these phases can result in male infertility. The production of mutant mouse models often yields an unexpected male infertility phenotype. It is with this in mind that the current review has been written. The review aims to act as a guide to the 'non-reproductive biologist' to facilitate a systematic analysis of sterile or subfertile mice and to assist in extracting the maximum amount of information from each model. This is a review of the original literature on defects in the processes that take a mouse spermatogonial stem cell through to a fully functional spermatozoon, which result in male infertility. Based on literature searches and personal experience, we have outlined a step-by-step strategy for the analysis of an infertile male mouse line. A wide range of methods can be used to define the phenotype of an infertile male mouse. These methods range from histological methods such as electron microscopy and immunohistochemistry, to hormone analyses and methods to assess sperm maturation status and functional competence. With the increased rate of genetically modified mouse production, the generation of mouse models with unexpected male infertility is increasing. This manuscript will help to ensure that the maximum amount of information is obtained from each mouse model and, by extension, will facilitate the knowledge of both normal fertility processes and the causes of human infertility.

SRY protein is expressed in ovotestis and streak gonads from human sex-reversal.

PubMed

Salas-Cortés, L; Jaubert, F; Nihoul-Feketé, C; Brauner, R; Rosemblatt, M; Fellous, M

2000-01-01

In mammals, a master gene located on the Y chromosome, the testis-determining gene SRY, controls sex determination. SRY protein is expressed in the genital ridge before testis determination, and in the testis it is expressed in Sertoli and germ cells. Completely sex-reversed patients are classified as either 46,XX males or 46,XY females. SRY mutations have been described in only 15% of patients with 46,XY complete or partial gonadal dysgenesis. However, although incomplete or partial sex-reversal affects 46,XX true hermaphrodites, 46,XY gonadal dysgenesis, and 46,XX/46,XY mosaicism, only 15% of the 46,XX true hermaphrodites analyzed have the SRY gene. Here, we demonstrate that the SRY protein is expressed in the tubules of streak gonads and rete testis, indicating that the SRY protein is normally expressed early during testis determination. Based on these results, we propose that some factors downstream from SRY may be mutated in these 46,XY sex-reversal patients. We have also analyzed SRY protein expression in the ovotestis from 46,XX true hermaphrodites and 46,XX/46,XY mosaicism, demonstrating SRY protein expression in both testicular and ovarian portions in these patients. This suggests that the SRY protein does not inhibit ovary development. These results confirm that other factors are needed for complete testis development, in particular, those downstream of the SRY protein. Copyright 2001 S. Karger AG, Basel

ATRX has a critical and conserved role in mammalian sexual differentiation

PubMed Central

2011-01-01

Background X-linked alpha thalassemia, mental retardation syndrome in humans is a rare recessive disorder caused by mutations in the ATRX gene. The disease is characterised by severe mental retardation, mild alpha-thalassemia, microcephaly, short stature, facial, skeletal, genital and gonadal abnormalities. Results We examined the expression of ATRX and ATRY during early development and gonadogenesis in two distantly related mammals: the tammar wallaby (a marsupial) and the mouse (a eutherian). This is the first examination of ATRX and ATRY in the developing mammalian gonad and fetus. ATRX and ATRY were strongly expressed in the developing male and female gonad respectively, of both species. In testes, ATRY expression was detected in the Sertoli cells, germ cells and some interstitial cells. In the developing ovaries, ATRX was initially restricted to the germ cells, but was present in the granulosa cells of mature ovaries from the primary follicle stage onwards and in the corpus luteum. ATRX mRNA expression was also examined outside the gonad in both mouse and tammar wallaby whole embryos. ATRX was detected in the developing limbs, craniofacial elements, neural tissues, tail and phallus. These sites correspond with developmental deficiencies displayed by ATR-X patients. Conclusions There is a complex expression pattern throughout development in both mammals, consistent with many of the observed ATR-X syndrome phenotypes in humans. The distribution of ATRX mRNA and protein in the gonads was highly conserved between the tammar and the mouse. The expression profile within the germ cells and somatic cells strikingly overlaps with that of DMRT1, suggesting a possible link between these two genes in gonadal development. Taken together, these data suggest that ATRX has a critical and conserved role in normal development of the testis and ovary in both the somatic and germ cells, and that its broad roles in early mammalian development and gonadal function have remained unchanged for over 148 million years of mammalian evolution. PMID:21672208

ATRX has a critical and conserved role in mammalian sexual differentiation.

PubMed

Huyhn, Kim; Renfree, Marilyn B; Graves, Jennifer A; Pask, Andrew J

2011-06-14

X-linked alpha thalassemia, mental retardation syndrome in humans is a rare recessive disorder caused by mutations in the ATRX gene. The disease is characterised by severe mental retardation, mild alpha-thalassemia, microcephaly, short stature, facial, skeletal, genital and gonadal abnormalities. We examined the expression of ATRX and ATRY during early development and gonadogenesis in two distantly related mammals: the tammar wallaby (a marsupial) and the mouse (a eutherian). This is the first examination of ATRX and ATRY in the developing mammalian gonad and fetus. ATRX and ATRY were strongly expressed in the developing male and female gonad respectively, of both species. In testes, ATRY expression was detected in the Sertoli cells, germ cells and some interstitial cells. In the developing ovaries, ATRX was initially restricted to the germ cells, but was present in the granulosa cells of mature ovaries from the primary follicle stage onwards and in the corpus luteum. ATRX mRNA expression was also examined outside the gonad in both mouse and tammar wallaby whole embryos. ATRX was detected in the developing limbs, craniofacial elements, neural tissues, tail and phallus. These sites correspond with developmental deficiencies displayed by ATR-X patients. There is a complex expression pattern throughout development in both mammals, consistent with many of the observed ATR-X syndrome phenotypes in humans. The distribution of ATRX mRNA and protein in the gonads was highly conserved between the tammar and the mouse. The expression profile within the germ cells and somatic cells strikingly overlaps with that of DMRT1, suggesting a possible link between these two genes in gonadal development. Taken together, these data suggest that ATRX has a critical and conserved role in normal development of the testis and ovary in both the somatic and germ cells, and that its broad roles in early mammalian development and gonadal function have remained unchanged for over 148 million years of mammalian evolution.

Perfluorooctanoic acid affects endocytosis involving clathrin light chain A and microRNA-133b-3p in mouse testes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Yin; Wang, Jianshe

Perfluorooctanoic acid (PFOA) is an abundant perfluoroalkyl substance widely applied in industrial and consumer products. Among its potential health hazards, testicular toxicity is of major concern. To explore the potential effect of miRNA on post-translational regulation after PFOA exposure, changes in miRNAs were detected via miRNA array. Seventeen miRNAs were differentially expressed (eight upregulated, nine downregulated) in male mouse testes after exposure to 5 mg/kg/d of PFOA for 28 d (> 1.5-fold and P < 0.05 compared with the control). Eight of these miRNAs were further selected for TaqMan qPCR analysis. Proteomic profile analysis indicated that many changed proteins aftermore » PFOA treatment, including intersectin 1 (ITSN1), serine protease inhibitor A3K (Serpina3k), and apolipoprotein a1 (APOA1), were involved in endocytosis and blood-testis barrier (BTB) processes. These changes were further verified by immunohistochemical and Western blot analyses. Endocytosis-related genes were selected for qPCR analysis, with many found to be significantly changed after PFOA treatment, including epidermal growth factor receptor pathway substrate 8 (Eps8), Eps15, cortactin, cofilin, espin, vinculin, and zyxin. We further predicted the potential interaction between changed miRNAs and proteins, which indicated that miRNAs might play a role in the post-translational regulation of gene expression after PFOA treatment in mouse testes. Among them, miR-133b-3p/clathrin light chain A (CLTA) was selected and verified in vitro by transfection and luciferase activity assay. Results showed that PFOA exposure affects endocytosis in mouse testes and that CLTA is a potential target of miR-133b-3p. - Highlights: • Endocytosis and blood-testis barrier proteins were changed after PFOA exposure. • Seventeen miRNAs were differentially expressed in testes after PFOA exposure. • MiRNAs might play a role in gene regulation in testes after PFOA exposure.CLTA is a potential target of miR-133b-3p.« less

[Morphological verification problems of Chernobyl factor influence on the testis of coal miners of Donbas-liquidators of Chernobyl accident].

PubMed

Danylov, Iu V; Motkov, K V; Shevchenko, T I

2013-01-01

Problem of a diagnostic of Chernobyl factor influences on different organs and systems of Chernobyl accident liquidators are remain actually until now. Though morbidly background which development at unfavorable work conditions in underground coalminers prevents from objective identification features of Chernobyl factor influences. The qualitative and quantitative histological and immunohistochemical law of morphogenesis changes in testis of Donbas's coalminer - non-liquidators Chernobyl accident in comparison with the group of Donbas's coalminers-liquidators Chernobyl accident, which we were stationed non determined problem. This reason stipulates to development and practical use of mathematical model of morphogenesis of a testis changes.

Intratubular transplantation as a strategy for establishing animal models of testicular germ cell tumours

PubMed Central

Li, Yunmin; Kido, Tatsuo; Luo, Jinping; Fukuda, Michiko; Dobrinski, Ina; Lau, Yun-Fai Chris

2008-01-01

Testicular germ cell tumours (TGCTs) are prevalent cancers among young men. Currently, there is no reliable animal model for TGCTs. To establish such animal models, we have explored the possibility of intratubular testicular transplantation as means to deliver tumour cells into the seminiferous tubules of host animals. Our results demonstrated that transplanted cells could effectively populate the testis of a recipient mouse and develop into TGCTs. In addition, the donor cells could be transfected with a specific transgene before transplantation, thereby providing an approach to evaluate the specific effects of gene functions in the oncogenic processes. Hence, depending on selection of specific donor cells or mixtures of donor cells, transplantation models of TGCTs could be significant for studies on the pathogenesis, diagnosis and therapies of such a prevalent and important cancer in men. PMID:18808526

Filamin A Is a Regulator of Blood-Testis Barrier Assembly during Postnatal Development in the Rat Testis

PubMed Central

Su, Wenhui; Mruk, Dolores D.; Lie, Pearl P. Y.; Lui, Wing-yee

2012-01-01

The blood-testis barrier (BTB) is an important ultrastructure in the testis. A delay in its assembly during postnatal development leads to meiotic arrest. Also, a disruption of the BTB by toxicants in adult rats leads to a failure in spermatogonial differentiation. However, the regulation of BTB assembly remains unknown. Herein, filamin A, an actin filament cross-linker that is known to maintain and regulate cytoskeleton structure and function in other epithelia, was shown to be highly expressed during the assembly of Sertoli cell BTB in vitro and postnatal development of BTB in vivo, perhaps being used to maintain the actin filament network at the BTB. A knockdown of filamin A by RNA interference was found to partially perturb the Sertoli cell tight junction (TJ) permeability barrier both in vitro and in vivo. Interestingly, this down-regulating effect on the TJ barrier function after the knockdown of filamin A was associated with a mis-localization of both TJ and basal ectoplasmic specialization proteins. Filamin A knockdown also induced a disorganization of the actin filament network in Sertoli cells in vitro and in vivo. Collectively, these findings illustrate that filamin A regulates BTB assembly by recruiting these proteins to the microenvironment in the seminiferous epithelium to serve as the building blocks. In short, filamin A participates in BTB assembly by regulating protein recruitment during postnatal development in the rat testis. PMID:22872576

Recombinant Chimpanzee Adenovirus Vaccine AdC7-M/E Protects against Zika Virus Infection and Testis Damage.

PubMed

Xu, Kun; Song, Yufeng; Dai, Lianpan; Zhang, Yongli; Lu, Xuancheng; Xie, Yijia; Zhang, Hangjie; Cheng, Tao; Wang, Qihui; Huang, Qingrui; Bi, Yuhai; Liu, William J; Liu, Wenjun; Li, Xiangdong; Qin, Chuan; Shi, Yi; Yan, Jinghua; Zhou, Dongming; Gao, George F

2018-03-15

The recent outbreak of Zika virus (ZIKV) has emerged as a global health concern. ZIKV can persist in human semen and be transmitted by sexual contact, as well as by mosquitoes, as seen for classical arboviruses. We along with others have previously demonstrated that ZIKV infection leads to testis damage and infertility in mouse models. So far, no prophylactics or therapeutics are available; therefore, vaccine development is urgently demanded. Recombinant chimpanzee adenovirus has been explored as the preferred vaccine vector for many pathogens due to the low preexisting immunity against the vector among the human population. Here, we developed a ZIKV vaccine based on recombinant chimpanzee adenovirus type 7 (AdC7) expressing ZIKV M/E glycoproteins. A single vaccination of AdC7-M/E was sufficient to elicit potent neutralizing antibodies and protective immunity against ZIKV in both immunocompetent and immunodeficient mice. Moreover, vaccinated mice rapidly developed neutralizing antibody with high titers within 1 week postvaccination, and the elicited antiserum could cross-neutralize heterologous ZIKV strains. Additionally, ZIKV M- and E-specific T cell responses were robustly induced by AdC7-M/E. Moreover, one-dose inoculation of AdC7-M/E conferred mouse sterilizing immunity to eliminate viremia and viral burden in tissues against ZIKV challenge. Further investigations showed that vaccination with AdC7-M/E completely protected against ZIKV-induced testicular damage. These data demonstrate that AdC7-M/E is highly effective and represents a promising vaccine candidate for ZIKV control. IMPORTANCE Zika virus (ZIKV) is a pathogenic flavivirus that causes severe clinical consequences, including congenital malformations in fetuses and Guillain-Barré syndrome in adults. Vaccine development is a high priority for ZIKV control. In this study, to avoid preexisting anti-vector immunity in humans, a rare serotype chimpanzee adenovirus (AdC7) expressing the ZIKV M/E glycoproteins was used for ZIKV vaccine development. Impressively, AdC7-M/E exhibited exceptional performance as a ZIKV vaccine, as follows: (i) protective efficacy by a single vaccination, (ii) rapid development of a robust humoral response, (iii) durable immune responses, (iv) robust T cell responses, and (v) sterilizing immunity achieved by a single vaccination. These advantages of AdC7-M/E strongly support its potential application as a promising ZIKV vaccine in the clinic. Copyright © 2018 American Society for Microbiology.

A novel ENU-induced mutation, peewee, causes dwarfism in the mouse

PubMed Central

Bon-Ryon, Lee; Kano, Kiyoshi; Young, Jay; John, Simon; Nishina, Patsy M; Naggert, Jurgen K; Naito, Kunihiko

2010-01-01

We identified a novel fertile, autosomal recessive mutation, called peewee and that results in dwarfing, in a region-specific ENU-induced mutagenesis. These mice at litter size were smaller those of other strains. Histological analysis revealed that the major organs appear normal, but abnormalities in cellular proliferation were observed in bone, liver and testis. Haplotype analysis localized the peewee gene to a 3.3-Mb region between D5Mit83 and D5Mit356.3. There are 18 genes in this linkage area, and we also performed in silico mapping using the PosMed℠ program, which searches for connections among keywords and genes in an interval, but no similar phenotype descriptions were found for these genes. In the peewee mutant compared to the normal, C57BL/6J mouse, only Slc10a4 expression was lower. Our preliminary mutation analysis examining the nucleotide sequence of three exons, two introns and an untranslated region of Slc10a4 did not find any sequence difference between the peewee mouse and the C57BL/6J mouse. Detailed analysis of peewee mice might provide novel molecular insights into the complex mechanisms regulating body growth. PMID:19513787

Toxicological assessment of Diplazium esculentum on the reproductive functions of male Swiss albino mouse.

PubMed

Roy, Subhrajyoti; Chaudhuri, Tapas Kumar

2017-04-01

Diplazium esculentum, a commonly consumed seasonal vegetable, has been reported to have some pathological effects in some animals. But, its effect on the male reproductive function has not yet been studied. To investigate the effects of boiled D. esculentum (BDE), the form which human consumes, on male reproductive functions of Swiss albino mice. Male (120 in no.) and female (80 in no.) Swiss albino mice (6-8 weeks of age) were fed orally with 80, 160 and 320 mg/kg bw of BDE within a span of 180 d. After the treatment, body weight, absolute- and relative-testis weight, relative-weight of other organs, their biochemical parameters, hypo-osmotic swelling test (HOST) of spermatozoa, testis histology and fertility and fecundity tests were performed to justify the toxic effects of D. esculentum on male reproductive functions. Significant dose- and time-dependent decreases were observed in body weight, absolute- and relative-testis weight, relative-weights of other organs and their biochemical parameters, percentage of live spermatozoa and percentage of fertility and fecundity in BDE fed mice. Significant decreases were observed in diameter, perimeter and area of the seminiferous tubules of mice treated for 180 d. The percentage of empty seminiferous tubules was increased significantly in BDE treated mice when compared to the controls. These results suggest that the intake of D. esculentum, even after cooking, may induce infertility by altering the male reproductive function, and therefore, should be evaluated further as a potential antifertility agent.

MALFORMATIONS IN GUBERNACULAR LIGAMENT DEVELOPMENT INDUCED BY DEHP, DBP, AND BBP ARE ASSOCIATED WITH DECREASES IN INSL3 GENE EXPRESSION IN THE FETAL RAT TESTIS

EPA Science Inventory

Malformations in gubernacular ligament development induced by DEHP, DBP, and BBP are associated with decreases in insl3 gene expression in the fetal rat testis.
Vickie S.Wilson, Christy Lambright, Johnathan Furr, Carmen Wood, Gary Held, L. Earl Gray Jr. U.S. EPA, ORD, NHEER...

Trace elemental analysis in cancer-afflicted tissues of penis and testis by PIXE technique

NASA Astrophysics Data System (ADS)

Naga Raju, G. J.; John Charles, M.; Bhuloka Reddy, S.; Sarita, P.; Seetharami Reddy, B.; Rama Lakshmi, P. V. B.; Vijayan, V.

2005-04-01

PIXE technique was employed to estimate the trace elemental concentrations in the biological samples of cancerous penis and testis. A 3 MeV proton beam was employed to excite the samples. From the present results it can be seen that the concentrations of Cl, Fe and Co are lower in the cancerous tissue of the penis when compared with those in normal tissue while the concentrations of Cu, Zn and As are relatively higher. The concentrations of K, Ca, Ti, Cr, Mn, Br, Sr and Pb are in agreement within standard deviations in both cancerous and normal tissues. In the cancerous tissue of testis, the concentrations of K, Cr and Cu are higher while the concentrations of Fe, Co and Zn are lower when compared to those in normal tissue of testis. The concentrations of Cl, Ca, Ti and Mn are in agreement in both cancerous and normal tissues of testis. The higher levels of Cu lead to the development of tumor. Our results also support the underlying hypothesis of an anticopper, antiangiogenic approach to cancer therapy. The Cu/Zn ratios of both penis and testis were higher in cancer tissues compared to that of normal.

Di(2-ethylhexyl)phthalate Alters the Synthesis and β-Oxidation of Fatty Acids and Hinders ATP Supply in Mouse Testes via UPLC-Q-Exactive Orbitrap MS-Based Metabonomics Study.

PubMed

Shen, Guolin; Zhou, Lili; Liu, Wei; Cui, Yuan; Xie, Wenping; Chen, Huiming; Yu, Wenlian; Li, Wentao; Li, Haishan

2017-06-21

Di(2-ethylhexyl) phthalate (DEHP) is considered to be an environmental endocrine disruptor at high levels of general exposure. Studies show that DEHP may cause testicular toxicity on human being. In this study, metabonomics techniques were used to identify differential endogenous metabolites, draw the network metabolic pathways, and conduct network analysis, to determine the underlying mechanisms of testicular toxicity induced by DEHP. The results showed that DEHP inhibited synthesis and accelerated β-oxidation of fatty acids and impaired the tricarboxylic acid cycle (TCA cycle) and gluconeogenesis, resulting in lactic acid accumulation and an insufficient ATP supply in the microenvironment of the testis. These alterations led to testicular atrophy and, thus, may be the underlying causes of testicular toxicity. DEHP also inhibited peroxisome proliferator activated receptors in the testis, which may be another potential reason for the testicular atrophy. These findings provided new insights to better understand the mechanisms of testicular toxicity induced by DEHP exposure.

Effects of In Utero Exposure to Bisphenol A or Diethylstilbestrol on the Adult Male Reproductive System

PubMed Central

LaRocca, Jessica; Boyajian, Alanna; Brown, Caitlin; Smith, Stuart Duncan; Hixon, Mary

2011-01-01

The objective of this study was to determine if in utero exposure to Bisphenol A (BPA) induced reproductive tract abnormalities in the adult male testis. Using the C57/Bl6 mouse, we examined sex-organ weights, anogenital distance (AGD), and testis histopathology in adult males exposed in utero via oral gavage to sesame oil, 50 μg/kg BPA, 1,000 μg/kg BPA, or 2 μg/kg diethylstilbestrol (DES) as a positive control from gestational days 10–16. No changes in sperm production or germ cell apoptosis were observed in adult testes following exposure to either chemical. Adult mRNA levels of genes associated with sexual maturation and differentiation, GATA4 and ID2, were significantly lower only in DES-exposed testes. In summary, the data indicate no gross alterations in spermatogenesis following in utero exposure to BPA or DES. At the molecular level, in utero exposure to DES, but not BPA, leads to decreased mRNA expression of genes associated with Sertoli cell differentiation. PMID:21922642

Zika virus infection damages the testes in mice.

PubMed

Govero, Jennifer; Esakky, Prabagaran; Scheaffer, Suzanne M; Fernandez, Estefania; Drury, Andrea; Platt, Derek J; Gorman, Matthew J; Richner, Justin M; Caine, Elizabeth A; Salazar, Vanessa; Moley, Kelle H; Diamond, Michael S

2016-12-15

Infection of pregnant women with Zika virus (ZIKV) can cause congenital malformations including microcephaly, which has focused global attention on this emerging pathogen. In addition to transmission by mosquitoes, ZIKV can be detected in the seminal fluid of affected males for extended periods of time and transmitted sexually. Here, using a mouse-adapted African ZIKV strain (Dakar 41519), we evaluated the consequences of infection in the male reproductive tract of mice. We observed persistence of ZIKV, but not the closely related dengue virus (DENV), in the testis and epididymis of male mice, and this was associated with tissue injury that caused diminished testosterone and inhibin B levels and oligospermia. ZIKV preferentially infected spermatogonia, primary spermatocytes and Sertoli cells in the testis, resulting in cell death and destruction of the seminiferous tubules. Less damage was caused by a contemporary Asian ZIKV strain (H/PF/2013), in part because this virus replicates less efficiently in mice. The extent to which these observations in mice translate to humans remains unclear, but longitudinal studies of sperm function and viability in ZIKV-infected humans seem warranted.

Zika virus infection damages the testes in mice

PubMed Central

Govero, Jennifer; Esakky, Prabagaran; Scheaffer, Suzanne M.; Fernandez, Estefania; Drury, Andrea; Platt, Derek J.; Gorman, Matthew J.; Richner, Justin M.; Caine, Elizabeth A.; Salazar, Vanessa; Moley, Kelle H.; Diamond, Michael S.

2017-01-01

Zika virus (ZIKV) infection of pregnant women can cause congenital malformations including microcephaly, which has focused global attention on this emerging pathogen1. In addition to transmission by mosquitoes, ZIKV can be detected in the seminal fluid of affected males for extended periods of time and transmitted sexually2. Here, using a mouse-adapted African ZIKV strain (Dakar 41519) we evaluated the consequences of infection in the male reproductive tract of mice. We observed persistence of ZIKV, but not the closely related Dengue virus (DENV), in the testis and epididymis of male mice, and this was associated with tissue injury that caused diminished testosterone and inhibin B levels, and oligospermia. ZIKV preferentially infected spermatogonia, primary spermatocytes, and Sertoli cells in the testis, resulting in cell death and destruction of the seminiferous tubules. Less damage was observed with a contemporary Asian ZIKV strain (H/PF/2013), in part because this virus replicates less efficiently in mice. The extent to which these observations in mice translate to humans remains unclear, but longitudinal studies of sperm function and viability in ZIKV-infected humans seem warranted. PMID:27798603

Cancer-associated variant expression and interaction of CIZ1 with cyclin A1 in differentiating male germ cells.

PubMed

Greaves, Erin A; Copeland, Nikki A; Coverley, Dawn; Ainscough, Justin F X

2012-05-15

CIZ1 is a nuclear-matrix-associated DNA replication factor unique to higher eukaryotes, for which alternatively spliced isoforms have been associated with a range of disorders. In vitro, the CIZ1 N-terminus interacts with cyclin E and cyclin A at distinct sites, enabling functional cooperation with cyclin-A-Cdk2 to promote replication initiation. C-terminal sequences anchor CIZ1 to fixed sites on the nuclear matrix, imposing spatial constraint on cyclin-dependent kinase activity. Here we demonstrate that CIZ1 is predominantly expressed as a predicted full-length product throughout mouse development, consistent with a ubiquitous role in cell and tissue renewal. CIZ1 is expressed in proliferating stem cells of the testis, but is notably downregulated following commitment to differentiation. Significantly, CIZ1 is re-expressed at high levels in non-proliferative spermatocytes before meiotic division. Sequence analysis identifies at least seven alternatively spliced variants, including a dominant cancer-associated form and a set of novel isoforms. Furthermore, we show that in these post-replicative cells, CIZ1 interacts with germ-cell-specific cyclin A1, which has been implicated in the repair of DNA double-strand breaks. Consistent with this role, antibody depletion of CIZ1 reduces the capacity for testis extract to repair digested plasmid DNA in vitro. Together, the data imply post-replicative roles for CIZ1 in germ cell differentiation that might include meiotic recombination - a process intrinsic to genome stability and diversification.

Duplication of SOX9 is not a common cause of 46,XX testicular or 46,XX ovotesticular DSD.

PubMed

Seeherunvong, Tossaporn; Ukarapong, Supamit; McElreavey, Kenneth; Berkovitz, Gary D; Perera, Erasmo M

2012-01-01

Translocation of the SRY gene to the paternal X chromosome is the explanation for testis development in the majority of subjects with 46,XX testicular disorder of sexual development (DSD). However, nearly all subjects with 46,XX ovotesticular DSD and up to one third of subjects with 46,XX testicular DSD lack SRY. SRY-independent expression of SOX9 has been implicated in the etiology of testis development in some individuals. We amplified microsatellite markers in the region of SOX9 from a cohort of 30 subjects with either 46,XX testicular or 46,XX ovotesticular DSD to detect SOX9 duplications. Duplication of the SOX9 region in 17q was not detected in any subject. Duplication in the region of 17q that contains SOX9 is not a common cause of testis development in subjects with SRY-negative 46,XX testicular or ovotesticular DSD.

Alterations in the developing testis transcriptome following embryonic vinclozolin exposure.

PubMed

Clement, Tracy M; Savenkova, Marina I; Settles, Matthew; Anway, Matthew D; Skinner, Michael K

2010-11-01

The current study investigates the direct effects of in utero vinclozolin exposure on the developing F1 generation rat testis transcriptome. Previous studies have demonstrated that exposure to vinclozolin during embryonic gonadal sex determination induces epigenetic modifications of the germ line and transgenerational adult onset disease states. Microarray analyses were performed to compare control and vinclozolin treated testis transcriptomes at embryonic days 13, 14 and 16. A total of 576 differentially expressed genes were identified and the major cellular functions and pathways associated with these altered transcripts were examined. The sets of regulated genes at the different development periods were found to be transiently altered and distinct. Categorization by major known functions of altered genes was performed. Specific cellular process and pathway analyses suggest the involvement of Wnt and calcium signaling, vascular development and epigenetic mechanisms as potential mediators of the direct F1 generation actions of vinclozolin. Copyright © 2010 Elsevier Inc. All rights reserved.

ALTERATIONS IN THE DEVELOPING TESTIS TRANSCRIPTOME FOLLOWING EMBRYONIC VINCLOZOLIN EXPOSURE

PubMed Central

Clement, Tracy M.; Savenkova, Marina I.; Settles, Matthew; Anway, Matthew D.; Skinner, Michael K.

2010-01-01

The current study investigates the direct effects of in utero vinclozolin exposure on the developing F1 generation rat testis transcriptome. Previous studies have demonstrated that exposure to vinclozolin during embryonic gonadal sex determination induces epigenetic modifications of the germ line and transgenerational adult onset disease states. Microarray analyses were performed to compare control and vinclozolin treated testis transcriptomes at embryonic day 13, 14 and 16. A total of 576 differentially expressed genes were identified and the major cellular functions and pathways associated with these altered transcripts were examined. The sets of regulated genes at the different development periods were found to be transiently altered and distinct. Categorization by major known functions of altered genes was performed. Specific cellular process and pathway analyses suggest the involvement of Wnt and calcium signaling, vascular development and epigenetic mechanisms as potential mediators of the direct F1 generation actions of vinclozolin. PMID:20566332

Practical selection methods for rat and mouse round spermatids without DNA staining by flow cytometric cell sorting.

PubMed

Hayama, Tomonari; Yamaguchi, Tomoyuki; Kato-Itoh, Megumi; Ishii, Yumiko; Mizuno, Naoaki; Umino, Ayumi; Sato, Hideyuki; Sanbo, Makoto; Hamanaka, Sanae; Masaki, Hideki; Hirabayashi, Masumi; Nakauchi, Hiromitsu

2016-06-01

Round spermatid injection (ROSI) into unfertilized oocytes enables a male with a severe spermatogenesis disorder to have children. One limitation of the application of this technique in the clinic is the identification and isolation of round spermatids from testis tissue. Here we developed an efficient and simple method to isolate rodent haploid round spermatids using flow cytometric cell sorting, based on DNA content (stained with Hoechst 33342 or Dye Cycle Violet) or by cell diameter and granularity (forward and side scatter). ROSI was performed with round spermatids selected by flow cytometry, and we obtained healthy offspring from unstained cells. This non-invasive method could therefore be an effective option for breeding domestic animals and human male infertility treatment. Mol. Reprod. Dev. 83: 488-496, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Identification and Characterization of MicroRNAs in Ovary and Testis of Nile Tilapia (Oreochromis niloticus) by Using Solexa Sequencing Technology

PubMed Central

Zhou, Yi; Yu, Fan; Gao, Yun; Luo, Yongju; Tang, Zhanyang; Guo, Zhongbao; Guo, Enyan; Gan, Xi; Zhang, Ming; Zhang, Yaping

2014-01-01

MicroRNAs (miRNAs) are endogenous non-coding small RNAs which play important roles in the regulation of gene expression by cleaving or inhibiting the translation of target gene transcripts. Thereinto, some specific miRNAs show regulatory activities in gonad development via translational control. In order to further understand the role of miRNA-mediated posttranscriptional regulation in Nile tilapia (Oreochromis niloticus) ovary and testis, two small RNA libraries of Nile tilapia were sequenced by Solexa small RNA deep sequencing methods. A total of 9,731,431 and 8,880,497 raw reads, representing 5,407,800 and 4,396,281 unique sequences were obtained from the sexually mature ovaries and testes, respectively. After comparing the small RNA sequences with the Rfam database, 1,432,210 reads in ovaries and 984,146 reads in testes were matched to the genome sequence of Nile tilapia. Bioinformatic analysis identified 764 mature miRNA, 209 miRNA-5p and 202 miRNA-3p were found in the two libraries, of which 525 known miRNAs are both expressed in the ovary and testis of Nile tilapia. Comparison of expression profiles of the testis, miR-727, miR-129 and miR-29 families were highly expressed in tilapia ovary. Additionally, miR-132, miR-212, miR-33a and miR-135b families, showed significant higher expression in testis compared with that in ovary. Furthermore, the expression patterns of the miRNAs were analyzed in different developmental stages of gonad. The result showed different expression patterns were observed during development of testis and ovary. In addition, the identification and characterization of differentially expressed miRNAs in the ovaries and testis of Nile tilapia provides important information on the role of miRNA in the regulation of the ovarian and testicular development and function. This data will be helpful to facilitate studies on the regulation of miRNAs during teleosts reproduction. PMID:24466258

Activation of GPER-1 estradiol receptor downregulates production of testosterone in isolated rat Leydig cells and adult human testis.

PubMed

Vaucher, Laurent; Funaro, Michael G; Mehta, Akanksha; Mielnik, Anna; Bolyakov, Alexander; Prossnitz, Eric R; Schlegel, Peter N; Paduch, Darius A

2014-01-01

Estradiol (E2) modulates testicular functions including steroidogenesis, but the mechanisms of E2 signaling in human testis are poorly understood. GPER-1 (GPR30), a G protein-coupled membrane receptor, mediates rapid genomic and non-genomic response to estrogens. The aim of this study was to evaluate GPER-1 expression in the testis, and its role in estradiol dependent regulation of steroidogenesis in isolated rat Leydig cells and human testis. Isolated Leydig cells (LC) from adult rats and human testicular tissue were used in this study. Expression and localization studies of GPER-1 were performed with qRT-PCR, immunofluorescence, immunohistochemistry and Western Blot. Luteinizing Hormone (LH) -stimulated, isolated LC were incubated with estradiol, G-1 (GPER-1-selective agonist), and estrogen receptor antagonist ICI 182,780. Testosterone production was measured with radioimmunoassay. LC viability after incubation with G-1 was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. GPER-1 mRNA is abundantly expressed in rat LC and human testis. Co-localization experiments showed high expression levels of GPER-1 protein in LC. E2-dependent activation of GPER-1 lowers testosterone production in isolated rats LCs and in human testis, with statistically and clinically significant drops in testosterone production by 20-30% as compared to estradiol-naïve LC. The exposure to G-1 does not affect viability of isolated LCs. Our results indicate that activation of GPER-1 lowers testosterone levels in the rat and human testis. The expression of GPER-1 in human testis, which lack ERα, makes it an exciting target for developing new agents affecting testosterone production in men.

Ultrasonographic Assessment of Testicular Viability Using Heterogeneity Levels in Torsed Testicles.

PubMed

Samson, Patrick; Hartman, Christopher; Palmerola, Ricardo; Rahman, Zara; Siev, Michael; Palmer, Lane S; Ghorayeb, Sleiman R

2017-03-01

Gross testicular heterogeneity on ultrasound has been associated with testis loss following testicular torsion in children. We aimed to quantify the extent of temporal heterogeneity associated with testis loss in testicular torsion cases using a noninvasive technique to determine a HI (heterogeneity index) on ultrasound images. We retrospectively studied the records of patients who presented with acute scrotal pain to the Pediatric Emergency Department over a 6-year period. Ultrasound images of the affected testis and the unaffected contralateral testis were examined using a proprietary program to determine the extent of heterogeneity of each image. The difference between the HI of the torsed testis and that of the contralateral normal testis was termed ΔHI. Receiver operating characteristics curve analysis was performed to determine the ΔHI threshold for nonviability. Among 529 patients who presented with acute scrotal pain 147 had testicular torsion based on surgical findings. Of these 147 patients 110 (74.8%) were found to have a viable testis while 37 (25.2%) had a nonviable testis. Using the ΔHI cutoff of 0.394 or greater for nonviability, sensitivity and specificity were 100% and 94.5%, respectively. Positive and negative predictive values were 86% and 100%, respectively. Our results demonstrate that a quantifiable temporal gradation of heterogeneity exists and the heterogeneity index can be used as an objective parameter to determine the viability of a torsed testicle. By developing the technology to measure the heterogeneity index in real time, we could potentially identify which patients with testicular torsion have a nonviable testicle and, thus, would not require immediate surgical exploration. Copyright © 2017 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

A gene expression resource generated by genome-wide lacZ profiling in the mouse

PubMed Central

Tuck, Elizabeth; Estabel, Jeanne; Oellrich, Anika; Maguire, Anna Karin; Adissu, Hibret A.; Souter, Luke; Siragher, Emma; Lillistone, Charlotte; Green, Angela L.; Wardle-Jones, Hannah; Carragher, Damian M.; Karp, Natasha A.; Smedley, Damian; Adams, Niels C.; Bussell, James N.; Adams, David J.; Ramírez-Solis, Ramiro; Steel, Karen P.; Galli, Antonella; White, Jacqueline K.

2015-01-01

ABSTRACT Knowledge of the expression profile of a gene is a critical piece of information required to build an understanding of the normal and essential functions of that gene and any role it may play in the development or progression of disease. High-throughput, large-scale efforts are on-going internationally to characterise reporter-tagged knockout mouse lines. As part of that effort, we report an open access adult mouse expression resource, in which the expression profile of 424 genes has been assessed in up to 47 different organs, tissues and sub-structures using a lacZ reporter gene. Many specific and informative expression patterns were noted. Expression was most commonly observed in the testis and brain and was most restricted in white adipose tissue and mammary gland. Over half of the assessed genes presented with an absent or localised expression pattern (categorised as 0-10 positive structures). A link between complexity of expression profile and viability of homozygous null animals was observed; inactivation of genes expressed in ≥21 structures was more likely to result in reduced viability by postnatal day 14 compared with more restricted expression profiles. For validation purposes, this mouse expression resource was compared with Bgee, a federated composite of RNA-based expression data sets. Strong agreement was observed, indicating a high degree of specificity in our data. Furthermore, there were 1207 observations of expression of a particular gene in an anatomical structure where Bgee had no data, indicating a large amount of novelty in our data set. Examples of expression data corroborating and extending genotype-phenotype associations and supporting disease gene candidacy are presented to demonstrate the potential of this powerful resource. PMID:26398943

Sry and SoxE genes: How they participate in mammalian sex determination and gonadal development?

PubMed

She, Zhen-Yu; Yang, Wan-Xi

2017-03-01

In mammals, sex determination defines the differentiation of the bipotential genital ridge into either testes or ovaries. Sry, the mammalian Y-chromosomal testis-determining gene, is a master regulator of male sex determination. It acts to switch the undifferentiated genital ridge towards testis development, triggering the adoption of a male fate. Sry initiates a cascade of gene networks through the direct regulation of Sox9 expression and promotes supporting cell differentiation, Leydig cell specification, vasculature formation and testis cord development. In the absence of Sry, alternative genetic cascades, including female sex-determining genes RSPO1, Wnt4/β-catenin and Foxl2, are involved in the formation of female genitalia and the maintenance of female ovarian development. The mutual antagonisms between male and female sex-determining pathways are crucial in not just the initiation but also the maintenance of the somatic sex of the gonad throughout the organism's lifetime. Any imbalances in above sex-determining genes can cause disorders of sex development in humans and mice. In this review, we provide a detailed summary of the expression profiles, biochemical properties and developmental functions of Sry and SoxE genes in embryonic testis development and adult gonadal development. We also briefly summarize the dedicate balances between male and female sex-determining genes in mammalian sex development, with particular highlights on the molecular actions of Sry and Sox9 transcription factors. Copyright © 2016 Elsevier Ltd. All rights reserved.

piRNA biogenesis during adult spermatogenesis in mice is independent of the ping-pong mechanism.

PubMed

Beyret, Ergin; Liu, Na; Lin, Haifan

2012-10-01

piRNAs, a class of small non-coding RNAs associated with PIWI proteins, have broad functions in germline development, transposon silencing, and epigenetic regulation. In diverse organisms, a subset of piRNAs derived from repeat sequences are produced via the interplay between two PIWI proteins. This mechanism, termed "ping-pong" cycle, operates among the PIWI proteins of the primordial mouse testis; however, its involvement in postnatal testes remains elusive. Here we show that adult testicular piRNAs are produced independent of the ping-pong mechanism. We identified and characterized large populations of piRNAs in the adult and postnatal developing testes associated with MILI and MIWI, the only PIWI proteins detectable in these testes. No interaction between MILI and MIWI or sequence feature for the ping-pong mechanism among their piRNAs was detected in the adult testis. The majority of MILI- and MIWI-associated piRNAs originate from the same DNA strands within the same loci. Both populations of piRNAs are biased for 5' Uracil but not for Adenine on the 10th nucleotide position, and display no complementarity. Furthermore, in Miwi mutants, MILI-associated piRNAs are not downregulated, but instead upregulated. These results indicate that the adult testicular piRNAs are predominantly, if not exclusively, produced by a primary processing mechanism instead of the ping-pong mechanism. In this primary pathway, biogenesis of MILI- and MIWI-associated piRNAs may compete for the same precursors; the types of piRNAs produced tend to be non-selectively dictated by the available precursors in the cell; and precursors with introns tend to be spliced before processed into piRNAs.

Contrasting Levels of Molecular Evolution on the Mouse X Chromosome

PubMed Central

Larson, Erica L.; Vanderpool, Dan; Keeble, Sara; Zhou, Meng; Sarver, Brice A. J.; Smith, Andrew D.; Dean, Matthew D.; Good, Jeffrey M.

2016-01-01

The mammalian X chromosome has unusual evolutionary dynamics compared to autosomes. Faster-X evolution of spermatogenic protein-coding genes is known to be most pronounced for genes expressed late in spermatogenesis, but it is unclear if these patterns extend to other forms of molecular divergence. We tested for faster-X evolution in mice spanning three different forms of molecular evolution—divergence in protein sequence, gene expression, and DNA methylation—across different developmental stages of spermatogenesis. We used FACS to isolate individual cell populations and then generated cell-specific transcriptome profiles across different stages of spermatogenesis in two subspecies of house mice (Mus musculus), thereby overcoming a fundamental limitation of previous studies on whole tissues. We found faster-X protein evolution at all stages of spermatogenesis and faster-late protein evolution for both X-linked and autosomal genes. In contrast, there was less expression divergence late in spermatogenesis (slower late) on the X chromosome and for autosomal genes expressed primarily in testis (testis-biased). We argue that slower-late expression divergence reflects strong regulatory constraints imposed during this critical stage of sperm development and that these constraints are particularly acute on the tightly regulated sex chromosomes. We also found slower-X DNA methylation divergence based on genome-wide bisulfite sequencing of sperm from two species of mice (M. musculus and M. spretus), although it is unclear whether slower-X DNA methylation reflects development constraints in sperm or other X-linked phenomena. Our study clarifies key differences in patterns of regulatory and protein evolution across spermatogenesis that are likely to have important consequences for mammalian sex chromosome evolution, male fertility, and speciation. PMID:27317678

A Novel Dynamic Expression of vasa in Male Germ Cells during Spermatogenesis in the Chinese Soft-Shell Turtle (Pelidiscus sinensis).

PubMed

Li, Wei; Zhang, Piaoyi; Wu, Xuling; Zhu, Xinping; Xu, Hongyan

2017-05-01

vasa gene encodes a highly conserved DEAD-box RNA helicase, required for germ cell development across animal kingdom. Vasa mutations cause male infertility in mammals. It has been widely used as a biomarker for studying animal fertility or manipulating germ cells in organisms. However, in reptilians, the functions of vasa gene involved in germ cell differentiation are largely unclear; this hampers the development of biological techniques and the improvement of the productivity in these species. Here a vasa cDNA was isolated in Chinese soft-shell turtle and it predicts a protein of 691 amino acid residues, which is 72%, 69%, 58%, 59%, and 54-56% identical to its homolog from mouse, platypus, frog, chicken, and fish, respectively, and named as PsVasa. The Psvasa mRNA was detected exclusively in the gonads of both sexes by RT-PCR. Chromogenic RNA in situ hybridization revealed that the Psvasa mRNA was restricted to germ cells in the testis: The psvasa mRNA is undetectable in resting spermatogonia, appears in proliferating spermatogonia, and becomes abundant in spermatocytes and detectable in spermatozoa. Immunofluorescence staining demonstrated that the PsVasa in the testis is also restricted to the germ cells, rich in spermatocytes and elongated spermatids but hardly detectable in spermatogonia and spermatozoa. Taken together, Psvasa is potentially a reliable germ cell marker in the Chinese soft-shell turtle; its RNA expression could distinguish the different spermatogenic stages of germ cells. These findings shed new insights into understanding the evolutionary conservations and divergences of vasa gene's functions in male germ cell differentiation in metazoans. © 2017 Wiley Periodicals, Inc.

Identification of potential mechanisms of toxicity after di-(2-ethylhexyl)-phthalate (DEHP) adult exposure in the liver using a systems biology approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eveillard, Alexandre; Lasserre, Frederic; Tayrac, Marie de

2009-05-01

Phthalates are industrial additives widely used as plasticizers. In addition to deleterious effects on male genital development, population studies have documented correlations between phthalates exposure and impacts on reproductive tract development and on the metabolic syndrome in male adults. In this work we investigated potential mechanisms underlying the impact of DEHP on adult mouse liver in vivo. A parallel analysis of hepatic transcript and metabolic profiles from adult mice exposed to varying DEHP doses was performed. Hepatic genes modulated by DEHP are predominantly PPAR{alpha} targets. However, the induction of prototypic cytochrome P450 genes strongly supports the activation of additional NRmore » pathways, including Constitutive Androstane Receptor (CAR). Integration of transcriptomic and metabonomic profiles revealed a correlation between the impacts of DEHP on genes and metabolites related to heme synthesis and to the Rev-erb{alpha} pathway that senses endogenous heme level. We further confirmed the combined impact of DEHP on the hepatic expression of Alas1, a critical enzyme in heme synthesis and on the expression of Rev-erb{alpha} target genes involved in the cellular clock and in energy metabolism. This work shows that DEHP interferes with hepatic CAR and Rev-erb{alpha} pathways which are both involved in the control of metabolism. The identification of these new hepatic pathways targeted by DEHP could contribute to metabolic and endocrine disruption associated with phthalate exposure. Gene expression profiles performed on microdissected testis territories displayed a differential responsiveness to DEHP. Altogether, this suggests that impacts of DEHP on adult organs, including testis, could be documented and deserve further investigations.« less

EFFECT OF THE ANTI-ANDROGENIC ENDOCRINE DISRUPTOR VINCLOZOLIN ON EMBRYONIC TESTIS CORD FORMATION AND POSTNATAL TESTIS DEVELOPMENT AND FUNCTION. (R827405)

EPA Science Inventory

Vinclozolin is a systemic dicarboximide fungicide that is used on fruits, vegetables, ornamental plants, and turf grass. Vinclozolin and its metabolites are known to be endocrine disruptors and act as androgen receptor antagonists. The hypothesis tested in the current study is...

Organogenesis of the Ovary

PubMed Central

Ditewig, Amy C

2005-01-01

The general perspective of ovary organogenesis is that the ovary is the default organ which develops in the absence of testis-promoting factors. Testis formation, on the other hand, is a male-specific event promoted by active components that override the default ovarian process. However, when comparing the sex determination mechanism among different vertebrate species, it is apparent that this default view of ovary formation can only be applied to mammals. In species such as reptiles and birds, ovary formation is an active process stimulated by estrogen. Remnants of this estrogen-dominant pathway are still present in marsupials, a close relative of eutherian mammals, like humans and mice. Although initial formation of the mammalian ovary has become strictly regulated by genetic components and is therefore independent of estrogen, the feminizing effect of estrogen regains its command in adult ovaries. When estrogen production, or its signaling, is inhibited, transdifferentiation of ovarian tissues to testis structures occur in adult females. Taken together, these observations prompt us to reconsider the process of ovary organogenesis as the default organ and question if testis development is actually the default pathway. PMID:19521565

Differential lactate and cholesterol synthetic activities in XY and XX Sertoli cells.

PubMed

Shishido, Yurina; Baba, Takashi; Sato, Tetsuya; Shima, Yuichi; Miyabayashi, Kanako; Inoue, Miki; Akiyama, Haruhiko; Kimura, Hiroshi; Kanai, Yoshiakira; Ishihara, Yasuhiro; Haraguchi, Shogo; Miyazaki, Akira; Rozman, Damjana; Yamazaki, Takeshi; Choi, Man-Ho; Ohkawa, Yasuyuki; Suyama, Mikita; Morohashi, Ken-Ichirou

2017-02-02

SRY, a sex-determining gene, induces testis development in chromosomally female (XX) individuals. However, mouse XX Sertoli cells carrying Sry (XX/Sry Sertoli cells) are incapable of fully supporting germ cell development, even when the karyotype of the germ cells is XY. While it has therefore been assumed that XX/Sry Sertoli cells are not functionally equivalent to XY Sertoli cells, it has remained unclear which specific functions are affected. To elucidate the functional difference, we compared the gene expression of XY and XX/Sry Sertoli cells. Lactate and cholesterol metabolisms, essential for nursing the developing germ cells, were down-regulated in XX/Sry cells, which appears to be caused at least in part by the differential expression of histone modification enzymes SMCX/SMCY (H3K4me3 demethylase) and UTX/UTY (H3K27me3 demethylase) encoded by the sex chromosomes. We suggest that down-regulation of lactate and cholesterol metabolism that may be due to altered epigenetic modification affects the nursing functions of XX/Sry Sertoli cells.

Differential lactate and cholesterol synthetic activities in XY and XX Sertoli cells

PubMed Central

Shishido, Yurina; Baba, Takashi; Sato, Tetsuya; Shima, Yuichi; Miyabayashi, Kanako; Inoue, Miki; Akiyama, Haruhiko; Kimura, Hiroshi; Kanai, Yoshiakira; Ishihara, Yasuhiro; Haraguchi, Shogo; Miyazaki, Akira; Rozman, Damjana; Yamazaki, Takeshi; Choi, Man-Ho; Ohkawa, Yasuyuki; Suyama, Mikita; Morohashi, Ken-ichirou

2017-01-01

SRY, a sex-determining gene, induces testis development in chromosomally female (XX) individuals. However, mouse XX Sertoli cells carrying Sry (XX/Sry Sertoli cells) are incapable of fully supporting germ cell development, even when the karyotype of the germ cells is XY. While it has therefore been assumed that XX/Sry Sertoli cells are not functionally equivalent to XY Sertoli cells, it has remained unclear which specific functions are affected. To elucidate the functional difference, we compared the gene expression of XY and XX/Sry Sertoli cells. Lactate and cholesterol metabolisms, essential for nursing the developing germ cells, were down-regulated in XX/Sry cells, which appears to be caused at least in part by the differential expression of histone modification enzymes SMCX/SMCY (H3K4me3 demethylase) and UTX/UTY (H3K27me3 demethylase) encoded by the sex chromosomes. We suggest that down-regulation of lactate and cholesterol metabolism that may be due to altered epigenetic modification affects the nursing functions of XX/Sry Sertoli cells. PMID:28150810

Germ cell differentiation and proliferation in the developing testis of the South American plains viscacha, Lagostomus maximus (Mammalia, Rodentia).

PubMed

Gonzalez, C R; Muscarsel Isla, M L; Fraunhoffer, N A; Leopardo, N P; Vitullo, A D

2012-08-01

Cell proliferation and cell death are essential processes in the physiology of the developing testis that strongly influence the normal adult spermatogenesis. We analysed in this study the morphometry, the expression of the proliferation cell nuclear antigen (PCNA), cell pluripotency marker OCT-4, germ cell marker VASA and apoptosis in the developing testes of Lagostomus maximus, a rodent in which female germ line develops through abolished apoptosis and unrestricted proliferation. Morphometry revealed an increment in the size of the seminiferous cords with increasing developmental age, arising from a significant increase of PCNA-positive germ cells and a stable proportion of PCNA-positive Sertoli cells. VASA showed a widespread cytoplasmic distribution in a great proportion of proliferating gonocytes that increased significantly at late development. In the somatic compartment, Leydig cells increased at mid-development, whereas peritubular cells showed a stable rate of proliferation. In contrast to other mammals, OCT-4 positive gonocytes increased throughout development reaching 90% of germ cells in late-developing testis, associated with a conspicuous increase in circulating FSH from mid- to late-gestation. TUNEL analysis was remarkable negative, and only a few positive cells were detected in the somatic compartment. These results show that the South American plains viscacha displays a distinctive pattern of testis development characterized by a sustained proliferation of germ cells throughout development, with no signs of apoptosis cell demise, in a peculiar endocrine in utero ambiance that seems to promote the increase of spermatogonial number as a primary direct effect of FSH.

Developmentally regulated expression of APG-1, a member of heat shock protein 110 family in murine male germ cells.

PubMed

Kaneko, Y; Kimura, T; Nishiyama, H; Noda, Y; Fujita, J

1997-04-07

Apg-1 encodes a heat shock protein belonging to the heat shock protein 110 family, and is inducible by a 32 degrees C to 39 degrees C heat shock. Northern blot analysis of the testis from immature and adult mice, and of the purified germ cells revealed the quantitative change of the apg-1 transcripts during germ cell development. By in situ hybridization histochemistry the expressions of the apg-1 transcripts were detected in germ cells at specific stages of development including spermatocytes and spermatids. Although heat-induction of the apg-1 transcripts was observed in W/Wv mutant testis lacking germ cells, it was not detected in wild-type testis nor in the purified germ cells. Thus, the apg-1 expression is not heat-regulated but developmentally regulated in germ cells, suggesting that APG-1 plays a role in normal development of germ cells.

Stage-dependency of apoptosis and the blood-testis barrier in the dogfish shark (Squalus acanthias): cadmium-induced changes as assessed by vital fluorescence techniques.

PubMed

McClusky, Leon M

2006-09-01

Naturally occurring heavy metals and synthetic compounds are potentially harmful for testicular function but evidence linking heavy metal exposure to reduced semen parameters is inconclusive. Elucidation of the exact stage at which the toxicant interferes with spermatogenesis is difficult because the various germ cell stages may have different sensitivities to any given toxicant, germ cell development is influenced by supporting testicular somatic cells and the presence of inter-Sertoli cell tight junctions create a blood-testis barrier, sequestering meiotic and postmeiotic germ cells in a special microenvironment. Sharks such as Squalus acanthias provide a suitable model for studying aspects of vertebrate spermatogenosis because of their unique features: spermatogenesis takes place within spermatocysts and relies mainly on Sertoli cells for somatic cell support; spermatocysts are linearly arranged in a maturational order across the diameter of the elongated testis; spermatocysts containing germ cells at different stages of development are topographically separated, resulting in visible zonation in testicular cross sections. We have used the vital dye acridine orange and a novel fluorescence staining technique to study this model to determine (1) the efficacy of these methods in assays of apoptosis and blood-testis barrier function, (2) the sensitivity of the various spermatogonial generations in Squalus to cadmium (as an illustrative spermatotoxicant) and (3) the way that cadmium might affect more mature spermatogenic stages and other physiological processes in the testis. Our results show that cadmium targets early spermatogenic stages, where it specifically activates a cell death program in susceptible (mature) spermatogonial clones, and negatively affects blood-testis barrier function. Since other parameters are relatively unaffected by cadmium, the effects of this toxicant on apoptosis are presumably process-specific and not attributable to general toxicity.

HPLC/UV quantitation of retinal, retinol, and retinyl esters in serum and tissues

PubMed Central

Kane, Maureen A.; Folias, Alexandra E.; Napoli, Joseph L.

2008-01-01

We report robust HPLC/UV methods for quantifying retinyl esters (RE), retinol (ROL) and retinal (RAL) applicable to diverse biological samples, with lower limits of detection of 0.7 pmol, 0.2 pmol, and 0.2 pmol, respectively, and linear ranges >3 orders of magnitude. These assays function well with small, complex biological samples (10–20 mg tissue). Coefficients of variation range from: intra-day, 5.9–10.0%; inter-day, 5.9–11.0%. Quantification of endogenous RE, ROL, and RAL in mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, skin, brain, and brain regions) reveals utility. Ability to discriminate spatial concentrations of ROL and RE is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum.) We also developed a method to distinguish isomeric forms of ROL to investigate precursors of retinoic acid. The ROL isomer assay has limits of detection between 3.5–4.5 pmol and a similar linear range and % CV as the ROL/RE and RAL assays. The assays described here provide for sensitive and rigorous quantification of endogenous RE, ROL, and RAL to elucidate retinoid homeostasis in disease states, such as Alzheimer’s disease, type 2 diabetes, obesity, and cancer. PMID:18410739

Conditions in utero and cancer risk.

PubMed

Grotmol, Tom; Weiderpass, Elisabete; Tretli, Steinar

2006-01-01

There is increasing recognition that conditions in utero are of importance for later cancer risk in several organs, particularly the testis and breast. A review of the most recent literature on this topic is therefore warranted. The PubMed database was searched for relevant recent literature on intrauterine conditions associated with cancer risk later in life, with particular emphasis on the testis, breast, but also studies pertaining to other organs were included. Epidemiological and experimental data support the hypothesis that factors acting in utero play a role in the development of cancer in the testis and breast. For other organs, such as the prostate, urinary system and colorectum, the results are inconclusive. While conditions during foetal life are associated with later cancer risk in the testis and breast, the biological mechanisms are for the most part elusive. They are, however, likely to involve hormonal disturbances, number of cells at risk, and genetic or epigenetic events.

CHANGES IN FETAL TESTIS GENE EXPRESSION AND STEROID HORMONE SYNTHESIS INDUCED IN MALE OFFSPRING AFTER MATERNAL TREATMENT WITH PHTHALATE ESTERS

EPA Science Inventory

Targeted inactivation of the insulin-like hormone 3 (insl3) gene in male mice results in altered gubernacular development, disrupted testis decent, and cryptorchidism. Cryptorchidism is a fairly common human malformation, being displayed in 1-3% of males at birth. Since only a s...

Structure of the 5' region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites.

PubMed Central

Scieglinska, D; Widłak, W; Konopka, W; Poutanen, M; Rahman, N; Huhtaniemi, I; Krawczyk, Z

2001-01-01

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5' region of the transcription unit of these genes as well as localization of the 'cis' elements conferring their testis-specific expression gave contradictory results [Widlak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191-200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310-321]. In the present paper we solve these controversies and show that the 5' untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of Hst70 gene transcripts with similar sizes but different 5' UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360 bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on 'cis' regulatory elements localized within exon 1 and the intron sequences. PMID:11563976

Polysome Profiling in Leishmania, Human Cells and Mouse Testis.

PubMed

Karamysheva, Zemfira N; Tikhonova, Elena B; Grozdanov, Petar N; Huffman, James C; Baca, Kristen R; Karamyshev, Alexander; Denison, R Brian; MacDonald, Clinton C; Zhang, Kai; Karamyshev, Andrey L

2018-04-08

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Sex cord-gonadal stromal tumor of the rete testis.

PubMed

Sajadi, Kamran P; Dalton, Rory R; Brown, James A

2009-01-01

A 34-year-old tetraplegic patient with suppurative epididymitis was found on follow-up examination and ultrasonography to have a testicular mass. The radical orchiectomy specimen contained an undifferentiated spindled sex cord-stromal tumor arising in the rete testis. Testicular sex cord-stromal tumors are far less common than germ cell neoplasms and are usually benign. The close relationship between sex cords and ductules of the rete testis during development provides the opportunity for these uncommon tumors to arise anatomically within the rete tesis. This undifferentiated sex cord-stromal tumor, occurring in a previously unreported location, is an example of an unusual lesion mimicking an intratesticular malignant neoplasm.

Roles of CD34+ cells and ALK5 signaling in the reconstruction of seminiferous tubule-like structures in 3-D re-aggregate culture of dissociated cells from neonatal mouse testes.

PubMed

Abe, Shin-Ichi; Abe, Kazuko; Zhang, Jidong; Harada, Tomoaki; Mizumoto, Go; Oshikawa, Hiroki; Akiyama, Haruhiko; Shimamura, Kenji

2017-01-01

Tissue reconstruction in vitro can provide, if successful, a refined and simple system to analyze the underlying mechanisms that drive the morphogenesis and maintain the ordered structure. We have recently succeeded in reconstruction of seminiferous cord-like and tubule-like structures using 3-D re-aggregate culture of dissociated testicular cells. In testis formation, endothelial cells that migrated from mesonephroi to embryonic gonads have been shown to be critical for development of testis cords, but how endothelial cells contribute to testis cord formation remains unknown. To decipher the roles of endothelial and peritubular cells in the reconstruction of cord-like and tubule-like structures, we investigated the behavior of CD34+ endothelial and p75+ cells, and peritubular myoid cells (PTMCs) in 3-D re-aggregate cultures of testicular cells. The results showed that these 3 types of cells had the capacity of re-aggregation on their own and with each other, and of segregation into 3 layers in a re-aggregate, which were very similar to interstitial and peritubular tissues in vivo. Observation of behaviors of fluorescent Sertoli cells and other non-fluorescent types of cells using testes from Sox9-EGFP transgenic mice showed dynamic cell movement and segregation in re-aggregate cultures. Cultures of testicular cells deprived of interstitial and peritubular cells resulted in dysmorphic structures, but re-addition of them restored tubule-like structures. Purified CD34+ cells in culture differentiated into p75+ cells and PTMCs. These results indicate that CD34+ cells differentiate into p75+ cells, which then differentiate into PTMCs. TGFβ signaling inhibitors, SB431542 and ALK5i, disturbed the reconstruction of cord-like and tubule-like structures, and the latter compromised re-construction of interstitial-like and peritubular-like structures, as well as the proliferation of CD34+, p75+, PTMCs, and Sertoli cells, and their movement and differentiation. These results indicate that CD34+ cells and signaling through ALK5 play pivotal roles in the morphogenesis of interstitial-like, peritubular-like and cord-like structures.

A ZFY-like sequence in fish, with comments on the evolution of the ZFY family of genes in vertebrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zimmerer, E.J.; Threlkeld, L.

1995-08-01

ZFY-like genes have been observed in a variety of vertebrate species. Although originally implicated as the primary testis-determining gene in humans and other placental mammals, more recent evidence indicates a role(s) outside that of testis determination. In this study, DNA from five species of fish, Carasius auratus, Rivulus marmoratus, Xiphophorus maculatus, X. milleri, and X. nigrensis was subjected to Southern blot analysis using a PCR-amplified fragment of mouse ZFY-like sequence as a probe. Restriction fragment patterns were not polymorphic between sexes in any one species but showed a different pattern for each species. With one exception, Rivulus, a 3.1-kb bandmore » from the EcoRI digestion was common to all. Sequence and open reading frame analysis of this fragment showed a strong homology to other known vertebrate ZFY-like genes. Of particular interest in this gene is a novel third finger domain similar to one human and one alligator ZFY-like gene. Our studies and others provide evidence for a family of vertebrate ZFY genes, with those having this novel third finger being representative of the ancestral condition. 30 refs., 3 figs., 3 tabs.« less

Heat shock protein 90 functions to stabilize and activate the testis-specific serine/threonine kinases, a family of kinases essential for male fertility.

PubMed

Jha, Kula N; Coleman, Alyssa R; Wong, Lily; Salicioni, Ana M; Howcroft, Elizabeth; Johnson, Gibbes R

2013-06-07

Spermiogenesis is characterized by a profound morphological differentiation of the haploid spermatid into spermatozoa. The testis-specific serine/threonine kinases (TSSKs) comprise a family of post-meiotic kinases expressed in spermatids, are critical to spermiogenesis, and are required for male fertility in mammals. To explore the role of heat shock protein 90 (HSP90) in regulation of TSSKs, the stability and catalytic activity of epitope-tagged murine TSSKs were assessed in 293T and COS-7 cells. TSSK1, -2, -4, and -6 (small serine/threonine kinase) were all found to associate with HSP90, and pharmacological inhibition of HSP90 function using the highly specific drugs 17-AAG, SNX-5422, or NVP-AUY922 reduced TSSK protein levels in cells. The attenuation of HSP90 function abolished the catalytic activities of TSSK4 and -6 but did not significantly alter the specific activities of TSSK1 and -2. Inhibition of HSP90 resulted in increased TSSK ubiquitination and proteasomal degradation, indicating that HSP90 acts to control ubiquitin-mediated catabolism of the TSSKs. To study HSP90 and TSSKs in germ cells, a mouse primary spermatid culture model was developed and characterized. Using specific antibodies against murine TSSK2 and -6, it was demonstrated that HSP90 inhibition resulted in a marked decrease of the endogenous kinases in spermatids. Together, our findings demonstrate that HSP90 plays a broad and critical role in stabilization and activation of the TSSK family of protein kinases.

Promyelocytic leukaemia zinc finger maintains self-renewal of male germline stem cells (mGSCs) and its expression pattern in dairy goat testis.

PubMed

Song, W; Zhu, H; Li, M; Li, N; Wu, J; Mu, H; Yao, X; Han, W; Liu, W; Hua, J

2013-08-01

Previous studies have shown that promyelocytic leukaemia zinc finger (PLZF) is a spermatogonia-specific transcription factor in the testis, required to regulate self-renewal and maintenance of the spermatogonia stem cell. Up to now, expression and function of PLZF in the goat testis has not been known. The objectives of this study were to investigate PLZF expression pattern in the dairy goat and its effect on male goat germline stem cell (mGSC) self-renewal and differentiation. Testis development and expression patterns of PLZF in the dairy goat were analysed by haematoxylin and eosin staining, immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, effects of PLZF overexpression on mGSC self-renewal and differentiation were evaluated by quantitative RT-PCR (QRT-PCR), immunofluorescence and BrdU incorporation assay. Promyelocytic leukaemia zinc finger was essential for dairy goat testis development and expression of several proliferation and pluripotency-associated proteins including OCT4, C-MYC were upregulated by PLZF overexpression. The study demonstrated that PLZF played a key role in maintaining self-renewal of mGSCs and its overexpression enhanced expression of proliferation-associated genes. Promyelocytic leukaemia zinc finger could function in the dairy goat as well as in other species in maintaining self-renewal of germline stem cells and this study provides a model to study the mechanism on self-renewal and differentiation of mGSCs in livestock. © 2013 John Wiley & Sons Ltd.

Cloning and characterization of full length of a novel zebrafish gene Zsrg abundantly expressed in the germline stem cells.

PubMed

Lv, Daoyuan; Song, Ping; Chen, Yungui; Gong, Wuming; Mo, Saijun

2005-04-08

Using the digital differential display program of the National Center for Biotechnology Information, we identified a contig of expression sequence tags (ESTs) (Accession No. BM316936), which came from zebrafish ovary and testis libraries. The full-length cDNA of this transcript was cloned and further confirmed by polymerase chain reaction and sequencing. The full-length cDNA of the novel gene is 807bp and encodes a novel protein of 187 amino acids, which shares no significant homology with any other known proteins. Characterization of genomic sequences of the gene revealed that it spans 6kb on the linkage group 3 and is composed of five exons and four introns. RT-PCR analysis showed that it was expressed in mature oocytes and one-cell stage, and persisted until 24h of development. RT-PCR also revealed that it is expressed in gonad and kidney, with the highest level of expression in the testis. The expression sites of the novel gene in adult gonad were further localized by in situ hybridization to oogonia and growing oocytes in ovary and to spermatogonia, spermatocytes but not to spermatids in testis. Based on its abundance in testis and the germline stem cell-spermatogonia and oogonia, we hypothesize that it may function as a testicular development and gametogenesis related gene that plays important roles in spermatogenesis, and named it Zsrg (zebrafish testis spermatogenesis related gene, Zsrg).

Does the cranial suspensory ligament have a role in cryptorchidism?

PubMed

Kassim, Normadiah M; Russell, D A; Payne, A P

2010-01-01

The cranial suspensory ligament (CSL) is a fibromuscular structure anchoring the embryonic gonad to the posterior abdominal wall in male and female mammals. Its persistence in females is believed to be responsible for retaining the ovaries within the abdomen, while its regression in males permits testis descent. Embryonic loss of the CSL in males is believed to be an androgen-dependent event, and failure of this process has been proposed as a cause of cryptorchidism. The present study demonstrates that the nuclei of mesenchymal cells in the caudal part of the CSL are immunoreactively positive for androgen receptor. We examined the effects of exposure of the non-steroidal antiandrogen flutamide during the period from gestational day 10 to birth on the development of the CSL and on testis descent. Exposure of male Albino Swiss rats to the antiandrogen flutamide during this period resulted in feminization of the external genitalia and the suppression of growth of the testes and male reproductive tracts. In adulthood, testes were found to be located in diverse positions including normal scrotal (50%), intra-abdominal (10%) and ectopic suprainguinal (40%). The CSL of the testis persisted into adulthood in all flutamide-treated males, regardless of testis location. In all cases, the ligament consisted of bundles of smooth muscle fibres in the retroperitoneal fat of the posterior abdominal wall. These findings suggest that androgen blockade during embryonic development interferes with testicular descent, but that maldescent cannot be correlated with either the persistence of the CSL of the testis or its structure.

Expression pattern of phb2 and its potential function in spermatogenesis of scallop ( Chlamys farreri)

NASA Astrophysics Data System (ADS)

Han, Tiantian; Ma, Xiaoshi; Liang, Shaoshuai; Gao, Beibei; Zhang, Zhifeng

2015-12-01

Prohibitin (PHB) participates in several biological processes including apoptosis, transcription regulation and suppression of cell proliferation in mammals. In this study, we cloned the full-length cDNA of prohibitin 2 ( Cf-phb2) from the testis of scallop ( Chlamys farreri). The deduced amino acid sequence presented a characteristic of PHB family with the PHB domain, and clustered with PHB2 of other species. Temporal and spatial expression of Cf-phb2 in testis during the reproductive cycle was detected by quantitative real-time PCR (qRT-PCR) and in situ hybridization. The expression of Cf-phb2 in the testis increased when testis developed from the resting stage to mature stage. The mRNA abundance of Cf-phb2 was the highest at mature stage, which was about 15-fold higher than that at proliferative stage. The expression of Cf-phb2 could be detected by in situ hybridization in all types of germ cells in testis, including spermatogonia, spermatocytes, spermatids and spermatozoa. The intensity of the signal increased with the spermatogenesis and was the highest in spermatids, which suggested that CF-PHB2 might affect the spermatogenesis of C. farreri.

Thyroid Hormone and Leptin in the Testis

PubMed Central

Ramos, Cristiane Fonte; Zamoner, Ariane

2014-01-01

Leptin is primarily expressed in white adipose tissue; however, it is expressed in the hypothalamus and reproductive tissues as well. Leptin acts by activating the leptin receptors (Ob-Rs). Additionally, the regulation of several neuroendocrine and reproductive functions, including the inhibition of glucocorticoids and enhancement of thyroxine and sex hormone concentrations in human beings and mice are leptin functions. It has been suggested that thyroid hormones (TH) could directly regulate leptin expression. Additionally, hypothyroidism compromises the intracellular integration of leptin signaling specifically in the arcuate nucleus. Two TH receptor isoforms are expressed in the testis, TRa and TRb, with TRa being the predominant one that is present in all stages of development. The effects of TH involve the proliferation and differentiation of Sertoli and Leydig cells during development, spermatogenesis, and steroidogenesis. In this context, TH disorders are associated with sexual dysfunction. An endocrine and/or direct paracrine effect of leptin on the gonads inhibits testosterone production in Leydig cells. Further studies are necessary to clarify the effects of both hormones in the testis during hypothyroidism. The goal of this review is to highlight the current knowledge regarding leptin and TH in the testis. PMID:25505448

NLRP3 in Somatic Non-Immune Cells of Rodent and Primate Testes.

PubMed

Walenta, Lena; Schmid, Nina; Schwarzer, J U; Köhn, Frank-Michael; Urbanski, Henryk; Behr, Ruediger; Strauss, Leena; Poutanen, Matti; Mayerhofer, Artur

2018-06-15

NLRP3 is part of the NLRP3 inflammasome and a global sensor of cellular damage. It was recently discovered in rodent Sertoli cells. We investigated NLRP3 in mouse, human and non-human primate (marmoset and rhesus macaque) testes, employing immunohistochemistry. Sertoli cells of all species expressed NLRP3, and the expression preceded puberty. In addition, peritubular cells of the adult human testes expressed NLRP3. NLRP3 and associated genes (ASC, CASP1, IL1B) were also found in isolated human testicular peritubular cells (HTPCs) and the mouse Sertoli cell line TM4. Male infertility due to impairments of spermatogenesis may be related to sterile inflammatory events. We observed that the expression of NLRP3 was altered in the testes of patients suffering from mixed atrophy syndrome, in which tubules with impairments of spermatogenesis showed prominent NLRP3 staining. In order to explore a possible role of NLRP3 in male infertility, associated with sterile testicular inflammation, we studied a mouse model of male infertility. These human aromatase expressing transgenic mice (AROM+) develop testicular inflammation and impaired spermatogenesis during aging, and the present data show that this is associated with strikingly elevated Nlrp3 expression in the testes compared to wild type controls. Interference by aromatase inhibitor treatment significantly reduced increased Nlrp3 levels. Thus, throughout species NLRP3 is expressed by somatic cells of the testis, which are involved in testicular immune surveillance. We conclude that NLRP3 may be a novel player in testicular immune regulation. .

Follitropin receptors in rat testis. Characterization with enzymatically 125I-labeled human follitropin.

PubMed

Ketelslegers, J M; Catt, K J

1978-07-03

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The 125I-labeled human follitropin exhibited high binding activity with specific binding of up to 17% in the presence of an excess of testis homogenate. Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (Ka) determined 24 degrees C were not significantly different in immature and adult rat testis, and the mean value for Ka was 3.9 . 10(9) M-1. At 37 degrees C, the Ka value obtained using immature rat testis was 1.3 . 10(10) M-1. The association of 125I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30--60% of the preformed hormone . receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnent mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.

Zika Virus Infection in Dexamethasone-immunosuppressed Mice Demonstrating Disseminated Infection with Multi-organ Involvement Including Orchitis Effectively Treated by Recombinant Type I Interferons.

PubMed

Chan, Jasper Fuk-Woo; Zhang, Anna Jinxia; Chan, Chris Chung-Sing; Yip, Cyril Chik-Yan; Mak, Winger Wing-Nga; Zhu, Houshun; Poon, Vincent Kwok-Man; Tee, Kah-Meng; Zhu, Zheng; Cai, Jian-Piao; Tsang, Jessica Oi-Ling; Chik, Kenn Ka-Heng; Yin, Feifei; Chan, Kwok-Hung; Kok, Kin-Hang; Jin, Dong-Yan; Au-Yeung, Rex Kwok-Him; Yuen, Kwok-Yung

2016-12-01

Disseminated or fatal Zika virus (ZIKV) infections were reported in immunosuppressed patients. Existing interferon-signaling/receptor-deficient mouse models may not be suitable for evaluating treatment effects of recombinant interferons. We developed a novel mouse model for ZIKV infection by immunosuppressing BALB/c mice with dexamethasone. Dexamethasone-immunosuppressed male mice (6-8weeks) developed disseminated infection as evidenced by the detection of ZIKV-NS1 protein expression and high viral loads in multiple organs. They had ≥10% weight loss and high clinical scores soon after dexamethasone withdrawal (10dpi), which warranted euthanasia at 12dpi. Viral loads in blood and most tissues at 5dpi were significantly higher than those at 12dpi (P<0.05). Histological examination revealed prominent inflammatory infiltrates in multiple organs, and CD45+ and CD8+ inflammatory cells were seen in the testis. These findings suggested that clinical deterioration occurred during viral clearance by host immune response. Type I interferon treatments improved clinical outcome of mice (100% vs 0% survival). Besides virus dissemination, inflammation of various tissues, especially orchitis, may be potential complications of ZIKV infection with significant implications on disease transmission and male fertility. Interferon treatment should be considered in patients at high risks for ZIKV-associated complications when the potential benefits outweigh the side effects of treatment. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Genomic structure, promoter identification, and chromosomal mapping of a mouse nuclear orphan receptor expressed in embryos and adult testes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, C.H.; Wei, Li-Na; Copeland, N.G.

We have isolated and characterized overlapping genomic clones containing the complete transcribed region of a newly isolated mouse cDNA encoding an orphan receptor expressed specifically in midgestation embryos and adult testis. This gene spans a distance of more than 50 kb and is organized into 13 exons. The transcription initiation site is located at the 158th nucleotide upstream from the translation initiation codon. All the exon/intron junction sequences follow the GT/AG rule. Based upon Northern blot analysis and the size of the transcribed region of the gene, its transcript was determined to be approximately 2.5 kb. Within approximately 500 hpmore » upstream from the transcription initiation site, several immune response regulatory elements were identified but no TATA box was located. This gene was mapped to the distal region of mouse chromosome 10 and its locus has been designated Tr2-11. Immunohistochemical studies show that the Tr2-11 protein is present mainly in advanced germ cell populations of mature testes and that Tr2-11 gene expression is dramatically decreased in vitamin A-depleted animals. 23 refs., 7 figs.« less

The longest telomeres: a general signature of adult stem cell compartments

PubMed Central

Flores, Ignacio; Canela, Andres; Vera, Elsa; Tejera, Agueda; Cotsarelis, George; Blasco, María A.

2008-01-01

Identification of adult stem cells and their location (niches) is of great relevance for regenerative medicine. However, stem cell niches are still poorly defined in most adult tissues. Here, we show that the longest telomeres are a general feature of adult stem cell compartments. Using confocal telomere quantitative fluorescence in situ hybridization (telomapping), we find gradients of telomere length within tissues, with the longest telomeres mapping to the known stem cell compartments. In mouse hair follicles, we show that cells with the longest telomeres map to the known stem cell compartments, colocalize with stem cell markers, and behave as stem cells upon treatment with mitogenic stimuli. Using K15-EGFP reporter mice, which mark hair follicle stem cells, we show that GFP-positive cells have the longest telomeres. The stem cell compartments in small intestine, testis, cornea, and brain of the mouse are also enriched in cells with the longest telomeres. This constitutes the description of a novel general property of adult stem cell compartments. Finally, we make the novel finding that telomeres shorten with age in different mouse stem cell compartments, which parallels a decline in stem cell functionality, suggesting that telomere loss may contribute to stem cell dysfunction with age. PMID:18283121

A new link between transcriptional initiation and pre-mRNA splicing: The RNA binding histone variant H2A.B

PubMed Central

Hart-Smith, Gene; Tay, Ying Jin; Tng, Wei-Quan; Wilkins, Marc; Ryan, Daniel

2017-01-01

The replacement of histone H2A with its variant forms is critical for regulating all aspects of genome organisation and function. The histone variant H2A.B appeared late in evolution and is most highly expressed in the testis followed by the brain in mammals. This raises the question of what new function(s) H2A.B might impart to chromatin in these important tissues. We have immunoprecipitated the mouse orthologue of H2A.B, H2A.B.3 (H2A.Lap1), from testis chromatin and found this variant to be associated with RNA processing factors and RNA Polymerase (Pol) II. Most interestingly, many of these interactions with H2A.B.3 (Sf3b155, Spt6, DDX39A and RNA Pol II) were inhibited by the presence of endogenous RNA. This histone variant can bind to RNA directly in vitro and in vivo, and associates with mRNA at intron—exon boundaries. This suggests that the ability of H2A.B to bind to RNA negatively regulates its capacity to bind to these factors (Sf3b155, Spt6, DDX39A and RNA Pol II). Unexpectedly, H2A.B.3 forms highly decompacted nuclear subdomains of active chromatin that co-localizes with splicing speckles in male germ cells. H2A.B.3 ChIP-Seq experiments revealed a unique chromatin organization at active genes being not only enriched at the transcription start site (TSS), but also at the beginning of the gene body (but being excluded from the +1 nucleosome) compared to the end of the gene. We also uncover a general histone variant replacement process whereby H2A.B.3 replaces H2A.Z at intron-exon boundaries in the testis and the brain, which positively correlates with expression and exon inclusion. Taken together, we propose that a special mechanism of splicing may occur in the testis and brain whereby H2A.B.3 recruits RNA processing factors from splicing speckles to active genes following its replacement of H2A.Z. PMID:28234895

Identification and characterization of alternatively transcribed form of peroxiredoxin IV gene that is specifically expressed in spermatids of postpubertal mouse testis.

PubMed

Yim, Sun Hee; Kim, Yoo-Jin; Oh, Sue Young; Fujii, Junichi; Zhang, Yan; Gladyshev, Vadim N; Rhee, Sue Goo

2011-11-11

2-Cysteine (Cys) peroxiredoxins (Prxs), which include mammalian Prxs I-IV, possess two conserved Cys residues that are readily oxidized by H(2)O(2) to form a disulfide. In the case of Prx I-III, the disulfide is reduced by thioredoxin, thus enabling these proteins to function as peroxidases. Prx IV was shown previously to be synthesized as a 31-kDa polypeptide with an NH(2)-terminal signal peptide that is subsequently cleaved to generate a 27-kDa form of the protein that is localized to the endoplasmic reticulum. A form of Prx IV, larger than 27 kDa revealed by immunoblot analysis was suggested to represent the unprocessed, 31-kDa form, but this larger form was detected only in spermatids of the postpubertal testis. We now show that the larger form of Prx IV (here designated Prx IV-L) detected in the testis is actually a product of alternative transcription of the Prx IV gene that is encoded by newly identified exon 1A together with exons 2-7 that are shared with the 27-kDa form (designated Prx IV-S). Prx IV-L was detected in spermatids but not in mature sperm, it could form disulfide-linked dimers but not higher order oligomers via oxidation, and it was resistant to hyperoxidation unless additional reductant was added, suggesting that its peroxidase activity is limited in vivo. Phylogenetic analysis showed that the Prx IV-S gene is present in all vertebrates examined, whereas the Prx IV-L gene was detected only in placental mammals. We suggest that Prx IV-L functions as an H(2)O(2) sensor that mediates protein thiol oxidation required for the maturation of spermatozoa in placental mammals.

Effects of environmental Bisphenol A exposures on germ cell development and Leydig cell function in the human fetal testis

PubMed Central

Guerquin, Marie-Justine; Matilionyte, Gabriele; Kilcoyne, Karen; N’Tumba-Byn, Thierry; Messiaen, Sébastien; Deceuninck, Yoann; Pozzi-Gaudin, Stéphanie; Benachi, Alexandra; Livera, Gabriel; Antignac, Jean-Philippe; Mitchell, Rod; Rouiller-Fabre, Virginie

2018-01-01

Background Using an organotypic culture system termed human Fetal Testis Assay (hFeTA) we previously showed that 0.01 μM BPA decreases basal, but not LH-stimulated, testosterone secreted by the first trimester human fetal testis. The present study was conducted to determine the potential for a long-term antiandrogenic effect of BPA using a xenograft model, and also to study the effect of BPA on germ cell development using both the hFETA and xenograft models. Methods Using the hFeTA system, first trimester testes were cultured for 3 days with 0.01 to 10 μM BPA. For xenografts, adult castrate male nude mice were injected with hCG and grafted with first trimester testes. Host mice received 10 μM BPA (~ 500 μg/kg/day) in their drinking water for 5 weeks. Plasma levels of total and unconjugated BPA were 0.10 μM and 0.038 μM respectively. Mice grafted with second trimester testes received 0.5 and 50 μg/kg/day BPA by oral gavage for 5 weeks. Results With first trimester human testes, using the hFeTA model, 10 μM BPA increased germ cell apoptosis. In xenografts, germ cell density was also reduced by BPA exposure. Importantly, BPA exposure significantly decreased the percentage of germ cells expressing the pluripotency marker AP-2γ, whilst the percentage of those expressing the pre-spermatogonial marker MAGE-A4 significantly increased. BPA exposure did not affect hCG-stimulated androgen production in first and second trimester xenografts as evaluated by both plasma testosterone level and seminal vesicle weight in host mice. Conclusions Exposure to BPA at environmentally relevant concentrations impairs germ cell development in first trimester human fetal testis, whilst gonadotrophin-stimulated testosterone production was unaffected in both first and second trimester testis. Studies using first trimester human fetal testis demonstrate the complementarity of the FeTA and xenograft models for determining the respective short-term and long term effects of environmental exposures. PMID:29385186

Bioluminescent Imaging of Trypanosoma brucei Shows Preferential Testis Dissemination Which May Hamper Drug Efficacy in Sleeping Sickness

PubMed Central

Claes, Filip; Vodnala, Suman K.; van Reet, Nick; Boucher, Nathalie; Lunden-Miguel, Hilda; Baltz, Theo; Goddeeris, Bruno Maria; Büscher, Philippe; Rottenberg, Martin E.

2009-01-01

Monitoring Trypanosoma spread using real-time imaging in vivo provides a fast method to evaluate parasite distribution especially in immunoprivileged locations. Here, we generated monomorphic and pleomorphic recombinant Trypanosoma brucei expressing the Renilla luciferase. In vitro luciferase activity measurements confirmed the uptake of the coelenterazine substrate by live parasites and light emission. We further validated the use of Renilla luciferase-tagged trypanosomes for real-time bioluminescent in vivo analysis. Interestingly, a preferential testis tropism was observed with both the monomorphic and pleomorphic recombinants. This is of importance when considering trypanocidal drug development, since parasites might be protected from many drugs by the blood-testis barrier. This hypothesis was supported by our final study of the efficacy of treatment with trypanocidal drugs in T. brucei-infected mice. We showed that parasites located in the testis, as compared to those located in the abdominal cavity, were not readily cleared by the drugs. PMID:19621071

Hyperthyroidism in the developing rat testis is associated with oxidative stress and hyperphosphorylated vimentin accumulation.

PubMed

Zamoner, Ariane; Barreto, Kátia Padilha; Filho, Danilo Wilhelm; Sell, Fabíola; Woehl, Viviane Mara; Guma, Fátima Costa Rodrigues; Silva, Fátima Regina Mena Barreto; Pessoa-Pureur, Regina

2007-03-15

Hyperthyroidism was induced in rats and somatic indices and metabolic parameters were analyzed in testis. In addition, the morphological analysis evidenced testes maturation and intense protein synthesis and processing, supporting the enhancement in vimentin synthesis in hyperthyroid testis. Furthermore, vimentin phosphorylation was increased, indicating an accumulation of phosphorylated vimentin associated to the cytoskeleton, which could be a consequence of the extracellular-regulated kinase (ERK) activation regulating the cytoskeleton. Biomarkers of oxidative stress demonstrated an increased basal metabolic rate measured by tissue oxygen consumption, as well as, increased TBARS levels. In addition, the enzymatic and non-enzymatic antioxidant defences appeared to respond according to the augmented oxygen consumption. We observed decreased total glutathione levels, with enhancement of reduced glutathione, whereas most of the antioxidant enzyme activities were induced. Otherwise, superoxide dismutase activity was inhibited. These results support the idea that an increase in mitochondrial ROS generation, underlying cellular oxidative damage, is a side effect of hyperthyroid-induced biochemical changes by which rat testis increase their metabolic capacity.

Somatic ACE regulates self-renewal of mouse spermatogonial stem cells via the MAPK signaling pathway.

PubMed

Gao, Tingting; Zhao, Xin; Liu, Chenchen; Shao, Binbin; Zhang, Xi; Li, Kai; Cai, Jinyang; Wang, Su; Huang, Xiaoyan

2018-05-24

Spermatogonial stem cell (SSC) self-renewal is an indispensable part of spermatogenesis. Angiotensin I-converting enzyme (ACE) is a zinc dipeptidyl carboxypeptidase that plays a critical role in regulation of the renin-angiotensin system. Here, we used RT-PCR and Western blot analysis to confirm that somatic ACE (sACE) but not testicular ACE (tACE) is highly expressed in mouse testis before postpartum day 7 and in cultured SSCs. Our results revealed that sACE is located on the membrane of SSCs. Treating cultured SSCs with the ACE competitive inhibitor captopril was found to inhibit sACE activity, and significantly reduced the proliferation rate of SSCs. Microarray analysis identified 651 genes with significant differential expression. KEGG pathway analysis showed that these differentially expressed genes are mainly involved in the mitogen-activated protein kinase (MAPK) signaling pathway and cell cycle. sACE was found to play an important role in SSC self-renewal via the regulation of MAPK-dependent cell proliferation.

Localization of peroxisome proliferator-activated receptor in mouse and rat-tissues and demonstration of its nuclear translocation in transfected cv-1 cells.

PubMed

Huang, Q; Yeldandi, A; Alvares, K; Ide, H; Reddy, J; Rao, M

1995-02-01

Hepatocarcinogenesis in rodents induced by nongenotoxic peroxisome proliferators is postulated to be a receptor-mediated process. The peroxisome proliferator-activated receptors (PPAR) are members of the steroid hormone receptor superfamily, which participate in ligand-dependent transcriptional activation of peroxisomal fatty acid beta oxidation enzyme system genes in liver parenchymal cells of rats and mice. In order to study the tissue distribution and cellular localization of PPAR, we raised polyclonal antibodies against PPAR using a recombinant rat PPAR (rPPAR) expressed as a glutathione-S-transferase-rPPAR fusion protein. On immunoblot analysis the antibodies specifically recognized a 55 kDa PPAR protein in rat, mouse and human liver homogenates. Immunoblotting also showed that in the mouse and rat, PPAR is expressed in liver, kidney and heart, and only weakly in brain and testis. Immunohistochemical localization in the rat and mouse revealed that PPAR is highly expressed in perivenular (i.e., those surrounding hepatic vein) hepatocytes and very weakly in the cytoplasm of remaining hepatocytes. In the kidney, PPAR was visualized predominantly in the p(3) segments of proximal convoluted tubular epithelium. CV-1 cells transiently transfected with rPPAR cDNA construct showed predominant cytoplasmic fluorescence; treatment of these cells with ciprofibrate, a peroxisome proliferator, resulted in the nuclear translocation of PPAR signal.

CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFκB signaling pathways

PubMed Central

Wang, Chaoqun; Fok, Kin Lam; Cai, Zhiming; Chen, Hao; Chan, Hsiao Chang

2017-01-01

CD147 null mutant male mice are infertile with arrested spermatogenesis and increased apoptotic germ cells. Our previous studies have shown that CD147 prevents apoptosis in mouse spermatocytes but not spermatogonia. However, the underlying mechanism remains elusive. In the present study, we aim to determine the CD147-regulated apoptotic pathway in mouse spermatocytes. Our results showed that immunodepletion of CD147 triggered apoptosis through extrinsic apoptotic pathway in mouse testis and spermatocyte cell line (GC-2 cells), accompanied by activation of non-canonical NFκB signaling and suppression of canonical NFκB signaling. Furthermore, CD147 was found to interact with TRAF2, a factor known to regulate NFκB and extrinsic apoptotic signaling, and interfering CD147 led to the decrease of TRAF2. Consistently, depletion of CD147 by CRISPR/Cas9 technique in GC-2 cells down-regulated TRAF2 and resulted in cell death with suppressed canonical NFκB and activated non-canonical NFκB signaling. On the contrary, interfering of CD147 had no effect on NFκB signaling pathways as well as TRAF2 protein level in mouse spermatogonia cell line (GC-1 cells). Taken together, these results suggested that CD147 plays a key role in reducing extrinsic apoptosis in spermatocytes, but not spermatogonia, through modulating NFκB signaling pathway. PMID:27902973

CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFκB signaling pathways.

PubMed

Wang, Chaoqun; Fok, Kin Lam; Cai, Zhiming; Chen, Hao; Chan, Hsiao Chang

2017-01-10

CD147 null mutant male mice are infertile with arrested spermatogenesis and increased apoptotic germ cells. Our previous studies have shown that CD147 prevents apoptosis in mouse spermatocytes but not spermatogonia. However, the underlying mechanism remains elusive. In the present study, we aim to determine the CD147-regulated apoptotic pathway in mouse spermatocytes. Our results showed that immunodepletion of CD147 triggered apoptosis through extrinsic apoptotic pathway in mouse testis and spermatocyte cell line (GC-2 cells), accompanied by activation of non-canonical NFκB signaling and suppression of canonical NFκB signaling. Furthermore, CD147 was found to interact with TRAF2, a factor known to regulate NFκB and extrinsic apoptotic signaling, and interfering CD147 led to the decrease of TRAF2. Consistently, depletion of CD147 by CRISPR/Cas9 technique in GC-2 cells down-regulated TRAF2 and resulted in cell death with suppressed canonical NFκB and activated non-canonical NFκB signaling. On the contrary, interfering of CD147 had no effect on NFκB signaling pathways as well as TRAF2 protein level in mouse spermatogonia cell line (GC-1 cells). Taken together, these results suggested that CD147 plays a key role in reducing extrinsic apoptosis in spermatocytes, but not spermatogonia, through modulating NFκB signaling pathway.

Identification of multiple dmrt1s in catfish: localization, dimorphic expression pattern, changes during testicular cycle and after methyltestosterone treatment.

PubMed

Raghuveer, K; Senthilkumaran, B

2009-05-01

The double sex and mab-3 related (DM) transcription factor 1 (dmrt1) plays an important role in testicular differentiation. Here, we report cloning of multiple dmrt1s, a full-length and two alternative spliced forms from adult catfish (Clarias gariepinus) testis, which encode predicted proteins of 287 (dmrt1a), 253 (dmrt1b) and 233 (dmrt1c) amino acid residues respectively. Interestingly, dmrt1c lacks the majority of the DM domain. Multiple dmrt1s (dmrt1a and dmrt1c) were obtained from Clarias batrachus as well. Tissue distribution (transcript and protein) of catfish dmrt1 revealed exclusive expression in testis. Semi-quantitative RT-PCR revealed the presence of multiple dmrt1s with high levels of dmrt1a in adult testis but not in ovary. Real-time RT-PCR analysis during testicular cycle showed higher levels of dmrt1 transcripts in preparatory and pre-spawning when compared with spawning and post-spawning phases. Immunocytochemical and immunofluorescence localization revealed the presence of catfish Dmrt1 protein in spermatogonia and spermatocytes, which indicates plausible role in spermatogenesis. Histological analysis indicated initiation of gonadal sex differentiation in catfish around 40-50 days after hatching. The potential role for dmrt1 in testicular differentiation is evident from its stage-dependent elevated expression in developing testis. Furthermore, dimorphic expressions of dmrt1s were evident at different stages of gonadal development or recrudescence in catfish. Treatment of methyl testosterone (MT) during early stages of gonadal sex differentiation resulted in adult males. Interestingly, we also obtained MT-treated fishes having ova-testis gonads. Analysis of dmrt1, sox9a, foxl2 and cyp19a1 expression patterns in MT-treated gonads revealed tissue-specific pattern. These results together suggest that multiple dmrt1s are testis-specific markers in catfish.

Function and regulation of heat shock factor 2 during mouse embryogenesis

PubMed Central

Rallu, M.; Loones, Mt.; Lallemand, Y.; Morimoto, R.; Morange, M.; Mezger, V.

1997-01-01

The spontaneous expression of heat shock genes during development is well documented in many animal species, but the mechanisms responsible for this developmental regulation are only poorly understood. In vertebrates, additional heat shock transcription factors, distinct from the heat shock factor 1 (HSF1) involved in the stress response, were suggested to be involved in this developmental control. In particular, the mouse HSF2 has been found to be active in testis and during preimplantation development. However, the role of HSF2 and its mechanism of activation have remained elusive due to the paucity of data on its expression during development. In this study, we have examined HSF2 expression during the postimplantation phase of mouse development. Our data show a developmental regulation of HSF2, which is expressed at least until 15.5 days of embryogenesis. It becomes restricted to the central nervous system during the second half of gestation. It is expressed in the ventricular layer of the neural tube which contains mitotically active cells but not in postmitotic neurons. Parallel results were obtained for mRNA, protein, and activity levels, demonstrating that the main level of control was transcriptional. The detailed analysis of the activity of a luciferase reporter gene under the control of the hsp70.1 promoter, as well as the description of the protein expression patterns of the major heat shock proteins in the central nervous system, show that HSF2 and heat shock protein expression domains do not coincide. This result suggests that HFS2 might be involved in other regulatory developmental pathways and paves the way to new functional approaches. PMID:9122205

Modeling human infertility with pluripotent stem cells.

PubMed

Chen, Di; Gell, Joanna J; Tao, Yu; Sosa, Enrique; Clark, Amander T

2017-05-01

Human fertility is dependent upon the correct establishment and differentiation of the germline. This is because no other cell type in the body is capable of passing a genome and epigenome from parent to child. Terminally differentiated germline cells in the adult testis and ovary are called gametes. However, the initial specification of germline cells occurs in the embryo around the time of gastrulation. Most of our knowledge regarding the cell and molecular events that govern human germline specification involves extrapolating scientific principles from model organisms, most notably the mouse. However, recent work using next generation sequencing, gene editing and differentiation of germline cells from pluripotent stem cells has revealed that the core molecular mechanisms that regulate human germline development are different from rodents. Here, we will discuss the major molecular pathways required for human germline differentiation and how pluripotent stem cells have revolutionized our ability to study the earliest steps in human embryonic lineage specification in order to understand human fertility. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Sexy transgenes: the impact of gene transfer and gene inactivation technologies on the understanding of mammalian sex determination.

PubMed

Vaiman, Daniel

2003-06-01

Amongst the various developmental pathways ending in a sound mammal, sex determination presents the peculiarity of a choice between two equally viable options: female or male. Therefore, destroying a 'male-determining gene' or a 'female-determining gene' should generally not be lethal. Genetic sex determination is divided into two consecutive steps: construction of the bipotential gonad, and then sex determination per se. The genes involved in the first step are in fact involved in the development of various body compartments, and their mutation is generally far from innocuous. From transgenic and inactivation studies carried out on the laboratory mouse, a complete picture of the two steps is beginning to emerge, where the gonad itself and the necessary ducts are shown to evolve in a very coordinate way, with well-defined sex-specificities. Compared with testis determination, the ovarian side of the picture is still relatively empty, but this situation can change rapidly as candidate ovarian genes for inactivation studies are beginning to be identified.

Niche players

PubMed Central

Seandel, Marco; Falciatori, Ilaria; Shmelkov, Sergey V.; Kim, Jiyeon; James, Daylon; Rafii, Shahin

2010-01-01

The undifferentiated spermatogonia of adult mouse testes are composed of both true stem cells and committed progenitors. It is unclear what normally prevents these adult germ cells from manifesting multipotency. The critical elements of the spermatogonial stem cell niche, while poorly understood, are thought to be composed of Sertoli cells with several other somatic cell types in close proximity. We recently discovered a novel orphan G-protein coupled receptor (GPR125) that is restricted to undifferentiated spermatogonia within the testis. GPR125 expression was maintained when the progenitor cells were extracted from the in vivo niche and propagated under growth conditions that recapitulate key elements of the niche. Such conditions preserved the ability of the cells to generate multipotent derivatives, known as multipotent adult spermatogonial derived progenitor cells (MASCs). Upon differentiation, the latter produced a variety tissues including functional endothelium, illustrating the potential applications of such cells. Thus, GPR125 represents a novel target for purifying adult stem and progenitors from tissues, with the goal of developing autologous multipotent cell lines. PMID:18256534

Spermatogonial stem cells: Current biotechnological advances in reproduction and regenerative medicine.

PubMed

Aponte, Pedro Manuel

2015-05-26

Spermatogonial stem cells (SSCs) are the germ stem cells of the seminiferous epithelium in the testis. Through the process of spermatogenesis, they produce sperm while concomitantly keeping their cellular pool constant through self-renewal. SSC biology offers important applications for animal reproduction and overcoming human disease through regenerative therapies. To this end, several techniques involving SSCs have been developed and will be covered in this article. SSCs convey genetic information to the next generation, a property that can be exploited for gene targeting. Additionally, SSCs can be induced to become embryonic stem cell-like pluripotent cells in vitro. Updates on SSC transplantation techniques with related applications, such as fertility restoration and preservation of endangered species, are also covered on this article. SSC suspensions can be transplanted to the testis of an animal and this has given the basis for SSC functional assays. This procedure has proven technically demanding in large animals and men. In parallel, testis tissue xenografting, another transplantation technique, was developed and resulted in sperm production in testis explants grafted into ectopical locations in foreign species. Since SSC culture holds a pivotal role in SSC biotechnologies, current advances are overviewed. Finally, spermatogenesis in vitro, already demonstrated in mice, offers great promises to cope with reproductive issues in the farm animal industry and human clinical applications.

Expression profiles of relaxin family peptides and their receptors indicate their influence on spermatogenesis in the domestic cat (Felis catus).

PubMed

Braun, B C; Müller, K; Jewgenow, K

2015-07-01

Disturbed spermatogenesis is a common problem in felines. Studying spermatogenesis in the domestic cat can improve the understanding of the biological background and help to counteract fertility problems in other feline species. Here, we analyzed 3 relaxin family peptides (relaxin, relaxin-3, and INSL3) and their receptors (RXFP1, RXFP2, and RXFP3) as potential spermatogenic factors involving their expression in the testis at different stages of its development. It may be concluded from its stage-dependent expression that relaxin, together with RXFP1, appears to be involved in the first stage of spermatogenesis, whereas relaxin-3 via binding to RXFP3 influences spermiogenesis. Furthermore, correlations were observed between relaxin, relaxin-3, RXFP1, RXFP2 and RXFP3 messenger RNA expression, and the relative numbers of haploid cells in testes. The peptide INSL3 was highly expressed at all testis development stages. Because of the low and stage-independent expression of its receptor RXFP2, an auto- and/or paracrine function of INSL3 in spermatogenesis seems unlikely. In the adult testis, messenger RNA expression of relaxin, RXFP1, and RXFP3 predominantly occurs in the tubular testis compartment, whereas INLS3 is mainly expressed in the interstitium. Copyright © 2015 Elsevier Inc. All rights reserved.

Gene expression profile during testicular development in patients with SRY-negative 46,XX testicular disorder of sex development.

PubMed

Mizuno, Kentaro; Kojima, Yoshiyuki; Kamisawa, Hideyuki; Moritoki, Yoshinobu; Nishio, Hidenori; Kohri, Kenjiro; Hayashi, Yutaro

2013-12-01

To elucidate alternative pathways in testicular development, we attempted to clarify the genetic characteristics of SRY-negative XX testes. We previously reported 5 cases of SRY-negative 46,XX testicular disorders of sex development and demonstrated that coordinated expression of genes such as SOX9, SOX3, and DAX1 was associated with testicular development. We performed a case-control study between the aforementioned boy with 46,XX testicular disorders of sex development and an age-matched patient with hydrocele testis (46,XY). During their consecutive surgeries, testicular biopsy specimens were obtained. Genes with differential expression compared with XY testis were identified using polymerase chain reaction (PCR)-based subtractive hybridization and sequencing. For validation of differential gene expression, real-time RT-PCR was performed using gene-specific primers. The distribution of candidate proteins in the testicular tissue was clarified by immunohistochemistry in human and rodent specimens. Moreover, in vitro inhibitory assays were performed. We identified 13 upregulated and 7 downregulated genes in XX testis. Among the candidate genes, we focused on ROCK1 (Rho-associated, coiled-coil protein kinase 1) in the upregulated gene group, because high expression in XX testis was validated by real-time RT-PCR. ROCK1 protein was detected in germ cells, Leydig cells, and Sertoli cells by immunohistochemistry. Moreover, the addition of specific ROCK1 inhibitor to Sertoli cells decreased SOX9 gene expression. On the basis of in vitro inhibitory assay, it is suggested that ROCK1 phosphorylates and activates SOX9 in Sertoli cells. Testes formation might be initiated by an alternative signaling pathway attributed to ROCK1, not SRY, activation in XX testes. Copyright © 2013 Elsevier Inc. All rights reserved.

Interaction between DMRT1 function and genetic background modulates signaling and pluripotency to control tumor susceptibility in the fetal germ line

PubMed Central

Krentz, Anthony D.; Murphy, Mark W.; Zhang, Teng; Sarver, Aaron L.; Jain, Sanjay; Griswold, Michael D.; Bardwell, Vivian J.; Zarkower, David

2013-01-01

Dmrt1(doublesex and mab-3 related transcription factor 1) is a regulator of testis development in vertebrates that has been implicated in testicular germ cell tumors of mouse and human. In the fetal mouse testis Dmrt1 regulates germ cell pluripotency in a strain-dependent manner. Loss of Dmrt1 in 129Sv strain mice results in a >90% incidence of testicular teratomas, tumors consisting cells of multiple germ layers; by contrast, these tumors have never been observed in Dmrt1 mutants of C57BL/6J (B6) or mixed genetic backgrounds. To further investigate the interaction between Dmrt1 and genetic background we compared mRNA expression in wild type and Dmrt1 mutant fetal testes of 129Sv and B6 mice at embryonic day 15.5 (E15.5), prior to overt tumorigenesis. Loss of Dmrt1 caused misexpression of overlapping but distinct sets of mRNAs in the two strains. The mRNAs that were selectively affected included some that changed expression only in one strain or the other and some that changed in both strains but to a greater degree in one versus the other. In particular, loss of Dmrt1 in 129Sv testes caused a more severe failure to silence regulators of pluripotency than in B6 testes. A number of genes misregulated in 129Sv mutant testes also are misregulated in human testicular germ cell tumors (TGCTs), suggesting similar etiology between germ cell tumors in mouse and man. Expression profiling showed that DMRT1 also regulates pluripotency genes in the fetal ovary, although Dmrt1 mutant females do not develop teratomas. Pathway analysis indicated disruption of several signaling pathways in Dmrt1 mutant fetal testes, including Nodal, Notch, and GDNF. We used a Nanos3-cre knock-in allele to perform conditional gene targeting, testing the GDNF coreceptors Gfra1 and Ret for effects on teratoma susceptibility. Conditional deletion of Gfra1 but not Ret in fetal germ cells of animals outcrossed to 129Sv caused a modest but significant elevation in tumor incidence. Despite some variability in genetic background in these crosses, this result is consistent with previous genetic mapping of teratoma susceptibility loci to the region containing Gfra1. Using Nanos3-cre we also uncovered a strong genetic interaction between Dmrt1 and Nanos3, suggesting parallel functions for these two genes in fetal germ cells. Finally, we used chromatin immunoprecipitation (ChIP-seq) analysis to identify a number of potentially direct DMRT1 targets. This analysis suggested that DMRT1 controls pluripotency via transcriptional repression of Esrrb, Nr5a2/Lrh1, and Sox2. Given the strong evidence for involvement of DMRT1 in human TGCT, the downstream genes and pathways identified in this study provide potentially useful candidates for roles in the human disease. PMID:23473982

Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Qiuling; Ma, Qi; Shehadeh, Lina A.

Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressedmore » in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.« less

Sperm flagella protein components: Human meichroacidin constructed by the membrane occupation and recognition nexus motif

PubMed Central

MATSUOKA, YASUHIRO; NISHIMURA, HIROMI; NUMAZAWA, KAHORI; TSUCHIDA, JUNJI; MIYAGAWA, YASUSHI; TSUJIMURA, AKIRA; MATSUMIYA, KIYOMI; OKUYAMA, AKIHIKO; NISHIMUNE, YOSHITAKE

2005-01-01

Background and Aims:   In a previous study, the authors of the present study cloned mouse meichroacidin (MCA), which is expressed in stages of spermatogenesis from pachytene spermatocytes through round spermatid germ cells. MCA protein contains the membrane occupation and recognition nexus (MORN) motif and localizes to a male meiotic metaphase chromosome. Recently, a MCA homolog of carp (Cyprinus carpio), MORN motif‐containing sperm‐specific axonemal protein (MSAP), was reportedly identified and localized in sperm flagella. Present knowledge of human spermiogenesis requires the identification of proteins in human sperm. The present study identified the human orthologue of MCA. Methods:   Colony hybridization using a human testis plasmid cDNA library was carried out to clone human MCA (h‐MCA) cDNA. Northern blot, Western blot, and immunohistochemical analyses were carried out. Results:   h‐MCA was found to be specifically expressed in the testes. The h‐MCA amino acid sequence shared 79.8% identity with mouse MCA and contained MORN motifs. h‐MCA localized in the sperm flagellum and basal body, as does MSAP in carp. Conclusion:   Expression and localization analyses showed that h‐MCA is a component of the sperm flagellum and basal body and might play an important role in the development of the sperm flagellum in humans. (Reprod Med Biol 2005; 4: 213–219) PMID:29699225

Inhibitory effect of tributyltin on expression of steroidogenic enzymes in mouse testis.

PubMed

Kim, Suel-Kee; Kim, Jong-Hoon; Han, Jung Ho; Yoon, Yong-Dal

2008-01-01

Tributyltin (TBT) is known to disrupt the development of reproductive organs, thereby reducing fertility. The aim of this study was to evaluate the acute toxicity of TBT on the testicular development and steroid hormone production. Immature (3-week-old) male mice were given a single administration of 25, 50, or 100 mg/kg of TBT by oral gavage. Lumen formation in seminiferous tubule was remarkably delayed, and the number of apoptotic germ cells found inside the tubules was increased in the TBT-exposed animals, whereas no apoptotic signal was observed in interstitial Leydig cells. Reduced serum testosterone concentration and down-regulated expressions of the mRNAs for cholesterol side-chain cleavage enzyme (P450scc), 17alpha -hydroxylase/C(17-20) lyase (P450(17alpha)), 3beta -hydroxysteroid-dehydrogenase (3beta -HSD), and 17beta -hydroxysteroid-dehydrogenase (17beta -HSD) were also observed after TBT exposure. Altogether, these findings demonstrate that exposure to TBT is associated with induced apoptosis of testicular germ cells and inhibition of steroidogenesis by reduction in the expression of steroidogenic enzymes in interstitial Leydig cells. These adverse effects of TBT would cause serious defects in testicular development and function.

Human brain factor 1, a new member of the fork head gene family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, D.B.; Wiese, S.; Burfeind, P.

1994-06-01

Analysis of cDNA clones that cross-hybridized with the fork head domain of the rat HNF-3 gene family revealed 10 cDNAs from human fetal brain and human testis cDNA libraries containing this highly conserved DNA-binding domain. Three of these cDNAs (HFK1, HFK2, and HFK3) were further analyzed. The cDNA HFK1 has a length of 2557 nucleotides and shows strong homology at the nucleotide level (91.2%) to brain factor 1 (BF-1) from rat. The HFK1 cDNA codes for a putative 476 amino acid protein. The homology to BF-1 from rat in the coding region at the amino acid level is 87.5%. Themore » fork head homologous region includes 111 amino acids starting at amino acid 160 and has a 97.5% homology to BF-1. Southern hybridization revealed that HFK1 is highly conserved among mammalian species and possibly birds. Northern analysis with total RNA from human tissues and poly(A)-rich RNA from mouse revealed a 3.2-kb transcript that is present in human and mouse fetal brain and in adult mouse brain. In situ hybridization with sections of mouse embryo and human fetal brain reveals that HFK1 expression is restricted to the neuronal cells in the telencepthalon, with strong expression being observed in the developing dentate gyrus and hippocampus. HFK1 was chromosomally localized by in situ hybridization to 14q12. The cDNA clones HFK2 and HFK3 were analyzed by restriction analysis and sequencing. HFK2 and HFK3 were found to be closely related but different from HFK1. Therefore, it would appear that HFK1, HFK2, HFK3, and BF-1 form a new fork head related subfamily. 33 refs., 6 figs.« less

Hydroxysteroid dehydrogenase HSD1L is localised to the pituitary–gonadal axis of primates

PubMed Central

Bird, A Daniel; Greatorex, Spencer; Reser, David; Lavery, Gareth G

2017-01-01

Steroid hormones play clinically important and specific regulatory roles in the development, growth, metabolism, reproduction and brain function in human. The type 1 and 2 11-beta hydroxysteroid dehydrogenase enzymes (11β-HSD1 and 2) have key roles in the pre-receptor modification of glucocorticoids allowing aldosterone regulation of blood pressure, control of systemic fluid and electrolyte homeostasis and modulation of integrated metabolism and brain function. Although the activity and function of 11β-HSDs is thought to be understood, there exists an open reading frame for a distinct 11βHSD-like gene; HSD11B1L, which is present in human, non-human primate, sheep, pig and many other higher organisms, whereas an orthologue is absent in the genomes of mouse, rat and rabbit. We have now characterised this novel HSD11B1L gene as encoded by 9 exons and analysis of EST library transcripts indicated the use of two alternate ATG start sites in exons 2 and 3, and alternate splicing in exon 9. Relatively strong HSD11B1L gene expression was detected in human, non-human primate and sheep tissue samples from the brain, ovary and testis. Analysis in non-human primates and sheep by immunohistochemistry localised HSD11B1L protein to the cytoplasm of ovarian granulosa cells, testis Leydig cells, and gonadatroph cells in the anterior pituitary. Intracellular localisation analysis in transfected human HEK293 cells showed HSD1L protein within the endoplasmic reticulum and sequence analysis suggests that similar to 11βHSD1 it is membrane bound. The endogenous substrate of this third HSD enzyme remains elusive with localisation and expression data suggesting a reproductive hormone as a likely substrate. PMID:28871060

Sorafenib Action in Hepatitis B Virus X-Activated Oncogenic Androgen Pathway in Liver through SHP-1.

PubMed

Wang, Sheng-Han; Yeh, Shiou-Hwei; Shiau, Chung-Wai; Chen, Kuen-Feng; Lin, Wei-Hsiang; Tsai, Ting-Fen; Teng, Yuan-Chi; Chen, Ding-Shinn; Chen, Pei-Jer

2015-10-01

Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) shows a higher incidence in men, mainly because of hepatitis B X (HBx)-mediated enhancement of androgen receptor (AR) activity. We aimed to examine this pathway in hepatocarcinogenesis and to identify drug(s) specifically blocking this carcinogenic event in the liver. HBx transgenic mice that spontaneously develop HCC (n = 28-34 per group) were used, either by knockout of hepatic AR or by castration. Efficacy of several HCC-targeted drugs in suppressing HBx-induced AR activity was evaluated, and cellular factors mediating suppression were investigated in cultured cells. Tissue specificity of the candidate drug was validated using mouse tissues. Data were analyzed with Chi-square and Student's t tests. All statistical tests were two-sided. The androgen pathway was shown to be important in early stage hepatocarcinogenesis of HBx transgenic mice. The tumor incidence was decreased from 80% to 32% by AR knockout (P < .001) and from 90% to 25% by early castration (P < .001). Sorafenib markedly inhibited the HBx-enhanced AR activity through activating the SHP-1 phosphatase, which antagonized the activation of Akt/GSK3β and c-Src by HBx. Moreover, SHP-1 protein level was much higher in the liver than in testis, which enabled sorafenib to inhibit aberrant AR activity in the HBx-expressing liver, while not affecting the physiological AR function in normal liver or testis. The androgen pathway may be a druggable target for the chemoprevention of HBV-related HCC, and sorafenib might be used as a tissue- and disease-specific regimen for the chemoprevention of HBV-related HCC. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Occurrence and degree of intersex (testis-ova) in darters (Etheostoma SPP.) across an urban gradient in the Grand River, Ontario, Canada.

PubMed

Tanna, Rajiv N; Tetreault, Gerald R; Bennett, Charles J; Smith, Brendan M; Bragg, Leslie M; Oakes, Ken D; McMaster, Mark E; Servos, Mark R

2013-09-01

The variability and extent of the intersex condition (oocytes in testes, or testis-ova) was documented in fish along an urban gradient in the Grand River, Ontario, Canada, that included major wastewater treatment plant outfalls. A method for rapid enumeration of testis-ova was developed and applied that increased the capacity to quantify intersex prevalence and severity. Male rainbow darters (Etheostoma caeruleum) sampled downstream of the first major wastewater outfall (Waterloo) had a significant increase, relative to 4 upstream reference sites, in the mean proportion of fish with at least 1 testis-oocyte per lobe of testes (9-20% proportion with ≤ 1 testis-oocyte/lobe vs 32-53% and >1.4 testis-oocyte/lobe). A much higher mean incidence of intersex proportion and degree was observed immediately downstream of the second wastewater outfall (Kitchener; 73-100% and 8-70 testis-oocyte/lobe); but only 6.3 km downstream of the Kitchener outfall, the occurrence of intersex dropped to those of the reference sites. In contrast, downstream of a tertiary treated wastewater outfall on a small tributary, intersex was similar to reference sites. Estrogenicity, measured using a yeast estrogen screen, followed a similar pattern, increasing from 0.81 ± 0.02 ng/L 17b-estradiol equivalents (EEq) (Guelph), to 4.32 ± 0.07 ng/L (Waterloo), and 16.99 ± 0.40 ng/L (Kitchener). Female rainbow darter downstream of the Kitchener outfall showed significant decreases in gonadosomatic index and liver somatic index, and increases in condition factor (k) relative to corresponding reference sites. The prevalence of intersex and alterations in somatic indices suggest that exposure to municipal wastewater effluent discharges can impact endocrine function, energy use, and energy storage in wild fish. Copyright © 2013 SETAC.

Identification of an HLA-A24-restricted OY-TES-1 epitope recognized by cytotoxic T-cells.

PubMed

Okumura, Hideo; Noguchi, Yuji; Uenaka, Akiko; Aji, Toshiki; Ono, Toshiro; Nakagawa, Kazuhiko; Aoe, Motoi; Shimizu, Nobuyoshi; Nakayama, Eiichi

2005-01-01

OY-TES-1 was identified as a human homologue of the mouse, guinea pig, and pig proacrosin binding protein sp32 precursor. Differential expression levels of OY-TES-1 mRNA between testis and other normal tissues, and its expression in cancers indicated that OY-TES-1 would be classified as a cancer/testis antigen and considered to be a candidate of target antigen for cancer immunotherapy. In this study, we showed identification of HLA-A24-binding OY-TES-1 peptide, TES(401-409) (KTPFVSPLL) recognized by CD8 T-cells. Purified CD8 T-cells from healthy donors stimulated in vitro with the peptide-pulsed autologous DC and PBMC produced IFNgamma in response to the peptide-pulsed PBMC and showed cytotoxicity against the peptide-pulsed autologous EBV-B specifically. Furthermore, cytotoxicity was also observed against an OY-TES-1 mRNA-expressing tumor line, LK79. The retention time of the fraction in HPLC of the acid eluate from LK79 cells that showed positive sensitization against autologous EBV-B cells in recognition by CD8 CTL was the same as that of the fraction of the TES(401-409) peptide itself, suggesting that the TES(401-409) was a naturally processed peptide on LK79.

The NOTCH Ligand JAG1 Regulates GDNF Expression in Sertoli Cells

PubMed Central

Garcia, Thomas X.; Parekh, Parag; Gandhi, Pooja; Sinha, Krishna

2017-01-01

In the seminiferous epithelium of the testis, Sertoli cells are key niche cells directing proliferation and differentiation of spermatogonial stem cells (SSCs) into spermatozoa. Sertoli cells produce glial cell line-derived neurotrophic factor (GDNF), which is essential for SSC self-renewal and progenitor expansion. While the role of GDNF in the testis stem cell niche is established, little is known about how this factor is regulated. Our previous studies on NOTCH activity in Sertoli cells demonstrated a role of this pathway in limiting stem/progenitor cell numbers, thus ultimately downregulating sperm cell output. In this study we demonstrate through a double-mutant mouse model that NOTCH signaling in Sertoli cells functions solely through the canonical pathway. Further, we demonstrate through Dual luciferase assay and chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) analysis that the NOTCH targets HES1 and HEY1, which are transcriptional repressors, directly downregulate GDNF expression by binding to the Gdnf promoter, thus antagonizing the effects of FSH/cAMP. Finally, we demonstrate that testicular stem/progenitors cells are activating NOTCH signaling in Sertoli cells in vivo and in vitro through the NOTCH ligand JAG1 at their surface, indicating that these cells may ensure their own homeostasis through negative feedback regulation. PMID:28051360

TCF21 is related to testis growth and development in broiler chickens.

PubMed

Zhang, Hui; Na, Wei; Zhang, Hong-Li; Wang, Ning; Du, Zhi-Qiang; Wang, Shou-Zhi; Wang, Zhi-Peng; Zhang, Zhiwu; Li, Hui

2017-02-24

Large amounts of fat deposition often lead to loss of reproductive efficiency in humans and animals. We used broiler chickens as a model species to conduct a two-directional selection for and against abdominal fat over 19 generations, which resulted in a lean and a fat line. Direct selection for abdominal fat content also indirectly resulted in significant differences (P < 0.05) in testis weight (TeW) and in TeW as a percentage of total body weight (TeP) between the lean and fat lines. A total of 475 individuals from the generation 11 (G 11 ) were genotyped. Genome-wide association studies revealed two regions on chicken chromosomes 3 and 10 that were associated with TeW and TeP. Forty G 16 individuals (20 from each line), were further profiled by focusing on these two chromosomal regions, to identify candidate genes with functions that may be potentially related to testis growth and development. Of the nine candidate genes identified with database mining, a significant association was confirmed for one gene, TCF21, based on mRNA expression analysis. Gene expression analysis of the TCF21 gene was conducted again across 30 G 19 individuals (15 individuals from each line) and the results confirmed the findings on the G 16 animals. This study revealed that the TCF21 gene is related to testis growth and development in male broilers. This finding will be useful to guide future studies to understand the genetic mechanisms that underlie reproductive efficiency.

Building the mammalian testis: origins, differentiation, and assembly of the component cell populations

PubMed Central

Svingen, Terje; Koopman, Peter

2013-01-01

Development of testes in the mammalian embryo requires the formation and assembly of several cell types that allow these organs to achieve their roles in male reproduction and endocrine regulation. Testis development is unusual in that several cell types such as Sertoli, Leydig, and spermatogonial cells arise from bipotential precursors present in the precursor tissue, the genital ridge. These cell types do not differentiate independently but depend on signals from Sertoli cells that differentiate under the influence of transcription factors SRY and SOX9. While these steps are becoming better understood, the origins and roles of many testicular cell types and structures—including peritubular myoid cells, the tunica albuginea, the arterial and venous blood vasculature, lymphatic vessels, macrophages, and nerve cells—have remained unclear. This review synthesizes current knowledge of how the architecture of the testis unfolds and highlights the questions that remain to be explored, thus providing a roadmap for future studies that may help illuminate the causes of XY disorders of sex development, infertility, and testicular cancers. PMID:24240231

Application of testis germ cell transplantation in breeding systems of food producing species: a review.

PubMed

Herrid, Muren; McFarlane, James R

2013-01-01

A major benefit of advanced reproduction technologies (ART) in animal breeding is the ability to produce more progeny per individual parent. This is particularly useful with animals of high genetic merit. Testis germ cell transplantation (TGCT) is emerging as a novel reproductive technology with application in animal breeding systems, including the potential for use as an alternative to artificial insemination (AI), an alternative to transgenesis, part of an approach to reducing generation intervals, or an approach toward development of interspecies hybrids. There is one major difference in TGCT between rodents and some other species associated with immunotolerance in heterologous transplantation. In particular, livestock and aquatic species do not require an immunesuppression procedure to allow donor cell survival in recipient testis. Testicular stem cells from a genetically elite individual transplanted into others can develop and produce a surrogate male-an animal that produces the functional sperm of the original individual. Spermatozoa produced from testis stem cells are the only cells in the body of males that can transmit genetic information to the offspring. The isolation and genetic manipulation of testis stem cells prior to transplantation has been shown to create transgenic animals. However, the current success rate of the transplantation procedure in livestock and aquatic species is low, with a corresponding small proportion of donor spermatozoa in the recipient's semen. The propagation of donor cells in culture and preparation of recipient animals are the two main factors that limit the commercial application of this technique. The current paper reviews and compares recent progress and examines the difficulties of TGCT in both livestock and aquatic species, thereby providing new insights into the application of TGCT in food producing animals.

Homozygous carnitine palmitoyltransferase 1a (liver isoform) deficiency is lethal in the mouse.

PubMed

Nyman, Lara R; Cox, Keith B; Hoppel, Charles L; Kerner, Janos; Barnoski, Barry L; Hamm, Doug A; Tian, Liqun; Schoeb, Trenton R; Wood, Philip A

2005-01-01

To better understand carnitine palmitoyltransferase 1a (liver isoform, gene=Cpt-1a, protein=CPT-1a) deficiency in human disease, we developed a gene knockout mouse model. We used a replacement gene targeting strategy in ES cells that resulted in the deletion of exons 11-18, thus producing a null allele. Homozygous deficient mice (CPT-1a -/-) were not viable. There were no CPT-1a -/- pups, embryos or fetuses detected from day 10 of gestation to term. FISH analysis demonstrated targeting vector recombination at the expected single locus on chromosome 19. The inheritance pattern from heterozygous matings was skewed in both C57BL/6NTac, 129S6/SvEvTac (B6;129 mixed) and 129S6/SvEvTac (129 coisogenic) genetic backgrounds biased toward CPT-1a +/- mice (>80%). There was no sex preference with regard to germ-line transmission of the mutant allele. CPT-1a +/- mice had decreased Cpt-1a mRNA expression in liver, heart, brain, testis, kidney, and white fat. This resulted in 54.7% CPT-1 activity in liver from CPT-1a +/- males but no significant difference in females as compared to CPT-1a +/+ controls. CPT-1a +/- mice showed no fatty change in liver and were cold tolerant. Fasting free fatty acid concentrations were significantly elevated, while blood glucose concentrations were significantly lower in 6-week-old CPT-1a +/- mice compared to controls. Although the homozygous mutants were not viable, we did find some aspects of haploinsufficiency in the CPT-1a +/- mutants, which will make them an important mouse model for studying the role of CPT-1a in human disease.

[Sclerotherapy with 3% polidocanol for hydrocele testis].

PubMed

Mizoguchi, H; Imagawa, M; Fukunaga, Y; Nomura, Y; Kubota, M; Okita, J

1995-12-01

We studied the clinical efficacy of sclerotherapy with injection of 3% polidocanol for hydrocele testis. From July, 1992 to March, 1995 sclerotherapy with single injection of polidocanol was performed for 11 patients with 12 hydrocele testis on an outpatient basis. We instilled 3 or 5ml of 3% polidocanol after complete removal of fluid in the hydrocele testis. Complete disappearance on ultrasonography was observed in 75% of the hydrocele testis 6 months after this sclerotherapy. There was neither pain during instillation of 3% polidocanol nor any other complication. Two patients with fluid reaccumulation underwent hydrocelectomy 16 and 6 months after sclerotherapy, respectively. This procedure seems to be a safe and useful technique as primary treatment for hydrocele testis.

A Critical Point of Male Gonad Development: Neuroendocrine Correlates of Accelerated Testicular Growth in Rats during Early Life

PubMed Central

Dygalo, Nikolay N.; Shemenkova, Tatjana V.; Kalinina, Tatjana S.; Shishkina, Galina T.

2014-01-01

Testis growth during early life is important for future male fertility and shows acceleration during the first months of life in humans. This acceleration coincides with the peak in gonadotropic hormones in the blood, while the role of hypothalamic factors remains vague. Using neonatal rats to assess this issue, we found that day 9 of life is likely critical for testis development in rats. Before this day, testicular growth was proportional to body weight gain, but after that the testes showed accelerated growth. Hypothalamic kisspeptin and its receptor mRNA levels begin to elevate 2 days later, at day 11. A significant increase in the mRNA levels for gonadotropin-releasing hormone (GnRH) receptors in the hypothalamus between days 5 and 7 was followed by a 3-fold decrease in GnRH mRNA levels in this brain region during the next 2 days. Starting from day 9, hypothalamic GnRH mRNA levels increased significantly and positively correlated with accelerated testicular growth. Triptorelin, an agonist of GnRH, at a dose that had no effect on testicular growth during “proportional” period, increased testis weights during the period of accelerated growth. The insensitivity of testicular growth to GnRH during “proportional” period was supported by inability of a 2.5-fold siRNA knockdown of GnRH expression in the hypothalamus of the 7-day-old animals to produce any effect on their testis weights. GnRH receptor blockade with cetrorelix was also without effect on testis weights during “proportional” period but the same doses of this GnRH antagonist significantly inhibited “accelerated” testicular growth. GnRH receptor mRNA levels in the pituitary as well as plasma LH concentrations were higher during “accelerated” period of testicular growth than during “proportional” period. In general, our data defined two distinct periods in rat testicular development that are primarily characterized by different responses to GnRH signaling. PMID:24695464

The relationship between the testis and tunica vaginalis changes with age.

PubMed

Lopez-Marambio, Francisco A; Hutson, John M

2015-12-01

Anatomy of the testis and tunica vaginalis (TV) is taught to pediatric surgeons from adult postmortem material. Textbooks describe the testis as 'behind' the TV, but at pediatric orchidopexy it appears to be inside the TV. We aimed to study whether testis and TV anatomy changes with age. After ethical approval, postmortem photographs and measurements of testis length, width, and mesenteric attachment length (mm) in 37 adults (22-92years), one infant (4/12), and one fetus (19/52) were compared with intraoperative orchidopexies (x6) after opening TV (n=4; 7/12-14years). Testis length, area and perimeter and ratios for mesentery attachment were plotted against age. The fetal and pediatric testes were intraperitoneal with a mesentery (mesorchium), but after 50years secondary adhesions between TV and testis obliterated the mesorchium, so in advanced age the testis appeared to be behind the TV. These results show that in childhood testes were 'intraperitoneal', but after 50years of age the TV progressively shrinks and adheres to the testis, making it appear to be behind the TV. This difference between anatomical texts and childhood anatomy suggests that pediatric surgery may need anatomy texts that specifically highlight age differences. Copyright © 2015 Elsevier Inc. All rights reserved.

A time-course study of long term over-expression of ARR19 in mice

PubMed Central

Qamar, Imteyaz; Ahmad, Mohammad Faiz; Narayanasamy, Arul

2015-01-01

A leucine-rich protein, ARR19 (androgen receptor corepressor-19 kDa), is highly expressed in male reproductive organs and moderately in others. Previously, we have reported that ARR19 is differentially expressed in adult Leydig cells during the testis development and inhibits steroidogenesis by reducing the expression of steroidogenic enzymes. Whereas in prostate, ARR19 represses the transcriptional activity of AR (androgen receptor), it is important for male sexual differentiation and maturation in prostate and epididymis, through the recruitment of HDAC4. In this study we show that long term adenovirus mediated overexpression of ARR19 in mice testis has the potential of inhibiting the differentiation of testicular and prostatic cells by reducing the size of testis and prostate but has no effect on the growth of seminal vesicles. Further, it reduces the level of progesterone and testosterone by reducing the steroidogenic enzymes such as 3HSD, P450c17 and StAR. This is the first study reporting a time-course analysis of the implications of long term overexpression of ARR19 in mice testis and its effect on other organs such as prostate and seminal vesicles. Taken together, these results suggest that ARR19 may play an important role in the differentiation of male reproductive organs such as testis and prostate. PMID:26260329

Histological observation of germ cell development and discovery of spermatophores in ovoviviparous black rockfish ( Sebastes schlegeli Hilgendorf) in reproductive season

NASA Astrophysics Data System (ADS)

Feng, Junrong; Liu, Liming; Jiang, Haibin; Wang, Maojian; Du, Rongbin

2014-10-01

Black rockfish ( Sebastes schlegeli) is an important species for culture; however, its reproductive characteristics have not been fully documented. In this study, we investigated the morphology and developmental process of germ cells in this ovoviviparous rockfish in reproductive season (October 2011-November 2012) with histological methods. We found that the gonad of mature fish showed notable seasonal changes in developmental characteristics and morphological structure. The sperm cells matured during a period lasting from October to December, significantly earlier than the oocytes did. A large number of spermatozoa and other cells occurred in testis at different developmental stages. Vitellogenesis in oocytes began in October, and gestation appeared in April next year. Spermatophores were discovered for the first time in Sebastes, which assembled in testis, main sperm duct, oviduct and genital tract, as well as ovarian cavity in October and April. These organs may serve either as production or hiding places for spermatophores and spermatozoa which were stored and transported in form of spermatophores. Testicular degeneration started from the distal part of testis in April, with spermatophores assembled in degenerating testis and waiting for transportation. The copulation probably lasted for a long period, during which the spermatozoa were discharged in batches as spermatophores. These spermatophores were coated with sticky materials secreted from the interstitial areas of testis and the main sperm duct, then transported into ovary.

Dmrt1 Expression Is Regulated by Follicle-Stimulating Hormone and Phorbol Esters in Postnatal Sertoli Cells*

PubMed Central

CHEN, JIANG KAI; HECKERT, LESLIE L.

2006-01-01

Dmrt1 is a recently described gene that is expressed exclusively in the testis and is required for postnatal testis differentiation. Here we describe the expression of Dmrt1 in postnatal rat testis and Sertoli cells. RNase protection analysis was used to examine Dmrt1 messenger RNA (mRNA) levels in intact testis during postnatal development and in primary cultures of Sertoli cells under various culture conditions. We show that Dmrt1 mRNA levels rise significantly beginning approximately 10 days after birth and remain elevated until after the third postnatal week. Thereafter, mRNA levels drop coincident with the proliferation of germ cells in the testis. In freshly isolated Sertoli cells, Dmrt1 mRNA levels were robust but decreased significantly when the cells were placed in culture for 24 h. Treatment of Sertoli cells with either FSH or 8-bromo-cAMP resulted in a significant rise in Dmrt1 mRNA levels. This cAMP response was sensitive to treatment with the transcriptional inhibitor actinomycin D but not to the translational inhibitor cycloheximide. The cAMP-dependent rise in Dmrt1 mRNA also required activation of protein kinase A, as mRNA induction was sensitive to the inhibitor H89. Studies also show that Dmrt1 expression was inhibited by phorbol esters (PMA) but only modestly effected by serum. PMID:11181532

SLC52A3, A Brown–Vialetto–van Laere syndrome candidate gene is essential for mouse development, but dispensable for motor neuron differentiation

PubMed Central

Intoh, Atsushi; Suzuki, Naoki; Koszka, Kathryn; Eggan, Kevin

2016-01-01

Riboflavin, also known as vitamin B2, is essential for cellular reduction-oxidation reactions, but is not readily synthesized by mammalian cells. It has been proposed that riboflavin absorption occurs through solute carrier family 52 members (SLC52) A1, A2 and A3. These transporters are also candidate genes for the childhood onset-neural degenerative syndrome Brown–Vialetto–Van Laere (BVVL). Although riboflavin is an essential nutrient, why mutations in its transporters result in a neural cell-specific disorder remains unclear. Here, we provide evidence that Slc52a3 is the mouse ortholog of SLC52A3 and show that Slc52a3 deficiency results in early embryonic lethality. Loss of mutant embryos was associated with both defects in placental formation and increased rates of apoptosis in embryonic cells. In contrast, Slc52a3 −/− embryonic stem cell lines could be readily established and differentiated into motor neurons, suggesting that this transporter is dispensable for neural differentiation and short-term maintenance. Consistent with this finding, examination of Slc52a3 gene products in adult tissues revealed expression in the testis and intestine but little or none in the brain and spinal cord. Our results suggest that BVVL patients with SCL52A3 mutations may be good candidates for riboflavin replacement therapy and suggests that either the mutations these individuals carry are hypomorphic, or that in these cases alternative transporters act during human embryogenesis to allow full-term development. PMID:26976849

Comparative modular analysis of gene expression in vertebrate organs.

PubMed

Piasecka, Barbara; Kutalik, Zoltán; Roux, Julien; Bergmann, Sven; Robinson-Rechavi, Marc

2012-03-29

The degree of conservation of gene expression between homologous organs largely remains an open question. Several recent studies reported some evidence in favor of such conservation. Most studies compute organs' similarity across all orthologous genes, whereas the expression level of many genes are not informative about organ specificity. Here, we use a modularization algorithm to overcome this limitation through the identification of inter-species co-modules of organs and genes. We identify such co-modules using mouse and human microarray expression data. They are functionally coherent both in terms of genes and of organs from both organisms. We show that a large proportion of genes belonging to the same co-module are orthologous between mouse and human. Moreover, their zebrafish orthologs also tend to be expressed in the corresponding homologous organs. Notable exceptions to the general pattern of conservation are the testis and the olfactory bulb. Interestingly, some co-modules consist of single organs, while others combine several functionally related organs. For instance, amygdala, cerebral cortex, hypothalamus and spinal cord form a clearly discernible unit of expression, both in mouse and human. Our study provides a new framework for comparative analysis which will be applicable also to other sets of large-scale phenotypic data collected across different species.

A single, mild, transient scrotal heat stress causes DNA damage, subfertility and impairs formation of blastocysts in mice.

PubMed

Paul, Catriona; Murray, Alison A; Spears, Norah; Saunders, Philippa T K

2008-07-01

Infertility represents a major clinical problem and 50% of cases are attributable to the male partner. Testicular function is temperature dependent, and in both man and mouse the position of the testes in the scrotum ensures that they are kept at between 2 and 8 degrees C below core body temperature. We used a mouse model to investigate the impact of a single, transient, mild, scrotal heat stress (38, 40 or 42 degrees C for 30 min) on testicular function, sperm DNA integrity and embryo survival. We detected temperature-dependent changes in testicular architecture, number of apoptotic cells and a significant reduction in testis weight 7 and 14 days after heat stress at 42 degrees C. We report for the first time that DNA strand breaks (gamma-H2AX-positive foci) were present in spermatocytes recovered from testes subjected to 40 or 42 degrees C. Fertility of heat-stressed males was tested 23-28 d after treatment (sperm at this time would have been spermatocytes at time of heating). Paternal heat stress at 42 degrees C resulted in reduced pregnancy rate, placental weight and litter size; pregnancies from the 40 degrees C group had increased resorptions at e14.5. Abnormalities in embryonic development were detected at e3.5 and in vitro fertilisation with sperm recovered 16 h or 23 d after scrotal stress at 42 degrees C revealed a block in development between the 4-cell and blastocyst stages. This study has provided evidence of temperature-dependent effects on germ cell DNA integrity and highlighted the importance of an intact paternal genome for normal embryo development.

Male-specific expression of Sox9 during gonad development of crocodile and mouse is mediated by alternative splicing of its proline-glutamine-alanine rich domain.

PubMed

Agrawal, Raman; Wessely, Oliver; Anand, Amit; Singh, Lalji; Aggarwal, Ramesh K

2009-08-01

The initial trigger for sexual differentiation is regulated by multiple ways during embryonic development. In vertebrates, chromosome-based mechanisms generally known as genetic sex determination are prevalent; however, some species, such as many reptilians, display temperature-dependent sex determination. The Sry-related transcription factor, Sox9, which is expressed by an evolutionary conserved gene, has been shown to be a key player in the process of sex determination. In the present study, we report the identification and expression of crocodile homolog of Sox9 (cpSox9) from the Indian Mugger, Crocodylus palustris. We show that cpSox9 undergoes extensive alternative splicing around the proline-glutamine-alanine rich transactivation domain that results in cpSox9 variants with presumably impaired or reduced transactivation potential. The multiple isoforms were also detected in various embryonic tissues, with some of them displaying a differential expression profile. With respect to sex differentiation, a putative unspliced full-length cpSox9 could be detected only in the genital ridge-adrenal-mesonephros complex of male, but not female embryos during the temperature-sensitive period. Importantly, we further show that this phenomenon was not restricted to the temperature-dependent sex determination species C. palustris, but was also observed in the mouse, a species exhibiting genetic sex determination. Thus, the present study describes, for the first time, a complete coding locus of Sox9 homolog from a temperature-dependent sex determination species. More importantly, we demonstrate an evolutionarily conserved role of alternative splicing resulting in transcriptional diversity and male-sex specific expression of Sox9 during testis development in vertebrates (i.e. irrespective of their underlying sex-determination mechanisms).

A long-term high-fat diet changes iron distribution in the body, increasing iron accumulation specifically in the mouse spleen.

PubMed

Yamano, Noriko; Ikeda, Yasumasa; Sakama, Minoru; Izawa-Ishizawa, Yuki; Kihira, Yoshitaka; Ishizawa, Keisuke; Miyamoto, Licht; Tomita, Shuhei; Tsuchiya, Koichiro; Tamaki, Toshiaki

2015-01-01

Although iron is an essential trace metal, its presence in excess causes oxidative stress in the human body. Recent studies have indicated that iron storage is a risk factor for type 2 diabetes mellitus. Dietary iron restriction or iron chelation ameliorates symptoms of type 2 diabetes in mouse models. However, whether iron content in the body changes with the development of diabetes is unknown. Here, we investigated the dynamics of iron accumulation and changes in iron absorption-related genes in mice that developed obesity and diabetes by consuming a high-fat diet (HFD-fed mice). HFD-fed mice (18-20 wk) were compared with control mice for hematologic features, serum ferritin levels, and iron contents in the gastrocnemius muscle, heart, epididymal fat, testis, liver, duodenum, and spleen. In addition, the spleen was examined histologically. Iron absorption-related gene expression in the liver and duodenum was also examined. Hemoglobin and serum ferritin levels were increased in HFD-fed mice. The HFD-fed mice showed iron accumulation in the spleen, but not in the heart or liver. Increased percentages of the splenic red pulp and macrophages were observed in HFD-fed mice and iron accumulation in the spleen was found mainly in the splenic red pulp. The HFD-fed mice also showed decreased iron content in the duodenum. The mRNA expression of divalent metal transporter-1 (DMT-1), an iron absorption-related gene, was elevated in the duodenum of HFD-fed mice. These results indicate that iron accumulation (specifically accumulation in the spleen) is enhanced by the development of type 2 diabetes induced by HFD.

Effect of Letrozole, a selective aromatase inhibitor, on testicular activities in adult mice: Both in vivo and in vitro study.

PubMed

Verma, Rachna; Krishna, Amitabh

2017-01-15

The aim of present study was to evaluate the significance of estradiol (E2) in testicular activities and to find out the mechanism by which E2 regulates spermatogenesis in mice. To achieve this, both in vivo and in vitro effect of Letrozole on testis of adult mice was investigated. Letrozole-induced changes in testicular histology, cell proliferation (proliferating cell nuclear antigen; PCNA), cell survival (B cell lymphoma factor-2; Bcl2), apoptotic (cysteine-aspartic proteases; caspase-3), steroidogenic (side chain cleavage; SCC, 3β-hydroxy steroid dehydrogenase enzyme; 3β HSD, steroidogenic acute regulatory protein; StAR, aromatase and luteinizing hormone receptor; LH-R) markers, glucose level, and rate of expression of glucose transporter (GLUT) 8 and insulin receptor (IR) proteins in the testis along with changes in serum E2 and testosterone (T) levels were evaluated. Letrozole acts on testis and caused significant decrease in E2 synthesis, but increase in testosterone level and showed regressive changes in the spermatogenesis. Letrozole-induced changes in various testicular markers were compared with the changes in serum E2 level. The correlation study showed that decreased circulating E2 level may be responsible for decreased insulin receptor (IR) level in the testis. The decreased effects of insulin inhibited the glucose transport in the testis by suppressing GLUT8. The decreased level of testicular glucose may produce less lactate as energy support to developing germ cells consequently resulting in decreased cell proliferation and cell survival, but increased apoptosis. Thus, Letrozole suppresses spermatogenesis by reducing insulin sensitivity and glucose transport in the testis, but significantly increased testosterone level by promoting gonadotrophin release by decreased E2. Copyright © 2016 Elsevier Inc. All rights reserved.

Juvenile granulosa cell tumor of the testis: a bilateral and synchronous case. Should testis-sparing surgery be mandatory?

PubMed

Cosentino, Marco; Algaba, Ferran; Saldaña, Lily; Bujons, Ana; Caffaratti, Jorge; Garat, Jose M; Villavicencio, Humberto

2014-09-01

Granulosa cell tumor of the testis is an infrequent stromal cell tumor that can be distinguished into adult and juvenile, the latter being more common. Juvenile granulosa cell tumor of the testis is a rare pathologic finding, accounting for 1.2%-3.9% of prepubertal testicular tumors. It is considered as a benign stromal sex cord tumor and is usually unilateral. Although radical surgery was previously considered the treatment of choice, testis-sparing surgery is now recommended in all cases where applicable. We report a bilateral synchronous juvenile granulosa cell tumor in a 6-month-old child treated with testis-sparing surgery and provide a review of the literature. Copyright © 2014 Elsevier Inc. All rights reserved.

Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function

PubMed Central

Gibbs, Gerard M.; Orta, Gerardo; Reddy, Thulasimala; Koppers, Adam J.; Martínez-López, Pablo; Luis de la Vega-Beltràn, José; Lo, Jennifer C. Y.; Veldhuis, Nicholas; Jamsai, Duangporn; McIntyre, Peter; Darszon, Alberto; O'Bryan, Moira K.

2011-01-01

The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function. PMID:21482758

Phenotypic screening of hepatocyte nuclear factor (HNF) 4-{gamma} receptor knockout mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gerdin, Anna Karin; Surve, Vikas V.; Joensson, Marie

2006-10-20

Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-{gamma}. HNF4-{gamma} is expressed in the kidneys,more » gut, pancreas, and testis. First level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-{gamma}{sup +/+}), the HNF4-{gamma} knockout (HNF4-{gamma}{sup -/-}) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-{gamma}{sup -/-} mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test.« less

Adjudin disrupts spermatogenesis by targeting drug transporters

PubMed Central

Qian, Xiaojing; Cheng, Yan-ho; Jenardhanan, Pranitha; Mruk, Dolores D.; Mathur, Premendu P.; Xia, Weiliang; Silvestrini, Bruno; Cheng, C. Yan

2013-01-01

For non-hormonal male contraceptives that exert their effects in the testis locally instead of via the hypothalamic-pituitary-testicular axis, such as adjudin that disrupts germ cell adhesion, a major hurdle in their development is to improve their bioavailability so that they can be efficiently delivered to the seminiferous epithelium by transporting across the blood-testis barrier (BTB). If this can be done, it would widen the gap between their efficacy and general toxicity. However, Sertoli cells that constitute the BTB, peritubular myoid cells in the tunica propria, germ cells at different stages of their development, as well as endothelial cells that constitute the microvessels in the interstitium are all equipped with multiple drug transporters, most notably efflux drug transporters, such as P-glycoprotein, multidrug resistance-related protein 1 (MRP1) and breast cancer resistance protein (BCRP) that can actively prevent drugs (e.g., adjudin) from entering the seminiferous epithelium to exert their effects. Recent studies have shown that BCRP is highly expressed by endothelial cells of the microvessels in the interstitium in the testis and also peritubular myoid cells in tunica propria even though it is absent from Sertoli cells at the site of the BTB. Furthermore, BCRP is also expressed spatiotemporally by Sertoli cells and step 19 spermatids in the rat testis and stage-specifically, limiting to stage VII‒VIII of the epithelial cycle, and restricted to the apical ectoplasmic specialization [apical ES, a testis-specific F-actin-rich adherens junction (AJ)]. Interestingly, adjudin was recently shown to be capable of downregulating BCRP expression at the apical ES. In this Opinion article, we critically discuss the latest findings on BCRP; in particular, we provide some findings utilizing molecular modeling to define the interacting domains of BCRP with adjudin. Based on this information, it is hoped that the next generation of adjudin analogs to be synthesized can improve their efficacy in downregulating BCRP and perhaps other drug efflux transporters in the testis to improve their efficacy to traverse the BTB by modifying their interacting domains. PMID:23885306

Bilateral cryptorchidism in a dog with persistent cranial testis suspensory ligaments and inverted gubernacula: report of a case with implications for understanding normal and aberrant testis descent.

PubMed Central

Kersten, W; Molenaar, G J; Emmen, J M; van der Schoot, P

1996-01-01

The genital system of a dog with bilateral intra-abdominal testes is described. External virilisation was normal except for an empty scrotum. Internally there was a prostate of normal macroscopic and histological appearances and, bilaterally, a fully developed male genital tract. Testicular vasculature was normal. Cranial to each testis, there was a strong ligament lying at the free edge of the gonadal/genital mesentery and running between the cranial tip of the testis/epididymis and the area craniolateral of the ipsilateral kidney. It was impossible to push the testes into the inguinal canal because of this strong ligament. Caudal to each testis, there was an elongated whitish structure between the caudal pole of the epididymis and the area of the internal inguinal ring. On closer inspection this structure appeared to be the inverted and elongated processus vaginalis sac. There was a minor ligament at the free border of the inguinal fold of the genital mesentery between the tip of this inverted processus vaginalis and the adjacent junction of the cauda epididymidis and vas deferens. The findings suggest that persistence of the fetal cranial gonadal suspensory ligaments could have been the major aetiological factor in this case of cryptorchidism. Their persistence could have prevented caudal outgrowth of the processus vaginalis with its consequent development into an intra-abdominal papilla-like structure. Inappropriate persistence of the cranial suspensory ligaments in male rodents, pig, and cattle has been associated with insufficient exposure of their primordia to androgen during fetal life. It is uncertain whether a similar deficiency could underlie persistence of these structures in the present specimen. The findings add further weight to the hypothesis that regression of the cranial gonadal suspensory ligament in males is a key event in the process of testis descent. The human homologue of this ligament deserves more attention in the analysis and treatment of human cryptorchidism. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:8771408

Correlation between expression of CatSper family and sperm profiles in the adult mouse testis following Iranian Kerack abuse.

PubMed

Amini, M; Shirinbayan, P; Behnam, B; Roghani, M; Farhoudian, A; Joghataei, M T; Koruji, M

2014-05-01

Illicit drug use can be an important cause of male infertility. The aim of this study was to investigate the effects of an Iranian illicit drug, Kerack, on sperm parameters, testicular structure and CatSper genes expression of mice. In this study, 25 male mice were divided into five groups consisting of control, sham and three experimental groups. All animal in experimental groups were addicted to Kerack for 7 days. These experimental groups include experimental I which was given Kerack at a dose of 5 mg/kg, experimental II, 35 mg/kg and experimental III, 70 mg/kg, intraperitoneally twice a day for a period of 35 days. Mice were then sacrificed and spermatozoas were removed from cauda epididymis and analyzed for count, motility, morphology (normal/abnormal) and viability. Right testes were removed, weighed and processed for light microscopic studies whereas left testes removed were subjected to total mRNA extraction for using in real-time PCR (RT-PCR). The results were analyzed by performing anova (Tukey's tests) and Pearson correlation coefficient. Sperm parameters and seminiferous epithelium thickness were decreased in experimental groups (dose-dependently) vs. sham and control groups (p < 0.05). RT-PCR results showed that CatSper 2, 3, 4 genes expressions were reduced with 35 and 70 mg/kg injected Kerack when compared with control testes (p ≤ 0.05). However, CatSper1 expression was only reduced with high dose injected Kerack (70 mg/kg) in comparison to control testes (p ≤ 0.05). This study shows the deleterious effects of Kerack used in Iran on testis structure and sperm parameters in general, and particularly sperm morphology in adult mouse. It could down-regulate the expression of CatSper genes, resulting in depression of sperm motility. © 2014 American Society of Andrology and European Academy of Andrology.

The oligosaccharidic content of the glycoconjugates of the prepubertal descended and undescended testis: lectin histochemical study.

PubMed

Gheri, Gherardo; Sgambati, Eleonora; Thyrion, Giorgia D Zappoli; Vichi, Debora; Orlandini, Giovanni E

2004-01-01

The saccharidic content of the glycoconjugates has been studied in the descended the undescended testes of a 8 years old boy. For this purpose, a battery of seven HRP-conjugated lectins (SBA, DBA,PNA,WGA,UEAI, LTA and ConA) was used. D-galactose-N-acetyl-D-galactosamine and alpha-L-fucose sugar residues, which were present in the cytoplasm of the Sertoli cells of the normally positioned prepubertal testis, were not detected in the same cells of the undescended testis. The Leydig's cells of the descended testis appeared characterized by N-acetyl-D-glucosamine which was absent in the rare and atrophic Leydig's cells of the cryptorchid testis. Differences in sugar residues distribution between the descended and the undescended testis were also detected in the lamina propria of the seminiferous tubules. Peritubular myoid cells in the undescended testis only reacted with PNA, after neuraminidase digestion, thus revealing the presence of D-galactose (beta1-->3)-N-acetyl-D-galactosamine and sialic acid. In this study a complete distributional map of the sugar residues of the glycoconjugates in the descended and undescended prepubertal testis is reported.

Cold exposure inhibits hypothalamic Kiss-1 gene expression, serum leptin concentration, and delays reproductive development in male Brandt's vole ( Lasiopodomys brandtii)

NASA Astrophysics Data System (ADS)

Zhang, Qiang; Lin, Yi; Zhang, Xue-Ying; Wang, De-Hua

2015-06-01

Cold commonly affects growth and reproductive development in small mammals. Here, we test the hypothesis that low ambient temperature will affect growth and puberty onset, associated with altered hypothalamic Kiss-1 gene expression and serum leptin concentration in wild rodents. Male Brandt's voles ( Lasiopodomys brandtii) were exposed to cold (4 ± 1 °C) and warm (23 ± 1 °C) conditions from the birth and sacrificed on different developmental stages (day 26, day 40, day 60, and day 90, respectively). Brandt's voles increased the thermogenic capacity of brown adipose tissue, mobilized body fat, decreased serum leptin levels, and delayed the reproductive development especially on day 40 in the cold condition. They increased food intake to compensate for the high energy demands in the cold. The hypothalamic Kiss-1 gene expression on day 26 was decreased, associated with lower wet testis mass and testis testosterone concentration on day 40, in the cold-exposed voles compared to that in the warm. Serum leptin was positively correlated with body fat, testis mass, and testosterone concentration. These data suggested that cold exposure inhibited hypothalamic Kiss-1 gene expression during the early stage of development, decreased serum leptin concentration, and delayed reproductive development in male Brandt's voles.

Painful ovulation in a 46,XX SRY −ve adult male with SOX9 duplication

PubMed Central

Kean, Anne-Maree; Ewans, Lisa; Ohnesorg, Thomas; Ayers, Katie L; Watson, Geoff; Vasilaras, Arthur; Sinclair, Andrew H; Twigg, Stephen M; Handelsman, David J

2017-01-01

46,XX disorders of sexual development (DSDs) occur rarely and result from disruptions of the genetic pathways underlying gonadal development and differentiation. We present a case of a young phenotypic male with 46,XX SRY-negative ovotesticular DSD resulting from a duplication upstream of SOX9 presenting with a painful testicular mass resulting from ovulation into an ovotestis. We present a literature review of ovulation in phenotypic men and discuss the role of SRY and SOX9 in testicular development, including the role of SOX9 upstream enhancer region duplication in female-to-male sex reversal. Learning points: In mammals, the early gonad is bipotent and can differentiate into either a testis or an ovary. SRY is the master switch in testis determination, responsible for differentiation of the bipotent gonad into testis. SRY activates SOX9 gene, SOX9 as a transcription factor is the second major gene involved in male sex determination. SOX9 drives the proliferation of Sertoli cells and activates AMH/MIS repressing the ovary. SOX9 is sufficient to induce testis formation and can substitute for SRY function. Assessing karyotype and then determination of the presence or absence of Mullerian structures are necessary serial investigations in any case of DSD, except for mixed gonadal dysgenesis identified by karyotype alone. Treatment is ideal in a multidisciplinary setting with considerations to genetic (implications to family and reproductive recurrence risk), psychological aspects (sensitive individualized counseling including patient gender identity and preference), endocrinological (hormone replacement), surgical (cosmetic, prophylactic gonadectomy) fertility preservation and reproductive opportunities and metabolic health (cardiovascular and bones). PMID:28620497

Painful ovulation in a 46,XX SRY -ve adult male with SOX9 duplication.

PubMed

Shankara Narayana, Nandini; Kean, Anne-Maree; Ewans, Lisa; Ohnesorg, Thomas; Ayers, Katie L; Watson, Geoff; Vasilaras, Arthur; Sinclair, Andrew H; Twigg, Stephen M; Handelsman, David J

2017-01-01

46,XX disorders of sexual development (DSDs) occur rarely and result from disruptions of the genetic pathways underlying gonadal development and differentiation. We present a case of a young phenotypic male with 46,XX SRY-negative ovotesticular DSD resulting from a duplication upstream of SOX9 presenting with a painful testicular mass resulting from ovulation into an ovotestis. We present a literature review of ovulation in phenotypic men and discuss the role of SRY and SOX9 in testicular development, including the role of SOX9 upstream enhancer region duplication in female-to-male sex reversal. In mammals, the early gonad is bipotent and can differentiate into either a testis or an ovary. SRY is the master switch in testis determination, responsible for differentiation of the bipotent gonad into testis.SRY activates SOX9 gene, SOX9 as a transcription factor is the second major gene involved in male sex determination. SOX9 drives the proliferation of Sertoli cells and activates AMH/MIS repressing the ovary. SOX9 is sufficient to induce testis formation and can substitute for SRY function.Assessing karyotype and then determination of the presence or absence of Mullerian structures are necessary serial investigations in any case of DSD, except for mixed gonadal dysgenesis identified by karyotype alone.Treatment is ideal in a multidisciplinary setting with considerations to genetic (implications to family and reproductive recurrence risk), psychological aspects (sensitive individualized counseling including patient gender identity and preference), endocrinological (hormone replacement), surgical (cosmetic, prophylactic gonadectomy) fertility preservation and reproductive opportunities and metabolic health (cardiovascular and bones).

A Novel igf3 Gene in Common Carp (Cyprinus carpio): Evidence for Its Role in Regulating Gonadal Development.

PubMed

Song, Feibiao; Wang, Lanmei; Zhu, Wenbin; Fu, Jianjun; Dong, Juanjuan; Dong, Zaijie

2016-01-01

Since the insulin-like growth factor 3 (igf3) gene was recently discovered in fish ovary, its function in the gonads has received much attention. In this study, we isolated two igf3 subtypes from common carp (Cyprinus carpio), which comprised full-length cDNA of 707 and 1153 nucleotides encoding 205 and 198 amino acids (aa), respectively. The Igf3 aa sequence had the highest gene homology of 72% with the corresponding sequence in zebrafish (Danio rerio). Phylogenetic tree construction revealed that the C. carpio igf3 gene was first clustered with D. rerio and then with other teleost species. Igf3 mRNA was widely expressed, with expression being highest in the gonads and blood. In the gonad development stage, igf3a mRNA expression was highest in the maturity and recession stage of the ovary, and decline phase of the testis, while igf3b was highest in the recession and fully mature periods of the ovaries and testes, respectively. Western blotting of testis protein samples showed two bands of approximately 21 kDa and 34 kDa corresponding to the calculated molecular mass of the two Igf3 subtypes; no signal was detected in the ovary. The Igf3 protein was localized in the ovary granulosa cells and testis spermatogonium and spermatids. 17β-Ethinylestradiol treatment increased both ovary and testis igf3 mRNA expression. These findings suggest that Igf3 may play an important role in C. carpio gonadal development.

A Novel igf3 Gene in Common Carp (Cyprinus carpio): Evidence for Its Role in Regulating Gonadal Development

PubMed Central

Zhu, Wenbin; Fu, Jianjun; Dong, Juanjuan; Dong, Zaijie

2016-01-01

Since the insulin-like growth factor 3 (igf3) gene was recently discovered in fish ovary, its function in the gonads has received much attention. In this study, we isolated two igf3 subtypes from common carp (Cyprinus carpio), which comprised full-length cDNA of 707 and 1153 nucleotides encoding 205 and 198 amino acids (aa), respectively. The Igf3 aa sequence had the highest gene homology of 72% with the corresponding sequence in zebrafish (Danio rerio). Phylogenetic tree construction revealed that the C. carpio igf3 gene was first clustered with D. rerio and then with other teleost species. Igf3 mRNA was widely expressed, with expression being highest in the gonads and blood. In the gonad development stage, igf3a mRNA expression was highest in the maturity and recession stage of the ovary, and decline phase of the testis, while igf3b was highest in the recession and fully mature periods of the ovaries and testes, respectively. Western blotting of testis protein samples showed two bands of approximately 21 kDa and 34 kDa corresponding to the calculated molecular mass of the two Igf3 subtypes; no signal was detected in the ovary. The Igf3 protein was localized in the ovary granulosa cells and testis spermatogonium and spermatids. 17β-Ethinylestradiol treatment increased both ovary and testis igf3 mRNA expression. These findings suggest that Igf3 may play an important role in C. carpio gonadal development. PMID:28002497

Dimorphic expression of sex-related genes in different gonadal development stages of sterlet, Acipenser ruthenus, a primitive fish species.

PubMed

Wang, Wei; Zhu, Hua; Dong, Ying; Tian, ZhaoHui; Dong, Tian; Hu, HongXia; Niu, CuiJuan

2017-12-01

Molecular mechanism of sex determination and differentiation of sturgeon, a primitive fish species, is extraordinarily important due to the valuable caviar; however, it is still poorly known. The present work aimed to identify the major genes involved in regulating gonadal development of sterlet, a small species of sturgeon, from 13 candidate genes which have been shown to relate to gonadal differentiation and development in other teleost fish. The sex and gonadal development of sterlets were determined by histological observation and levels of sex steroids testosterone (T), 11-ketotestosterone (11-KT), and 17β-estradiol (E2) in serum. Sexually dimorphic gene expressions were investigated. The results revealed that gonadal development were asynchronous in 2-year-old male and female sterlets with the testes in early or mid-spermatogenesis and the ovaries in chromatin nucleolus stage or perinucleolus stage, respectively. The levels of T and E2 were not significantly different between sexes or different gonadal development stages while 11-KT had the higher level in mid-spermatogenesis testis stage. In all the investigated gonadal development stages, gene dmrt1 and hsd11b2 were expressed higher in male whereas foxl2 and cyp19a1 were expressed higher in female. Thus, these genes provided the promising markers for sex identification of sterlet. It was unexpected that dkk1 and dax1 had significantly higher expression in ovarian perinucleolus stage than in ovarian chromatin nucleolus stage and in the testis, suggesting that these two genes had more correlation with ovarian development than with the testis, contrary to the previous reports in other vertebrates. Testicular development-related genes (gsdf and amh) and estrogen receptor genes (era and erb) differentially expressed at different testis or ovary development stages, but their expressions were not absolutely significantly different in male and female, depending on the gonadal development stage. Expression of androgen receptor gene ar or rspo, which was supposed to be related to ovarian development, presented no difference between gonadal development stages investigated in this study whenever in male or female.

An Ancient Transcription Factor Initiates the Burst of piRNA Production During Early Meiosis in Mouse Testes

PubMed Central

Li, Xin Zhiguo; Roy, Christian K.; Dong, Xianjun; Bolcun-Filas, Ewelina; Wang, Jie; Han, Bo W.; Xu, Jia; Moore, Melissa J.; Schimenti, John C.; Weng, Zhiping; Zamore, Phillip D.

2013-01-01

SUMMARY Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during post-natal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors, including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feed-forward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals. PMID:23523368

Maternally expressed PGK-Cre transgene as a tool for early and uniform activation of the Cre site-specific recombinase.

PubMed

Lallemand, Y; Luria, V; Haffner-Krausz, R; Lonai, P

1998-03-01

A transgenic mouse strain with early and uniform expression of the Cre site-specific recombinase is described. In this strain, PGK-Crem, Cre is driven by the early acting PGK-1 promoter, but, probably due to cis effects at the integration site, the recombinase is under dominant maternal control. When Cre is transmitted by PGK-Crem females mated to males that carry a reporter transgene flanked by loxP sites, even offspring that do not inherit PGK-Cre delete the target gene. It follows that in the PGK-Crem female Cre activity commences in the diploid phase of oogenesis. In PGK-Crem crosses complete recombination was observed in all organs, including testis and ovary. We prepared a mouse stock that is homozygous for PGK-Crem and at the albino (c) locus. This strain will be useful for the early and uniform induction of ectopic and dominant negative mutations, for the in vivo removal of selective elements from targeted mutations and in connection with the manipulation of targeted loci in 'knock in' and related technologies.

PW1 gene/paternally expressed gene 3 (PW1/Peg3) identifies multiple adult stem and progenitor cell populations

PubMed Central

Besson, Vanessa; Smeriglio, Piera; Wegener, Amélie; Relaix, Frédéric; Nait Oumesmar, Brahim; Sassoon, David A.; Marazzi, Giovanna

2011-01-01

A variety of markers are invaluable for identifying and purifying stem/progenitor cells. Here we report the generation of a murine reporter line driven by Pw1 that reveals cycling and quiescent progenitor/stem cells in all adult tissues thus far examined, including the intestine, blood, testis, central nervous system, bone, skeletal muscle, and skin. Neurospheres generated from the adult PW1-reporter mouse show near 100% reporter-gene expression following a single passage. Furthermore, epidermal stem cells can be purified solely on the basis of reporter-gene expression. These cells are clonogenic, repopulate the epidermal stem-cell niches, and give rise to new hair follicles. Finally, we demonstrate that only PW1 reporter-expressing epidermal cells give rise to follicles that are capable of self-renewal following injury. Our data demonstrate that PW1 serves as an invaluable marker for competent self-renewing stem cells in a wide array of adult tissues, and the PW1-reporter mouse serves as a tool for rapid stem cell isolation and characterization. PMID:21709251

Maternal exposure to a mixture of di(2-ethylhexyl) phthalate (DEHP) and polychlorinated biphenyls (PCBs) causes reproductive dysfunction in adult male mouse offspring.

PubMed

Fiandanese, Nadia; Borromeo, Vitaliano; Berrini, Anna; Fischer, Bernd; Schaedlich, Kristina; Schmidt, Juliane-Susanne; Secchi, Camillo; Pocar, Paola

2016-10-01

We investigated the effects of maternal exposure to the plasticizer di(2-ethylhexyl) phthalate (DEHP) and the organic industrial compounds polychlorinated biphenyls (PCBs), singly and combined, on the reproductive function of male mouse offspring. Mice dams were exposed throughout pregnancy and lactation to 1μg PCBs (101+118)/kg/day, 50μg DEHP/kg/day, or the DEHP/PCB mixture in the diet. The mixture induced permanent alterations in adult F1 males' reproductive health in a way, differently from the single compounds. Depending on the endpoint, we observed: (1) synergy in altering the gross and histological morphology of the testis; (2) antagonism on the expression levels of genes involved in pituitary-gonadal cross-talk; (3) non-interactions on sperm parameters and testosterone production. This study illustrates the complex action of a DEHP/PCB mixture, leading to a unique panel of effects on the male reproductive system, indicating the need for research on the reproductive hazards of combined endocrine disruptors. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of Genes Uniquely Expressed in the Germ-Line Tissues of the Jewel Wasp Nasonia vitripennis

PubMed Central

Ferree, Patrick M.; Fang, Christopher; Mastrodimos, Mariah; Hay, Bruce A.; Amrhein, Henry; Akbari, Omar S.

2015-01-01

The jewel wasp Nasonia vitripennis is a rising model organism for the study of haplo-diploid reproduction characteristic of hymenopteran insects, which include all wasps, bees, and ants. We performed transcriptional profiling of the ovary, the female soma, and the male soma of N. vitripennis to complement a previously existing transcriptome of the wasp testis. These data were deposited into an open-access genome browser for visualization of transcripts relative to their gene models. We used these data to identify the assemblies of genes uniquely expressed in the germ-line tissues. We found that 156 protein-coding genes are expressed exclusively in the wasp testis compared with only 22 in the ovary. Of the testis-specific genes, eight are candidates for male-specific DNA packaging proteins known as protamines. We found very similar expression patterns of centrosome associated genes in the testis and ovary, arguing that de novo centrosome formation, a key process for development of unfertilized eggs into males, likely does not rely on large-scale transcriptional differences between these tissues. In contrast, a number of meiosis-related genes show a bias toward testis-specific expression, despite the lack of true meiosis in N. vitripennis males. These patterns may reflect an unexpected complexity of male gamete production in the haploid males of this organism. Broadly, these data add to the growing number of genomic and genetic tools available in N. vitripennis for addressing important biological questions in this rising insect model organism. PMID:26464360

Do retractile testes have anatomical anomalies?

PubMed Central

Anderson, Kleber M.; Costa, Suelen F.; Sampaio, Francisco J.B.; Favorito, Luciano A.

2016-01-01

ABSTRACT Objectives: To assess the incidence of anatomical anomalies in patients with retractile testis. Materials and Methods: We studied prospectively 20 patients (28 testes) with truly retractile testis and compared them with 25 human fetuses (50 testes) with testis in scrotal position. We analyzed the relations among the testis, epididymis and patency of the processus vaginalis (PV). To analyze the relations between the testis and epididymis, we used a previous classification according to epididymis attachment to the testis and the presence of epididymis atresia. To analyze the structure of the PV, we considered two situations: obliteration of the PV and patency of the PV. We used the Chi-square test for contingency analysis of the populations under study (p <0.05). Results: The fetuses ranged in age from 26 to 35 weeks post-conception (WPC) and the 20 patients with retractile testis ranged in ages from 1 to 12 years (average of 5.8). Of the 50 fetal testes, we observed complete patency of the PV in 2 cases (4%) and epididymal anomalies (EAs) in 1 testis (2%). Of the 28 retractile testes, we observed patency of the PV in 6 cases (21.4%) and EA in 4 (14.28%). When we compared the incidence of EAs and PV patency we observed a significantly higher prevalence of these anomalies in retractile testes (p=0.0116). Conclusions: Retractile testis is not a normal variant with a significant risk of patent processus vaginalis and epididymal anomalies. PMID:27564294

Integrative testis transcriptome analysis reveals differentially expressed miRNAs and their mRNA targets during early puberty in Atlantic salmon.

PubMed

Skaftnesmo, K O; Edvardsen, R B; Furmanek, T; Crespo, D; Andersson, E; Kleppe, L; Taranger, G L; Bogerd, J; Schulz, R W; Wargelius, A

2017-10-18

Our understanding of the molecular mechanisms implementing pubertal maturation of the testis in vertebrates is incomplete. This topic is relevant in Atlantic salmon aquaculture, since precocious male puberty negatively impacts animal welfare and growth. We hypothesize that certain miRNAs modulate mRNAs relevant for the initiation of puberty. To explore which miRNAs regulate mRNAs during initiation of puberty in salmon, we performed an integrated transcriptome analysis (miRNA and mRNA-seq) of salmon testis at three stages of development: an immature, long-term quiescent stage, a prepubertal stage just before, and a pubertal stage just after the onset of single cell proliferation activity in the testis. Differentially expressed miRNAs clustered into 5 distinct expression profiles related to the immature, prepubertal and pubertal salmon testis. Potential mRNA targets of these miRNAs were predicted with miRmap and filtered for mRNAs displaying negatively correlated expression patterns. In summary, this analysis revealed miRNAs previously known to be regulated in immature vertebrate testis (miR-101, miR-137, miR-92b, miR-18a, miR-20a), but also miRNAs first reported here as regulated in the testis (miR-new289, miR-30c, miR-724, miR-26b, miR-new271, miR-217, miR-216a, miR-135a, miR-new194 and the novel predicted n268). By KEGG enrichment analysis, progesterone signaling and cell cycle pathway genes were found regulated by these differentially expressed miRNAs. During the transition into puberty we found differential expression of miRNAs previously associated (let7a/b/c), or newly associated (miR-15c, miR-2184, miR-145 and the novel predicted n7a and b) with this stage. KEGG enrichment analysis revealed that mRNAs of the Wnt, Hedgehog and Apelin signaling pathways were potential regulated targets during the transition into puberty. Likewise, several regulated miRNAs in the pubertal stage had earlier been associated (miR-20a, miR-25, miR-181a, miR-202, let7c/d/a, miR-125b, miR-222a/b, miR-190a) or have now been found connected (miR-2188, miR-144, miR-731, miR-8157 and the novel n2) to the initiation of puberty. This study has - for the first time - linked testis maturation to specific miRNAs and their inversely correlated expressed targets in Atlantic salmon. The study indicates a broad functional conservation of already known miRNAs and associated pathways involved in the transition into puberty in vertebrates. The analysis also reveals miRNAs not previously associated with testis tissue or its maturation, which calls for further functional studies in the testis.

Expression analysis of cyp11a1 during gonadal development, recrudescence and after hCG induction and sex steroid analog treatment in the catfish, Clarias batrachus.

PubMed

Rajakumar, Anbazhagan; Senthilkumaran, Balasubramanian

2014-10-01

In teleosts, the levels of steroids are critical for sexual development and hence, expression of steroidogenic enzyme genes and specific substrate availability are indispensable for gonadal steroidogenesis. Early stages of steroidogenesis specifically cholesterol to pregnenolone conversion by Cyp11a1 is crucial for estradiol and testosterone biosynthesis. Based on this, in this study, full length cDNA of cyp11a1 (2581bp) was cloned from catfish testis to investigate the importance of Cyp11a1 by analyzing the expression of cyp11a1 during gonadal development, seasonal reproductive cycle, after human chorionic gonadotropin (hCG) induction and sex steroid analog treatment. Phylogenetic analysis revealed that the Cyp11a1 is more conserved across teleosts. Tissue distribution analysis showed that the cyp11a1 expression was higher in the testis followed by the brain, head kidney, muscle and ovary compared to other tissues analyzed. High expression of cyp11a1 in the head kidney and muscle revealed that Cyp11a1 could potentially regulate the extra-gonadal and/or circulating steroid levels in teleosts. Developing and mature testes showed higher expression of cyp11a1 than the ovary of corresponding age group. Further, cyp11a1 expression was found to be higher during pre-spawning and spawning phases of testicular cycle and was upregulated by hCG, in vivo and in vitro, which indicates the possible regulation by gonadotropin. Exposure of methyltestosterone (1μg/L) and ethinylestradiol (1μg/L) for 21days during catfish testicular development showed lower cyp11a1 expression levels in the testis and brain indicating a certain feedback intervention. These results suggest possible role for Cyp11a1 in the testis development and recrudescence. Copyright © 2014 Elsevier Inc. All rights reserved.

Functional complementation between a novel mammalian polygenic transport complex and an evolutionarily ancient organic solute transporter, OSTalpha-OSTbeta.

PubMed

Seward, David J; Koh, Albert S; Boyer, James L; Ballatori, Nazzareno

2003-07-25

These studies identify an organic solute transporter (OST) that is generated when two novel gene products are co-expressed, namely human OSTalpha and OSTbeta or mouse OSTalpha and OSTbeta. The results also demonstrate that the mammalian proteins are functionally complemented by evolutionarily divergent Ostalpha-Ostbeta proteins recently identified in the little skate, Raja erinacea, even though the latter exhibit only 25-41% predicted amino acid identity with the mammalian proteins. Human, mouse, and skate OSTalpha proteins are predicted to contain seven transmembrane helices, whereas the OSTbeta sequences are predicted to have a single transmembrane helix. Human OSTalpha-OSTbeta and mouse Ostalpha-Ostbeta cDNAs were cloned from liver mRNA, sequenced, expressed in Xenopus laevis oocytes, and tested for their ability to functionally complement the corresponding skate proteins by measuring transport of [3H]estrone 3-sulfate. None of the proteins elicited a transport signal when expressed individually in oocytes; however, all nine OSTalpha-OSTbeta combinations (i.e. OSTalpha-OSTbeta pairs from human, mouse, or skate) generated robust estrone 3-sulfate transport activity. Transport was sodium-independent, saturable, and inhibited by other steroids and anionic drugs. Human and mouse OSTalpha-OSTbeta also were able to mediate transport of taurocholate, digoxin, and prostaglandin E2 but not of estradiol 17beta-d-glucuronide or p-aminohippurate. OSTalpha and OSTbeta were able to reach the oocyte plasma membrane when expressed either individually or in pairs, indicating that co-expression is not required for proper membrane targeting. Interestingly, OSTalpha and OSTbeta mRNAs were highly expressed and widely distributed in human tissues, with the highest levels occurring in the testis, colon, liver, small intestine, kidney, ovary, and adrenal gland.

Testosterone Regulates NUCB2 mRNA Expression in Male Mouse Hypothalamus and Pituitary Gland

PubMed Central

Seon, Sojeong; Jeon, Daun; Kim, Heejeong; Chung, Yiwa; Choi, Narae; Yang, Hyunwon

2017-01-01

ABSTRACT Nesfatin-1/NUCB2 is known to take part in the control of the appetite and energy metabolism. Recently, many reports have shown nesfatin-1/NUCB2 expression and function in various organs. We previously demonstrated that nesfatin-1/NUCB2 expression level is higher in the pituitary gland compared to other organs and its expression is regulated by 17β-estradiol and progesterone secreted from the ovary. However, currently no data exist on the expression of nesfatin-1/NUCB2 and its regulation mechanism in the pituitary of male mouse. Therefore, we examined whether nesfatin-1/NUCB2 is expressed in the male mouse pituitary and if its expression is regulated by testosterone. As a result of PCR and western blotting, we found that a large amount of nesfatin-1/NUCB2 was expressed in the pituitary and hypothalamus. The NUCB2 mRNA expression level in the pituitary was decreased after castration, but not in the hypothalamus. In addition, its mRNA expression level in the pituitary was increased after testosterone treatment in the castrated mice, whereas, the expression level in the hypothalamus was significantly decreased after the treatment with testosterone. The in vitro experiment to elucidate the direct effect of testosterone on NUCB2 mRNA expression showed that NUCB2 mRNA expression was significantly decreased with testosterone in cultured hypothalamus tissue, but increased with testosterone in cultured pituitary gland. The present study demonstrated that nesfatin-1/NUCB2 was highly expressed in the male mouse pituitary and was regulated by testosterone. This data suggests that reproductive-endocrine regulation through hypothalamus-pituitary-testis axis may contribute to NUCB2 mRNA expression in the mouse hypothalamus and pituitary gland. PMID:28484746

Identification of an anti-sperm auto-monoclonal antibody (Ts4)-recognized molecule in the mouse sperm acrosomal region and its inhibitory effect on fertilization in vitro.

PubMed

Yoshitake, Hiroshi; Oda, Risako; Yanagida, Mitsuaki; Kawasaki, Yu; Sakuraba, Mayumi; Takamori, Kenji; Hasegawa, Akiko; Fujiwara, Hiroshi; Araki, Yoshihiko

2016-06-01

We previously established an anti-mouse sperm auto-monoclonal antibody, Ts4, which shows immunoreactivity against several kinds of glycoproteins in the acrosomal region of epididymal spermatozoa, testicular germ cells, and early embryo, via binding to an epitope containing a common N-linked oligosaccharide (OS) chain on the molecules. In mice, we have already demonstrated that the OS chain in the epitope for Ts4 is a fucosylated agalacto-complex-type biantennary glycan carrying bisecting N-acetylglucosamine. In the testis, one of the specific OS chain-conjugated molecules is TEX101, a germ cell-marker glycoprotein, which is expressed in spermatocytes, spermatids, and testicular spermatozoa, but not in epididymal spermatozoa. In this study, we identified a Ts4-reactive glycoprotein in mouse cauda epididymal sperm. An immunoprecipitation method together with liquid chromatography-tandem mass spectrometry showed that alpha-N-acetylglucosaminidase (Naglu; a degradation enzyme of heparan sulfate) is one of the glycoproteins recognized by Ts4 in the epididymal spermatozoa. Western blot and immunohistochemical analyses revealed that mouse Naglu exists in two forms (82 and 77kDa) and is expressed in the acrosomal region and the flagellum of cauda epididymal sperm. Of the two Naglu-forms expressed in sperm, Ts4 immunoreacted against only the 82-kDa form located on the acrosomal region. The Ts4 mAb and anti-Naglu pAb negatively affected mouse fertilization in vitro. In addition, Ts4 inhibited sperm acrosome reaction induced by heparan sulfate. The Ts4-recognized fucosylated agalactobiantennary complex-type glycan with bisecting N-acetylglucosamine and Naglu on cauda epididymal spermatozoa may play a role in the process of fertilization. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Giant malignant fibrous histiocytoma of the testis/spermatic cord: psychologic and possible etiologic complications of unethical Nazi medical experimentation.

PubMed

Staiman, V R; O'Toole, K M; Rubin, M A; Lowe, F C

1996-12-01

This case of malignant fibrous histiocytoma of the testis/spermatic cord was found in a Holocaust survivor who was injected with an unknown substance in the left testicle while in Auschwitz concentration camp in 1943. Because malignant fibrous histiocytoma is a neoplasm rarely found in this location, with only 26 previously reported cases, a review of this entity was performed. A review of Nazi medical practices in the literature and through the Holocaust Museum research department was undertaken in an attempt to ascertain identification of the unknown substance injected into this patient; however, exact identification of the Auschwitz experiment or experimenter could not be determined. A left radical orchiectomy was performed, and subsequent histolopathologic review revealed a well-encapsulated 27 x 22 x 17-cm malignant fibrous histiocytoma. The left testis was not clearly identified due to necrosis of the tumor. This is the largest malignant fibrous histiocytoma of the spermatic cord/testis recorded in the literature to date. Based on the unusual location and size, the intratesticular injection probably contributed to the tumor development and certainly caused the patient's delay in seeking medical treatment.

Wi-Fi (2.45 GHz)- and mobile phone (900 and 1800 MHz)-induced risks on oxidative stress and elements in kidney and testis of rats during pregnancy and the development of offspring.

PubMed

Özorak, Alper; Nazıroğlu, Mustafa; Çelik, Ömer; Yüksel, Murat; Özçelik, Derviş; Özkaya, Mehmet Okan; Çetin, Hasan; Kahya, Mehmet Cemal; Kose, Seyit Ali

2013-12-01

The present study was designed to determine the effects of both Wi-Fi (2.45 GHz)- and mobile phone (900 and 1800 MHz)-induced electromagnetic radiation (EMR) on oxidative stress and trace element levels in the kidney and testis of growing rats from pregnancy to 6 weeks of age. Thirty-two rats and their 96 newborn offspring were equally divided into four different groups, namely, control, 2.45 GHz, 900 MHz, and 1800 MHz groups. The 2.45 GHz, 900 MHz, and 1,800 MHz groups were exposed to EMR for 60 min/day during pregnancy and growth. During the fourth, fifth, and sixth weeks of the experiment, kidney and testis samples were taken from decapitated rats. Results from the fourth week showed that the level of lipid peroxidation in the kidney and testis and the copper, zinc, reduced glutathione (GSH), glutathione peroxidase (GSH-Px), and total antioxidant status (TAS) values in the kidney decreased in the EMR groups, while iron concentrations in the kidney as well as vitamin A and vitamin E concentrations in the testis increased in the EMR groups. Results for fifth-week samples showed that iron, vitamin A, and β-carotene concentrations in the kidney increased in the EMR groups, while the GSH and TAS levels decreased. The sixth week results showed that iron concentrations in the kidney and the extent of lipid peroxidation in the kidney and testis increased in the EMR groups, while copper, TAS, and GSH concentrations decreased. There were no statistically significant differences in kidney chromium, magnesium, and manganese concentrations among the four groups. In conclusion, Wi-Fi- and mobile phone-induced EMR caused oxidative damage by increasing the extent of lipid peroxidation and the iron level, while decreasing total antioxidant status, copper, and GSH values. Wi-Fi- and mobile phone-induced EMR may cause precocious puberty and oxidative kidney and testis injury in growing rats.

Claudin 5 Expression in Mouse Seminiferous Epithelium Is Dependent upon the Transcription Factor Ets Variant 5 and Contributes to Blood-Testis Barrier Function1

PubMed Central

Morrow, Carla M.K.; Tyagi, Gaurav; Simon, Liz; Carnes, Kay; Murphy, Kenneth M.; Cooke, Paul S.; Hofmann, Marie-Claude C.; Hess, Rex A.

2009-01-01

The blood-testis barrier (BTB) is formed by tight junctions between Sertoli cells. Results of previous studies suggested that the barrier is deficient in ets variant 5 (ETV5) gene-deleted mice; therefore, microarray data were examined for changes in tight junction-associated genes. The tight junctional protein claudin 5 (CLDN5) was decreased in testes of 8-day-old Etv5−/− pups. The study reported herein examined the expression of CLDN5 in wild-type (WT) and Etv5−/− mice and evaluated its contribution to BTB function. CLDN5 protein expression was evaluated in 8-day-old WT and Etv5−/− and adult WT, Etv5−/−, and W/Wv testes by immunohistochemistry and in 8-day-old WT Sertoli cell-enriched and germ cell-enriched fractions by immunocytochemistry. Cldn5 mRNA expression was evaluated in 0- to 20-day-old and adult WT mice and in 8-day-old and adult Etv5−/− mice via quantitative PCR. Tracer studies were performed in adult WT, Etv5−/−, and W/Wv mice. The results indicate the following: 1) CLDN5 was expressed in Sertoli cells, spermatogonia, and preleptotene spermatocytes. 2) Seminiferous epithelial CLDN5 expression depended upon both the presence of germ cells and ETV5. 3) CLDN5 expression in testicular vascular endothelium and rete testis epithelium was ETV5 independent. 4) Cldn5 mRNA expression increased in the testes of juvenile mice at the time of BTB formation. 5) Testes of Etv5−/− and W/Wv mice, which are both deficient in seminiferous epithelial CLDN5 expression, had biotin tracer leakage from the interstitial space into the seminiferous tubule lumen. In conclusion, CLDN5 is expressed in the seminiferous epithelium, appears to be regulated by multiple influences, and contributes to BTB function. PMID:19571261

A novel hsp110-related gene, apg-1, that is abundantly expressed in the testis responds to a low temperature heat shock rather than the traditional elevated temperatures.

PubMed

Kaneko, Y; Nishiyama, H; Nonoguchi, K; Higashitsuji, H; Kishishita, M; Fujita, J

1997-01-31

We isolated a novel hsp110-related gene, apg-1, from a testis cDNA library. The apg-1 transcripts were constitutively expressed in the testicular germ cells and, in some degree, most tissues examined. In a mouse TAMA26 Sertoli cell line, apg-1 transcripts were induced in 2 h by a temperature shift from 32 to 39 degrees C, but not by a shift from 37 to 42 degrees C, the traditional heat stress, or a shift from 32 to 42 degrees C. The heat response pattern of hsp110 expression was similar to that of apg-1. Although induction of a hsp70 transcript was observed in 2 h by a shift from 32 to 39 degrees C, the induction was more apparent by a shift from 37 to 42 degrees C or from 32 to 42 degrees C. Essentially similar differential response patterns were observed among these genes in NIH/3T3 fibroblasts as well. The nuclear run-on assay and the native gel mobility shift assay demonstrated that, by the 32 to 39 degrees C temperature shift, the apg-1 gene was transcriptionally activated, and heat shock factor 1 bound to the heat shock elements in the 5'-flanking region of the apg-1 gene. These results demonstrated that expressions of apg-1, hsp110, and hsp70 could be heat-induced at a temperature lower than the traditional elevated temperatures in somatic cells of both testis and nontestis origin and suggest that the mechanisms regulating the transcript levels of apg-1 and hsp110 are different from those of hsp70. Furthermore, the constitutive expression in germ cells suggests that APG-1 plays a specific role in spermatogenesis as well as in stress response.

[Epidermoid cyst of the testis difficult to make a preoperative diagnosis on the echoic examination: a case report].

PubMed

Yamamoto, Keisuke; Takada, Tsuyoshi; Momohara, Chikahiro; Komori, Kazuhiko; Honda, Masahito; Fujioka, Hideki

2003-04-01

A case of epidermoid cyst of the testis is presented. The patient was a 64-year-old man who complained of a painless mass in the left scrotum. Physical examination revealed a hen-egg sized enlargement of the left scrotal contents. The ultrasonographic appearance did not show a hyperechoic partition, which is called echogenic rim, a characteristic of this tumor on the echoic examination, and was homogeneous, almost similar to that of a normal testis. Because malignant testicular tumors could not be excluded preoperatively, excisional biopsy of the left testis was performed first. Histological diagnosis was an epidermoid cyst of the testis. As the left testis was almost completely occupied by the tumor and no normal testicular tissue was recognized, we performed orchiectomy additionally. Epidermoid cyst of the testis is a rare benign tumor that accounts for about 1 percent of all testicular tumors. It clinically resembles malignant testicular tumors, and orchiectomy is often performed for treatment. About 154 cases of testicular epidermoid cyst have been reported in the Japanese literature and are reviewed briefly here.

Development of new stem cell-based technologies for carnivore reproduction research.

PubMed

Travis, A J; Kim, Y; Meyers-Wallen, V

2009-07-01

New reproductive technologies based on stem cells offer several potential benefits to carnivore species. For example, development of lines of embryonic stem cells in cats and dogs would allow for the generation of transgenic animal models, which could be used to advance both veterinary and human health. Techniques such as spermatogonial stem cell transplantation (SSCT) and testis xenografting offer new approaches to propagate genetically valuable individual males, even if they should die before producing sperm. These techniques might therefore have application to the conservation of endangered species of carnivores, as well as to biomedical research. Recently, our laboratory has successfully performed SSCT in the dog, with a recipient dog producing sperm of donor genetic origin. Testis xenografting has been used to produce sperm from pre-pubertal testis tissue from both cats and ferrets. These early steps reinforce the need not only for research on stem cell technologies, but also for additional research into complementary technologies of assisted reproduction in carnivores, so that the widest array of research and clinical benefits can be realized.

Testicular cell junction: a novel target for male contraception.

PubMed

Lee, Nikki P Y; Wong, Elissa W P; Mruk, Dolores D; Cheng, C Yan

2009-01-01

Even though various contraceptive methods are widely available, the number of unwanted pregnancies is still on the rise in developing countries, pressurizing the already resource limited nations. One of the major underlying reasons is the lack of effective, low cost, and safe contraceptives for couples. During the past decade, some studies were performed using animal models to decipher if the Sertoli-germ cell junction in the testis is a target for male fertility regulation. Some of these study models were based on the use of hormones and/or chemicals to disrupt the hypothalamic-pituitary-testicular axis (e.g., androgen-based implants or pills) and others utilized a panel of chemical entities or synthetic peptides to perturb spermatogenesis either reversibly or non-reversibly. Among them, adjudin, a potential male contraceptive, is one of the compounds exerting its action on the unique adherens junctions, known as ectoplasmic specializations, in the testis. Since the testis is equipped with inter-connected cell junctions, an initial targeting of one junction type may affect the others and these accumulative effects could lead to spermatogenic arrest. This review attempts to cover an innovative theme on how male infertility can be achieved by inducing junction instability and defects in the testis, opening a new window of research for male contraceptive development. While it will still take much time and effort of intensive investigation before a product can reach the consumable market, these findings have provided hope for better family planning involving men.

Rare presentation of a testicular angiofibroma treated with testis sparing surgery.

PubMed

Leone, Luca; Fulvi, Paola; Sbrollini, Giulia; Filosa, Alessandra; Caraceni, Enrico; Marronaro, Angelo; Galosi, Andrea B

2016-12-30

Testicular benign tumors are very rare (< 5%). Testicular Angiofibroma (AF) is one of those, however the gold standard of treatment and follow-up is still unclear. A 47 years-old man with only one functioning testis was referred to our clinic for a palpable right testicular mass and atrophic contralateral testis. Patient underwent testis-sparing surgery with inguinal approach and intraoperative frozen sections examination with diagnosis of AF. Final histology confirmed AF. Post-operative follow-up was uneventful. Clinical and ultrasonographic follow-up was negative after 8 months. We report a conservative surgery in a patient with AF of the solitary testis. AF is a benign para-testicular fibrous neoplasm that could be misinterpreted as malignant tumor and treated with orchiectomy. Testis-sparing surgery is recommended in this case with intraoperative pathological examination. The excision of the mass is enough but in front of a possible recurrence a long follow-up is advisable.

An oncological view on the blood-testis barrier.

PubMed

Bart, Joost; Groen, Harry J M; van der Graaf, Winette T A; Hollema, Harry; Hendrikse, N Harry; Vaalburg, Willem; Sleijfer, Dirk T; de Vries, Elisabeth G E

2002-06-01

The function of the blood-testis barrier is to protect germ cells from harmful influences; thus, it also impedes the delivery of chemotherapeutic drugs to the testis. The barrier has three components: first, a physicochemical barrier consisting of continuous capillaries, Sertoli cells in the tubular wall, connected together with narrow tight junctions, and a myoid-cell layer around the seminiferous tubule. Second, an efflux-pump barrier that contains P-glycoprotein in the luminal capillary endothelium and on the myoid-cell layer; and multidrug-resistance associated protein 1 located basolaterally on Sertoli cells. Third, an immunological barrier, consisting of Fas ligand on Sertoli cells. Inhibition of P-glycoprotein function offers the opportunity to increase the delivery of cytotoxic drugs to the testis. In the future, visualisation of function in the blood-testis barrier may also be helpful to identify groups of patients in whom testis conservation is safe or to select drugs that are less harmful to fertility.

Persistent DNA Damage in Spermatogonial Stem Cells After Fractionated Low-Dose Irradiation of Testicular Tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grewenig, Angelika; Schuler, Nadine; Rübe, Claudia E., E-mail: claudia.ruebe@uks.eu

Purpose: Testicular spermatogenesis is extremely sensitive to radiation-induced damage, and even low scattered doses to testis from radiation therapy may pose reproductive risks with potential treatment-related infertility. Radiation-induced DNA double-strand breaks (DSBs) represent the greatest threat to the genomic integrity of spermatogonial stem cells (SSCs), which are essential to maintain spermatogenesis and prevent reproduction failure. Methods and Materials: During daily low-dose radiation with 100 mGy or 10 mGy, radiation-induced DSBs were monitored in mouse testis by quantifying 53 binding protein 1 (53BP-1) foci in SSCs within their stem cell niche. The accumulation of DSBs was correlated with proliferation, differentiation, and apoptosis ofmore » testicular germ cell populations. Results: Even very low doses of ionizing radiation arrested spermatogenesis, primarily by inducing apoptosis in spermatogonia. Eventual recovery of spermatogenesis depended on the survival of SSCs and their functional ability to proliferate and differentiate to provide adequate numbers of differentiating spermatogonia. Importantly, apoptosis-resistant SSCs resulted in increased 53BP-1 foci levels during, and even several months after, fractionated low-dose radiation, suggesting that surviving SSCs have accumulated an increased load of DNA damage. Conclusions: SSCs revealed elevated levels of DSBs for weeks after radiation, and if these DSBs persist through differentiation to spermatozoa, this may have severe consequences for the genomic integrity of the fertilizing sperm.« less

Molecular characterization of the equine testis-specific protein 1 (TPX1) and acidic epididymal glycoprotein 2 (AEG2) genes encoding members of the cysteine-rich secretory protein (CRISP) family.

PubMed

Giese, Alexander; Jude, Rony; Kuiper, Heidi; Raudsepp, Terje; Piumi, Francois; Schambony, Alexandra; Guérin, Gérard; Chowdhary, Bhanu P; Distl, Ottmar; Töpfer-Petersen, Edda; Leeb, Tosso

2002-10-16

The cysteine-rich secretory protein (CRISP) family consists of three members called acidic epididymal glycoprotein 1 (AEG1), AEG2, and testis-specific protein 1 (TPX1), which share 16 conserved cysteine residues at their C-termini. The CRISP proteins are primarily expressed in different sections of the male genital tract and are thought to mediate cell-cell interactions of male germ cells with other cells during sperm maturation or during fertilization. Therefore, their genes are of interest as candidate genes for inherited male fertility dysfunctions and as putative quantitative trait loci for male fertility traits. In this report, the cloning and DNA sequence of 137 kb of horse genomic DNA from equine chromosome 20q22 containing the closely linked equine TPX1 and AEG2 genes are described. The equine TPX1 gene consists of ten exons spanning 18 kb while the AEG2 gene consists of eight exons that are spread over 24 kb. The expression of these two genes was investigated in several tissues by reverse transcription polymerase chain reaction analysis and Western blotting. Comparative genome analysis between horse, human, and mouse indicates that all three CRISP genes are clustered on one chromosomal location, which shows conserved synteny between these species.

Altered Expression of ZO-1 and ZO-2 in Sertoli Cells and Loss of Blood-Testis Barrier Integrity in Testicular Carcinoma In Situ1

PubMed Central

Fink, Cornelia; Weigel, Roswitha; Hembes, Tanja; Lauke-Wettwer, Heidrun; Kliesch, Sabine; Bergmann, Martin; Brehm, Ralph H

2006-01-01

Abstract Carcinoma in situ (CIS) is the noninvasive precursor of most human testicular germ cell tumors. In normal seminiferous epithelium, specialized tight junctions between Sertoli cells constitute the major component of the blood-testis barrier. Sertoli cells associated with CIS exhibit impaired maturation status, but their functional significance remains unknown. The aim was to determine whether the blood-testis barrier is morphologically and/or functionally altered. We investigated the expression and distribution pattern of the tight junction proteins zonula occludens (ZO) 1 and 2 in normal seminiferous tubules compared to tubules showing CIS. In normal tubules, ZO-1 and ZO-2 immunostaining was observed at the blood-testis barrier region of adjacent Sertoli cells. Within CIS tubules, ZO-1 and ZO-2 immunoreactivity was reduced at the blood-testis barrier region, but spread to stain the Sertoli cell cytoplasm. Western blot analysis confirmed ZO-1 and ZO-2, and their respective mRNA were shown by RT-PCR. Additionally, we assessed the functional integrity of the blood-testis barrier by lanthanum tracer study. Lanthanum permeated tight junctions in CIS tubules, indicating disruption of the blood-testis barrier. In conclusion, Sertoli cells associated with CIS show an altered distribution of ZO-1 and ZO-2 and lose their blood-testis barrier function. PMID:17217619

Estrogen alters gonadal soma-derived factor (Gsdf)/Foxl2 expression levels in the testes associated with testis-ova differentiation in adult medaka, Oryzias latipes.

PubMed

Kobayashi, Tohru; Chiba, Ayaka; Sato, Tadashi; Myosho, Taijun; Yamamoto, Jun; Okamura, Tetsuro; Onishi, Yuta; Sakaizumi, Mitsuru; Hamaguchi, Satoshi; Iguchi, Taisen; Horie, Yoshifumi

2017-10-01

Testis-ova differentiation in sexually mature male medaka (Oryzias latipes) is easily induced by estrogenic chemicals, indicating that spermatogonia persist in sexual bipotentiality, even in mature testes in medaka. By contrast, the effects of estrogen on testicular somatic cells associated with testis-ova differentiation in medaka remain unclear. In this study, we focused on the dynamics of sex-related genes (Gsdf, Dmrt1, and Foxl2) expressed in Sertoli cells in the mature testes of adult medaka during estrogen-induced testis-ova differentiation. When mature male medaka were exposed to estradiol benzoate (EB; 800ng/L), testis-ova first appeared after EB treatment for 14days (observed as the first oocytes of the leptotene-zygotene stage). However, the testis remained structurally unchanged, even after EB treatment for 28days. Although Foxl2 is a female-specific sex gene, EB treatment for 7days induced Foxl2/FOXL2 expression in all Sertoli cell-enclosed spermatogonia before testis-ova first appeared; however, Foxl2 was not detected in somatic cells in control testes. Conversely, Sertoli-cell-specific Gsdf mRNA expression levels significantly decreased after EB treatment for 14days, and no changes were observed in DMRT1 localization following EB treatment, whereas Dmrt1 mRNA levels increased significantly. Furthermore, after EB exposure, FOXl2 and DMRT1 were co-localized in Sertoli cells during testis-ova differentiation, although FOXL2 localization was undetectable in Sertoli-cell-enclosed apoptotic testis-ova, whereas DMRT1 remained localized in Sertoli cells. These results indicated for the first time that based on the expression of female-specific sex genes, feminization of Sertoli cells precedes testis-ova differentiation induced by estrogen in mature testes in medaka; however, complete feminization of Sertoli cells was not induced in this study. Additionally, it is suggested strongly that Foxl2 and Gsdf expression constitute potential molecular markers for evaluating the effects of estrogenic chemicals on testicular somatic cells associated with estrogen-induced testis-ova differentiation in mature male medaka. Copyright © 2017 Elsevier B.V. All rights reserved.

Postnatal somatic cell proliferation and seminiferous tubule maturation in pigs: A non-random event

PubMed Central

Avelar, Gleide F.; Oliveira, Carolina F.A.; Soares, Jaqueline M.; Silva, Israel J.; Dobrinski, Ina; Hess, Rex A.; França, Luiz R.

2015-01-01

Although seminiferous tubule maturation in horses begins in the central area of the testis, this process is thought to occur randomly throughout the testis in most mammals. Studies in our laboratory revealed that the establishment of spermatogenesis may not be a synchronous event in the testicular parenchyma of pigs. The objectives of the present study were to evaluate the pattern of seminiferous cord/tubule maturation and the morphological and functional characteristics of testicular somatic cells during postnatal development in three regions of the pig testis: a) near the tunica albuginea (TA); b) in the transitional area between the seminiferous tubules and mediastinum (TR); and c) in the intermediate area (ID) between the TA and TR. Based on the diameter of seminiferous cords/tubules, nucleus size of Sertoli cells and fluid secretion, mainly at 90 and 120 d of age, seminiferous tubule maturation was more advanced in the ID and TR. The mitotic activity of Sertoli cells was higher (P < 0.05) in the TR than the ID and TA at 7 and 120 d. Except for the mitotic index of the Leydig cells, which was lower (P < 0.05) in the ID at 7, 30, and 180 d than in the TA and TR, other Leydig cell ebd points, e.g., individual cell size, nuclear volume, and cytoplasmic volume, were consistently higher (P < 0.05) in the ID, suggesting that steroidogenesis was more active in this region during the period investigated. Overall, we inferred that Leydig cells in the ID may play a pivotal role in postnatal testis development in pigs and this type of cell is likely related to asynchronous testicular parenchyma development, with the transitional area providing the primary zone for growth of seminiferous tubules. PMID:20189235

Sexually Dimorphic Expression of Foxl2 and Ftz-F1 in Chinese Giant Salamander Andrias Davidianus.

PubMed

Hu, Qiaomu; Meng, Yan; Tian, Haifeng; Zhang, Y U; Xiao, Hanbing

2016-09-01

Foxl2 and FTZ-F1 play a crucial role in the regulation of gonad development in fish and mammals, but studies of their function in amphibians are scarce. We isolated the full length of Foxl2 (adFoxl2) and Ftz-F1 (adFtz-f1) cDNA from the Chinese giant salamander Andrias davidianus and quantified its expression in various tissues and developing gonads. The adFoxl2 gene encodes 301aa including a conserved forkhead box, and the adFtz-f1 gene encodes 467aa containing an Ftz-F1 box. The amino acid sequences showed high homology with other amphibians. adFoxl2 expression was high in ovary, whereas adFtz-f1 was higher in testis, moderate in pituitary, ovary, and kidney; and low in the remaining tested tissues. Expression of adFoxl2 gradually increased from 1Y to 5Y in ovary, whereas adFtz-f1 expression gradually decreased in testis. In addition, adFoxl2 and adFtz-f1 were detected in granulosa cell in ovary and in spermatocytes in testis. The adFoxl2 transcription was inhibited in brain and ovary after treatment with methyltestosterone and with letrozole, whereas adFtz-f1 expression was upregulated. High-temperature suppressed the expression of adFxl2 in ovary and enhanced the transcription of adFtz-f1. These results suggest that adFoxl2 functioned in ovary differentiation, whereas adFtz-f1 played a role in testis development, which lays a foundation for study of the sex differentiation mechanism in A. davidianus. © 2016 Wiley Periodicals, Inc.

[Subcutaneous transplants of juvenile rat testicular tissues continue to develop and secret androgen in adult rats].

PubMed

Yu, Zhou; Wang, Tong; Cui, Jiangbo; Song, Yajuan; Ma, Xianjie; Su, Yingjun; Peng, Pai

2017-12-01

Objective To explore the effects of subcutaneous microenvironment of adult rats on survival, development and androgen secretion of Leydig cells of transplanted juvenile rat testis. Methods Healthy adult SD rats were randomly divided into control group, sham group, castrated group and non-castrated group. Rats in the control group were kept intact, no testis was transplanted subcutaneously after adult recipients were castrated in the sham group; 5-7-day juvenile rat testes were transplanted subcutaneously in the castrated group, with one testis per side; Testes resected from juvenile rats were directly transplanted subcutaneously on both sides of the recipients in the non-castrated group. The grafts were obtained and weighed 4 weeks later. Then the histological features of the grafts were examined by HE staining; the expression and distribution of hydroxysteroid 17-beta dehydrogenase 1 (HSD-17β1) were investigated by immunohistochemistry; and the serum androgen level was determined by ELISA. Results The average mass of grafts obtained from the castrated group was significantly higher than that of the non-castrated group. Immunohistochemistry indicated that Leydig cells were visible in the tissues from both the castrated and non-castrated groups, but the number of HSD-17β1-posotive cells in the castrated group was larger than that in the non-castrated group. ELISA results showed that the serum androgen level was higher in the control group and non-castrated group than in the sham group and castrated group, and compared with the sham group, the serum androgen level in the castrated group was significantly higher. Conclusion The juvenile rat testis subcutaneously transplanted could further develop under the adult recipient rat skin, and the Leydig cells of grafts harbored the ability to produce and secret androgen.

Analgesic use in pregnancy and male reproductive development

PubMed Central

Hurtado-Gonzalez, Pablo; Mitchell, Rod T.

2017-01-01

Purpose of review Male reproductive disorders are common and increasing in incidence in many countries. Environmental factors (including pharmaceuticals) have been implicated in the development of these disorders. This review aims to summarise the emerging epidemiological and experimental evidence for a potential role of in-utero exposure to analgesics in the development of male reproductive disorders. Recent findings A number of epidemiological studies have demonstrated an association between in-utero exposure to analgesics and the development of cryptorchidism, although these findings are not consistent across all studies. Where present, these associations primarily relate to exposure during the second trimester of pregnancy. In-vivo and in-vitro experimental studies have demonstrated variable effects of exposure to analgesics on Leydig cell function in the fetal testis of rodents, particularly in terms of testosterone production. These effects frequently involve exposures that are in excess of those to which humans are exposed. Investigation of the effects of analgesics on human fetal testis have also demonstrated effects on Leydig cell function. Variation in species, model system, dosage and timing of exposure is likely to contribute to differences in the findings between studies. Summary There is increasing evidence for analgesic effects on the developing testis that have the potential to impair reproductive function. However, the importance of these findings in relation to human-relevant exposures and the risk of male reproductive disorders remains unclear. PMID:28277341

Oct4-GFP expression during transformation of gonocytes into spermatogonial stem cells in the perinatal mouse testis.

PubMed

Li, Ruili; Vannitamby, Amanda; Zhang, Jian-Guo; Fehmel, Emma L; Southwell, Bridget R; Hutson, John M

2015-12-01

In cryptorchidism perinatal failure to switch off Oct4, a germ cell (GC) marker, may lead to carcinoma in situ. We aimed to analyze Oct4 expression during mouse gonocyte transformation into spermatogonial stem cells (SSC). Testes from OG2 (Oct4-promoter driven eGFP) mice at embryonic day (E) 17 and postnatal day P0-10 underwent immunohistochemistry and immunoblotting. Antibodies against MVH, AMH, Ki67, and c-Kit were visualized by confocal microscopy. Numbers of Oct4-GFP(+) GC and Oct4-GFP(-) GC/tubule were counted using ImageJ. Data were analyzed using nonparametric one-way ANOVA. GC from E17-P4 were Oct4-GFP(+). Numbers of Oct4-GFP(-) GC/tubule increased from P6-10, whereas Oct4-GFP(+) GC/tubule numbers remained similar between P6 and P10. Sertoli cells proliferated from E17-P10, whereas GC only proliferated from P2. Gonocytes (Oct4-GFP(+)/c-Kit(-)) central in tubules migrated to the basement membrane to become prospermatogonia (Oct4-GFP(+)/c-Kit(-)) and then SSC (Oct4-GFP(+)/c-Kit(+)) from day 4 and further developed into Oct4-GFP(-)/c-Kit(+) at P6. In Oct4-GFP mice both centrally located gonocytes and prospermatogonia located at the tubular basement membrane were Oct4-GFP(+)/c-Kit(-) before further developing into SSC (Oct4-GFP(+)/c-Kit(+)). This indicates that Oct4 is important in gonocyte transformation into SSC. Understanding this process will aid GC tumor diagnostics and fertility potential in boys with UDT undergoing orchidopexy. Copyright © 2015 Elsevier Inc. All rights reserved.

Sex determination in mythology and history.

PubMed

Mittwoch, Ursula

2005-02-01

The history of ideas on how the sexes became divided spans at least three thousand years. The biblical account of the origin of Eve, and the opinions of the philosophers of classical Greece, have unexpected bearings on present-day ideas. The scientific study of sex determination can be said to have begun in the 17th century with the discovery of spermatozoa, but the origin and function of the "spermatic animalcules" eluded investigators until 1841. The mammalian egg was discovered in 1827, and in the last quarter of the century fertilization was observed. The view current at that time, that sex determination was under environmental control, gave way to the idea of chromosomal determination in the first quarter of the 20th century. The study of human and other mammalian chromosomes during the third quarter of the century, and the discovery of sex-chromosome abnormalities, emphasized the importance of the Y chromosome for male sex determination. The last quarter of the century witnessed a hunt for the "testis-determining" gene, thought to be responsible for the differentiation of Sertoli cells, and culminating in the isolation of SRY (Sry in the mouse). However, an increasing number of additional genes and growth factors were found to be required for the establishment of male sex. During the same period evidence emerged that male development was accompanied by enhanced growth, both of gonads and whole embryos. An unexpected finding was the demonstration of temperature-dependent sex determination in reptiles. With the advent of the 21st century, it was shown that Sry induces cell proliferation in fetal mouse gonads, and it has been suggested that male sex differentiation in mammals requires a higher metabolic rate. These insights could lead to a better understanding and improved treatment of abnormalities of sexual development.

Hanging drop cultures of human testis and testis cancer samples: a model used to investigate activin treatment effects in a preserved niche.

PubMed

Jørgensen, A; Young, J; Nielsen, J E; Joensen, U N; Toft, B G; Rajpert-De Meyts, E; Loveland, K L

2014-05-13

Testicular germ cell tumours of young adults, seminoma or non-seminomas, are preceded by a pre-invasive precursor, carcinoma in situ (CIS), understood to arise through differentiation arrest of embryonic germ cells. Knowledge about the malignant transformation of germ cells is currently limited by the lack of experimental models. The aim of this study was to establish an experimental tissue culture model to maintain normal and malignant germ cells within their niche and allow investigation of treatment effects. Human testis and testis cancer specimens from orchidectomies were cultured in 'hanging drops' and effects of activin A and follistatin treatment were investigated in seminoma cultures. Testis fragments with normal spermatogenesis or CIS cells were cultured for 14 days with sustained proliferation of germ cells and CIS cells and without increased apoptosis. Seminoma cultures survived 7 days, with proliferating cells detectable during the first 5 days. Activin A treatment significantly reduced KIT transcript and protein levels in seminoma cultures, thereby demonstrating a specific treatment response. Hanging drop cultures of human testis and testis cancer samples can be employed to delineate mechanisms governing growth of normal, CIS and tumorigenic germ cells retained within their niche.

[Cloning and characterization of a novel rat gene RSD-7 differentially expressed in testis].

PubMed

Zhang, Xiao-dong; Gou, Da-wei; Miao, Shi-ying; Zhang, Jian-chao; Zong, Shu-dong; Wang, Lin-fang

2003-06-01

To isolate and identify the differentially expressed genes in spermatogenesis for the understanding molecular mechanism of spermatogenesis. Screening of the cDNA library, Northern blot, expression and purification in E. coli with GST expression system, immunocytochemical staining of testis sections were used. (1) A cDNA fragment designated as RSD-7 was isolated from rat testis cDNA library. It was 1,238 bp in length, coding a protein of 232 amino acids with the GenBank accession number AF315467. The encoding protein of RSD-7 cDNA had a Ubiquitin-like domain. (2) Northern blot indicated that RSD-7 was uniquely expressed in rat testis, and in the testis RSD-7 emerged on the 30th postnatal day and expressed until 120th postnatal day. (3) Expression and purification of RSD-7 protein in E. coli with GST expression system and were used to obtain anti-RSD-7 antibody. (4) Immunolocalization of RSD-7 in rat testis revealed that it is expressed only in Sertoli cells. Transcription pattern of RSD-7 and localization of RSD-7 protein in testis have been made, which established the base for the functional study of RSD-7.

Secreted Frizzled-related protein 1 (sFRP1) regulates spermatid adhesion in the testis via dephosphorylation of focal adhesion kinase and the nectin-3 adhesion protein complex

PubMed Central

Wong, Elissa W. P.; Lee, Will M.; Cheng, C. Yan

2013-01-01

Development of spermatozoa in adult mammalian testis during spermatogenesis involves extensive cell migration and differentiation. Spermatogonia that reside at the basal compartment of the seminiferous epithelium differentiate into more advanced germ cell types that migrate toward the apical compartment until elongated spermatids are released into the tubule lumen during spermiation. Apical ectoplasmic specialization (ES; a testis-specific anchoring junction) is the only cell junction that anchors and maintains the polarity of elongating/elongated spermatids (step 8–19 spermatids) in the epithelium. Little is known regarding the signaling pathways that trigger the disassembly of the apical ES at spermiation. Here, we show that secreted Frizzled-related protein 1 (sFRP1), a putative tumor suppressor gene that is frequently down-regulated in multiple carcinomas, is a crucial regulatory protein for spermiation. The expression of sFRP1 is tightly regulated in adult rat testis to control spermatid adhesion and sperm release at spermiation. Down-regulation of sFRP1 during testicular development was found to coincide with the onset of the first wave of spermiation at approximately age 45 d postpartum, implying that sFRP1 might be correlated with elongated spermatid adhesion conferred by the apical ES before spermiation. Indeed, administration of sFRP1 recombinant protein to the testis in vivo delayed spermiation, which was accompanied by down-regulation of phosphorylated (p)-focal adhesion kinase (FAK)-Tyr397 and retention of nectin-3 adhesion protein at the apical ES. To further investigate the functional relationship between p-FAK-Tyr397 and localization of nectin-3, we overexpressed sFRP1 using lentiviral vectors in the Sertoli-germ cell coculture system. Consistent with the in vivo findings, overexpression of sFRP1 induced down-regulation of p-FAK-Tyr397, leading to a decline in phosphorylation of nectin-3. In summary, this report highlights the critical role of sFRP1 in regulating spermiation via its effects on the FAK signaling and retention of nectin-3 adhesion complex at the apical ES.—Wong, E. W. P., Lee, W. M., Cheng, C. Y. Secreted Frizzled-related protein 1 (sFRP1) regulates spermatid adhesion in the testis via dephosphorylation of focal adhesion kinase and the nectin-3 adhesion protein complex. PMID:23073828

Transcriptome analysis reveals differentially expressed genes associated with germ cell and gonad development in the Southern bluefin tuna (Thunnus maccoyii).

PubMed

Bar, Ido; Cummins, Scott; Elizur, Abigail

2016-03-10

Controlling and managing the breeding of bluefin tuna (Thunnus spp.) in captivity is an imperative step towards obtaining a sustainable supply of these fish in aquaculture production systems. Germ cell transplantation (GCT) is an innovative technology for the production of inter-species surrogates, by transplanting undifferentiated germ cells derived from a donor species into larvae of a host species. The transplanted surrogates will then grow and mature to produce donor-derived seed, thus providing a simpler alternative to maintaining large-bodied broodstock such as the bluefin tuna. Implementation of GCT for new species requires the development of molecular tools to follow the fate of the transplanted germ cells. These tools are based on key reproductive and germ cell-specific genes. RNA-Sequencing (RNA-Seq) provides a rapid, cost-effective method for high throughput gene identification in non-model species. This study utilized RNA-Seq to identify key genes expressed in the gonads of Southern bluefin tuna (Thunnus maccoyii, SBT) and their specific expression patterns in male and female gonad cells. Key genes involved in the reproductive molecular pathway and specifically, germ cell development in gonads, were identified using analysis of RNA-Seq transcriptomes of male and female SBT gonad cells. Expression profiles of transcripts from ovary and testis cells were compared, as well as testis germ cell-enriched fraction prepared with Percoll gradient, as used in GCT studies. Ovary cells demonstrated over-expression of genes related to stem cell maintenance, while in testis cells, transcripts encoding for reproduction-associated receptors, sex steroids and hormone synthesis and signaling genes were over-expressed. Within the testis cells, the Percoll-enriched fraction showed over-expression of genes that are related to post-meiosis germ cell populations. Gonad development and germ cell related genes were identified from SBT gonads and their expression patterns in ovary and testis cells were determined. These expression patterns correlate with the reproductive developmental stage of the sampled fish. The majority of the genes described in this study were sequenced for the first time in T. maccoyii. The wealth of SBT gonadal and germ cell-related gene sequences made publicly available by this study provides an extensive resource for further GCT and reproductive molecular biology studies of this commercially valuable fish.

Effects of a simulated microgravity model on cell structure and function in rat testis and epididymis

NASA Technical Reports Server (NTRS)

Hadley, Jill A.; Hall, Joseph C.; O'Brien, Ami; Ball, Richard

1992-01-01

The effect of simulated microgravity on the structure and function of the testis and epididymis cells was investigated in rats subjected to 7 days of tail suspension. Results of a histological examination revealed presence of disorganized seminiferous tubules and accumulation of large multinucleated cells and spermatids in the lumen of the epididymis. In addition, decreases in the content of testis protein and in testosterone levels in the testis, the interstitial fluid, and the epididymis were observed.

Misuse of ultrasound for palpable undescended testis by primary care providers: A prospective study

PubMed Central

Wong, Nathan C.; Bansal, Rahul K.; Lorenzo, Armando J.; DeMaria, Jorge; Braga, Luis H.

2015-01-01

Introduction: Although previous evidence has shown that ultrasound is unreliable to diagnose undescended testis, many primary care providers (PCP) continue to misuse it. We assessed the performance of ultrasound as a diagnostic tool for palpable undescended testis, as well as the diagnostic agreement between PCP and pediatric urologists. Methods: We performed a prospective observational cohort study between 2011 and 2013 for consecutive boys referred with a diagnosis of undescended testis to our tertiary pediatric hospital. Patients referred without an ultrasound and those with non-palpable testes were excluded. Data on referring diagnosis, pediatric urology examination and ultrasound reports were analyzed. Results: Our study consisted of 339 boys. Of these, patients without an ultrasound (n = 132) and those with non-palpable testes (n = 38) were excluded. In the end, there were 169 pateints in this study. Ultrasound was performed in 50% of referred boys showing 256 undescended testis. The mean age at time of referral was 45 months. When ultrasound was compared to physical examination by the pediatric urologist, agreement was only 34%. The performance of ultrasound for palpable undescended testis was: sensitivity = 100%; specificity = 16%; positive predictive value = 34%; negative predictive value = 100%; positive likelihood ratio = 1.2; and negative likelihood ratio = 0. Diagnosis of undescended testis by PCP was confirmed by physical examination in 30% of cases, with 70% re-diagnosed with normal or retractile testes. Conclusion: Ultrasound performed poorly to assess for palpable undescended testis in boys and should not be used. Although the study has important limitations, there is an increasing need for education and evidence-based guidelines for PCP in the management of undescended testis. PMID:26788226

HISTOLOGICAL AND HISTOPATHOLOGICAL EVALUATION OF THE TESTIS

EPA Science Inventory

This book, the first to describe how the testis is evaluated in research and toxicology testing settings, is a resource for individuals who wish to perform a systematic evaluation of the testis. he book contains 728 illustrations and drawings. The book begins with a description o...

Testisimmune privilege - Assumptions versus facts

PubMed Central

Kaur, G.; Mital, P.; Dufour, J.M.

2013-01-01

The testis has long enjoyed a reputation as an immunologically privileged site based on its ability to protect auto-antigenic germ cells and provide an optimal environment for the extended survival of transplanted allo- or xeno-grafts. Exploration of the role of anatomical, physiological, immunological and cellular components in testis immune privilege revealed that the tolerogenic environment of the testis is a result of the immunomodulatory factors expressed or secreted by testicular cells (mainly Sertoli cells, peritubular myoid cells, Leydig cells, and resident macrophages). The blood-testis barrier/Sertoli cell barrier, is also important to seclude advanced germ cells but its requirement in testis immune privilege needs further investigation. Testicular immune privilege is not permanent, as an effective immune response can be mounted against transplanted tissue, and bacterial/viral infections in the testis can be effectively eliminated. Overall, the cellular components control the fate of the immune response and can shift the response from immunodestructive to immunoprotective, resulting in immune privilege. PMID:25309630

Anatomy and histology of the scrotal ligament in adults: inconsistency and variability of the gubernaculum testis.

PubMed

Cavalie, G; Bellier, Alexandre; Marnas, G; Boisson, B; Robert, Y; Rabattu, P Y; Chaffanjon, P

2018-04-01

The anatomy of gubernaculum testis (GT) is often discussed; however, the postnatal anatomy of the GT or scrotal ligament (SL) is rarely described. Hence, we performed an anatomical and histological study to analyze histologically the structures between testis and scrotum. We performed anatomical dissections on 25 human fresh cadavers' testes. Each testis was removed with its envelopes and macroscopically analyzed. Then samples were included for histological study. Finally, they were analyzed under microscope, looking for attachments between testis, epididymis and scrotal envelopes. The absence of proximal and distal attachment was found in 56.0% of cases. Looking at the proximal attachment of the SL, the main one found is the epididymal attachment (28.0%), whereas no cases of testis attachment was found. Distally, there are more variations with scrotal attachment (12%) and cremaster attachment (12.0%). We found a significant prevalence of multiple adherences in 16.0% of cases too. Finally, in 15 cases (57.7%) an attachment is present between testis and epididymis, as it is commonly described. In the majority of cases there is no attachment of the lower pole of the testis and epididymis and these structures remain free. So it seems that the SL disappears with aging. Moreover, there is not only one kind of ligamentous attachment, but a high variability of attachments at the lower pole of the testiculo-epididymal structure. When it exists, this structure is never a real ligament and it seems more appropriate to use the term "attachments".

Characteristics of PCR-SSCP and RAPD-HPCE methods for identifying authentication of Penis et testis cervi in Traditional Chinese Medicine based on cytochrome b gene.

PubMed

Li, Mingcheng; Gao, Lijun; Qu, Li; Sun, Jingyu; Yuan, Guangxin; Xia, Wei; Niu, Jiamu; Fu, Guilian; Zhang, Lihua

2016-07-01

The use of Penis et testis cervi, as a kind of precious Traditional Chinese Medicine (TCM), which is derived from dry deer's testis and penis, has been recorded for many years in China. There are abundant species of deer in China, the Penis et testis from species of Cervus Nippon and Cervus elaphusL were authentic, others species were defined as adulterant (different subspecies of deer) or counterfeits (different species). Identification of their origins or authenticity becomes a key in controlling the herbal products. A modified column chromatography was used to extract mitochondrial DNA of dried deer's testis and penis from sika deer (C. Nippon) and red deer (C. elaphusL) in addition to adulterants and counterfeits. Column chromatography requires for a short time to extract mitochondrial DNA of high purity with little damage of DNA molecules, which provides the primary structure of guarantee for the specific PCR; PCR-SSCP method showed a clear intra-specific difference among patterns of single-chain fragments, and completely differentiate Penis et testis origins from C. Nippon and C. elaphusL. RAPD-HPCE was based on the standard electropherograms to compute a control spectrum curve as similarity reference (R) among different samples. The similarity analysis indicated that there were significant inter-species differences among Penis et testis' adulterant or counterfeits. Both techniques provide a fast, simple, and accurate way to directly identify among inter-species or intra-species of Penis et testis.

A single-dose live-attenuated vaccine prevents Zika virus pregnancy transmission and testis damage.

PubMed

Shan, Chao; Muruato, Antonio E; Jagger, Brett W; Richner, Justin; Nunes, Bruno T D; Medeiros, Daniele B A; Xie, Xuping; Nunes, Jannyce G C; Morabito, Kaitlyn M; Kong, Wing-Pui; Pierson, Theodore C; Barrett, Alan D; Weaver, Scott C; Rossi, Shannan L; Vasconcelos, Pedro F C; Graham, Barney S; Diamond, Michael S; Shi, Pei-Yong

2017-09-22

Zika virus infection during pregnancy can cause congenital abnormities or fetal demise. The persistence of Zika virus in the male reproductive system poses a risk of sexual transmission. Here we demonstrate that live-attenuated Zika virus vaccine candidates containing deletions in the 3' untranslated region of the Zika virus genome (ZIKV-3'UTR-LAV) prevent viral transmission during pregnancy and testis damage in mice, as well as infection of nonhuman primates. After a single-dose vaccination, pregnant mice challenged with Zika virus at embryonic day 6 and evaluated at embryonic day 13 show markedly diminished levels of viral RNA in maternal, placental, and fetal tissues. Vaccinated male mice challenged with Zika virus were protected against testis infection, injury, and oligospermia. A single immunization of rhesus macaques elicited a rapid and robust antibody response, conferring complete protection upon challenge. Furthermore, the ZIKV-3'UTR-LAV vaccine candidates have a desirable safety profile. These results suggest that further development of ZIKV-3'UTR-LAV is warranted for humans.Zika virus infection can result in congenital disorders and cause disease in adults, and there is currently no approved vaccine. Here Shan et al. show that a single dose of a live-attenuated Zika vaccine prevents infection, testis damage and transmission to the fetus during pregnancy in different animal models.

Effects of the antiandrogens, vinclozolin and cyproterone acetate on gonadal development in the Japanese medaka (Oryzias latipes).

PubMed

Kiparissis, Yiannis; Metcalfe, Tracy L; Balch, Gordon C; Metcalfe, Chris D

2003-05-29

This study was focused on determining the effects of exposure to antiandrogens on the gonadal development of Japanese medaka (Oryzias latipes). Test compounds included the fungicide, vinclozolin and the clinical antiandrogen, cyproterone acetate. Newly hatched medaka were exposed to aqueous solutions of vinclozolin (2500 microg/l) and the vinclozolin fungicide formulation, Ronilan (1000 and 5000 microg/l) and cyproterone acetate (1 and 10 microg/l), for 3 months. Histological evaluation of the gonadal tissues of exposed fish indicated that the 5000 microg/l concentration of the vinclozolin formulation (Ronilan) induced a low incidence of intersex (i.e. testis-ova) and the 2500 microg/l concentration of vinclozolin-affected spermatogenesis in males. Also, the vinclozolin treatments induced moderate ovarian atresia. Cyproterone acetate also induced a low incidence of testis-ova, but in contrast to the vinclozolin treatment the amount of ovarian tissue in the testis-ova was equal to or greater than the amount of testicular tissue. In the cyproterone acetate treatments, both oogenesis and spermatogenesis were moderately inhibited at all test concentrations. The results of this study indicate that antiandrogens have the potential to alter testicular development and gametogenesis in fish. However, research is needed to determine the mechanisms by which antiandrogens affect fish.

Interaction of oligomeric breast cancer resistant protein (BCRP) with adjudin: a male contraceptive with anti-cancer activity.

PubMed

Cheng, Yan Ho; Jenardhanan, Pranitha; Mathur, Premendu P; Qian, Xiaojing; Xia, Weiliang; Silvestrini, Bruno; Cheng, Chuen Yan

2014-01-01

Breast cancer resistant protein (BCRP, ABCG2) is an ATP-binding cassette (ABC) transporter, which together with two other ABC efflux drug pumps, namely P-glycoprotein (P-gp, ABCB1) and multidrug resistance-related protein 1 (MRP1, ABCC1) is the most important multidrug resistance protein foun d in eukaryotic cells including cells in the testis. However, unlike P-gp and MRP1, which are components of the Sertoli cell blood-testis barrier (BTB), BCRP is not expressed at the BTB in rodents and human testes. Instead, BCRP is expressed by peritubular myoid cells and endothelial cells of the lymphatic vessel in the tunica propria, residing outside the BTB. As such, the testis is equipped with two levels of defense against xenobiotics or drugs, preventing these harmful substances from entering the adluminal compartment to perturb meiosis and post-meiotic spermatid development: one at the level of the BTB conferred by P-gp and MRP1 and one at the tunica propria conferred by BCRP. The presence of drug transporters at the tunica propria as well as at the Sertoli cell BTB thus poses significant obstacles in developing non-hormonal contraceptives if these drugs (e.g., adjudin) exert their effects in germ cells behind the BTB, such as in the adluminal (apical) compartment of the seminiferous epithelium. Herein, we summarize recent findings pertinent to adjudin, a non-hormonal male contraceptive, and molecular interactions of adjudin with BCRP so that this information can be helpful to devise delivery strategies to evade BCRP in the tunica propria to improve its bioavailability in the testis.

Specific Tandem 3'UTR Patterns and Gene Expression Profiles in Mouse Thy1+ Germline Stem Cells

PubMed Central

Lin, Zhuoheng; Feng, Xuyang; Jiang, Xue; Songyang, Zhou; Huang, Junjiu

2015-01-01

A recently developed strategy of sequencing alternative polyadenylation (APA) sites (SAPAS) with second-generation sequencing technology can be used to explore complete genome-wide patterns of tandem APA sites and global gene expression profiles. spermatogonial stem cells (SSCs) maintain long-term reproductive abilities in male mammals. The detailed mechanisms by which SSCs self-renew and generate mature spermatozoa are not clear. To understand the specific alternative polyadenylation pattern and global gene expression profile of male germline stem cells (GSCs, mainly referred to SSCs here), we isolated and purified mouse Thy1+ cells from testis by magnetic-activated cell sorting (MACS) and then used the SAPAS method for analysis, using pluripotent embryonic stem cells (ESCs) and differentiated mouse embryonic fibroblast cells (MEFs) as controls. As a result, we obtained 99,944 poly(A) sites, approximately 40% of which were newly detected in our experiments. These poly(A) sites originated from three mouse cell types and covered 17,499 genes, including 831 long non-coding RNA (lncRNA) genes. We observed that GSCs tend to have shorter 3'UTR lengths while MEFs tend towards longer 3'UTR lengths. We also identified 1337 genes that were highly expressed in GSCs, and these genes were highly consistent with the functional characteristics of GSCs. Our detailed bioinformatics analysis identified APA site-switching events at 3'UTRs and many new specifically expressed genes in GSCs, which we experimentally confirmed. Furthermore, qRT-PCR was performed to validate several events of the 334 genes with distal-to-proximal poly(A) switch in GSCs. Consistently APA reporter assay confirmed the total 3'UTR shortening in GSCs compared to MEFs. We also analyzed the cis elements around the proximal poly(A) site preferentially used in GSCs and found C-rich elements may contribute to this regulation. Overall, our results identified the expression level and polyadenylation site profiles and these data provide new insights into the processes potentially involved in the GSC life cycle and spermatogenesis. PMID:26713853

Effects of dietary omega-3/omega-6 fatty acid ratios on reproduction in the young breeder rooster.

PubMed

Feng, Yun; Ding, Yu; Liu, Juan; Tian, Ye; Yang, Yanzhou; Guan, Shuluan; Zhang, Cheng

2015-03-21

Polyunsaturated fatty acids (PUFAs) are necessary for the body's metabolism, growth and development. Although PUFAs play an important role in the regulation of reproduction, their role in testis development in the rooster is unknown. The present study was conducted to investigate the effects of omega-3/omega-6 (n-3/n-6, PUFAs) ratios on reproductive performance in young breeder roosters. Plasma levels of reproductive hormones, testis development, and reproductive hormone receptor and StAR mRNA expression were also assessed. Although PUFAs (n-3/n-6: 1/4.15) had no significant effect on the testis index (P > 0.05), the spermatogonial development and germ cell layers were increased. Moreover, serum levels of hormones (GnRH, FSH, LH and T) on day 35 were also significantly increased by PUFAs (n-3/n-6: 1/4.15). To investigate whether PUFAs regulate the expression of hormone receptors and StAR, real time-PCR was used to measure GnRHR, FSHR, LHR and StAR mRNA levels. PUFAs significantly increased the mRNA levels of all of these genes. These results indicate that PUFAs enhance the reproductive performance of young roosters by increasing hormone secretion and function, the latter by up-regulating receptor expression. These findings provide a sound basis for a balanced n-3/n-6 PUFA ratio being beneficial to young rooster reproduction.

Effects of gestational exposure to di-n-butyl phthalate and mineral oil on testis development of the Mongolian gerbil.

PubMed

Christante, C M; Pinto-Fochi, M E; Negrin, A C; Taboga, S R; Góes, R M

2018-06-14

Phthalate esters are endocrine disrupters that can affect the development of the testis in a species-specific manner. However, their interference in the male gonads of the Mongolian gerbil is unknown. The aim of the present study was to evaluate whether gestational exposure to di-n-butyl phthalate (DBP) interferes with the development of the gerbil testis during the first six weeks of life. Males were evaluated at 1, 7, 14, 28, 35 and 42 days of age in an untreated (control) group or groups exposed from 8 to 23 days gestation to DBP (100mgkg-1day-1 in mineral oil) or vehicle by maternal gavage. DBP exposure impaired cell proliferation within the seminiferous cords at birth, but increased proliferation at the end of the first week, when higher testosterone concentrations were observed. The vehicle (mineral oil) reduced the total number of gonocytes and attenuated the decrease in testosterone concentrations at 7 days. The vehicle also altered gonocyte relocation at 14 days and increased oestrogen concentrations at 28 days by approximately 112%. In summary, both DBP and oil interfered in gonadal development and testosterone plasma concentrations in the first week of postnatal life. However, the changes observed at the beginning of puberty were not seen after exposure to DBP, indicating a more harmful effect of mineral oil in this period.

Hanging drop cultures of human testis and testis cancer samples: a model used to investigate activin treatment effects in a preserved niche

PubMed Central

Jørgensen, A; Young, J; Nielsen, J E; Joensen, U N; Toft, B G; Rajpert-De Meyts, E; Loveland, K L

2014-01-01

Background: Testicular germ cell tumours of young adults, seminoma or non-seminomas, are preceded by a pre-invasive precursor, carcinoma in situ (CIS), understood to arise through differentiation arrest of embryonic germ cells. Knowledge about the malignant transformation of germ cells is currently limited by the lack of experimental models. The aim of this study was to establish an experimental tissue culture model to maintain normal and malignant germ cells within their niche and allow investigation of treatment effects. Methods: Human testis and testis cancer specimens from orchidectomies were cultured in ‘hanging drops' and effects of activin A and follistatin treatment were investigated in seminoma cultures. Results: Testis fragments with normal spermatogenesis or CIS cells were cultured for 14 days with sustained proliferation of germ cells and CIS cells and without increased apoptosis. Seminoma cultures survived 7 days, with proliferating cells detectable during the first 5 days. Activin A treatment significantly reduced KIT transcript and protein levels in seminoma cultures, thereby demonstrating a specific treatment response. Conclusions: Hanging drop cultures of human testis and testis cancer samples can be employed to delineate mechanisms governing growth of normal, CIS and tumorigenic germ cells retained within their niche. PMID:24781282

Selective deletion of Smad4 in postnatal germ cells does not affect spermatogenesis or fertility in mice.

PubMed

Hao, Xiao-Xia; Chen, Su-Ren; Tang, Ji-Xin; Li, Jian; Cheng, Jin-Mei; Jin, Cheng; Wang, Xiu-Xia; Liu, Yi-Xun

2016-07-01

SMAD4 is the central component of canonical signaling in the transforming growth factor beta (TGFβ) superfamily. Loss of Smad4 in Sertoli cells affects the expansion of the fetal testis cords, whereas selective deletion of Smad4 in Leydig cells alone does not appreciably alter fetal or adult testis development. Loss of Smad4 in Sertoli and Leydig cells, on the other hand, leads to testicular dysgenesis, and tumor formation in mice. Within the murine testes, Smad4 is also expressed in germ cells of the seminiferous tubules. We therefore, crossed Ngn3-Cre or Stra8-Cre transgenic mice with Smad4-flox mice to generate conditional knockout animals in which Smad4 was specifically deleted in postnatal germ cells to further uncover cell type-specific requirement of Smad4. Unexpectedly, these germ-cell-knockout mice were fertile and did not exhibit any detectable abnormalities in spermatogenesis, indicating that Smad4 is not required for the production of sperm; instead, these data indicate a cell type-specific requirement of Smad4 primarily during testis development. Mol. Reprod. Dev. 83: 615-623, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Nodavirus Colonizes and Replicates in the Testis of Gilthead Seabream and European Sea Bass Modulating Its Immune and Reproductive Functions

PubMed Central

Valero, Yulema; Arizcun, Marta; Esteban, M. Ángeles; Bandín, Isabel; Olveira, José G.; Patel, Sonal; Cuesta, Alberto; Chaves-Pozo, Elena

2015-01-01

Viruses are threatening pathogens for fish aquaculture. Some of them are transmitted through gonad fluids or gametes as occurs with nervous necrosis virus (NNV). In order to be transmitted through the gonad, the virus should colonize and replicate inside some cell types of this tissue and avoid the subsequent immune response locally. However, whether NNV colonizes the gonad, the cell types that are infected, and how the immune response in the gonad is regulated has never been studied. We have demonstrated for the first time the presence and localization of NNV into the testis after an experimental infection in the European sea bass (Dicentrarchus labrax), and in the gilthead seabream (Sparus aurata), a very susceptible and an asymptomatic host fish species, respectively. Thus, we localized in the testis viral RNA in both species using in situ PCR and viral proteins in gilthead seabream by immunohistochemistry, suggesting that males might also transmit the virus. In addition, we were able to isolate infective particles from the testis of both species demonstrating that NNV colonizes and replicates into the testis of both species. Blood contamination of the tissues sampled was discarded by completely fish bleeding, furthermore the in situ PCR and immunocytochemistry techniques never showed staining in blood vessels or cells. Moreover, we also determined how the immune and reproductive functions are affected comparing the effects in the testis with those found in the brain, the main target tissue of the virus. Interestingly, NNV triggered the immune response in the European sea bass but not in the gilthead seabream testis. Regarding reproductive functions, NNV infection alters 17β-estradiol and 11-ketotestosterone production and the potential sensitivity of brain and testis to these hormones, whereas there is no disruption of testicular functions according to several reproductive parameters. Moreover, we have also studied the NNV infection of the testis in vitro to assess local responses. Our in vitro results show that the changes observed on the expression of immune and reproductive genes in the testis of both species are different to those observed upon in vivo infections in most of the cases. PMID:26691348

Aspiration and tetracycline sclerotherapy of primary vaginal hydrocoele of testis in adults.

PubMed

Ali, Johar; Anwar, Waqas; Akbar, Mohammad; Akbar, Syed Ali; Zafar, Arshad

2008-01-01

Primary Vaginal Hydrocoele of Testis is a common condition which is primarily treated surgically. Many patients with Hydrocoele of testis are either not willing or are unfit for surgery. This study was done to know the safety, efficacy and out come of tetracycline induced sclerotherapy of Primary Vaginal Hydrocoele of Testis in adults. This quasi experimental study was done in Shahina Jamil Hospital, attached with Frontier Medical College and Ayub Teaching Hospital, Abbottabad from March 2006 to April, 2007. Thirty-seven patients with primary vaginal hydrocoele were included in the study. Aspiration and instillation of Tetracycline was done after spermatic cord block with 2% lignocaine. Procedure time, Peri and Post-procedure complications, number of injections for cure and patients' satisfaction with the procedure were recorded. Patients were discharged home 3 to 4 hours after the procedure and followed up after one week, one month, three months and six months. Direct admission and re-admissions were recorded. The mean age of patients was 47 years. Mean procedure time was 45 minutes. All patients were cured. Mild postoprocedure pain occurred in 12 (40%), moderate pain in 14 (46%) patients and severe pain in 4 (13.3%) patients. No patient developed haematoma or local infection. One patient (3.3%) had micturition problem. Two (6.6%) patients had minimal recurrence. One injection was sufficient for cure in all patients. 28 (93%) patients were satisfied while 2 (6.6%) patients were not satisfied with this procedure. No patient was admitted in the hospital after the procedure. Aspiration and injection of tetracycline in Primary vaginal Hydrocoele of Testis in adults is safe, effective and very economical procedure.

Breast cancer resistance protein regulates apical ectoplasmic specialization dynamics stage specifically in the rat testis

PubMed Central

Qian, Xiaojing; Mruk, Dolores D.; Wong, Elissa W. P.

2013-01-01

Drug transporters determine the bioavailability of drugs in the testis behind the blood-testis barrier (BTB). Thus, they are crucial for male contraceptive development if these drugs (e.g., adjudin) exert their effects behind the BTB. Herein breast cancer resistance protein (Bcrp), an efflux drug transporter, was found to be expressed by both Sertoli and germ cells. Interestingly, Bcrp was not a component of the Sertoli cell BTB. Instead, it was highly expressed by peritubular myoid cells at the tunica propria and also endothelial cells of the microvessels in the interstitium at all stages of the epithelial cycle. Unexpectedly, Bcrp was found to be expressed at the Sertoli-step 18–19 spermatid interface but limited to stage VI-early VIII tubules, and an integrated component of the apical ectoplasmic specialization (apical ES). Apparently, Bcrp is being used by late-stage spermatids to safeguard their completion of spermiogenesis by preventing harmful drugs to enter these cells while they transform to spermatozoa. Also, the association of Bcrp with actin, Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein), and Arp3 (actin-related protein 3, a component of the Arp2/3 complex known to induce branched actin polymerization) at the apical ES suggest that Bcrp may be involved in regulating the organization of actin filament bundles at the site. Indeed, a knockdown of Bcrp by RNAi in the testis perturbed the apical ES function, disrupting spermatid polarity and adhesion. In summary, Bcrp is a regulator of the F-actin-rich apical ES in the testis. PMID:23403943

[Triorchidism: which therapy?

PubMed

Piro, Eugenia; Abati, Laura; Zocca, Veronica; Brugnoni, Marta; D'Alessio, Antonio

2017-06-23

Polyorchidism is an anomaly characterized by more than two gonads; triorchidism is the most common variant. Its management is controversial, mostly when surgical treatment is occasional. CB, 14 year-old, came to the hospital due to right-sided testicular torsion. During surgery, testis was rotated and the contralateral testis, which presented as an anatomically continuum with a gonadic structure similar to the other testes but with a smaller diameter, was fixed. We performed biopsy on both left testes and decided to preserve the supernumerary one. Following the anatomic and functional classification of polyorchidism by Singer, preservation is justified on the grounds of the presence of a supernumerary testis that drains into the epididymis of the normal testis, merging into one single deferent duct (Singer Type 1). At biopsy, both testes had a valid spermatogenic asset. The diagnostic follow-up at 6 and 12 months did not show any pathological alteration. Diagnosis of polyorchidism is occasional. Its treatment varies depending on the site, dimension, and anatomy of the drainage system of the supernumerary testis. If the supernumerary testis is preserved, a standardized diagnostic follow-up is recommended.

Novel noncoding RNA from human Y distal heterochromatic block (Yq12) generates testis-specific chimeric CDC2L2

PubMed Central

Jehan, Zeenath; Vallinayagam, Sambandam; Tiwari, Shrish; Pradhan, Suman; Singh, Lalji; Suresh, Amritha; Reddy, Hemakumar M.; Ahuja, Y.R.; Jesudasan, Rachel A.

2007-01-01

The human Y chromosome, because it is enriched in repetitive DNA, has been very intractable to genetic and molecular analyses. There is no previous evidence for developmental stage- and testis-specific transcription from the male-specific region of the Y (MSY). Here, we present evidence for the first time for a developmental stage- and testis-specific transcription from MSY distal heterochromatic block. We isolated two novel RNAs, which localize to Yq12 in multiple copies, show testis-specific expression, and lack active X-homologs. Experimental evidence shows that one of the above Yq12 noncoding RNAs (ncRNAs) trans-splices with CDC2L2 mRNA from chromosome 1p36.3 locus to generate a testis-specific chimeric β sv13 isoform. This 67-nt 5′UTR provided by the Yq12 transcript contains within it a Y box protein-binding CCAAT motif, indicating translational regulation of the β sv13 isoform in testis. This is also the first report of trans-splicing between a Y chromosomal and an autosomal transcript. PMID:17095710

Pentacle gold-copper alloy nanocrystals: a new system for entering male germ cells in vitro and in vivo

NASA Astrophysics Data System (ADS)

Lin, Yu; He, Rong; Sun, Liping; Yang, Yushan; Li, Wenqing; Sun, Fei

2016-12-01

Gold-based nanocrystals have attracted considerable attention for drug delivery and biological applications due to their distinct shapes. However, overcoming biological barriers is a hard and inevitable problem, which restricts medical applications of nanomaterials in vivo. Seeking for an efficient transportation to penetrate biological barriers is a common need. There are three barriers: blood-testis barrier, blood-placenta barrier, and blood-brain barrier. Here, we pay close attention to the blood-testis barrier. We found that the pentacle gold-copper alloy nanocrystals not only could enter GC-2 cells in vitro in a short time, but also could overcome the blood-testis barrier and enter male germ cells in vivo. Furthermore, we demonstrated that the entrance efficiency would become much higher in the development stages. The results also suggested that the pentacle gold-copper alloy nanocrystals could easier enter to germ cells in the pathological condition. This system could be a new method for theranostics in the reproductive system.

Identification of a testis-enriched heat shock protein and fourteen members of Hsp70 family in the swamp eel.

PubMed

He, Yan; Luo, Majing; Yi, Minhan; Sheng, Yue; Cheng, Yibin; Zhou, Rongjia; Cheng, Hanhua

2013-01-01

Gonad differentiation is one of the most important developmental events in vertebrates. Some heat shock proteins are associated with gonad development. Heat shock protein 70 (Hsp70) in the teleost fish and its roles in sex differentiation are poorly understood. We have identified a testis-enriched heat shock protein Hspa8b2 in the swamp eel using Western blot analysis and Mass Spectrometry (MS). Fourteen Hsp70 family genes were further identified in this species based on transcriptome information. The phylogenetic tree of Hsp70 family was constructed using the Maximum Likelihood method and their expression patterns in the swamp eel gonads were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). There are fourteen gene members in the Hsp70 family in the swamp eel genome. Hsp70 family, particularly Hspa8, has expanded in the species. One of the family members Hspa8b2 is predominantly expressed in testis of the swamp eel.

Structural and quantitative expression analyses of HERV gene family in human tissues.

PubMed

Ahn, Kung; Kim, Heui-Soo

2009-08-31

Human endogenous retroviruses (HERVs) have been implicated in the pathogenesis of several human diseases as multi-copy members in the human genome. Their gene expression profiling could provide us with important insights into the pathogenic relationship between HERVs and cancer. In this study, we have evaluated the genomic structure and quantitatively determined the expression patterns in the env gene of a variety of HERV family members located on six specific loci by the RetroTector 10 program, as well as real-time RT-PCR amplification. The env gene transcripts evidenced significant differences in the human tumor/normal adjacent tissues (colon, liver, uterus, lung and testis). As compared to the adjacent normal tissues, high levels of expression were noted in testis tumor tissues for HERV-K, in liver and lung tumor tissues for HERV-R, in liver, lung, and testis tumor tissues for HERV-H, and in colon and liver tumor tissues for HERV-P. These data warrant further studies with larger groups of patients to develop biomarkers for specific human cancers.

Lipogenesis and lipid peroxidation in rat testes after long-term treatment with sucrose and tannic acid in drinking water.

PubMed

Mašek, T; Starčević, K

2017-05-01

We studied the influence of long-term treatment with sucrose and tannic acid in drinking water on the fatty acid profile and lipid peroxidation in rat testes. Male Wistar rats were supplemented with sucrose (30% w/v) or with sucrose and tannic acid (sucrose 30% w/v, tannic acid 0.1% w/v) in drinking water. The treatment with sucrose elevated blood glucose levels in the plasma (p < .05) and decreased the testis weight (p < .05) and testis index (p < .05) of the rats. Sucrose treatment increased monounsaturated fatty acids (MUFA) and C22:6n3, and decreased n6 fatty acids in testis tissue. Lipid peroxidation was significantly increased after sucrose administration in plasma (p < .05) and testis tissue (p < .01). The addition of tannic acid led to the decrease in lipid peroxidation in the plasma (p < .05) and testis (p < .05), a further increase in MUFA and decrease in n6 fatty acids. In conclusion, sucrose significantly altered the testis fatty acid profile with an increase in MUFA and C22:6n3, and a decrease in n6 fatty acids. Tannic acid attenuated oxidative stress and hyperglycaemia, but it did not improve pathological changes in the fatty acid composition of the testis. © 2016 Blackwell Verlag GmbH.

TDRP deficiency contributes to low sperm motility and is a potential risk factor for male infertility.

PubMed

Mao, Shanhua; Wu, Fei; Cao, Xinyi; He, Min; Liu, Naijia; Wu, Huihui; Yang, Zhihong; Ding, Qiang; Wang, Xuanchun

2016-01-01

TDRP (Testis Development-Related Protein), a nuclear factor, might play an important role in spermatogenesis. However, the molecular mechanisms of TDRP underlying these fundamental processes remain elusive. In this study, a Tdrp-deficient mouse model was generated. Fertility tests and semen analysis were performed. Tdrp-deficient mice were not significantly different from wild-type littermates in development of testes, genitourinary tract, or sperm count. Morphologically, spermatozoa of the Tdrp-deficient mice was not significantly different from the wild type. Several sperm motility indexes, i.e. the average path velocity (VAP), the straight line velocity (VSL) and the curvilinear velocity (VCL) were significantly decreased in Tdrp-deficient mice (p<0.05). The proportion of slow velocity sperm also increased significantly in the mutant mice (p<0.05). However, fertility tests showed that no significant difference inaverage offspring amount (AOA), frequency of copulatory plug (FCP), and frequency of conception (FC). Furthermore, TDRP1 could interact with PRM2, which might be the molecular mechanism of its nuclear function in spermatozoa. In conclusion, these data collectively demonstrated that Tdrp deficiency impaired the sperm motility, but Tdrp deficiency alone was not sufficient to cause male infertility in mice. Additionally, TDRP1 might participate in spermatogenes is through interaction with PRM2.

Desert hedgehog (Dhh) gene is required in the mouse testis for formation of adult-type Leydig cells and normal development of peritubular cells and seminiferous tubules.

PubMed

Clark, A M; Garland, K K; Russell, L D

2000-12-01

Testes from adult and prepubertal mice lacking the Desert hedgehog (DHH:) gene were examined in order to describe further the role of Dhh in spermatogenesis because, in a previous report, DHH:-null male mice were shown to be sterile. Dhh is a signaling molecule expressed by Sertoli cells. Its receptor, patched (Ptc), has been previously localized to Leydig cells and is herein described as being localized also to peritubular cells. Two phenotypes of the mice were observed: masculinized (7.5% of DHH:-null males) and feminized (92.5%), both of which displayed abnormal peritubular tissue and severely restricted spermatogenesis. Testes from adult feminized animals lacked adult-type Leydig cells and displayed numerous undifferentiated fibroblastic cells in the interstitium that produced abundant collagen. The basal lamina, normally present between the myoid cells and Sertoli cells, was focally absent. We speculate that the abnormal basal lamina contributed to other characteristics, such as extracordal gonocytes, apolar Sertoli cells, and anastomotic seminiferous tubules. The two DHH:-null phenotypes described have common peritubular cell defects that may be indicative of the essential role of peritubular cells in development of tubular morphology, the differentiation of Leydig cells, and the ultimate support of spermatogenesis.

Knockdown of the GnRH-II receptor in the porcine testis impairs the biosynthesis of 10 gonadal steroids

USDA-ARS?s Scientific Manuscript database

The second mammalian GnRH isoform (GnRH-II) and its cognate receptor (GnRHR-II) are poor modulators of gonadotropin secretion in swine. However, both are abundantly produced within the porcine testis suggesting an autocrine/paracrine role. Within the boar testis, GnRHR-II immunolocalizes to the plas...

Immunohistochemical localization of GLUT3, MCT1, and MCT2 in the testes of mice and rats: the use of different energy sources in spermatogenesis.

PubMed

Kishimoto, Ayuko; Ishiguro-Oonuma, Toshina; Takahashi, Ritei; Maekawa, Mamiko; Toshimori, Kiyotaka; Watanabe, Masahiko; Iwanaga, Toshihiko

2015-01-01

Lactate represents a preferential energy substrate of germ cells rather than glucose. Testicular Sertoli cells are believed to produce lactate and pyruvate and to supply these to germ cells, particularly spermatocytes and spermatids. Monocarboxylate transporter (MCT), responsible for the transport of lactate and other monocarboxylates via the cell membrane, is abundant in the testes and sperm (MCT1, MCT2, and MCT4). For the uptake of glucose, germ cells within the seminiferous tubules and sperm have been known to intensely express GLUT3. The present study investigated expression profiles of MCTs and GLUTs and revealed their cellular and subcellular localization in the mouse and rat testis. An in situ hybridization analysis showed significant expressions of MCT1, MCT2, and GLUT3 mRNA in the testis. Immunohistochemically, spermatogonia, spermatocytes, and spermatids expressed MCT1 on their cell surfaces in a stage-dependent manner: in some seminiferous tubules, an intense expression of MCT1 was unique to the spermatogonia. MCT2 was restricted to the tails of elongated spermatids and sperm. An intense immunoreactivity for GLUT3 was shared by spermatocytes, spermatids, and sperm. Sertoli cells were devoid of any immunoreactivities for MCT1, MCT2, and GLUT3. The predominant energy source of germ cells may be lactate and other monocarboxylates--especially for spermatogonia, but glucose and other hexoses may be responsible for an energy supply to spermatocytes and spermatids.

Prolyl Endopeptidase (PREP) is Associated With Male Reproductive Functions and Gamete Physiology in Mice.

PubMed

Dotolo, Raffaele; Kim, Jung Dae; Pariante, Paolo; Minucci, Sergio; Diano, Sabrina

2016-03-01

Prolyl endopeptidase (PREP) is a serine protease which has been implicated in many biological processes, such as the maturation and degradation of peptide hormones and neuropeptides, learning and memory, cell proliferation and differentiation, and glucose metabolism. A small number of reports have also suggested PREP participation in both male and female reproduction-associated processes. In the present work, we examined PREP distribution in male germ cells and studied the effects of its knockdown (Prep(gt/gt)) on testis and sperm in adult mice. The protein is expressed and localized in elongating spermatids and luminal spermatozoa of wild type (wt) mice, as well as Sertoli, Leydig, and peritubular cells. PREP is also expressed in the head and midpiece of epididymal spermatozoa, whereas the remaining tail region shows a weaker signal. Furthermore, testis weight, histology of seminiferous tubules, and epididymal sperm parameters were assessed in wt and Prep(gt/gt) mice: wild type testes have larger average tubule and lumen diameter; in addition, lumenal composition of seminiferous tubules is dissimilar between wt and Prep(gt/gt), as the percentage of spermiated tubules is much higher in wt. Finally, total sperm count, sperm motility, and normal morphology are also higher in wt than in Prep(gt/gt). These results show for the first time that the expression of PREP could be necessary for a correct reproductive function, and suggest that the enzyme may play a role in mouse spermatogenesis and sperm physiology. © 2015 Wiley Periodicals, Inc.

Avian infectious bronchitis virus: a possible cause of reduced fertility in the rooster.

PubMed

Boltz, David A; Nakai, Masaaki; Bahra, Janice M

2004-12-01

The formation of epididymal stones in the rooster epididymis is a widespread problem that has detrimental effects on sperm production and fertility. The cause of epididymal stones is unknown, but an infectious agent, the avian infectious bronchitis virus (AIBV), has been implicated. The goal of this study was to determine if administering the live attenuated AIBV vaccine to male chicks increases the incidence of stones in the epididymal region of the adult rooster. Specific pathogen free (SPF) Leghorn roosters were divided into two groups: a vaccine-free group (n = 7) and a group vaccinated with AIBV (n = 12). The vaccine was administered orally at 2, 4, 10, and 14 wk of age. Blood was drawn weekly to monitor antibodies to AIBV. At 26 wk of age, blood was obtained to determine testosterone concentrations, and reproductive tracts were removed to analyze daily sperm production and to detect epididymal stones. Nine of 12 vaccinated roosters developed stones, whereas those not given the vaccine did not develop stones. Serum testosterone concentrations were significantly (P < 0.05) reduced in vaccinated roosters with epididymal stones (3.6 +/- 0.30 ng/ml) when compared with nonvaccinated roosters that did not have epididymal stones (7.0 +/- 1.63 ng/ml). Testis weight was significantly (P < 0.05) reduced in vaccinated roosters with epididymal stones (12.1 +/- 0.76 g), as compared with nonvaccinated roosters without epididymal stones (15.2 +/- 0.81 g). Daily sperm production was significantly (P < 0.05) decreased in vaccinated roosters with epididymal stones (5.03 +/- 0.31 x 10(8) sperm/testis/day) when compared with nonvaccinated roosters without epididymal stones (7.43 +/- 0.52 x 10(8) sperm/testis/day). Comparing daily sperm production on a per gram basis, vaccinated roosters with epididymal stones had 4.38 +/- 0.14 x 10(7) sperm/g of testis, which was significantly (P < 0.05) smaller than nonvaccinated roosters without epididymal stones, which had 5.17 +/- 0.17 x 10(7) sperm/g of testis. We conclude that the use of a live attenuated AIBV vaccine increases the incidence of epididymal stones in roosters, resulting in decreased sperm production and decreased serum testosterone concentrations.

Perfluorooctanoic acid affects endocytosis involving clathrin light chain A and microRNA-133b-3p in mouse testes.

PubMed

Lu, Yin; Wang, Jianshe; Guo, Xuejiang; Yan, Shengmin; Dai, Jiayin

2017-03-01

Perfluorooctanoic acid (PFOA) is an abundant perfluoroalkyl substance widely applied in industrial and consumer products. Among its potential health hazards, testicular toxicity is of major concern. To explore the potential effect of miRNA on post-translational regulation after PFOA exposure, changes in miRNAs were detected via miRNA array. Seventeen miRNAs were differentially expressed (eight upregulated, nine downregulated) in male mouse testes after exposure to 5mg/kg/d of PFOA for 28d (>1.5-fold and P<0.05 compared with the control). Eight of these miRNAs were further selected for TaqMan qPCR analysis. Proteomic profile analysis indicated that many changed proteins after PFOA treatment, including intersectin 1 (ITSN1), serine protease inhibitor A3K (Serpina3k), and apolipoprotein a1 (APOA1), were involved in endocytosis and blood-testis barrier (BTB) processes. These changes were further verified by immunohistochemical and Western blot analyses. Endocytosis-related genes were selected for qPCR analysis, with many found to be significantly changed after PFOA treatment, including epidermal growth factor receptor pathway substrate 8 (Eps8), Eps15, cortactin, cofilin, espin, vinculin, and zyxin. We further predicted the potential interaction between changed miRNAs and proteins, which indicated that miRNAs might play a role in the post-translational regulation of gene expression after PFOA treatment in mouse testes. Among them, miR-133b-3p/clathrin light chain A (CLTA) was selected and verified in vitro by transfection and luciferase activity assay. Results showed that PFOA exposure affects endocytosis in mouse testes and that CLTA is a potential target of miR-133b-3p. Copyright © 2017 Elsevier Inc. All rights reserved.

Mouse cysteine-rich secretory protein 4 (CRISP4): a member of the Crisp family exclusively expressed in the epididymis in an androgen-dependent manner.

PubMed

Jalkanen, Jenni; Huhtaniemi, Ilpo; Poutanen, Matti

2005-05-01

The final maturation of spermatozoa produced in the testis takes place during their passage through the epididymis. In this process, the proteins secreted into the epididymal lumen along with changes in the pH and salt composition of the epididymal fluid cause several biochemical changes and remodeling of the sperm plasma membrane. The Crisp family is a group of cysteine-rich secretory proteins that previously consisted of three members, one of which-CRISP1-is an epididymal protein shown to attach to the sperm surface in the epididymal lumen and to inhibit gamete membrane fusion. In the present paper, we introduce a new member of the Crisp protein family, CRISP4. The new gene was discovered through in silico analysis of the epididymal expressed sequence tag library deposited in the UniGene database. The peptide sequence of CRISP4 has a signal sequence suggesting that it is secreted into the epididymal lumen and might thus interact with sperm. Unlike the other members of the family, Crisp4 is located on chromosome 1 in a cluster of genes encoding for cysteine-rich proteins. Crisp4 is expressed in the mouse exclusively in epithelial cells of the epididymis in an androgen-dependent manner, and the expression of the gene starts at puberty along with the onset of sperm maturation. The identified murine CRISP4 peptide has high homology with human CRISP1, and the homology is higher than that between murine and human CRISP1, suggesting that CRISP4 represents the mouse counterpart of human CRISP1 and could have similar effects on sperm membrane as mouse and human CRISP1.

[Perineal ectopic testis: report of four paediatric cases].

PubMed

Jlidi, Said; Echaieb, Anis; Ghorbel, Sofiene; Khemakhem, Rachid; Ben Khalifa, Sonia; Chaouachi, Béji

2004-09-01

Perineal ectopic testis is a rare congenital malformation in which the testis is abnormally situated between the penoscrotal raphe and the genitofemoral fold. The authors report four new cases in children aged 2 months, 6 months, 2 years and 5 years. The abnormality was associated with an inguinal hernia in one case. The diagnosis was based on the presence of an empty scrotum or perineal swelling. Treatment, via an inguinal incision, consisted of orchidopexy in a dartos pouch with a favourable course in every case. The aetiopathogenesis of perineal ectopic testis is controversial. It can be easily diagnosed by palpation of the testis in the perineal region. Orchidopexy in a dartos pouch must be performed early, and does not raise any particular problems because of the sufficient length of the spermatic cord. The functional prognosis, always difficult to define, appears to be identical to that of other sites.

A case of adenocarcinoma of the rete testis accompanied by focal adenomatous hyperplasia

PubMed Central

2013-01-01

Abstract Adenocarcinoma of the rete testis is very rare. There is still little knowledge about its etiology and pathogenesis. Herein, we present a case of rete testis adenocarcinoma in a 36-year-old Chinese male. The tumor was predominantly composed of irregular small tubules and papillary structures with cuboidal or polygonal cells. In peripheral area of the tumor, the remaining normal rete testis and adenomatous hyperplasia of the rete testis could also be seen, indicating the possible relationship between adenomatous hyperplasia and adenocarcinoma. In addition, the patient underwent a left hydrocelectomy because of the existence of hydrocele 3 years ago. But, it is unclear whether hydrocele and hydrocelectomy is its cause or just the early clinical presentation of the adenocarcinoma. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/6757609119625499 PMID:23800084

Specialized rules of gene transcription in male germ cells: the CREM paradigm.

PubMed

Monaco, Lucia; Kotaja, Noora; Fienga, Giulia; Hogeveen, Kevin; Kolthur, Ullas S; Kimmins, Sarah; Brancorsini, Stefano; Macho, Betina; Sassone-Corsi, Paolo

2004-12-01

Specialized transcription complexes that coordinate the differentiation programme of spermatogenesis have been found in germ cells, which display specific differences in the components of the general transcription machinery. The TATA-binding protein family and its associated cofactors, for example, show upregulated expression in testis. In this physiological context, transcriptional control mediated by the activator cAMP response element modulator (CREM) represents an established paradigm. Somatic cell activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CREB-binding protein. In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, activator of CREM in the testis (ACT), which confers a powerful, phosphorylation-independent activation capacity. The function of ACT was found to be regulated by the testis-specific kinesin KIF17b. Here we discuss some aspects of the testis-specific transcription machinery, whose function is essential for the process of spermatogenesis.

Interaction of Oligomeric Breast Cancer Resistant Protein (BCRP) with Adjudin: A Male Contraceptive with Anti-Cancer Activity

PubMed Central

Cheng, Yan Ho; Jenardhanan, Pranitha; Mathur, Premendu P.; Qian, Xiaojing; Xia, Weiliang; Silvestrini, Bruno; Cheng, Chuen Yan

2016-01-01

Breast cancer resistant protein (BCRP, ABCG2) is an ATP-binding cassette (ABC) transporter, which together with two other ABC efflux drug pumps, namely P-glycoprotein (P-gp, ABCB1) and multidrug resistance-related protein 1 (MRP1, ABCC1) is the most important multidrug resistance protein found in eukaryotic cells including cells in the testis. However, unlike P-gp and MRP1, which are components of the Sertoli cell blood-testis barrier (BTB), BCRP is not expressed at the BTB in rodents and human testes. Instead, BCRP is expressed by peritubular myoid cells and endothelial cells of the lymphatic vessel in the tunica propria, residing outside the BTB. As such, the testis is equipped with two levels of defense against xenobiotics or drugs, preventing these harmful substances from entering the adluminal compartment to perturb meiosis and post-meiotic spermatid development: one at the level of the BTB conferred by P-gp and MRP1 and one at the tunica propria conferred by BCRP. The presence of drug transporters at the tunica propria as well as at the Sertoli cell BTB thus poses significant obstacles in developing non-hormonal contraceptives if these drugs (e.g., adjudin) exert their effects in germ cells behind the BTB, such as in the adluminal (apical) compartment of the seminiferous epithelium. Herein, we summarize recent findings pertinent to adjudin, a non-hormonal male contraceptive, and molecular interactions of adjudin with BCRP so that this information can be helpful to devise delivery strategies to evade BCRP in the tunica propria to improve its bioavailability in the testis. PMID:25620224

Expression profiles of urbilaterian genes uniquely shared between honey bee and vertebrates

PubMed Central

Matsui, Toshiaki; Yamamoto, Toshiyuki; Wyder, Stefan; Zdobnov, Evgeny M; Kadowaki, Tatsuhiko

2009-01-01

Background Large-scale comparison of metazoan genomes has revealed that a significant fraction of genes of the last common ancestor of Bilateria (Urbilateria) is lost in each animal lineage. This event could be one of the underlying mechanisms involved in generating metazoan diversity. However, the present functions of these ancient genes have not been addressed extensively. To understand the functions and evolutionary mechanisms of such ancient Urbilaterian genes, we carried out comprehensive expression profile analysis of genes shared between vertebrates and honey bees but not with the other sequenced ecdysozoan genomes (honey bee-vertebrate specific, HVS genes) as a model. Results We identified 30 honey bee and 55 mouse HVS genes. Many HVS genes exhibited tissue-selective expression patterns; intriguingly, the expression of 60% of honey bee HVS genes was found to be brain enriched, and 24% of mouse HVS genes were highly expressed in either or both the brain and testis. Moreover, a minimum of 38% of mouse HVS genes demonstrated neuron-enriched expression patterns, and 62% of them exhibited expression in selective brain areas, particularly the forebrain and cerebellum. Furthermore, gene ontology (GO) analysis of HVS genes predicted that 35% of genes are associated with DNA transcription and RNA processing. Conclusion These results suggest that HVS genes include genes that are biased towards expression in the brain and gonads. They also demonstrate that at least some of Urbilaterian genes retained in the specific animal lineage may be selectively maintained to support the species-specific phenotypes. PMID:19138430

Expression profiles of urbilaterian genes uniquely shared between honey bee and vertebrates.

PubMed

Matsui, Toshiaki; Yamamoto, Toshiyuki; Wyder, Stefan; Zdobnov, Evgeny M; Kadowaki, Tatsuhiko

2009-01-12

Large-scale comparison of metazoan genomes has revealed that a significant fraction of genes of the last common ancestor of Bilateria (Urbilateria) is lost in each animal lineage. This event could be one of the underlying mechanisms involved in generating metazoan diversity. However, the present functions of these ancient genes have not been addressed extensively. To understand the functions and evolutionary mechanisms of such ancient Urbilaterian genes, we carried out comprehensive expression profile analysis of genes shared between vertebrates and honey bees but not with the other sequenced ecdysozoan genomes (honey bee-vertebrate specific, HVS genes) as a model. We identified 30 honey bee and 55 mouse HVS genes. Many HVS genes exhibited tissue-selective expression patterns; intriguingly, the expression of 60% of honey bee HVS genes was found to be brain enriched, and 24% of mouse HVS genes were highly expressed in either or both the brain and testis. Moreover, a minimum of 38% of mouse HVS genes demonstrated neuron-enriched expression patterns, and 62% of them exhibited expression in selective brain areas, particularly the forebrain and cerebellum. Furthermore, gene ontology (GO) analysis of HVS genes predicted that 35% of genes are associated with DNA transcription and RNA processing. These results suggest that HVS genes include genes that are biased towards expression in the brain and gonads. They also demonstrate that at least some of Urbilaterian genes retained in the specific animal lineage may be selectively maintained to support the species-specific phenotypes.

Cloning, Characterization, and Expression Analysis of the Novel Acetyltransferase Retrogene Ard1b in the Mouse1

PubMed Central

Pang, Alan Lap-Yin; Peacock, Stephanie; Johnson, Warren; Bear, Deborah H.; Rennert, Owen M.; Chan, Wai-Yee

2009-01-01

N-alpha-terminal acetylation is a modification process that occurs cotranslationally on most eukaryotic proteins. The major enzyme responsible for this process, N-alpha-terminal acetyltransferase, is composed of the catalytic subunit ARD1A and the auxiliary subunit NAT1. We cloned, characterized, and studied the expression pattern of Ard1b (also known as Ard2), a novel homolog of the mouse Ard1a. Comparison of the genomic structures suggests that the autosomal Ard1b is a retroposed copy of the X-linked Ard1a. Expression analyses demonstrated a testis predominance of Ard1b. A reciprocal expression pattern between Ard1a and Ard1b is also observed during spermatogenesis, suggesting that Ard1b is expressed to compensate for the loss of Ard1a starting from meiosis. Both ARD1A and ARD1B can interact with NAT1 to constitute a functional N-alpha-terminal acetyltransferase in vitro. The expression of ARD1B protein can be detected in mouse testes but is delayed until the first appearance of round spermatids. In a cell culture model, the inclusion of the long 3′ untranslated region of Ard1b leads to reduction of luciferase reporter activity, which implicates its role in translational repression of Ard1b during spermatogenesis. Our results suggest that ARD1B may have an important role in the later course of the spermatogenic process. PMID:19246321

Cloning, characterization, and expression analysis of the novel acetyltransferase retrogene Ard1b in the mouse.

PubMed

Pang, Alan Lap-Yin; Peacock, Stephanie; Johnson, Warren; Bear, Deborah H; Rennert, Owen M; Chan, Wai-Yee

2009-08-01

N-alpha-terminal acetylation is a modification process that occurs cotranslationally on most eukaryotic proteins. The major enzyme responsible for this process, N-alpha-terminal acetyltransferase, is composed of the catalytic subunit ARD1A and the auxiliary subunit NAT1. We cloned, characterized, and studied the expression pattern of Ard1b (also known as Ard2), a novel homolog of the mouse Ard1a. Comparison of the genomic structures suggests that the autosomal Ard1b is a retroposed copy of the X-linked Ard1a. Expression analyses demonstrated a testis predominance of Ard1b. A reciprocal expression pattern between Ard1a and Ard1b is also observed during spermatogenesis, suggesting that Ard1b is expressed to compensate for the loss of Ard1a starting from meiosis. Both ARD1A and ARD1B can interact with NAT1 to constitute a functional N-alpha-terminal acetyltransferase in vitro. The expression of ARD1B protein can be detected in mouse testes but is delayed until the first appearance of round spermatids. In a cell culture model, the inclusion of the long 3' untranslated region of Ard1b leads to reduction of luciferase reporter activity, which implicates its role in translational repression of Ard1b during spermatogenesis. Our results suggest that ARD1B may have an important role in the later course of the spermatogenic process.

Usherin expression is highly conserved in mouse and human tissues.

PubMed

Pearsall, Nicole; Bhattacharya, Gautam; Wisecarver, Jim; Adams, Joe; Cosgrove, Dominic; Kimberling, William

2002-12-01

Usher syndrome is an autosomal recessive disease that results in varying degrees of hearing loss and retinitis pigmentosa. Three types of Usher syndrome (I, II, and III) have been identified clinically with Usher type II being the most common of the three types. Usher type II has been localized to three different chromosomes 1q41, 3p, and 5q, corresponding to Usher type 2A, 2B, and 2C respectively. Usherin is a basement membrane protein encoded by the USH2A gene. Expression of usherin has been localized in the basement membrane of several tissues, however it is not ubiquitous. Immunohistochemistry detected usherin in the following human tissues: retina, cochlea, small and large intestine, pancreas, bladder, prostate, esophagus, trachea, thymus, salivary glands, placenta, ovary, fallopian tube, uterus, and testis. Usherin was absent in many other tissues such as heart, lung, liver, kidney, and brain. This distribution is consistent with the usherin distribution seen in the mouse. Conservation of usherin is also seen at the nucleotide and amino acid level when comparing the mouse and human gene sequences. Evolutionary conservation of usherin expression at the molecular level and in tissues unaffected by Usher 2a supports the important structural and functional role this protein plays in the human. In addition, we believe that these results could lead to a diagnostic procedure for the detection of Usher syndrome and those who carry an USH2A mutation.

A blood-testis barrier restricting passage from blood into rete testis fluid but not into lymph

PubMed Central

Setchell, B. P.; Voglmayr, J. K.; Waites, G. M. H.

1969-01-01

1. A permeability barrier in or around the seminiferous tubules of rams has been demonstrated by studying the rate of passage of a variety of substances from blood plasma into fluid collected from the rete testis and into testicular lymph. 2. All substances studied passed readily into testicular lymph. 3. Tritiated water, urea, ethanol and bicarbonate in rete testis fluid equilibrated with blood plasma within 3 hr; Na+, K+, Rb+, Cl-, I-, CNS-, creatinine and galactose entered slowly and p-aminohippurate (PAH), glutamate, iodinated albumin, inulin and [51Cr]EDTA did not appear in rete testis fluid at all. 4. Rubidium was excluded relative to iodoantipyrine from the testes of control and hypophysectomized rats and from rat testes heated to 37, 40, 43 and 45° C; no such exclusion was seen in testes of rats which had been given cadmium chloride 5 months earlier so as to destroy the seminiferous tubules. 5. It is suggested that this permeability barrier will regulate the access to the seminiferous epithelium of some constituents of blood plasma, isolate the germinal cells immunologically and help to maintain the concentration differences between rete testis fluid and lymph or blood plasma. PMID:4973530

PAGE-1, an X chromosome-linked GAGE-like gene that is expressed in normal and neoplastic prostate, testis, and uterus

PubMed Central

Brinkmann, Ulrich; Vasmatzis, George; Lee, Byungkook; Yerushalmi, Noga; Essand, Magnus; Pastan, Ira

1998-01-01

We have used a combination of computerized database mining and experimental expression analyses to identify a gene that is preferentially expressed in normal male and female reproductive tissues, prostate, testis, fallopian tube, uterus, and placenta, as well as in prostate cancer, testicular cancer, and uterine cancer. This gene is located on the human X chromosome, and it is homologous to a family of genes encoding GAGE-like proteins. GAGE proteins are expressed in a variety of tumors and in testis. We designate the novel gene PAGE-1 because the expression pattern in the Cancer Genome Anatomy Project libraries indicates that it is predominantly expressed in normal and neoplastic prostate. Further database analysis indicates the presence of other genes with high homology to PAGE-1, which were found in cDNA libraries derived from testis, pooled libraries (with testis), and in a germ cell tumor library. The expression of PAGE-1 in normal and malignant prostate, testicular, and uterine tissues makes it a possible target for the diagnosis and possibly for the vaccine-based therapy of neoplasms of prostate, testis, and uterus. PMID:9724777

PAGE-1, an X chromosome-linked GAGE-like gene that is expressed in normal and neoplastic prostate, testis, and uterus.

PubMed

Brinkmann, U; Vasmatzis, G; Lee, B; Yerushalmi, N; Essand, M; Pastan, I

1998-09-01

We have used a combination of computerized database mining and experimental expression analyses to identify a gene that is preferentially expressed in normal male and female reproductive tissues, prostate, testis, fallopian tube, uterus, and placenta, as well as in prostate cancer, testicular cancer, and uterine cancer. This gene is located on the human X chromosome, and it is homologous to a family of genes encoding GAGE-like proteins. GAGE proteins are expressed in a variety of tumors and in testis. We designate the novel gene PAGE-1 because the expression pattern in the Cancer Genome Anatomy Project libraries indicates that it is predominantly expressed in normal and neoplastic prostate. Further database analysis indicates the presence of other genes with high homology to PAGE-1, which were found in cDNA libraries derived from testis, pooled libraries (with testis), and in a germ cell tumor library. The expression of PAGE-1 in normal and malignant prostate, testicular, and uterine tissues makes it a possible target for the diagnosis and possibly for the vaccine-based therapy of neoplasms of prostate, testis, and uterus.

Cystic dysplasia of the testis: a very rare paediatric tumor of the testis.

PubMed

Eberli, Daniel; Gretener, Heini; Dommann-Scherrer, Corina; Pestalozzi, Dietegen; Fehr, Jean-Luc

2002-01-01

To describe a case of cystic dysplasia of the testis (CDT), an uncommon cause of scrotal swelling in the pediatric patient. Clinic, therapy, fertility, and radiographic and pathologic findings are discussed and the 30 previously reported cases are reviewed. A 9-year-old boy presented with asymptomatic scrotal swelling. A scrotal ultrasound showed a multicystic scrotal mass in the rete testis and an ipsilateral renal agenesis. The growth in size of the mass forced the authors to perform an operative exploration. Intraoperative findings included a multicystic mass in the rete testis of the right testicle. Testicle-sparing total removal of the multicystic mass was performed and the pathologic examination revealed a benign, multilobulated configuration of the cysts in the region of the rete testis. These findings were similar to those found in previously reported cases of CDT. Ipsilateral renal agenesis is the most common associated anomaly. As a pathogenetic factor, mal-junction of the Wolffian duct in the 5th week of gestation is most creditable. CDT is a rare cause of pediatric scrotal mass. When feasible, a testicle-sparing approach should be considered and all patients should undergo evaluation for associated urologic anomalies.

Effects of Trans-Resveratrol on hyperglycemia-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase signaling in rat testis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abdelali, Ala

Diabetes induces oxidative stress, DNA damage and alters several intracellular signaling pathways in organ systems. This study investigated modulatory effects of Trans-Resveratrol on type 1 diabetes mellitus (T1DM)-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase (PARP) signaling in rat testis. Trans-Resveratrol administration (5mg/kg/day, ip) to Streptozotocin-induced T1DM adult male Wistar rats from day 22–42 resulted in recovery of induced oxidative stress, abnormal spermatogenesis and inhibited DNA synthesis, and led to mitigation of 8-hydroxy-2'-deoxyguanosine formation in the testis and spermatozoa, and DNA double-strand breaks in the testis. Trans-Resveratrol aggravated T1DM-induced up-regulation of aminoacyl tRNA synthetase complex-interacting multifunctional proteinmore » 2 expression; however, it did not modify the up-regulated total PARP and down-regulated PARP1 expressions, but recovered the decreased SirT1 (Sirtuin 1) levels in T1DM rat testis. Trans-Resveratrol, when given alone, reduced the poly (ADP-ribosyl)ation (pADPr) process in the testis due to an increase in PAR glycohydrolase activity, but when given to T1DM rats it did not affect the pADPr levels. T1DM with or without Trans-Resveratrol did not induce nuclear translocation of apoptosis-inducing factor and the formation of 50 kb DNA breaks, suggesting to the lack of caspase-3-independent cell death called parthanatos. T1DM with or without Trans-Resveratrol did not increase necrotic cell death in the testis. Primary spermatocytes, Sertoli cells, Leydig cells and intra-testicular vessels showed the expression of PARP pathway related proteins. In conclusion, Trans-Resveratrol mitigates T1DM-induced sperm abnormality and DNA damage, but does not significantly modulate PARP signaling pathway, except the SirT1 expression, in the rat testis. - Highlights: • Resveratrol inhibits diabetes-induced abnormal sperm morphogenesis • Resveratrol recovers diabetes-induced DNA damage in testis and spermatozoa • Resveratrol does not normalize diabetes-induced increase in total PARP • Resveratrol does not modulate diabetes-induced decrease in PARP1 • Resveratrol normalizes diabetes-induced decrease in SirT1 levels in testis.« less

Effect of vitamin E supplement in diet on antioxidant ability of testis in Boer goat.

PubMed

Hong, Zhu; Hailing, Luo; Hui, Meng; Guijie, Zhang; Leyan, Yan; Dubing, Yue

2010-01-01

The aim of this study was to evaluate the supplementation of Vitamin E in diet on the antioxidant capacity of testis in Boer goat. Twenty-four healthy, Boer male kids of similar body weight (BW) were selected at 3 months of age from the kid flock. Kids were born from does treated with simultaneous flushing and artificial insemination technology. The Boer kids were divided into four groups randomly, supplemented with 0, 80, 320 and 880 IU kid(-1)d(-1) Vitamin E, which were labeled as Groups 1, 2, 3 and 4, respectively, for 150 days (5 months). Blood samples were collected at the 15th-, 30th-, 60th-, 90th-, 120th-, and 150th-day during the experimental period, and the serums were used to determine Vitamin E content. Three Boer goats in each group were slaughtered at the age of eight months at the end of the experiment. Liver and testis were collected to test the Vitamin E content and the antioxidant capacity of testis. Results showed that the content of Vitamin E in serum, liver and testis increased with the increasing addition of Vitamin E. However, the content of Vitamin E in the serum, liver and testis, in the control, was significantly lower than in Groups 2 and 3, respectively, but there was no significant difference between the control Group and Group 4. When high levels of Vitamin E (880 IU kid(-1)d(-1)) were added, contents of Vitamin E in serum, liver and testis were decreased and compared with the controls. Adding a low level (80 IU kid(-1)d(-1)) of Vitamin E can increase activity of total anti-oxidation competence (T-AOC) and superoxide dismutase (SOD), and decrease content of nitric oxide (NO) in testis. MDA (malondialdehyde) content was decreased significantly in Group 3 (P<0.05). Supplementing a low level (80 IU kid(-1)d(-1)) and middle level (320 IU kid(-1)d(-1)) of Vitamin E decreased activity of nitric oxide syntha (NOS) in testis (P<0.05). Vitamin E can increase activity of GSH-PX (glutathione peroxidase). These results indicate that supplementing Vitamin E protects testis from damage by preoxidation.

Merlin Isoforms 1 and 2 Both Act as Tumour Suppressors and Are Required for Optimal Sperm Maturation

PubMed Central

Zoch, Ansgar; Mayerl, Steffen; Schulz, Alexander; Greither, Thomas; Frappart, Lucien; Rübsam, Juliane; Heuer, Heike; Giovannini, Marco; Morrison, Helen

2015-01-01

The tumour suppressor Merlin, encoded by the gene NF2, is frequently mutated in the autosomal dominant disorder neurofibromatosis type II, characterised primarily by the development of schwannoma and other glial cell tumours. However, NF2 is expressed in virtually all analysed human and rodent organs, and its deletion in mice causes early embryonic lethality. Additionally, NF2 encodes for two major isoforms of Merlin of unknown functionality. Specifically, the tumour suppressor potential of isoform 2 remains controversial. In this study, we used Nf2 isoform-specific knockout mouse models to analyse the function of each isoform during development and organ homeostasis. We found that both isoforms carry full tumour suppressor functionality and can completely compensate the loss of the other isoform during development and in most adult organs. Surprisingly, we discovered that spermatogenesis is strictly dependent on the presence of both isoforms. While the testis primarily expresses isoform 1, we noticed an enrichment of isoform 2 in spermatogonial stem cells. Deletion of either isoform was found to cause decreased sperm quality as observed by maturation defects and head/midpiece abnormalities. These defects led to impaired sperm functionality as assessed by decreased sperm capacitation. Thus, we describe spermatogenesis as a new Nf2-dependent process. Additionally, we provide for the first time in vivo evidence for equal tumour suppressor potentials of Merlin isoform 1 and isoform 2. PMID:26258444

Cellular Signaling by Fibroblast Growth Factors (FGFs) and Their Receptors (FGFRs) in Male Reproduction

PubMed Central

Cotton, Leanne M.; O’Bryan, Moira K.; Hinton, Barry T.

2008-01-01

The major function of the reproductive system is to ensure the survival of the species by passing on hereditary traits from one generation to the next. This is accomplished through the production of gametes and the generation of hormones that function in the maturation and regulation of the reproductive system. It is well established that normal development and function of the male reproductive system is mediated by endocrine and paracrine signaling pathways. Fibroblast growth factors (FGFs), their receptors (FGFRs), and signaling cascades have been implicated in a diverse range of cellular processes including: proliferation, apoptosis, cell survival, chemotaxis, cell adhesion, motility, and differentiation. The maintenance and regulation of correct FGF signaling is evident from human and mouse genetic studies which demonstrate that mutations leading to disruption of FGF signaling cause a variety of developmental disorders including dominant skeletal diseases, infertility, and cancer. Over the course of this review, we will provide evidence for differential expression of FGFs/FGFRs in the testis, male germ cells, the epididymis, the seminal vesicle, and the prostate. We will show that this signaling cascade has an important role in sperm development and maturation. Furthermore, we will demonstrate that FGF/FGFR signaling is essential for normal epididymal function and prostate development. To this end, we will provide evidence for the involvement of the FGF signaling system in the regulation and maintenance of the male reproductive system. PMID:18216218

Cellular signaling by fibroblast growth factors (FGFs) and their receptors (FGFRs) in male reproduction.

PubMed

Cotton, Leanne M; O'Bryan, Moira K; Hinton, Barry T

2008-04-01

The major function of the reproductive system is to ensure the survival of the species by passing on hereditary traits from one generation to the next. This is accomplished through the production of gametes and the generation of hormones that function in the maturation and regulation of the reproductive system. It is well established that normal development and function of the male reproductive system is mediated by endocrine and paracrine signaling pathways. Fibroblast growth factors (FGFs), their receptors (FGFRs), and signaling cascades have been implicated in a diverse range of cellular processes including: proliferation, apoptosis, cell survival, chemotaxis, cell adhesion, motility, and differentiation. The maintenance and regulation of correct FGF signaling is evident from human and mouse genetic studies which demonstrate that mutations leading to disruption of FGF signaling cause a variety of developmental disorders including dominant skeletal diseases, infertility, and cancer. Over the course of this review, we will provide evidence for differential expression of FGFs/FGFRs in the testis, male germ cells, the epididymis, the seminal vesicle, and the prostate. We will show that this signaling cascade has an important role in sperm development and maturation. Furthermore, we will demonstrate that FGF/FGFR signaling is essential for normal epididymal function and prostate development. To this end, we will provide evidence for the involvement of the FGF signaling system in the regulation and maintenance of the male reproductive system.

Conserved form and function of the germinal epithelium through 500 million years of vertebrate evolution.

PubMed

Grier, Harry J; Uribe, Mari Carmen; Lo Nostro, Fabiana L; Mims, Steven D; Parenti, Lynne R

2016-08-01

The germinal epithelium, i.e., the site of germ cell production in males and females, has maintained a constant form and function throughout 500 million years of vertebrate evolution. The distinguishing characteristic of germinal epithelia among all vertebrates, males, and females, is the presence of germ cells among somatic epithelial cells. The somatic epithelial cells, Sertoli cells in males or follicle (granulosa) cells in females, encompass and isolate germ cells. Morphology of all vertebrate germinal epithelia conforms to the standard definition of an epithelium: epithelial cells are interconnected, border a body surface or lumen, are avascular and are supported by a basement membrane. Variation in morphology of gonads, which develop from the germinal epithelium, is correlated with the evolution of reproductive modes. In hagfishes, lampreys, and elasmobranchs, the germinal epithelia of males produce spermatocysts. A major rearrangement of testis morphology diagnoses osteichthyans: the spermatocysts are arranged in tubules or lobules. In protogynous (female to male) sex reversal in teleost fishes, female germinal epithelial cells (prefollicle cells) and oogonia transform into the first male somatic cells (Sertoli cells) and spermatogonia in the developing testis lobules. This common origin of cell types from the germinal epithelium in fishes with protogynous sex reversal supports the homology of Sertoli cells and follicle cells. Spermatogenesis in amphibians develops within spermatocysts in testis lobules. In amniotes vertebrates, the testis is composed of seminiferous tubules wherein spermatogenesis occurs radially. Emerging research indicates that some mammals do not have lifetime determinate fecundity. The fact emerged that germinal epithelia occur in the gonads of all vertebrates examined herein of both sexes and has the same form and function across all vertebrate taxa. Continued study of the form and function of the germinal epithelium in vertebrates will increasingly clarify our understanding of vertebrate reproduction. J. Morphol. 277:1014-1044, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Chronic pain has a negative impact on sexuality in testis cancer survivors.

PubMed

Pühse, Gerald; Wachsmuth, Julia Urte; Kemper, Sebastian; Husstedt, Ingo W; Evers, Stefan; Kliesch, Sabine

2012-01-01

Testis cancer is a disease that directly affects a man's sense of masculinity and involves treatments compromising sexual function. The aim of this study was to investigate the prevalence of sexual dysfunction and the influence of chronic pain on sexuality in long-term testis cancer survivors. Thus, we examined 539 patients after they had one testis removed because of a testicular germ cell tumor. Having completed oncologic therapy, all patients received a detailed questionnaire asking about the occurrence and clinical presentation of testis pain before and after orchiectomy. In addition, items from the abridged International Index of Erectile Function and Brief Sexual Function Inventory were used to gain precise information on individual sexual function. Overall, 34.5% of our testicular cancer survivors complained of reduced sexual desire, and sexual activity was reduced in 41.6%. Erectile dysfunction was present in up to 31.5% of patients. In 24.4%, the ability to maintain an erection during intercourse was impaired. Ejaculatory disorders (premature, delayed, retrograde, or anejaculation) occurred in 84.9% of our testis cancer survivors. A total of 32.4% of our participants experienced a reduced intensity of orgasm, and 95.4% experienced reduced overall sexual satisfaction. There was a significant correlation between the occurrence of chronic pain symptoms and the relative frequency and intensity of erectile dysfunction, inability to maintain an erection, ejaculation disorders, and reduced intensity of orgasm. In conclusion, chronic pain has a negative impact on sexuality in testis cancer survivors.

Dramatic changes in 67 miRNAs during initiation of first wave of spermatogenesis in Mus musculus testis: global regulatory insights generated by miRNA-mRNA network analysis.

PubMed

Sree, Sreesha; Radhakrishnan, Karthika; Indu, Sivankutty; Kumar, Pradeep G

2014-09-01

We mapped global changes in miRNA and mRNA profiles spanning the first wave of spermatogenesis using prepubertal (Postnatal Day 8 [P8]), pubertal (P16), and adolescent (P24) Mus musculus testes and identified the differential expression of 67 miRNAs and 8226 mRNAs. These two data sets were integrated into miRNA-dependent regulatory networks based on miRWalk predictions. In a network representing the P8 to P16 transition, downregulation of four miRNAs and upregulation of 19 miRNAs were linked with 81 upregulated target mRNAs and 228 downregulated target mRNAs, respectively. Furthermore, during the P16 to P24 transition, two miRNAs were downregulated, and eight miRNAs were upregulated, which linked with 64 upregulated mRNAs and 389 downregulated mRNAs, respectively. Only three of the miRNAs present in the network (miR-34b-5p, miR-34c, and miR-449a) showed a progressive increase from P8 through P16 to P24, while the remaining miRNAs in the network showed statistically significant changes in their levels either during the P8 to P16 transition or during the P16 to P24 transition. Analysis of the chromosomal location of these differentially expressed miRNAs showed that 14 out of 25 miRNAs upregulated from P8 to P16, and 18 out of 40 miRNAs upregulated from P8 to P24 were X-linked. This is suggestive of their escape from meiotic sex chromosome inactivation and postmeiotic sex chromatin. This integrated network of miRNA-level and mRNA-level changes in mouse testis during the first wave of spermatogenesis is expected to build a base for evaluating the role of miRNA-mediated gene expression regulation in maturing mammalian testis. © 2014 by the Society for the Study of Reproduction, Inc.

Canine cryptorchism and subsequent testicular neoplasia: case-control study with epidemiologic update.

PubMed

Hayes, H M; Wilson, G P; Pendergrass, T W; Cox, V S

1985-08-01

A retrospective study of 2,912 cryptorchid dogs identified 14 breeds with significantly high risk. Among six distinct closely interrelated breed groups (e.g., toy, miniature, and standard poodles), the risk in the smaller breed was always greater than that in the larger relative, suggesting that genetically influenced maldescent could be, in part, related to physical size or the rate of growth of the involved structures. Testicular tumors were diagnosed in 5.7% of the cryptorchid dogs; half had only Sertoli cell tumors, one-third had only seminomas. The relative risk for Sertoli cell tumor or seminoma was not directly related to a familial risk for cryptorchism. Using the health experience of a control population composed of male dogs with anal sac disease (N = 4,184), there is an estimated relative risk of 9.2 in cryptorchid dogs to develop a testis tumor (95% confidence interval, 5.9-14.3) and 4.2 in dogs with inguinal hernia (95% confidence interval, 1.8-9.5). Considering that the anatomical development of the genital tract, testis descent, and tunic relationships in dog are very similar to that in man, and that the associations of cryptorchism and inguinal hernia with testis neoplasms are also similar, the dog should be an excellent model system to further investigate the causes of human cryptorchism.

Spontaneous CD4+ and CD8+ T‐cell responses directed against cancer testis antigens are present in the peripheral blood of testicular cancer patients

PubMed Central

Pearce, Hayden; Hutton, Paul; Chaudhri, Shalini; Porfiri, Emilio; Patel, Prashant; Viney, Richard

2017-01-01

Cancer/testis antigen (CTAg) expression is restricted to spermatogenic cells in an immune‐privileged site within the testis. However, these proteins are expressed aberrantly by malignant cells and T‐cell responses against CTAgs develop in many cancer patients. We investigated the prevalence, magnitude and phenotype of CTAg‐specific T cells in the blood of patients with testicular germ cell tumors (TGCTs). CD8+ and CD4+ T‐cell responses against MAGE‐A family antigens were present in 44% (20/45) of patients’ samples assayed by ex vivo IFN‐γ ELISPOT. The presence of MAGE‐specific CD8+ T cells was further determined following short‐term in vitro expansion through the use of pMHC‐I multimers containing known immunogenic peptides. Longitudinal analysis revealed that the frequency of MAGE‐specific T cells decreased by 89% following orchidectomy suggesting that persistence of tumor antigen is required to sustain CTAg‐specific T‐cell immunity. Notably, this decrease correlated with a decline in the global effector/memory T‐cell pool following treatment. Spontaneous T‐cell immunity against CTAg proteins therefore develops in many patients with testicular cancer and may play an important role in the excellent clinical outcome of patients with this tumor subtype. PMID:28555838

Recent advances of in vitro culture systems for spermatogonial stem cells in mammals.

PubMed

Sahare, Mahesh G; Suyatno; Imai, Hiroshi

2018-04-01

Spermatogonial stem cells (SSCs) in the mammalian testis are unipotent stem cells for spermatozoa. They show unique cell characteristics as stem cells and germ cells after being isolated from the testis and cultured in vitro. This review introduces recent progress in the development of culture systems for the establishment of SSC lines in mammalian species, including humans. Based on the published reports, the isolation and purification of SSCs, identification and characteristics of SSCs, and culture system for mice, humans, and domestic animals have been summarized. In mice, cell lines from SSCs are established and can be reprogrammed to show pluripotent stem cell potency that is similar to embryonic stem cells. However, it is difficult to establish cell lines for animals other than mice because of the dearth of understanding about species-specific requirements for growth factors and mechanisms supporting the self-renewal of cultured SSCs. Among the factors that are associated with the development of culture systems, the enrichment of SSCs that are isolated from the testis and the combination of growth factors are essential. Providing an example of SSC culture in cattle, a rational consideration was made about how it can be possible to establish cell lines from neonatal and immature testes.

Characterisation of ATRX, DMRT1, DMRT7 and WT1 in the platypus (Ornithorhynchus anatinus).

PubMed

Tsend-Ayush, Enkhjargal; Lim, Shu Ly; Pask, Andrew J; Hamdan, Diana Demiyah Mohd; Renfree, Marilyn B; Grützner, Frank

2009-01-01

One of the most puzzling aspects of monotreme reproductive biology is how they determine sex in the absence of the SRY gene that triggers testis development in most other mammals. Although monotremes share a XX female/XY male sex chromosome system with other mammals, their sex chromosomes show homology to the chicken Z chromosome, including the DMRT1 gene, which is a dosage-dependent sex determination gene in birds. In addition, monotremes feature an extraordinary multiple sex chromosome system. However, no sex determination gene has been identified as yet on any of the five X or five Y chromosomes and there is very little knowledge about the conservation and function of other known genes in the monotreme sex determination and differentiation pathway. We have analysed the expression pattern of four evolutionarily conserved genes that are important at different stages of sexual development in therian mammals. DMRT1 is a conserved sex-determination gene that is upregulated in the male developing gonad in vertebrates, while DMRT7 is a mammal-specific spermatogenesis gene. ATRX, a chromatin remodelling protein, lies on the therian X but there is a testis-expressed Y-copy in marsupials. However, in monotremes, the ATRX orthologue is autosomal. WT1 is an evolutionarily conserved gene essential for early gonadal formation in both sexes and later in testis development. We show that these four genes in the adult platypus have the same expression pattern as in other mammals, suggesting that they have a conserved role in sexual development independent of genomic location.

Safety of intra-articular transplantation of lentivirally transduced mesenchymal stromal cells for haemophilic arthropathy in a non-human primate.

PubMed

Ohmori, Tsukasa; Mizukami, Hiroaki; Katakai, Yuko; Kawai, Sho; Nakamura, Hitoyasu; Inoue, Makoto; Shu, Tsugumine; Sugimoto, Hideharu; Sakata, Yoichi

2018-05-08

Joint bleeding and resultant arthropathy are major determinants of quality of life in haemophilia patients. We previously developed a mesenchymal stromal cell (MSC)-based treatment approach for haemophilic arthropathy in a mouse model of haemophilia A. Here, we evaluated the long-term safety of intra-articular injection of lentivirally transduced autologous MSCs in non-human primates. Autologous bone-marrow-derived MSCs transduced with a lentiviral vector expressing coagulation factor VIII (FVIII) were injected into the left knee joint of cynomolgus monkeys. We first conducted codon optimization to increase FVIII production in the cells. Lentiviral transduction of autologous MSCs resulted in a significant increase of FVIII in the culture supernatant before transplantation. We did not find any tumour generation around the knee structure at 11-16 months after injection by magnetic resonance imaging. The proviral sequence of the simian immunodeficiency virus lentiviral vector was not detected in the heart, lungs, spleen, liver, testis, or bone marrow by real-time quantitative PCR. We confirmed the long-term safety of intra-articular injection of transduced MSCs in a non-human primate. The procedure may be an attractive therapeutic approach for joint diseases in haemophilia patients.

Disorders of Sex Development with Testicular Differentiation in SRY-Negative 46,XX Individuals: Clinical and Genetic Aspects.

PubMed

Grinspon, Romina P; Rey, Rodolfo A

2016-01-01

Virilisation of the XX foetus is the result of androgen excess, resulting most frequently from congenital adrenal hyperplasia in individuals with typical ovarian differentiation. In rare cases, 46,XX gonads may differentiate into testes, a condition known as 46,XX testicular disorders of sex development (DSD), or give rise to the coexistence of ovarian and testicular tissue, a condition known as 46,XX ovotesticular DSD. Testicular tissue differentiation may be due to the translocation of SRY to the X chromosome or an autosome. In the absence of SRY, overexpression of other pro-testis genes, e.g. SOX family genes, or failure of pro-ovarian/anti-testis genes, such as WNT4 and RSPO1, may underlie the development of testicular tissue. Recent experimental and clinical evidence giving insight into SRY-negative 46,XX testicular or ovotesticular DSD is discussed. © 2016 S. Karger AG, Basel.

Blueberry extracts protect testis from hypobaric hypoxia induced oxidative stress in rats.

PubMed

Zepeda, Andrea; Aguayo, Luis G; Fuentealba, Jorge; Figueroa, Carolina; Acevedo, Alejandro; Salgado, Perla; Calaf, Gloria M; Farías, Jorge

2012-01-01

Exposure to hypobaric hypoxia causes oxidative damage to male rat reproductive function. The aim of this study was to evaluate the protective effect of a blueberry extract (BB-4) in testis of rats exposed to hypobaric hypoxia. Morphometric analysis, cellular DNA fragmentation, glutathione reductase (GR), and superoxide dismutase (SOD) activities were evaluated. Our results showed that supplementation of BB-4 reduced lipid peroxidation, decreased apoptosis, and increased GR and SOD activities in rat testis under hypobaric hypoxia conditions (P < 0.05). Therefore, this study demonstrates that blueberry extract significantly reduced the harmful effects of oxidative stress caused by hypobaric hypoxia in rat testis by affecting glutathione reductase and superoxide dismutase activities.

Transgenerational Epigenetic Programming of the Embryonic Testis Transcriptome

PubMed Central

Anway, Matthew D.; Rekow, Stephen S.; Skinner, Michael K.

2008-01-01

Embryonic exposure to the endocrine disruptor vinclozolin during gonadal sex determination appears to promote an epigenetic reprogramming of the male germ-line that is associated with transgenerational adult onset disease states. Transgenerational effects on the embryonic day 16 (E16) testis demonstrated reproducible changes in the testis transcriptome for multiple generations (F1-F3). The expression of 196 genes were found to be influenced, with the majority of gene expression being decreased or silenced. Dramatic changes in the gene expression of methyltransferases during gonadal sex determination were observed in the F1 and F2 vinclozolin generation (E16) embryonic testis, but the majority returned to control generation levels by the F3 generation. The most dramatic effects were on the germ-line associated Dnmt3A and Dnmt3L isoforms. Observations demonstrate that an embryonic exposure to vinclozolin appears to promote an epigenetic reprogramming of the male germ-line that correlates with transgenerational alterations in the testis transcriptome in subsequent generations. PMID:18042343

Developmental expression of the glutathione S-transferase Yo subunit in the rat testis and epididymis using light microscope immunocytochemistry.

PubMed

Papp, S; Robaire, B; Hermo, L

1994-11-01

Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione with various toxic electrophilic compounds. GSTs are composed of several classes based on the degree of sequence homology of their subunits. The Yo subunit, a member of the mu class, is expressed at high levels in the testis and epididymis. The purpose of this study was to immunolocalize the GST-Yo in these tissues during development. The testes and epididymides of rats aged 7, 15, 21, 28, 39, 42, 45, 49, and 56 days were fixed in Bouin's fixative, and immunostained for light microscopic analysis. In the testis the cytoplasm of all germ cells was unreactive until day 39. At that time, step 18 spermatids appeared moderately reactive, while the few observed step 19 spermatids were intensely reactive as were their residual bodies. The presence of residual bodies indicates that spermiation takes place as early as day 39; however, the number of step 19 spermatids is low at this age. A progressive increase in the size of the tubule and number of elongating spermatids was seen between days 42 and 49. In addition, by day 49, a weak staining was observed in steps 12-15, moderate in steps 16-17, and intense in steps 18-19 spermatids. In terms of the intensity of staining, cell types stained, size of the tubules, and number of elongating spermatids, no difference was noted between day 49, 56, and adult animals. Thus Yo protein expression in germ cells reached maturity by day 49. The epithelial cells of the rete testis were intensely reactive at day 7 and remained so throughout development. In contrast, while the epithelial cells of the efferent ducts at day 7 were intensely reactive, they were weakly reactive by day 39 and remained so at later ages. Along the entire epididymis, the columnar epithelial cells showed a moderate apical/supranuclear reaction from day 7 to 28. By day 39 principal cells of the initial segment became weakly reactive, while those in the caput and corpus were moderately stained, a situation seen at later ages including adults. Only by day 49 did principal cells of the proximal cauda become moderately stained as seen in adult animals. Thus the expression of the Yo protein in the principal cells of the proximal cauda may be regulated by different factors than those of the caput and corpus epididymidis. Alternatively, the expression of the Yo subunit in principal cells of the proximal cauda may develop later since this region would be the last to receive luminally derived testicular products. In the initial segment, the decrease in staining of principal cells at day 39 may be due to an inhibiting factor emanating from the testis. Spermatozoa appeared in the lumen of each epididymal region well after the expression of Yo had reached its adult staining pattern indicating that they are not a factor. Overall these results suggest that the expression of GST-Yo in the various cells of the testis and epididymis are controlled by different factors during postnatal development.

The production of glial cell line-derived neurotrophic factor by human sertoli cells is substantially reduced in sertoli cell-only testes.

PubMed

Singh, D; Paduch, D A; Schlegel, P N; Orwig, K E; Mielnik, A; Bolyakov, A; Wright, W W

2017-05-01

Do human Sertoli cells in testes that exhibit the Sertoli cell-only (SCO) phenotype produce substantially less glial cell line-derived neurotrophic factor (GDNF) than Sertoli cells in normal testes? In human SCO testes, both the amounts of GDNF mRNA per testis and the concentration of GDNF protein per Sertoli cell are markedly reduced as compared to normal testes. In vivo, GDNF is required to sustain the numbers and function of mouse spermatogonial stem cells (SSCs) and their immediate progeny, transit-amplifying progenitor spermatogonia. GDNF is expressed in the human testis, and the ligand-binding domain of the GDNF receptor, GFRA1, has been detected on human SSCs. The numbers and/or function of these stem cells are markedly reduced in some infertile men, resulting in the SCO histological phenotype. We determined the numbers of human spermatogonia per mm2 of seminiferous tubule surface that express GFRA1 and/or UCHL1, another marker of human SSCs. We measured GFRA1 mRNA expression in order to document the reduced numbers and/or function of SSCs in SCO testes. We quantified GDNF mRNA in testes of humans and mice, a species with GDNF-dependent SSCs. We also compared GDNF mRNA expression in human testes with normal spermatogenesis to that in testes exhibiting the SCO phenotype. As controls, we also measured transcripts encoding two other Sertoli cell products, kit ligand (KITL) and clusterin (CLU). Finally, we compared the amounts of GDNF per Sertoli cell in normal and SCO testes. Normal human testes were obtained from beating heart organ donors. Biopsies of testes from men who were infertile due to maturation arrest or the SCO phenotype were obtained as part of standard care during micro-testicular surgical sperm extraction. Cells expressing GFRA1, UCHL1 or both on whole mounts of normal human seminiferous tubules were identified by immunohistochemistry and confocal microscopy and their numbers were determined by image analysis. Human GDNF mRNA and GFRA1 mRNA were quantified by use of digital PCR and Taqman primers. Transcripts encoding mouse GDNF and human KITL, CLU and 18 S rRNA, used for normalization of data, were quantified by use of real-time PCR and Taqman primers. Finally, we used two independent methods, flow cytometric analysis of single cells and ELISA assays of homogenates of whole testis biopsies, to compare amounts of GDNF per Sertoli cell in normal and SCO testes. Normal human testes contain a large population of SSCs that express GFRA1, the ligand-binding domain of the GDNF receptor. In human SCO testes, GFRA1 mRNA was detected but at markedly reduced levels. Expression of GDNF mRNA and the amount of GDNF protein per Sertoli cell were also significantly reduced in SCO testes. These results were observed in multiple, independent samples, and the reduced amount of GDNF in Sertoli cells of SCO testes was demonstrated using two different analytical approaches. N/A. There currently are no approved protocols for the in vivo manipulation of human testis GDNF concentrations. Thus, while our data suggest that insufficient GDNF may be the proximal cause of some cases of human male infertility, our results are correlative in nature. We propose that insufficient GDNF expression may contribute to the infertility of some men with an SCO testicular phenotype. If their testes contain some SSCs, an approach that increases their testicular GDNF concentrations might expand stem cell numbers and possibly sperm production. This research was funded by the Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Centers for Translational Research in Reproduction and Infertility Program (NCTRI) Grant 1R01HD074542-04, as well as grants R01 HD076412-02 and P01 HD075795-02 and the U.S.-Israel Binational Science Foundation. Support for this research was also provided by NIH P50 HD076210, the Robert Dow Foundation, the Frederick & Theresa Dow Wallace Fund of the New York Community Trust and the Brady Urological Foundation. There are no competing interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Does testis weight decline towards the Subarctic? A case study on the common frog, Rana temporaria

NASA Astrophysics Data System (ADS)

Hettyey, Attila; Laurila, Anssi; Herczeg, Gábor; Jönsson, K. Ingemar; Kovács, Tibor; Merilä, Juha

2005-04-01

Interpopulation comparisons of variation in resource availability and in allocation patterns along altitudinal and latitudinal gradients allow insights into the mechanisms shaping the life history of animals. Patterns of between-population differences in female life history traits have been studied intensively across a wide range of taxa, but similar investigations in males have remained scarce. To study if testis weight—a measure of reproductive investment—varies on a geographical scale in anurans, we focussed on the variation in relative testis weight (RelTW) and asymmetry in 22 populations of the common frog Rana temporaria along a 1,600-km latitudinal transect across the Scandinavian peninsula. We found that RelTW decreased towards the north. Body mass and body length both had independent positive effects on testes mass. We found evidence for directional asymmetry (DA) in testis weight with the right testis being larger than the left. The level of DA in testis weight was not related to latitude, but both body mass and testes mass had independent positive effects on asymmetry. We discuss the northwards decrease in RelTW in terms of a decreased reproductive investment as a possible consequence of harsher environmental conditions, and perhaps also, weaker sexual selection in the north than in the south.

High doses of nandrolone decanoate reduce volume of testis and length of seminiferous tubules in rats.

PubMed

Noorafshan, Ali; Karbalay-Doust, Saied; Ardekani, Fakhrodin Mesbah

2005-02-01

Anabolic-androgenic steroid (AAS) compounds rank among the drugs most widely abused with the goal of improving athletic ability, appearance, or muscle mass. It has been shown that these compounds have adverse effects on human and animal physiology and sperm quality, but quantitative structural changes of the testis have received less attention. The present study was conducted to evaluate the effects of nandrolone decanoate, which is one of the AAS compounds, on testis weight and volume, diameter and length of seminiferous tubules in rats by unbiased stereological methods. Adult rats were divided into three groups. The first comprised control rats; the second and third groups received low and high doses of nandrolone decanoate for 14 weeks. The rats were then left untreated for 14 weeks. After removal of the testis, stereological study of these tissues showed that the mean volume of testis and length of the seminiferous tubules in the animals that received high doses of nandrolone decanoate were reduced approximately 32% (p<0.01) and approximately 31% (p<0.04), respectively, in comparison with the control group. It can be concluded that the high doses of nandrolone decanoate produce structural changes in the rat testis that remain 14 weeks after stopping injection of the drug.

Characteristics of boys with the so-called true undescended testis diagnosed at the third postnatal month--a population-based case-control study.

PubMed

Mavrogenis, Stelios; Urbán, Robert; Czeizel, Andrew E

2015-07-01

Undescended testis (cryptorchidism) is a common congenital abnormality of male genital organs diagnosed at birth followed with frequent postnatal descensus. However, the so-called isolated true undescended testis (ITUT) diagnosed at the third postnatal month seems to be an independent defect-entity, and this hypothesis was planned to confirm or reject in the study. The evaluation of birth outcomes and maternal socio-demographic data of cases with ITUT in the population-based large dataset of the Hungarian Congenital Abnormality Registry. There was a higher rate of preterm birth and particularly of low birthweight in 2052 cases with ITUT compared to 24,814 population male controls without any defects. The rate of twins was not higher in cases with older mothers, higher birth order and lower socio-economic status. The comparison of data of boys with undescended testis diagnosed at birth found in the previous study and with ITUT in this study confirmed our hypothesis. Undescended testis can be differentiated into two subgroups: boys with frequent postnatal descensus mainly after preterm delivery and boys with ITUT without postnatal testis descensus with frequent intrauterine growth restriction, older mothers with higher birth order and low socio-economic status.

Roles of piRNAs in microcystin-leucine-arginine (MC-LR) induced reproductive toxicity in testis on male offspring.

PubMed

Zhang, Ling; Zhang, Hui; Zhang, Huan; Benson, Mikael; Han, Xiaodong; Li, Dongmei

2017-07-01

In the present study, we evaluated the toxic effects on the testis of the male offspring of MC-LR exposure during fetal and lactational periods. Pregnant females were distributed into two experimental groups: control group and MC-LR group which were exposed to 0 and 10 μg/L of MC-LR, respectively, through drinking water separately during fetal and lactational periods. At the age of 30 days after birth, the male offspring were euthanized. The body weight, testis index, and histomorphology change were observed and the global changes of piwi-interacting RNA (piRNA) expression were evaluated. The results revealed that MC-LR was found in the testis of male offspring, body weight and testis index decreased significantly, and testicular tissue structure was damaged in the MC-LR group. In addition, the exposure to MC-LR resulted in an altered piRNA expression profile and an increase of the cell apoptosis and a decrease of the cell proliferation in the testis of the male offspring. It was reasonable to speculate that the toxic effects on reproductive system of the male offspring in MC-LR group might be mediated by piRNAs through the regulation of the target genes. As far as we are aware, this is the first report showing that MC-LR could play a role in disorder of proliferative and cell apoptosis in the testis of the male offspring by the maternal transmission effect of toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Use of testicular sperm for intracytoplasmic sperm injection in men with high sperm DNA fragmentation: a SWOT analysis

PubMed Central

Esteves, Sandro C; Roque, Matheus; Garrido, Nicolás

2018-01-01

Spermatozoa retrieved from the testis of men with high levels of sperm DNA fragmentation (SDF) in the neat semen tend to have better DNA quality. Given the negative impact of SDF on the outcomes of Assisted Reproductive Technology (ART), an increased interest has emerged about the use of testicular sperm for intracytoplasmic sperm injection (Testi-ICSI). In this article, we used a SWOT (strengths, weaknesses, opportunities, and threats) analysis to summarize the advantages and drawbacks of this intervention. The rationale of Testi-ICSI is bypass posttesticular DNA fragmentation caused by oxidative stress during sperm transit through the epididymis. Hence, oocyte fertilization by genomically intact testicular spermatozoa may be optimized, thus increasing the chances of creating a normal embryonic genome and the likelihood of achieving a live birth, as recently demonstrated in men with high SDF. However, there is still limited evidence as regards the clinical efficacy of Testi-ICSI, thus creating opportunities for further confirmatory clinical research as well as investigation of Testi-ICSI in clinical scenarios other than high SDF. Furthermore, Testi-ICSI can be compared to other laboratory preparation methods for deselecting sperm with damaged DNA. At present, the available literature supports the use of testicular sperm when performing ICSI in infertile couples whose male partners have posttesticular SDF. Due to inherent risks of sperm retrieval, Testi-ICSI should be offered when less invasive treatments for alleviating DNA damage have failed. A call for continuous monitoring is nonetheless required concerning the health of generated offspring and the potential complications of sperm retrieval. PMID:28440264

Use of testicular sperm for intracytoplasmic sperm injection in men with high sperm DNA fragmentation: a SWOT analysis.

PubMed

Esteves, Sandro C; Roque, Matheus; Garrido, Nicolás

2018-01-01

Spermatozoa retrieved from the testis of men with high levels of sperm DNA fragmentation (SDF) in the neat semen tend to have better DNA quality. Given the negative impact of SDF on the outcomes of Assisted Reproductive Technology (ART), an increased interest has emerged about the use of testicular sperm for intracytoplasmic sperm injection (Testi-ICSI). In this article, we used a SWOT (strengths, weaknesses, opportunities, and threats) analysis to summarize the advantages and drawbacks of this intervention. The rationale of Testi-ICSI is bypass posttesticular DNA fragmentation caused by oxidative stress during sperm transit through the epididymis. Hence, oocyte fertilization by genomically intact testicular spermatozoa may be optimized, thus increasing the chances of creating a normal embryonic genome and the likelihood of achieving a live birth, as recently demonstrated in men with high SDF. However, there is still limited evidence as regards the clinical efficacy of Testi-ICSI, thus creating opportunities for further confirmatory clinical research as well as investigation of Testi-ICSI in clinical scenarios other than high SDF. Furthermore, Testi-ICSI can be compared to other laboratory preparation methods for deselecting sperm with damaged DNA. At present, the available literature supports the use of testicular sperm when performing ICSI in infertile couples whose male partners have posttesticular SDF. Due to inherent risks of sperm retrieval, Testi-ICSI should be offered when less invasive treatments for alleviating DNA damage have failed. A call for continuous monitoring is nonetheless required concerning the health of generated offspring and the potential complications of sperm retrieval.

Histopathological effects of boldenone in cattle.

PubMed

Groot, M J; Biolatti, B

2004-03-01

Histopathology of male cattle previously found positive for béta-boldenone in urine in the Netherlands and in Italy was studied. The animals were derived from practice and several weeks had passed after the finding of béta-boldenone before the animals were examined. The animals consisted of 34 male veal calves and one finishing bull. In the prostate gland hypersecretion, cyst formation (45%) and hyperplasia of the urethral epithelium was observed, in the bulbo-urethral gland similar alterations were present. The testis showed reduced development and degeneration of the germinal epithelium (70%), leading to debris and syncytial cell formation in the lumina. Stromal proliferation was evident. In some animals the liver was sampled and showed periportal fibrosis, bile duct proliferation and sometimes necrosis. The bull also showed degeneration of the germinal epithelium of the testis and absence of sperm production, the prostate gland showed some secretion and had an atrophic appearance. It is concluded that béta-boldenone may lead to degeneration of the germinal epithelium of the testis and hypersecretion and cyst formation in the prostate and bulbo-urethral gland, which alterations may heal in time.

Effects of flutamide and finasteride on rat testicular descent.

PubMed

Spencer, J R; Torrado, T; Sanchez, R S; Vaughan, E D; Imperato-McGinley, J

1991-08-01

The endocrine control of descent of the testis in mammalian species is poorly understood. The androgen dependency of testicular descent was studied in the rat using an antiandrogen (flutamide) and an inhibitor of the enzyme 5 alpha-reductase (finasteride). Androgen receptor blockade inhibited testicular descent more effectively than inhibition of 5 alpha-reductase activity. Moreover, its inhibitory effect was limited to the outgrowth phase of the gubernaculum testis, particularly the earliest stages of outgrowth. Gubernacular size was also significantly reduced in fetuses exposed to flutamide during the outgrowth period. In contrast, androgen receptor blockade or 5 alpha-reductase inhibition applied after the initiation of gubernacular outgrowth or during the regression phase did not affect testicular descent. Successful inhibition of the development of epididymis and vas by prenatal flutamide did not correlate with ipsilateral testicular maldescent, suggesting that an intact epididymis is not required for descent of the testis. Plasma androgen assays confirmed significant inhibition of dihydrotestosterone formation in finasteride-treated rats. These data suggest that androgens, primarily testosterone, are required during the early phases of gubernacular outgrowth for subsequent successful completion of testicular descent.

Germ cell survival and differentiation after xenotransplantation of testis tissue from three endangered species: Iberian lynx (Lynx pardinus), Cuvier's gazelle (Gazella cuvieri) and Mohor gazelle (G. dama mhorr).

PubMed

Arregui, Lucía; Dobrinski, Ina; Roldan, Eduardo R S

2014-01-01

The use of assisted reproductive techniques for endangered species is a major goal for conservation. One of these techniques, testis tissue xenografting, allows for the development of spermatozoa from animals that die before reaching sexual maturity. To assess the potential use of this technique with endangered species, testis tissue from six Iberian lynxes (one fetus, two perinatal cubs, two 6-month-old and one 2-year-old lynx), two Cuvier's gazelle fetuses and one 8-month-old Mohor gazelle were transplanted ectopically into nude mice. Tissue from the lynx fetus, perinatal cubs and 2-year-old donors degenerated, whereas spermatogonia were present in 15% of seminiferous tubules more than 70 weeks after grafting in transplanted testis tissue from 6-month-old donors. Seminal vesicle weights (indicative of testosterone production) increased over time in mice transplanted with tissue from 6-month-old lynxes. Progression of spermatogenesis was observed in xenografts from gazelles and was donor age dependent. Tissue from Cuvier's gazelle fetuses contained spermatocytes 40 weeks after grafting. Finally, round spermatids were found 28 weeks after transplantation in grafts from the 8-month-old Mohor gazelle. This is the first time that xenotransplantation of testicular tissue has been performed with an endangered felid and the first successful xenotransplantation in an endangered species. Our results open important options for the preservation of biological diversity.

Nestin in the epididymis is expressed in vascular wall cells and is regulated during postnatal development and in case of testosterone deficiency.

PubMed

Reckmann, Ansgar N; Tomczyk, Claudia U M; Davidoff, Michail S; Michurina, Tatyana V; Arnhold, Stefan; Müller, Dieter; Mietens, Andrea; Middendorff, Ralf

2018-01-01

Vascular smooth muscle cells (SMCs), distinguished by the expression of the neuronal stem cell marker nestin, may represent stem cell-like progenitor cells in various organs including the testis. We investigated epididymal tissues of adult nestin-GFP mice, rats after Leydig cell depletion via ethane dimethane sulfonate (EDS), rats and mice during postnatal development and human tissues. By use of Clarity, a histochemical method to illustrate a three-dimensional picture, we could demonstrate nestin-GFP positive cells within the vascular network. We localized nestin in the epididymis in proliferating vascular SMCs by colocalization with both smooth muscle actin and PCNA, and it was distinct from CD31-positive endothelial cells. The same nestin localization was found in the human epididymis. However, nestin was not found in SMCs of the epididymal duct. Nestin expression is high during postnatal development of mouse and rat and down-regulated towards adulthood when testosterone levels increase. Nestin increases dramatically in rats after Leydig cell ablation with EDS and subsequently low testosterone levels. Interestingly, during this period, the expression of androgen receptor in the epididymis is low and increases until nestin reaches normal levels of adulthood. Here we show that nestin, a common marker for neuronal stem cells, is also expressed in the vasculature of the epididymis. Our results give new insights into the yet underestimated role of proliferating nestin-expressing vascular SMCs during postnatal development and repair of the epididymis.

Effect on embryos of injection of phosphorothioate-modified oligonucleotides into pregnant mice.

PubMed

Gaudette, M F; Hampikian, G; Metelev, V; Agrawal, S; Crain, W R

1993-01-01

Phosphorothioate-modified oligonucleotides were injected into pregnant female mice to assess the effect on developing embryos. Injections were carried out during two different time periods, one when embryos were in preimplantation stages of development (about 3.5 days of development) and the other after implantation, when both a fetus and placenta are present (from days 9.5 to 11.5 of development). Three different phosphorothioate-modified oligonucleotides were injected. One, which had a sequence not present in the mouse genome, was used to ask whether nonspecific toxic or teratogenic effects on embryos result from treatment of the mother. A second was complementary to the mRNA of the testis-determining factor gene Sry and was used to ask whether a specific developmental pathway (i.e., sex determination) could be disrupted in embryos in vivo. The third was the complement of the anti-Sry sequence. None of these oligonucleotides reduced the frequency of successful pregnancy after mating or the average litter size from that observed in controls animals. Furthermore, examination of 291 pups or fetuses from all oligonucleotide-injected pregnant females revealed no developmental defects regardless of which sequence was used. It is concluded that injection of phosphorothioate-modified oligonucleotides into pregnant females according to the protocols described here is not toxic or teratogenic to embryos in a nonspecific way. Also, anti-Sry oligonucleotides did not influence sex determination in embryos, although there are several possible explanations for this.

Systematic characterization of human testis-specific actin capping protein β3 as a possible biomarker for male infertility.

PubMed

Soda, T; Miyagawa, Y; Ueda, N; Takezawa, K; Okuda, H; Fukuhara, S; Fujita, K; Kiuchi, H; Uemura, M; Okamoto, Y; Tsujimura, A; Tanaka, H; Nonomura, N

2017-03-01

Is actin capping protein (CP) β3 involved in human spermatogenesis and male infertility? Human CPβ3 (hCPβ3) is expressed in testis, changes its localization dynamically during spermatogenesis, and has some association with male infertility. The testis-specific α subunit of CP (CPα3) was previously identified in human, and mutations in the cpα3 gene in mouse were shown to induce malformation of the sperm head and male infertility. However, CPβ3, which is considered to be a heterodimeric counterpart of CPα3, has been neither characterized in human nor reported in association with male infertility. To confirm the existence of CPβ3 in human testis, fresh semen samples from proven fertile men were analyzed. To investigate protein expression during spermatogenesis, cryopreserved testis obtained from men with obstructive azoospermia were examined by immunofluorescent analysis. To assess the association of CP with male infertility, we compared protein expression of human CPα3 (hCPα3) and hCPβ3 using immunofluorescent analysis of cryopreserved sperm between men with normozoospermia (volunteers: Normo group, n = 20) and infertile men with oligozoospermia and/or asthenozoospermia (O + A group, n = 21). The tissue-specific expression of hCPβ3 was investigated by RT-PCR and Western blot analysis. To investigate whether hCPα3 and hCPβ3 form a heterodimer, a tandem expression vector containing hcpα3 tagged with monomeric red fluorescent protein 1 and hcpβ3 tagged with enhanced green fluorescent protein in a single plasmid was constructed and analyzed by co-immunoprecipitation (Co-IP) assay. The protein expression profiles of hCPα3 and hCPβ3 during spermatogenesis were examined by immunohistochemical analysis using human spermatogenic cells. The protein expressions of hCPα3 and hCPβ3 in sperm were compared between the Normo and O + A groups by immunohistochemical analysis. RT-PCR showed that mRNA of hcpβ3 was expressed exclusively in testis. Western blot analysis detected hCPβ3 with anti-bovine CPβ3 antibody. Co-IP assay with recombinant protein showed that hCPα3 and hCPβ3 form a protein complex. At each step during spermatogenesis, the cellular localization of hCPβ3 changed dynamically. In spermatogonia, hCPβ3 showed a slight signal in cytoplasm. hCPβ3 expression was conspicuous mainly from spermatocytes, and hCPβ3 localization dynamically migrated from cytoplasm to the acrosomal cap and acrosome. In mature spermatozoa, hCPβ3 accumulated in the postacrosomal region and less so at the midpiece of the tail. Double-staining analysis revealed that hCPα3 localization was identical to hCPβ3 at every step in the spermatogenic cells. Most spermatozoa from the Normo group were stained homogenously by both hCPα3 and hCPβ3. In contrast, significantly more spermatozoa in the O + A versus Normo group showed heterogeneous or lack of staining for either hCPα3 or hCPβ3 (abnormal staining) (P < 0.001). The percentage of abnormal staining was higher in the O + A group (52.4 ± 3.0%) than in the Normo group (31.2 ± 2.5%). Even by confining the observations to morphologically normal spermatozoa selected in accordance with David's criteria, the percentage of abnormal staining was still higher in the O + A group (39.9 ± 2.9%) versus the Normo group (22.5 ± 2.1%) (P < 0.001). hCPβ3 in conjunction with hCPα3 seemed to play an important role in spermatogenesis and may be associated with male infertility. Not applicable. Owing to the difficulty of collecting fresh samples of human testis, we used cryopreserved samples from testicular sperm extraction. To examine the interaction of spermatogenic cells or localization in seminiferous tubules, fresh testis sample of healthy males are ideal. The altered expression of hCPα3 and hCPβ3 may not only be a cause of male infertility but also a prognostic factor for the results of ART. They may be useful biomarkers to determine the fertilization ability of human sperm in ART. This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JP16K20133). The authors declare no competing interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Dmrt1 is necessary for male sexual development in zebrafish

PubMed Central

Webster, Kaitlyn A.; Schach, Ursula; Ordaz, Angel; Steinfeld, Jocelyn S.; Draper, Bruce W.; Siegfried, Kellee R.

2018-01-01

The dmrt1 (doublesex and mab-3 related transcription factor 1) gene is a key regulator of sex determination and/or gonadal sex differentiation across metazoan animals. This is unusual given that sex determination genes are typically not well conserved. The mechanisms by which zebrafish sex is determined have remained elusive due to the lack of sex chromosomes and the complex polygenic nature of sex determination in domesticated strains. To investigate the role of dmrt1 in zebrafish sex determination and gonad development, we isolated mutations disrupting this gene. We found that the majority of dmrt1 mutant fish develop as fertile females suggesting a complete male-to-female sex reversal in mutant animals that would have otherwise developed as males. A small percentage of mutant animals became males, but were sterile and displayed testicular dysgenesis. Therefore zebrafish dmrt1 functions in male sex determination and testis development. Mutant males had aberrant gonadal development at the onset of gonadal sex-differentiation, displaying reduced oocyte apoptosis followed by development of intersex gonads and failed testis morphogenesis and spermatogenesis. By contrast, female ovaries developed normally. We found that Dmrt1 is necessary for normal transcriptional regulation of the amh (anti-Müllerian hormone) and foxl2 (forkhead box L2) genes, which are thought to be important for male or female sexual development respectively. Interestingly, we identified one dmrt1 mutant allele that cooperates with a linked segregation distorter locus to generate an apparent XY sex determination mechanism. We conclude that dmrt1 is dispensable for ovary development but necessary for testis development in zebrafish, and that dmrt1 promotes male development by transcriptionally regulating male and female genes as has been described in other animals. Furthermore, the strong sex-ratio bias caused by dmrt1 reduction-of-function points to potential mechanisms through which sex chromosomes may evolve. PMID:27940159

Fusion Imaging: A Novel Staging Modality in Testis Cancer

DTIC Science & Technology

2010-01-01

the anatomic precision of computed tomography. To the best of our knowledge, this represents the first study of the effectiveness using fusion...imaging in evaluation of patients with testis cancer. Methods: A prospective study of 49 patients presenting to Walter Reed Army Medical Center with...incidence of testis cancer has been increasing at an annual rate of 3%, leading to a doubling in cases world-wide over the last 40 years. With the advent

Gestational form of Selenium in Free-Choice Mineral Mixes Affects Transcriptome Profiles of the Neonatal Calf Testis, Including those of Steroidogenic and Spermatogenic Pathways.

PubMed

Cerny, K L; Garbacik, S; Skees, C; Burris, W R; Matthews, J C; Bridges, P J

2016-01-01

In areas where soils are deficient in Selenium (Se), dietary supplementation of this trace mineral directly to cattle is recommended. Because Se status affects testosterone synthesis and frequency of sperm abnormalities, and the form of Se supplemented to cows affects tissue-specific gene expression, the objective of this study was to determine whether the form of Se consumed by cows during gestation would affect the expression of mRNAs that regulate steroidogenesis and/or spermatogenesis in the neonatal calf testis. Twenty-four predominantly Angus cows were assigned randomly to have individual, ad libitum, access of a mineral mix containing 35 ppm of Se in free-choice vitamin-mineral mixes as either inorganic (ISe), organic (OSe), or a 50/50 mix of ISe and OSe (MIX), starting 4 months prior to breeding and continuing throughout gestation. Thirteen male calves were born over a 3-month period (ISe, n = 5; OSe, n = 4; MIX, n = 4), castrated within 2 days of birth, and extracted testis RNA subjected to transcriptomal analysis by microarray (Affymetrix Bovine 1.0 ST arrays) and targeted gene expression analysis by real-time reverse-transcription PCR (RT-PCR) of mRNAs encoding proteins known to affect steroidogenesis and/or spermatogenesis. The form of dam Se affected (P < 0.05) the expression of 853 annotated genes, including 17 mRNAs putatively regulating steroidogenesis and/or spermatogenesis. Targeted RT-PCR analysis indicated that the expression of mRNA encoding proteins CYP2S1 (cytochrome P450, family 2, subfamily S, polypeptide 1), HSD17B7 (hydroxysteroid (17β) dehydrogenase 7), SULT1E1 (sulfotransferase family 1E, estrogen preferring, member 1), LDHA (lactate dehydrogenase A), CDK5R1 (cyclin-dependent kinase 5, regulatory subunit 1), and LEP (leptin) was affected (P < 0.05) by form of Se consumed by dams of developing bull calves, while AKR1C4 (aldo-keto reductase family 1, member C4) and CCND2 (cyclin D2) tended (P < 0.09) to be affected. Our results indicate that form of Se fed to dams during gestation affected the transcriptome of the neonatal calf testis. If these profiles are maintained throughout maturation, then the form of Se fed to dams may impact bull fertility and the development of Se form-dependent mineral mixes that target gestational development of the testis are warranted.

Combined Leydig cell and Sertoli cell dysfunction in 46,XX males lacking the sex determining region Y gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Turner, B.; Vordermark, J.S.; Fechner, P.Y.

1995-07-03

We have evaluated 3 individuals with a rare form of 46,XX sex reversal. All of them had ambiguous external genitalia and mixed wolffian and muellerian structures, indicating both Leydig cell and Sertoli cell dysfunction, similar to that of patients with true hermaphroditism. However, gonadal tissue was not ovotesticular but testicular with varying degrees of dysgenesis. SRY sequences were absent in genomic DNA from peripheral leukocytes in all 3 subjects. Y centromere sequences were also absent, indicating that testis development did not occur because of a low level mosaicism of Y-bearing cells. The subjects in this report demonstrate that there ismore » a continuum in the extent of the testis determination in SRY-negative 46,XX sex reversal, ranging from nearly normal to minimal testicular development. 20 refs.« less

Thyroid hormone actions on male reproductive system of teleost fish.

PubMed

Tovo-Neto, Aldo; da Silva Rodrigues, Maira; Habibi, Hamid R; Nóbrega, Rafael Henrique

2018-04-17

Thyroid hormones (THs) play important roles in the regulation of many biological processes of vertebrates, such as growth, metabolism, morphogenesis and reproduction. An increasing number of studies have been focused on the involvement of THs in the male reproductive system of vertebrates, in particular of fish. Therefore, this mini-review aims to summarize the main findings on THs role in male reproductive system of fish, focusing on sex differentiation, testicular development and spermatogenesis. The existing data in the literature have demonstrated that THs exert their roles at the different levels of the hypothalamic-pituitary-gonadal (HPG) axis. In general a positive correlation has been shown between THs and fish reproductive status; where THs are associated with testicular development, growth and maturation. Recently, the molecular mechanisms underlying the role of THs in spermatogenesis have been unraveled in zebrafish testis. THs promote germ cell proliferation and differentiation by increasing a stimulatory growth factor of spermatogenesis produced by Sertoli cells. In addition, THs enhanced the gonadotropin-induced androgen release in zebrafish testis. Next to their functions in the adult testis, THs are involved in the gonadal sex differentiation through modulating sex-related gene expression, and testicular development via regulation of Sertoli cell proliferation. In conclusion, this mini-review showed that THs modulate the male reproductive system during the different life stages of fish. The physiological and molecular mechanisms showed a link between the thyroid and reproduction, suggesting a possibly co-evolution and interdependence of these two systems. Copyright © 2018 Elsevier Inc. All rights reserved.

Transforming Growth Factor Beta (TGFβ1, TGFβ2 and TGFβ3) Null-Mutant Phenotypes in Embryonic Gonadal Development

PubMed Central

Memon, Mushtaq A.; Anway, Matthew D.; Covert, Trevor R.; Uzumcu, Mehmet; Skinner, Michael K.

2008-01-01

The role transforming growth factor beta (TGFb) isoforms TGFb1, TGFb2 and TGFb3 have in the regulation of embryonic gonadal development was investigated with the use of null-mutant (i.e. knockout) mice for each of the TGFb isoforms. Late embryonic gonadal development was investigated because homozygote TGFb null-mutant mice generally die around birth, with some embryonic loss as well. In the testis, the TGFb1 null-mutant mice had a decrease in the number of germ cells at birth, postnatal day 0 (P0). In the testis, the TGFb2 null-mutant mice had a decrease in the number of seminiferous cords at embryonic day 15 (E15). In the ovary, the TGFb2 null-mutant mice had an increase in the number of germ cells at P0. TGFb isoforms appear to have a role in gonadal development, but interactions between the isoforms is speculated to compensate in the different TGFb isoform null-mutant mice. PMID:18790002

Successful xenogeneic germ cell transplantation from Jundia catfish (Rhamdia quelen) into adult Nile tilapia (Oreochromis niloticus) testes.

PubMed

Silva, M A; Costa, G M J; Lacerda, S M S N; Brandão-Dias, P F P; Kalapothakis, E; Silva Júnior, A F; Alvarenga, E R; França, L R

2016-05-01

Fish germ cell transplantation presents several important potential applications for aquaculture, including the preservation of germplasm from endangered fish species with high genetic and commercial values. Using this technique in studies developed in our laboratory with adult male Nile tilapias (Oreochromis niloticus), all the necessary procedures were successfully established, allowing the production of functional sperm and healthy progeny approximately 2months after allogeneic transplantation. In the present study, we evaluated the viability of the adult Nile tilapia testis to generate sperm after xenogeneic transplant of germ cells from sexually mature Jundia catfish (Rhamdia quelen) that belong to a different taxonomic order. Therefore, in order to investigate at different time-periods post-transplantation, the presence and development of donor PKH26 labeled catfish germ cells were followed in the tilapia seminiferous tubules. From 7 to 20days post-transplantation, only PKH26 labeled spermatogonia were observed, whereas spermatocytes at different stages of development were found at 70days. Germ cell transplantation success and progression of spermatogenesis were indicated by the presence of labeled PKH26 spermatids and sperm on days 90 and 120 post-transplantation, respectively. Confirming the presence of the catfish genetic material in the tilapia testis, all recipient tilapias evaluated (n=8) showed the genetic markers evaluated. Therefore, we demonstrated for the first time that the adult Nile tilapia testis offers the functional conditions for development of spermatogenesis with sperm production from a fish species belonging to a different order, which provides an important new venue for aquaculture advancement. Copyright © 2016 Elsevier Inc. All rights reserved.

Retinoic Acid Signalling and the Control of Meiotic Entry in the Human Fetal Gonad

PubMed Central

Kinnell, Hazel L.; Anderson, Richard A.; Saunders, Philippa T. K.

2011-01-01

The development of mammalian fetal germ cells along oogenic or spermatogenic fate trajectories is dictated by signals from the surrounding gonadal environment. Germ cells in the fetal testis enter mitotic arrest, whilst those in the fetal ovary undergo sex-specific entry into meiosis, the initiation of which is thought to be mediated by selective exposure of fetal ovarian germ cells to mesonephros-derived retinoic acid (RA). Aspects of this model are hard to reconcile with the spatiotemporal pattern of germ cell differentiation in the human fetal ovary, however. We have therefore examined the expression of components of the RA synthesis, metabolism and signalling pathways, and their downstream effectors and inhibitors in germ cells around the time of the initiation of meiosis in the human fetal gonad. Expression of the three RA-synthesising enzymes, ALDH1A1, 2 and 3 in the fetal ovary and testis was equal to or greater than that in the mesonephros at 8–9 weeks gestation, indicating an intrinsic capacity within the gonad to synthesise RA. Using immunohistochemistry to detect RA receptors RARα, β and RXRα, we find germ cells to be the predominant target of RA signalling in the fetal human ovary, but also reveal widespread receptor nuclear localization indicative of signalling in the testis, suggesting that human fetal testicular germ cells are not efficiently shielded from RA by the action of the RA-metabolising enzyme CYP26B1. Consistent with this, expression of CYP26B1 was greater in the human fetal ovary than testis, although the sexually-dimorphic expression patterns of the germ cell-intrinsic regulators of meiotic initiation, STRA8 and NANOS2, appear conserved. Finally, we demonstrate that RA induces a two-fold increase in STRA8 expression in cultures of human fetal testis, but is not sufficient to cause widespread meiosis-associated gene expression. Together, these data indicate that while local production of RA within the fetal ovary may be important in regulating the onset of meiosis in the human fetal ovary, mechanisms other than CYP26B1-mediated metabolism of RA may exist to inhibit the entry of germ cells into meiosis in the human fetal testis. PMID:21674038

Oxidative Stress Triggers Body-Wide Skipping of Multiple Exons of the Spinal Muscular Atrophy Gene

PubMed Central

Seo, Joonbae; Singh, Natalia N.; Ottesen, Eric W.; Sivanesan, Senthilkumar; Shishimorova, Maria; Singh, Ravindra N.

2016-01-01

Humans carry two nearly identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Loss of SMN1 leads to spinal muscular atrophy (SMA), the most frequent genetic cause of infant mortality. While SMN2 cannot compensate for the loss of SMN1 due to predominant skipping of exon 7, correction of SMN2 exon 7 splicing holds the promise of a cure for SMA. Previously, we used cell-based models coupled with a multi-exon-skipping detection assay (MESDA) to demonstrate the vulnerability of SMN2 exons to aberrant splicing under the conditions of oxidative stress (OS). Here we employ a transgenic mouse model and MESDA to examine the OS-induced splicing regulation of SMN2 exons. We induced OS using paraquat that is known to trigger production of reactive oxygen species and cause mitochondrial dysfunction. We show an overwhelming co-skipping of SMN2 exon 5 and exon 7 under OS in all tissues except testis. We also show that OS increases skipping of SMN2 exon 3 in all tissues except testis. We uncover several new SMN2 splice isoforms expressed at elevated levels under the conditions of OS. We analyze cis-elements and transacting factors to demonstrate the diversity of mechanisms for splicing misregulation under OS. Our results of proteome analysis reveal downregulation of hnRNP H as one of the potential consequences of OS in brain. Our findings suggest SMN2 as a sensor of OS with implications to SMA and other diseases impacted by low levels of SMN protein. PMID:27111068

In vitro effects of simulated microgravity on Sertoli cell function

NASA Astrophysics Data System (ADS)

Masini, M. A.; Prato, P.; Scarabelli, L.; Lanza, C.; Palmero, S.; Pointis, G.; Ricci, F.; Strollo, F.

2011-02-01

With the advent of space flights questions concerning the effects of microgravity (0×G) on human reproductive physiology have received great attention. The aim of this study was to evaluate the influence of 0×G on Sertoli cells. A Sertoli cell line from mouse testis (42GPA9) was analyzed for cytoskeletal and Sex Hormone Binding Globilin (SHBG) changes by immunohistochemistry, for antioxidant content by RT-PCR and for culture medium lactate concentrations by protein chemistry. Cells were cultured for 6, 24 and 48 h on a three-dimensional Random Positioning Machine (3D-RPM); static controls (1×G) were positioned on the supporting frame. At the end of each experiment, cultured cells were either fixed in paraformaldehyde or lysed and RNA-extracted or used for culture medium lactate measurements as needed. At 0×G, Sertoli cytoskeleton became disorganized, microtubules fragmented and SHBG undetectable already after 24 h, with alterations worsening by 48 h. It was evident that various antioxidant systems appreciably increased during the first 24 h but significantly decreased at 48 h. No changes occurred in the 1×G samples. Initially, 0×G seemed to disturb antioxidant protection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate production at 0×G slightly decreased after 24 h. Further experiments are needed in space to investigate upon steroidogenesis and germ cell differentiation within the testis, to rule out male infertility as a possible consequence, which could be a problem, as life expectancy increases.

Expression of MAGE--A restricted to testis and ovary or to various cancers in dogs.

PubMed

Chen, Yin-Chu; Hsu, Wei-Li; Chiu, Cheng-Yang; Liao, Jiunn-Wang; Chang, Chao-Chin; Chang, Shih-Chieh

2013-05-15

Expression of MAGE-A protein, a family of cancer/testis antigens, was investigated in normal and neoplastic canine tissues. Immunohistochemical analysis of cross-reactions between a mouse anti-human MAGE-A proteins including MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 monoclonal antibody and canine proteins, showed positive immunoreactivity only in testicular spermatogonia and spermatocytes, and ovary oocytes. The immunoreaction was negative in all other tissues tested, including normal tissues of the skin, gingiva, muscle, adipose, connective, salivary gland, lymph node, intestinal mucosa, mammary gland, liver, cartilage, oviduct, endometrium, cerebrum and cerebellum. Use of a scoring system in the investigated tumors showed positive immunoreactivity in 75% (21/28) of melanomas including oral, cutaneous, eyelid, and interdigital melanomas; in 68.7% (22/32) of oral and nasal tumors; in 52.5% (21/40) discrete round cell tumors; and in 40.5% (15/37) of soft tissue sarcomas. Different tumor types also showed large difference in percentage of MAGE-A expression. Although oral squamous cell carcinomas, multicentric lymphomas and extraosseous osteosarcomas showed no expression, overexpression occurred in oral melanomas (81.82%, 18/21), malignant nasal tumors (100%, 3/3) and in transmissible venereal tumors (100%, 10/10). Based on the characteristic expression of MAGE-A in canine germ cells and in various neoplasms, MAGE-A has potential use as an indicator of malignancy but is probably unsuitable for strictly diagnostic purposes (i.e., diagnosis of tumor type). Copyright © 2013 Elsevier B.V. All rights reserved.

Differences in the fatty-acid composition of rodent spermatozoa are associated to levels of sperm competition

PubMed Central

delBarco-Trillo, Javier; Mateo, Rafael; Roldan, Eduardo R. S.

2015-01-01

Sperm competition is a prevalent phenomenon that drives the evolution of sperm function. High levels of sperm competition lead to increased metabolism to fuel higher sperm velocities. This enhanced metabolism can result in oxidative damage (including lipid peroxidation) and damage to the membrane. We hypothesized that in those species experiencing high levels of sperm competition there are changes in the fatty-acid composition of the sperm membrane that makes the membrane more resistant to oxidative damage. Given that polyunsaturated fatty acids (PUFAs) are the most prone to lipid peroxidation, we predicted that higher sperm competition leads to a reduction in the proportion of sperm PUFAs. In contrast, we predicted that levels of sperm competition should not affect the proportion of PUFAs in somatic cells. To test these predictions, we quantified the fatty-acid composition of sperm, testis and liver cells in four mouse species (genus Mus) that differ in their levels of sperm competition. Fatty-acid composition in testis and liver cells was not associated to sperm competition levels. However, in sperm cells, as predicted, an increase in sperm competition levels was associated with an increase in the proportion of saturated fatty-acids (the most resistant to lipid peroxidation) and by a concomitant decrease in the proportion of PUFAs. Two particular fatty acids were most responsible for this pattern (arachidonic acid and palmitic acid). Our findings thus indicate that sperm competition has a pervasive influence in the composition of sperm cells that ultimately may have important effects in sperm function. PMID:25795911

[Male pseudohermaphroditism].

PubMed

Fukutani, K

1997-11-01

Male pseudohermaphroditism is a condition of sex differentiation disorders in which the gonads are tests, but the genital ducts and/or external genitalia are incompletely masculinized. This syndrome is caused by a failure of the sequential process in embryonal development of the testis. In the presence of functioning testis the Müllerian ducts regress, while the mesonephric ducts and urogenital sinus differentiate into the internal and external male genitalia. Male pseudohermaphroditism is classified to subtypes according to etiological factors: (1) testicular unresponsiveness to hCG and LH; (2) defect in testosterone biosynthesis; (3) end-organ resistance to androgen; (4) defects in the intracellular metabolism of testosterone; (5) aberrations in testicular organogenesis; (6) defects in anti-Müllerian hormone.

Organization of testicular interstitial tissue of an Australian rodent, the spinifex hopping mouse, Notomys alexis.

PubMed

Peirce, E J; Breed, W G

1990-05-01

The organization of testicular interstitial tissue of the spinifex hopping mouse, Notomys alexis differs from that of other rodents. It comprises between 10.3% and 17.3% (average 15.0%) of the total testicular volume, and is variable in its organization both at different locations within the testis of the one animal and among different individuals. Abundant, closely packed Leydig cells are usually present; however, in some regions large, thick-walled blood vessels and extensive peritubular lymphatic spaces, often lacking an endothelium adjacent to the Leydig cells, are also prominent. The Leydig cells in contact with the large blood vessels and lymphatics, unlike those in regions where lymph is sparse, are not densely packed and sometimes contain numerous lipid droplets. Ultrastructure of Leydig cells is typical of steroid-producing cells; however, mitochondria are often extremely large, unusual in shape or bizarrely arranged in relation to one another. Also electron-dense bodies displaying a paracrystalline-like internal structure of parallel, electron-dense filaments arranged in a lattice pattern occur in the cytoplasm of many cells. The significance of these unusual ultrastructural features and the organization of the interstitial tissue remain to be determined conclusively, but may relate to steroid synthesis, secretion and uptake.

Distributional map of the terminal and sub-terminal sugar residues of the glycoconjugates in the prepubertal and postpubertal testis of a subject affected by complete androgen insensitivity syndrome (Morris's syndrome): lectin histochemical study.

PubMed

Gheri, G; Vannelli, G B; Marini, M; Zappoli Thyrion, G D; Gheri, R G; Sgambati, E

2004-01-01

In the present research we have investigated the distribution of the sugar residues of the glycoconjugates in the prepubertal and postpubertal testes of a subject with Morris's syndrome (CAIS, Complete Androgen Insensitivity Syndrome). For this purpose a battery of six horseradish peroxidase-conjugated lectins was used (SBA, PNA, WGA, ConA, LTA and UEAI). We have obtained a complete distributional map of the terminal and sub-terminal oligosaccharides in the tunica albuginea, interstitial tissue, lamina propria of the seminiferous tubules, Leydig cells, Sertoli cells, spermatogonia, mastocytes and endothelial cells. Furthermore the present study has shown that a large amount of sugar residues were detectable in the prepubertal and postpubertal testes but that some differences exist with particular regard to the Sertoli cells. The Sertoli cells and the Leydig cells of the retained prepubertal testis of the patient affected by Morris's syndrome were characterized by the presence of alpha-L-fucose, which was absent in the retained prepubertal testis of the normal subjects. Comparing the results on the postpubertal testis with those obtained on the same aged testis of healthy subjects we have demonstrated that alpha-L-fucose in the Sertoli and Leydig cells and D-galactose-N-acetyl-D-galactosamine in the Leydig cells are a unique feature of the subject affected by Morris's syndrome. D-galactose (ss1,3)-N-acetyl-D-galactosamine and sialic acid, which are present in the Leydig cells of the normal testis were never observed in the same cells of the postpubertal testis of the CAIS patient.

Protective effects of polyunsatutared fatty acids supplementation against testicular damage induced by intermittent hypobaric hypoxia in rats.

PubMed

Castillo, Rodrigo L; Zepeda, Andrea B; Short, Stefania E; Figueroa, Elías; Bustos-Obregon, Eduardo; Farías, Jorge G

2015-01-23

Intermittent hypobaric hypoxia (IHH) induces changes in the redox status and structure in rat testis. These effects may be present in people at high altitudes, such as athletes and miners. Polyunsaturated fatty acids (PUFA) can be effective in counteracting these oxidative modifications due to their antioxidants properties. The aim of the work was to test whether PUFA supplementation attenuates oxidative damage in testis by reinforcing the antioxidant defense system. The animals were divided into four groups (7 rats per group): normobaric normoxia (~750 tor; pO2 156 mmHg; Nx); Nx + PUFA, supplemented with PUFA (DHA: EPA = 3:1; 0.3 g kg(-1) of body weight per day); hypoxic hypoxia (~428 tor; pO2 90 mmHg; Hx) and, Hx + PUFA. The hypoxic groups were exposed in 4 cycles to 96 h of HH followed by 96 h of normobaric normoxia for 32 days. Total antioxidant capacity (FRAP) and lipid peroxidation (malondialdehyde, MDA) in plasma and reduced (GSH)/oxidized glutathione (GSSG) ratio, tissue lipid peroxidation (TBARS) and antioxidant enzymes activity were assessed at the end of the study in testis. Also, SIRTUIN 1 and HIF-1 protein expression in testis were determined. IHH increased lipid peroxidation in plasma and HIF-1 protein levels in testis. In addition, IHH reduced FRAP levels in plasma, antioxidant enzymes activities and SIRTUIN 1 protein levels in testis. PUFA supplementation attenuated these effects, inducing the increases in FRAP, in the antioxidant enzymes activity and HIF-1 levels. These results suggest that the IHH model induces a prooxidant status in plasma and testis. The molecular protective effect of PUFA may involve the induction of an antioxidant mechanism.

Stage-specific expression of DDX4 and c-kit at different developmental stages of the porcine testis.

PubMed

Lee, Ran; Lee, Won-Young; Park, Hyun-Jung; Ha, Woo-Tae; Woo, Jae-Seok; Chung, Hak-Jae; Lee, Ji-Heon; Hong, Kwonho; Song, Hyuk

2018-03-01

Spermatogenesis begins with spermatogonial stem cells (SSCs), which are located in the basement membrane of the adult testes. Previous studies have described specific biomarkers for undifferentiated porcine spermatogonia or SSCs; however, these markers are not sufficient to understand spermatogenesis at different developmental stages. The objective of this study was characterize the expression of DEAD-Box polypeptide 4 (DDX4, also known as VASA) and tyrosine-protein kinase kit (c-kit), as potential markers of male germ cells in the porcine testis. In porcine testis tissue at prepubertal stages (5, 30, and 60 days), DDX4 and c-kit protein expression was detected in the most undifferentiated spermatogonia, which also express protein gene product 9.5 (PGP9.5). However, in porcine testis tissues from pubertal and postpubertal stages (90, 120, and 150 days), DDX4 and c-kit were not detected in PGP9.5-positive undifferentiated spermatogonia. The DDX4 expression pattern was similar to that of c-kit in the porcine testis. In adult porcine testes, DDX4-expressing cells were located on the lumenal side, compared to synaptonemal complex protein 3-positive primary spermatocytes, but DDX-4 was not co-expressed with acrosin, a known acrosome marker. In addition, DDX4 was detected in PGP9.5-expressing porcine SSCs in culture. Based on our results, we suggest that DDX4 and c-kit are putative markers of undifferentiated spermatogonia in the prepubertal porcine testis. While in the postpubertal porcine testis, they are markers of differentiated spermatocytes. These findings may facilitate future studies of porcine spermatogenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

Morphological and Surgical Overview of Adolescent Testis Affected by Varicocele

PubMed Central

Santoro, Giuseppe

2013-01-01

Varicocele is a common pathology of the testis frequently associated with infertility. For its management, a fine morphological study of the testis, both macroscopically and microscopically, and an accurate choice of surgical procedure are mandatory. The present review focuses its attention on the anatomic substrates of adolescent varicocele and its pathophysiologic modifications. The comprehensive assessment of all the reported alterations should be considered by the clinician before deciding the type of treatment and the timing. PMID:24348160

Effects of vitamin E on reproductive hormones and testis structure in chronic dioxin-treated mice.

PubMed

Yin, Hai-Ping; Xu, Jian-Ping; Zhou, Xian-Qing; Wang, Ying

2012-03-01

The purpose of this study was to investigate the effects of vitamin E on reproductive hormones and testis structure in mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Five experimental groups of a combination of TCDD and vitamin E were designed as follows: 0 ng/kg/d and 0 mg/kg/d (control group), 100 ng/kg/d and 0 mg/kg/d (Group I), 100 ng/kg/d and 20 mg/kg/d (Group II), 100 ng/kg/d and 100 mg/kg/d (Group III), and 100 ng/kg/d and 500 mg/kg/d (Group IV) respectively. Vitamin E and TCDD were given by oral gavage for 7 weeks. The results demonstrated that TCDD decreased the levels of brain gonadotropin releasing hormone (GnRH), testis luteinizing hormone (LH) and follicle stimulating hormone (FSH), serum testosterone and testis spermatozoa number, and damaged testis structure. Vitamin E at 20 mg/kg alleviated the decrease of GnRH; vitamin E at 20, 100, and 500 mg/kg antagonized the decline of LH and FSH; vitamin E at 20 and 100 mg/kg reversed the decrease of testosterone and spermatozoa number; and vitamin E at 100 mg/kg decreased the damage of the testis structure caused by TCDD. The results indicate that vitamin E antagonizes the reproductive endocrine toxicity and alleviates the changes in testicular structure caused by TCDD.

Protective effect of Zingiber officinale extract on rat testis after cyclophosphamide treatment.

PubMed

Mohammadi, F; Nikzad, H; Taghizadeh, M; Taherian, A; Azami-Tameh, A; Hosseini, S M; Moravveji, A

2014-08-01

Decreasing the side effects of chemotherapy in testis has been the subjects of many studies. In this study, the protective effects of Zingiber officinale extract on rat testis were investigated after chemotherapy with cyclophosphamide. Histological and biochemical parameters were compared in cyclophosphamide-treated rats with or without ginger extract intake. Wistar male rats were randomly divided into four groups each 10. The control group received a single injection of 1 ml isotonic saline intraperitoneally. The Cyclophosphamide (CP) group received a single dose of cyclophosphamide (100 mg kg(-1) BW) intraperitoneally. CP + 300 and CP + 600 groups received orally 300 or 600 mg of ginger extract, respectively, for a period of 6 weeks after cyclophosphamide injection. The morphologic and histological structure of the testis was compared in different groups of the rats. Also, factors like malondialdehyde, reactive oxygen species, total antioxidant capacity and testosterone level were assessed in blood serum as well. Our results showed that although ginger extract could not change testis weight, malondialdehyde (MDA) and ROS, but antioxidant and testosterone levels in serum were increased significantly. Also, an obvious improved histological change was seen in CP + 300 and CP + 600 groups in comparison with CP group. These protective effects of ginger on rat testis after cyclophosphamide treatment could be attributed to the higher serum level of antioxidants. © 2013 Blackwell Verlag GmbH.

Free radicals in adolescent varicocele testis.

PubMed

Romeo, Carmelo; Santoro, Giuseppe

2014-01-01

We examine the relationship between the structure and function of the testis and the oxidative and nitrosative stress, determined by an excessive production of free radicals and/or decreased availability of antioxidant defenses, which occur in the testis of adolescents affected by varicocele. Moreover, the effects of surgical treatment on oxidative stress were provided. We conducted a PubMed and Medline search between 1980 and 2014 using "adolescent," "varicocele," "free radicals," "oxidative and nitrosative stress," "testis," and "seminiferous tubules" as keywords. Cross-references were checked in each of the studies, and relevant articles were retrieved. We conclude that increased concentration of free radicals, generated by conditions of hypoxia, hyperthermia, and hormonal dysfunction observed in adolescent affected by varicocele, can harm germ cells directly or indirectly by influencing nonspermatogenic cells and basal lamina. With regard to few available data in current literature, further clinical trials on the pre- and postoperative ROS and RNS levels together with morphological studies of the cellular component of the testis are fundamental for complete comprehension of the role played by free radicals in the pathogenesis of adolescent varicocele and could justify its pharmacological treatment with antioxidants.

Oxidative alterations induced by D-aspartic acid in prepubertal rat testis in vitro: a mechanistic study.

PubMed

Chandrashekar, K N; Muralidhara

2008-07-01

The objective of the present study was to investigate the oxidative induction response following in vitro treatment with D-aspartic acid (DA) in prepubertal rat testis (homogenates, explants, and cell suspensions). In all three preparations, DA enhanced (P<0.001) lipid peroxidation, manifest as increased reactive oxygen species (ROS) and malondialdehyde (MDA). Further, DA-induced oxidative induction was potentiated (P<0.001) in the presence of iron (5 microM) and 3-amino triazole and mercaptosuccinate (P<0.001), known inhibitors of the peroxide metabolizing enzymes, catalase and glutathione peroxidase, respectively. Testis homogenates exposed to L-arginine (LA) per se had reduced (P<0.001) endogenous levels of ROS and MDA; furthermore, pre-incubation with L-arginine markedly suppressed (P<0.001) DA-induced oxidative induction, suggesting an antagonistic action, perhaps due to LA-derived nitric oxide. In conclusion, DA caused significant oxidative induction in prepubertal rat testis, but this action was abrogated by L-arginine. The relevance of this phenomenon in vivo merits further study, as both of these molecules have specific physiological functions in the testis.

Effect of sildenafil citrate (Viagra) and ethanol on the Albino rat testis: a scanning electron microscopic approach.

PubMed

Sivasankaran, T G; Udayakumar, R; Elanchezhiyan, C; Sabhanayakam, Selvi

2008-02-01

The effects of sildenafil citrate with ethanol on the rat testis was studied using scanning electron microscopy. Male Albino rats were divided into 8 groups, each being treated for a maximum of 45 days as follows. In the 4 short-term treatment groups, control rats were administered normal saline orally, whereas experimental animals were fed sildenafil citrate (Viagra) 1 microg/g with 18% ethanol (5 g/kg body weight), which was given orally as a single dose. After 1, 2.5, 4 and 24h the rats were killed. In the 4 long-term treatment groups, daily continuous doses of drug and ethanol with a single dosage were given for 15, 30 and 45 days and the animals killed 4h after the last dosage. Changes in the testis were compared with the normal healthy rat testis. The use of a scanning electron microscope for evaluation of the changes in the testis is more suitable for observation of the surface and morphological shapes of the tissue structures.

Operations-oriented performance measures for freeway management systems : final report.

DOT National Transportation Integrated Search

2008-12-01

This report describes the second and final year activities of the project titled Using Operations-Oriented Performance Measures to Support Freeway Management Systems. Work activities included developing a prototype system architecture for testi...

Renaissance in tumor immunotherapy: possible combination with phototherapy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hamblin, Michael R.

2016-03-01

Photodynamic therapy (PDT) uses the combination of non-toxic dyes and harmless visible light to produce highly toxic reactive oxygen species that destroy tumors. The ideal cancer treatment should target both the primary tumor and the metastases with minimal toxicity. This is best accomplished by educating the body's immune system to recognize the tumor as foreign so that after the primary tumor is destroyed, distant metastases will also be eradicated. PDT may accomplish this feat and stimulate long-term, specific anti-tumor immunity. PDT causes an acute inflammatory response, the rapid induction of large amounts of necrotic and apoptotic tumor cells, induction of damage-associated molecular patterns (DAMPS) including heat-shock proteins, stimulates tumor antigen presentation to naïve T-cells, and generation of cytotoxic T-cells that can destroy distant tumor metastases. By using various syngeneic mouse tumors in immunocompetent mice, we have studied specific PDT regimens related to tumor type as well as mouse genotype and phenotype. We have investigated the role of tumor-associated antigens in PDT-induced immune response by choosing mouse tumors that express: model defined antigen, naturally-occurring cancer testis antigen, and oncogenic virus-derived antigen. We studied the synergistic combination of low-dose cyclophosphamide and PDT that unmasks the PDT-induced immune response by depleting the immunosuppressive T-regulatory cells. PDT combined with immunostimulants (toll-like receptor ligands) can synergistically maximize the generation of anti-tumor immunity by activating dendritic cells and switching immunosuppressive macrophages to a tumor rejection phenotype. Tumors expressing defined tumor-associated antigens with known MHC class I peptides allows anti-tumor immunity to be quantitatively compared.

The interferon-inducible p47 (IRG) GTPases in vertebrates: loss of the cell autonomous resistance mechanism in the human lineage

PubMed Central

Bekpen, Cemalettin; Hunn, Julia P; Rohde, Christoph; Parvanova, Iana; Guethlein, Libby; Dunn, Diane M; Glowalla, Eva; Leptin, Maria; Howard, Jonathan C

2005-01-01

Background Members of the p47 (immunity-related GTPases (IRG) family) GTPases are essential, interferon-inducible resistance factors in mice that are active against a broad spectrum of important intracellular pathogens. Surprisingly, there are no reports of p47 function in humans. Results Here we show that the p47 GTPases are represented by 23 genes in the mouse, whereas humans have only a single full-length p47 GTPase and an expressed, truncated presumed pseudo-gene. The human full-length gene is orthologous to an isolated mouse p47 GTPase that carries no interferon-inducible elements in the promoter of either species and is expressed constitutively in the mature testis of both species. Thus, there is no evidence for a p47 GTPase-based resistance system in humans. Dogs have several interferon-inducible p47s, and so the primate lineage that led to humans appears to have lost an ancient function. Multiple p47 GTPases are also present in the zebrafish, but there is only a tandem p47 gene pair in pufferfish. Conclusion Mice and humans must deploy their immune resources against vacuolar pathogens in radically different ways. This carries significant implications for the use of the mouse as a model of human infectious disease. The absence of the p47 resistance system in humans suggests that possession of this resistance system carries significant costs that, in the primate lineage that led to humans, are not outweighed by the benefits. The origin of the vertebrate p47 system is obscure. PMID:16277747

Expression of the G72/G30 gene in transgenic mice induces behavioral changes

PubMed Central

Cheng, Lijun; Hattori, Eiji; Nakajima, Akira; Woehrle, Nancy S.; Opal, Mark D.; Zhang, Chunling; Grennan, Kay; Dulawa, Stephanie C.; Tang, Ya-Ping; Gershon, Elliot S.; Liu, Chunyu

2012-01-01

The G72/G30 gene complex is a candidate gene for schizophrenia and bipolar disorder. However, G72 and G30 mRNAs are expressed at very low levels in human brain, with only rare splicing forms observed. We report here G72/G30 expression profiles and behavioral changes in a G72/G30 transgenic mouse model. A human BAC clone containing the G72/G30 genomic region was used to establish the transgenic mouse model, on which gene expression studies, Western blot and behavioral tests were performed. Relative to their minimal expression in humans, G72 and G30 mRNAs were highly expressed in the transgenic mice, and had a more complex splicing pattern. The highest G72 transcript levels were found in testis, followed by cerebral cortex, with very low or undetectable levels in other tissues. No LG72 (the long putative isoform of G72) protein was detected in the transgenic mice. Whole-genome expression profiling identified 361 genes differentially-expressed in transgenic mice compared to wild-type, including genes previously implicated in neurological and psychological disorders. Relative to wild-type mice, the transgenic mice exhibited fewer stereotypic movements in the open field test, higher baseline startle responses in the course of the prepulse inhibition test, and lower hedonic responses in the sucrose preference test. The transcriptome profile changes and multiple mouse behavioral effects suggest that the G72 gene may play a role in modulating behaviors relevant to psychiatric disorders. PMID:23337943

In vitro functional screening as a means to identify new plasticizers devoid of reproductive toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boisvert, Annie; Jones, Steven; Issop, Leeyah

Plasticizers are indispensable additives providing flexibility and malleability to plastics. Among them, several phthalates, including di (2-ethylhexyl) phthalate (DEHP), have emerged as endocrine disruptors, leading to their restriction in consumer products and creating a need for new, safer plasticizers. The goal of this project was to use in vitro functional screening tools to select novel non-toxic plasticizers suitable for further in vivo evaluation. A panel of novel compounds with satisfactory plasticizer properties and biodegradability were tested, along with several commercial plasticizers, such as diisononyl-cyclohexane-1,2-dicarboxylate (DINCH®). MEHP, the monoester metabolite of DEHP was also included as reference compound. Because phthalates targetmore » mainly testicular function, including androgen production and spermatogenesis, we used the mouse MA-10 Leydig and C18-4 spermatogonial cell lines as surrogates to examine cell survival, proliferation, steroidogenesis and mitochondrial integrity. The most promising compounds were further assessed on organ cultures of rat fetal and neonatal testes, corresponding to sensitive developmental windows. Dose-response studies revealed the toxicity of most maleates and fumarates, while identifying several dibenzoate and succinate plasticizers as innocuous on Leydig and germ cells. Interestingly, DINCH®, a plasticizer marketed as a safe alternative to phthalates, exerted a biphasic effect on steroid production in MA-10 and fetal Leydig cells. MEHP was the only plasticizer inducing the formation of multinucleated germ cells (MNG) in organ culture. Overall, organ cultures corroborated the cell line data, identifying one dibenzoate and one succinate as the most promising candidates. The adoption of such collaborative approaches for developing new chemicals should help prevent the development of compounds potentially harmful to human health. - Highlights: • Phthalate plasticizers exert toxic effects on male reproduction. • Reproductive toxicity of new plasticizers was assessed by functional assays. • Mouse Leydig and germ cell lines, and rat perinatal testis cultures were used. • Survival, proliferation, steroidogenesis, abnormal germ cell formation were examined. • Reproductive toxic and innocuous plasticizer candidates were identified.« less

Insights into GABA receptor signalling in TM3 Leydig cells.

PubMed

Doepner, Richard F G; Geigerseder, Christof; Frungieri, Monica B; Gonzalez-Calvar, Silvia I; Calandra, Ricardo S; Raemsch, Romi; Fohr, Karl; Kunz, Lars; Mayerhofer, Artur

2005-01-01

Gamma-aminobutyric acid (GABA) is an emerging signalling molecule in endocrine organs, since it is produced by endocrine cells and acts via GABA(A) receptors in a paracrine/autocrine fashion. Testicular Leydig cells are producers and targets for GABA. These cells express GABA(A) receptor subunits and in the murine Leydig cell line TM3 pharmacological activation leads to increased proliferation. The signalling pathway of GABA in these cells is not known in this study. We therefore attempted to elucidate details of GABA(A) signalling in TM3 and adult mouse Leydig cells using several experimental approaches. TM3 cells not only express GABA(A )receptor subunits, but also bind the GABA agonist [(3)H]muscimol with a binding affinity in the range reported for other endocrine cells (K(d) = 2.740 +/- 0.721 nM). However, they exhibit a low B(max) value of 28.08 fmol/mg protein. Typical GABA(A) receptor-associated events, including Cl(-) currents, changes in resting membrane potential, intracellular Ca(2+) or cAMP, were not measurable with the methods employed in TM3 cells, or, as studied in part, in primary mouse Leydig cells. GABA or GABA(A) agonist isoguvacine treatment resulted in increased or decreased levels of several mRNAs, including transcription factors (c-fos, hsf-1, egr-1) and cell cycle-associated genes (Cdk2, cyclin D1). In an attempt to verify the cDNA array results and because egr-1 was recently implied in Leydig cell development, we further studied this factor. RT-PCR and Western blotting confirmed a time-dependent regulation of egr-1 in TM3. In the postnatal testis egr-1 was seen in cytoplasmic and nuclear locations of developing Leydig cells, which bear GABA(A) receptors and correspond well to TM3 cells. Thus, GABA acts via an atypical novel signalling pathway in TM3 cells. Further details of this pathway remain to be elucidated. Copyright (c) 2005 S. Karger AG, Basel.

Magnetic resonance angiography for the nonpalpable testis: a cost and cancer risk analysis.

PubMed

Eggener, S E; Lotan, Y; Cheng, E Y

2005-05-01

For the unilateral nonpalpable testis standard management is open surgical or laparoscopic exploration. An ideal imaging technique would reliably identify testicular nubbins and safely allow children to forgo surgical exploration without compromising future health or fertility. Our goal was to perform a cost and risk analysis of magnetic resonance angiography (MRA) for unilateral nonpalpable cryptorchid testes. A search of the English medical literature revealed 3 studies addressing the usefulness of MRA for the nonpalpable testicle. We performed a meta-analysis and applied the results to a hypothetical set of patients using historical testicular localization data. Analysis was then performed using 3 different management protocols-MRA with removal of testicular nubbin tissue, MRA with observation of testicular nubbin tissue and diagnostic laparoscopy. A cancer risk and cost analysis was then performed. MRA with observation of testicular nubbin tissue results in 29% of patients avoiding surgery without any increased cost of care. Among the 29% of boys with testicular nubbins left in situ and observed the highest estimated risk was 1 in 300 of cancer developing, and 1 in 5,300 of dying of cancer. A protocol using MRA with observation of inguinal nubbins results in nearly a third of boys avoiding surgical intervention at a similar cost to standard care without any significant increased risk of development of testis cancer.

Spontaneous CD4+ and CD8+ T-cell responses directed against cancer testis antigens are present in the peripheral blood of testicular cancer patients.

PubMed

Pearce, Hayden; Hutton, Paul; Chaudhri, Shalini; Porfiri, Emilio; Patel, Prashant; Viney, Richard; Moss, Paul

2017-07-01

Cancer/testis antigen (CTAg) expression is restricted to spermatogenic cells in an immune-privileged site within the testis. However, these proteins are expressed aberrantly by malignant cells and T-cell responses against CTAgs develop in many cancer patients. We investigated the prevalence, magnitude and phenotype of CTAg-specific T cells in the blood of patients with testicular germ cell tumors (TGCTs). CD8 + and CD4 + T-cell responses against MAGE-A family antigens were present in 44% (20/45) of patients' samples assayed by ex vivo IFN-γ ELISPOT. The presence of MAGE-specific CD8 + T cells was further determined following short-term in vitro expansion through the use of pMHC-I multimers containing known immunogenic peptides. Longitudinal analysis revealed that the frequency of MAGE-specific T cells decreased by 89% following orchidectomy suggesting that persistence of tumor antigen is required to sustain CTAg-specific T-cell immunity. Notably, this decrease correlated with a decline in the global effector/memory T-cell pool following treatment. Spontaneous T-cell immunity against CTAg proteins therefore develops in many patients with testicular cancer and may play an important role in the excellent clinical outcome of patients with this tumor subtype. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A potential target for organophosphate insecticides leading to spermatotoxicity.

PubMed

Suzuki, Himiko; Tomizawa, Motohiro; Ito, Yuki; Abe, Keisuke; Noro, Yuki; Kamijima, Michihiro

2013-10-16

Organophosphate (OP) insecticides as an anticholinesterase also act on the diverse serine hydrolase targets, thereby revealing secondary or unexpected toxic effects including male reproductive toxicity. The present investigation detects a possible target molecule(s) for OP-induced spermatotoxicity (sperm deformity, underdevelopment, and reduced motility) from a chemical standpoint. The activity-based protein profiling (ABPP) approach with a phosphonofluoridate fluorescent probe pinpointed the molecular target for fenitrothion (FNT, a major OP insecticide) oxon (bioactive metabolite of FNT) in the mouse testicular membrane proteome, i.e., FNT oxon phosphorylates the fatty acid amide hydrolase (FAAH), which plays pivotal roles in spermatogenesis and sperm motility acquirement. Subsequently, mice were treated orally with vehicle or FNT for 10 days, and FAAH activity in testis or epididymis cauda was markedly reduced by the subacute exposure. ABPP analysis revealed that FAAH was selectively inhibited among the FNT-treated testicular membrane proteome. Accordingly, FAAH is a potential target for OP-elicited spermatotoxicity.

Effects of diatrizoate and iopamidol on spermatogenesis.

PubMed

Yaghmai, V; Harapanhalli, R S; Patel, Y D; Baker, S R; Rao, D V

1993-12-01

The biological effects of iodinated contrast media were examined by using spermatogenesis in mouse testis as the experimental model. Spermhead survival and abnormality assays were used as the biological end points. Diatrizoate meglumine/diatrizoate sodium and iopamidol were administered intravenously at equal rates and concentrations. Testicular uptake and clearance of these contrast agents were examined by high-performance liquid chromatography techniques. Appropriate mannitol solutions were employed as osmolality controls. Intravenous administration of the contrast agent or its respective mannitol control resulted in approximately a 30% decrease in spermhead count. A dose-related experiment with mannitol demonstrated that the spermhead count decreased rapidly until 600 mOsm/kg was reached, beyond which this decrease was minimal. Clearance of both contrast media was complete in approximately 4 hours. No significant increase in the induction of spermhead abnormalities was observed. Osmotic substances, such as iodinated contrast agents, affect the process of spermatogenesis.

Integrity of the blood-testis barrier in healthy men after suppression of spermatogenesis with testosterone and levonorgestrel.

PubMed

Ilani, Niloufar; Armanious, Nancy; Lue, Yan-He; Swerdloff, Ronald S; Baravarian, Sima; Adler, Alex; Tsang, Christina; Jia, Yue; Cui, Yu-Gui; Wang, Xing-Hai; Zhou, Zuo-Min; Sha, Jia-Hao; Wang, Christina

2012-12-01

Do exogenous male hormonal contraceptives that suppress intratesticular testosterone and spermatogenesis interfere with the blood-testis barrier integrity in men? When spermatogenesis was suppressed by testosterone alone or combined with levonorgestrel (LNG) treatment in men, the structural appearance of Sertoli cell tight junctions remained intact in the human testis. Testosterone promotes the integrity of the blood-testis barrier. Intratesticular androgen deprivation induced by exogenous testosterone plus a progestin to suppress spermatogenesis in a contraceptive regimen may disturb the structural and functional integrity of the blood-testis barrier. Testicular biopsies were obtained from a sub-study of a randomized clinical trial of 36 healthy Chinese men who were treated for 18 weeks and followed for at least a 12-week recovery period. Healthy Chinese male volunteers (27-48 years) were randomized to two treatment groups (n = 18/group) for 18 weeks: (1) testosterone undecanoate (TU) 1000 mg i.m. injection followed by a 500 mg injection every 6 weeks and (2) TU + LNG 250 μg orally daily. Blood samples were obtained from all participants before and during treatment and at the end of the recovery phase. Open testicular biopsies for this study were obtained from four men before treatment and from four men in each of the TU and TU + LNG groups at 2 and 9 weeks of treatment. The presence of antisperm antibodies was checked in the archived serum samples of the subjects at baseline, during treatment and at the end of the recovery period. Stored testicular biopsy samples from cynomolgus monkeys treated with either sub-cutaneous testosterone or placebo for 12 weeks were used for additional protein expression studies. Expression of blood-testis barrier associated proteins quantified by immunohistochemistry (claudin 3, claudin 11, junctional adhesion molecule-A, zonula occludens-1) remained unchanged despite a significant decrease in the numbers of pachytene spermatocytes and round spermatids in the seminiferous tubules at 9 weeks in the TU + LNG group. This was confirmed by immunoblots showing a lack of quantitative change in these tight junction proteins in monkeys after testosterone treatment. There were no increases in serum antisperm antibodies in the volunteers during the study. The duration of the study was short and the long-term effects of male hormonal contraceptive treatments on the integrity of the blood-testis barrier remain to be determined. This study supports the safety of male hormonal contraceptive treatment and does not corroborate the previous findings of disturbed immunological integrity of the blood-testis barrier from animal studies such as androgen receptor knockout mice and exogenous hormonal treatment in rats. The study was supported by grants from the Contraceptive Research and Development Program and the Mellon Foundation (MFG-02-64, MFG-03-67), Endocrine, Metabolism and Nutrition Training Grant (T32 DK007571), the Clinical and Translational Science Institute at Los Angeles Biomedical and Harbor-UCLA Medical Center (UL1RR033176 and UL1TR000124) and the Los Angeles Biomedical Research Institute Summer High School Student Program.

Juvenile granulosa cell tumor of the testis: case report and review of literature.

PubMed

Nieto, Nieves; Torres-Valdivieso, Maria José; Aguado, Pablo; Mateos, Maria Elena; López-Pérez, Jesús; Melero, Carmen; Vivanco, José Luis; Gómez, Andrés

2002-01-01

Juvenile granulosa cell tumor of the testis is an infrequent tumor of the gonadal stroma characteristic of the pediatric age. It usually appears as a scrotal mass and less frequently as an abdominal or inguinal mass. It may be associated with ambiguous genitalia and/or abnormal sex chromosomes. The recommended treatment is orchiectomy alone because local recurrence or metastasis have never been observed. We describe a patient with a juvenile granulosa cell tumor of the testis and review the literature.

Copy number variation of two separate regulatory regions upstream of SOX9 causes isolated 46,XY or 46,XX disorder of sex development.

PubMed

Kim, Gwang-Jin; Sock, Elisabeth; Buchberger, Astrid; Just, Walter; Denzer, Friederike; Hoepffner, Wolfgang; German, James; Cole, Trevor; Mann, Jillian; Seguin, John H; Zipf, William; Costigan, Colm; Schmiady, Hardi; Rostásy, Moritz; Kramer, Mildred; Kaltenbach, Simon; Rösler, Bernd; Georg, Ina; Troppmann, Elke; Teichmann, Anne-Christin; Salfelder, Anika; Widholz, Sebastian A; Wieacker, Peter; Hiort, Olaf; Camerino, Giovanna; Radi, Orietta; Wegner, Michael; Arnold, Hans-Henning; Scherer, Gerd

2015-04-01

SOX9 mutations cause the skeletal malformation syndrome campomelic dysplasia in combination with XY sex reversal. Studies in mice indicate that SOX9 acts as a testis-inducing transcription factor downstream of SRY, triggering Sertoli cell and testis differentiation. An SRY-dependent testis-specific enhancer for Sox9 has been identified only in mice. A previous study has implicated copy number variations (CNVs) of a 78 kb region 517-595 kb upstream of SOX9 in the aetiology of both 46,XY and 46,XX disorders of sex development (DSD). We wanted to better define this region for both disorders. By CNV analysis, we identified SOX9 upstream duplications in three cases of SRY-negative 46,XX DSD, which together with previously reported duplications define a 68 kb region, 516-584 kb upstream of SOX9, designated XXSR (XX sex reversal region). More importantly, we identified heterozygous deletions in four families with SRY-positive 46,XY DSD without skeletal phenotype, which define a 32.5 kb interval 607.1-639.6 kb upstream of SOX9, designated XY sex reversal region (XYSR). To localise the suspected testis-specific enhancer, XYSR subfragments were tested in cell transfection and transgenic experiments. While transgenic experiments remained inconclusive, a 1.9 kb SRY-responsive subfragment drove expression specifically in Sertoli-like cells. Our results indicate that isolated 46,XY and 46,XX DSD can be assigned to two separate regulatory regions, XYSR and XXSR, far upstream of SOX9. The 1.9 kb SRY-responsive subfragment from the XYSR might constitute the core of the Sertoli-cell enhancer of human SOX9, representing the so far missing link in the genetic cascade of male sex determination. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testis.

PubMed

Wen, Qing; Li, Nan; Xiao, Xiang; Lui, Wing-Yee; Chu, Darren S; Wong, Chris K C; Lian, Qingquan; Ge, Renshan; Lee, Will M; Silvestrini, Bruno; Cheng, C Yan

2018-02-12

Germ cell differentiation during the epithelial cycle of spermatogenesis is accompanied by extensive remodeling at the Sertoli cell-cell and Sertoli cell-spermatid interface to accommodate the transport of preleptotene spermatocytes and developing spermatids across the blood-testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. The unique cell junction in the testis is the actin-rich ectoplasmic specialization (ES) designated basal ES at the Sertoli cell-cell interface, and the apical ES at the Sertoli-spermatid interface. Since ES dynamics (i.e., disassembly, reassembly and stabilization) are supported by actin microfilaments, which rapidly converts between their bundled and unbundled/branched configuration to confer plasticity to the ES, it is logical to speculate that actin nucleation proteins play a crucial role to ES dynamics. Herein, we reported findings that Spire 1, an actin nucleator known to polymerize actins into long stretches of linear microfilaments in cells, is an important regulator of ES dynamics. Its knockdown by RNAi in Sertoli cells cultured in vitro was found to impede the Sertoli cell tight junction (TJ)-permeability barrier through changes in the organization of F-actin across Sertoli cell cytosol. Unexpectedly, Spire 1 knockdown also perturbed microtubule (MT) organization in Sertoli cells cultured in vitro. Biochemical studies using cultured Sertoli cells and specific F-actin vs. MT polymerization assays supported the notion that a transient loss of Spire 1 by RNAi disrupted Sertoli cell actin and MT polymerization and bundling activities. These findings in vitro were reproduced in studies in vivo by RNAi using Spire 1-specific siRNA duplexes to transfect testes with Polyplus in vivo-jetPEI as a transfection medium with high transfection efficiency. Spire 1 knockdown in the testis led to gross disruption of F-actin and MT organization across the seminiferous epithelium, thereby impeding the transport of spermatids and phagosomes across the epithelium and perturbing spermatogenesis. In summary, Spire 1 is an ES regulator to support germ cell development during spermatogenesis.

A critical developmental role for tgfbr2 in myogenic cell lineages is revealed in mice expressing SM22-Cre, not SMMHC-Cre.

PubMed

Frutkin, Andrew D; Shi, Haikun; Otsuka, Goro; Levéen, Per; Karlsson, Stefan; Dichek, David A

2006-10-01

Smooth muscle cell (SMC)-specific deletion of transforming growth factor beta (TGF-beta) signaling would help elucidate the mechanisms through which TGF-beta signaling contributes to vascular development and disease. We attempted to generate mice with SMC-specific deletion of TGF-beta signaling by mating mice with a conditional ("floxed") allele for the type II TGF-beta receptor (tgfbr2flox) to mice with SMC-targeted expression of Cre recombinase. We bred male mice transgenic for smooth muscle myosin heavy chain (SMMHC)-Cre with females carrying tgfbr2flox. Surprisingly, SMMHC-Cre mice recombined tgfbr2flox at low levels in SMC and at high levels in the testis. Recombination of tgfbr2flox in testis correlated with high-level expression of SMMHC-Cre in testis and germline transmission of tgfbr2null. In contrast, mice expressing Cre from a SM22alpha promoter (SM22-Cre) efficiently recombined tgfbr2flox in vascular and visceral SMC and the heart, but not in testis. Use of the R26R reporter allele confirmed that Cre-mediated recombination in vascular SMC was inefficient for SMMHC-Cre mice and highly efficient for SM22-Cre mice. Breedings that introduced the SM22-Cre allele into tgfbr2flox/flox zygotes in order to generate adult mice that are hemizygous for SM22-Cre and homozygous for tgfbr2flox- and would have conversion of tgfbr2flox/flox to tgfbr2null/null in SMC-produced no live SM22-Cre : tgfbr2flox/flox pups (P<0.001). We conclude: (1) "SMC-targeted" Cre lines vary significantly in specificity and efficiency of Cre expression; (2) TGF-beta signaling in the subset of cells that express SM22alpha is required for normal development; (3) generation of adult mice with absent TGF-beta signaling in SMC remains a challenge.

White tea intake prevents prediabetes-induced metabolic dysfunctions in testis and epididymis preserving sperm quality.

PubMed

Dias, Tânia R; Alves, Marco G; Rato, Luís; Casal, Susana; Silva, Branca M; Oliveira, Pedro F

2016-11-01

Prediabetes has been associated with alterations in male reproductive tract, especially in testis and epididymis. Moreover, in vitro studies described a promising action of tea (Camellia sinensis L.) against metabolic dysfunctions. Herein, we hypothesized that white tea (WTEA) ingestion by prediabetic animals could ameliorate the metabolic alterations induced by the disease in testicular and epididymal tissues, preserving sperm quality. WTEA infusion was prepared and its phytochemical profile was evaluated by 1 H-NMR. A streptozotocin-induced prediabetic rat model was developed and three experimental groups were defined: control, prediabetic (PreDM) and prediabetic drinking WTEA (PreDM+WTEA). Metabolic profiles of testis and epididymis were evaluated by determining the metabolites content ( 1 H-NMR), protein levels (western blot) and enzymatic activities of key metabolic intervenient. The quality of spermatozoa from cauda epididymis was also assessed. Prediabetes increased glucose transporter 3 protein levels and decreased lactate dehydrogenase activity in testis, resulting in a lower lactate content. WTEA ingestion led to a metabolic adaptation to restore testicular lactate content. Concerning epididymis, prediabetes decreased the protein levels of several metabolic intervenient, resulting in decreased lactate and alanine content. WTEA consumption restored most of the evidenced alterations, however, not lactate content. WTEA also improved epididymal sperm motility and restored sperm viability. Prediabetes strongly affected testicular and epididymal metabolic status and most of these alterations were restored by WTEA consumption, resulting in the improvement of sperm quality. Our results suggest that WTEA consumption can be a cost-effective strategy to improve prediabetes-induced reproductive dysfunction. Copyright © 2016 Elsevier Inc. All rights reserved.

Duration of spermatogenesis and daily sperm production in the jaguar (Panthera onca).

PubMed

Costa, G M J; Chiarini-Garcia, H; Morato, R G; Alvarenga, R L L S; França, L R

2008-10-15

The jaguar, like most wild felids, is an endangered species. Since there are few data regarding reproductive biology for this species, our main goal was to investigate basic aspects of the testis and spermatogenesis. Four adult male jaguars were utilized; to determine the duration of spermatogenesis, two animals received an intratesticular injection of H(3)-thymidine. Mean (+/-SEM) testis weight and the gonadosomatic index were 17.7+/-2.2g and 0.05+/-0.01%, respectively, whereas the seminiferous tubules and the Leydig cells volume density were 74.7+/-3.8 and 16.7+/-1.6%. Eight stages of spermatogenesis were characterized, according to the tubular morphology system and acrosome development. Each spermatogenic cycle and the entire spermatogenic process (based on 4.5 cycles) lasted approximately 12.8+/-0.01 and 57.7+/-0.07 d. The number of Sertoli and Leydig cells per gram of testis was 29+/-4 x 10(6) and 107+/-12 x 10(6). Based on the number of round spermatids per pachytene spermatocyte (2.8+/-0.3:1; meiotic index); significant cell loss (30%) occurred during the two meiotic divisions. There were approximately eight spermatids for each Sertoli cell (Sertoli cell efficiency), whereas the daily sperm production per gram of testis was 16.9+/-1.2 x 10(6). We expect that in the near future, the knowledge obtained in the present investigation will facilitate, utilizing germ cell transplantation, preservation of the germinal epithelium and the ability to generate sperm from jaguars in testes of domestic cats.

Coexistence of Trisomy 13 and SRY (-) XX Ovotesticular Disorder of Sex Development.

PubMed

Ürel Demir, Gizem; Doğan, Özlem Akgün; Şimşek Kiper, Pelin Özlem; Utine, Gülen Eda; Boduroğlu, Koray; Gucer, Safak; Alikaşifoğlu, Mehmet

2017-12-01

Ovotesticular disorder of sex development (OT-DSD) is a rare disorder of sexual differentiation characterized by the presence of both testicular and ovarian tissue in an individual and the majority of cases have been reported with 46,XX karyotype. In 46,XX cases, testicular differentiation may occur due to the translocation of SRY to the X chromosome or to an autosome. Herein, we present a female newborn with a combination of trisomy 13 and SRY (-) XX OT-DSD. Trisomy 13 is a relatively common and well-known chromosomal disorder in which disorders of sexual differentiation are not frequent. In the absence of SRY, overexpression of pro-testis genes, or decreased expression of pro-ovarian/anti-testis genes have been suggested as underlying mechanisms of testicular formation. The findings in this patient were suggestive of an underlying genomic disorder associated with FGF9 and/or SPRY2.

New frontiers in nonhormonal male contraception

PubMed Central

Cheng, C. Yan; Mruk, Dolores D.

2015-01-01

The world’s population is nearing 6.8 billion, and we are in need of a male contraceptive that is safe, effective, reversible and affordable. Hormonal approaches, which employ different formulations of testosterone administered in combination with other hormones, have shown considerable promise in clinical trials, and they are currently at the forefront of research and development. However, the long-term effects of using hormones throughout a male’s reproductive life for contraception are unknown, and it may take decades before this information becomes available. Because of this, many investigators are aiming to bring a nonhormonal male contraceptive to the consumer market. Indeed, there are several distinct but feasible avenues in which fertility can be regulated without affecting the hypothalamus-pituitary-testis axis. In this review, we discuss several approaches for fertility control involving the testis that one day may lead to the development of a nonhormonal male contraceptive. PMID:20933122

New frontiers in nonhormonal male contraception.

PubMed

Cheng, C Yan; Mruk, Dolores D

2010-11-01

The world's population is nearing 6.8 billion, and we are in need of a male contraceptive that is safe, effective, reversible and affordable. Hormonal approaches, which employ different formulations of testosterone administered in combination with other hormones, have shown considerable promise in clinical trials, and they are currently at the forefront of research and development. However, the long-term effects of using hormones throughout a male's reproductive life for contraception are unknown, and it may take decades before this information becomes available. Because of this, many investigators are aiming to bring a nonhormonal male contraceptive to the consumer market. Indeed, there are several distinct but feasible avenues in which fertility can be regulated without affecting the hypothalamus-pituitary-testis axis. In this review, we discuss several approaches for fertility control involving the testis that one day may lead to the development of a nonhormonal male contraceptive. Copyright © 2010 Elsevier Inc. All rights reserved.

Development and clinical application of a new testicular prosthesis

PubMed Central

Ning, Ye; Cai, Zhikang; Chen, Huixing; Ping, Ping; Li, Peng; Wang, Zhong; Li, Zheng

2011-01-01

A new type of testicular prosthesis made of silastic with an elliptical shape to mimic a normal testis was developed by our team and submitted for patenting in China. The prosthesis was produced in different sizes to imitate the normal testis of the patient. To investigate the effects and safety of the testicular prosthesis, 20 patients receiving testicular prosthesis implantation were recruited for this study. Follow-up after 6 months revealed no complications in the patients. All the patients answered that they were satisfied with their body image and the position of the implants, 19 patients were satisfied with the size and 16 patients were satisfied with the weight. These results show that the testicular prosthesis used in this study can meet patient's expectations. Patients undergoing orchiectomy should be offered the option to receive a testicular prosthesis implantation. The dimensions and weight of the available prosthetic implants should be further addressed to improve patient satisfaction. PMID:21927041

Anchoring Junctions As Drug Targets: Role in Contraceptive Development

PubMed Central

Mruk, Dolores D.; Silvestrini, Bruno; Cheng, C. Yan

2010-01-01

In multicellular organisms, cell-cell interactions are mediated in part by cell junctions, which underlie tissue architecture. Throughout spermatogenesis, for instance, preleptotene leptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier to enter the adluminal compartment for continued development. At the same time, germ cells must also remain attached to Sertoli cells, and numerous studies have reported extensive restructuring at the Sertoli-Sertoli and Sertoli-germ cell interface during germ cell movement across the seminiferous epithelium. Furthermore, the proteins and signaling cascades that regulate adhesion between testicular cells have been largely delineated. These findings have unveiled a number of potential “druggable” targets that can be used to induce premature release of germ cells from the seminiferous epithelium, resulting in transient infertility. Herein, we discuss a novel approach with the aim of developing a nonhormonal male contraceptive for future human use, one that involves perturbing adhesion between Sertoli and germ cells in the testis. PMID:18483144

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

PubMed

Persson, H; Pelto-Huikko, M; Metsis, M; Söder, O; Brene, S; Skog, S; Hökfelt, T; Ritzén, E M

1990-09-01

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

PubMed Central

Persson, H; Pelto-Huikko, M; Metsis, M; Söder, O; Brene, S; Skog, S; Hökfelt, T; Ritzén, E M

1990-01-01

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility. Images PMID:1697032

Tissue-Specific Transcriptomics in the Field Cricket Teleogryllus oceanicus

PubMed Central

Bailey, Nathan W.; Veltsos, Paris; Tan, Yew-Foon; Millar, A. Harvey; Ritchie, Michael G.; Simmons, Leigh W.

2013-01-01

Field crickets (family Gryllidae) frequently are used in studies of behavioral genetics, sexual selection, and sexual conflict, but there have been no studies of transcriptomic differences among different tissue types. We evaluated transcriptome variation among testis, accessory gland, and the remaining whole-body preparations from males of the field cricket, Teleogryllus oceanicus. Non-normalized cDNA libraries from each tissue were sequenced on the Roche 454 platform, and a master assembly was constructed using testis, accessory gland, and whole-body preparations. A total of 940,200 reads were assembled into 41,962 contigs, to which 36,856 singletons (reads not assembled into a contig) were added to provide a total of 78,818 sequences used in annotation analysis. A total of 59,072 sequences (75%) were unique to one of the three tissues. Testis tissue had the greatest proportion of tissue-specific sequences (62.6%), followed by general body (56.43%) and accessory gland tissue (44.16%). We tested the hypothesis that tissues expressing gene products expected to evolve rapidly as a result of sexual selection—testis and accessory gland—would yield a smaller proportion of BLASTx matches to homologous genes in the model organism Drosophila melanogaster compared with whole-body tissue. Uniquely expressed sequences in both testis and accessory gland showed a significantly lower rate of matching to annotated D. melanogaster genes compared with those from general body tissue. These results correspond with empirical evidence that genes expressed in testis and accessory gland tissue are rapidly evolving targets of selection. PMID:23390599

Tissue-specific transcriptomics in the field cricket Teleogryllus oceanicus.

PubMed

Bailey, Nathan W; Veltsos, Paris; Tan, Yew-Foon; Millar, A Harvey; Ritchie, Michael G; Simmons, Leigh W

2013-02-01

Field crickets (family Gryllidae) frequently are used in studies of behavioral genetics, sexual selection, and sexual conflict, but there have been no studies of transcriptomic differences among different tissue types. We evaluated transcriptome variation among testis, accessory gland, and the remaining whole-body preparations from males of the field cricket, Teleogryllus oceanicus. Non-normalized cDNA libraries from each tissue were sequenced on the Roche 454 platform, and a master assembly was constructed using testis, accessory gland, and whole-body preparations. A total of 940,200 reads were assembled into 41,962 contigs, to which 36,856 singletons (reads not assembled into a contig) were added to provide a total of 78,818 sequences used in annotation analysis. A total of 59,072 sequences (75%) were unique to one of the three tissues. Testis tissue had the greatest proportion of tissue-specific sequences (62.6%), followed by general body (56.43%) and accessory gland tissue (44.16%). We tested the hypothesis that tissues expressing gene products expected to evolve rapidly as a result of sexual selection--testis and accessory gland--would yield a smaller proportion of BLASTx matches to homologous genes in the model organism Drosophila melanogaster compared with whole-body tissue. Uniquely expressed sequences in both testis and accessory gland showed a significantly lower rate of matching to annotated D. melanogaster genes compared with those from general body tissue. These results correspond with empirical evidence that genes expressed in testis and accessory gland tissue are rapidly evolving targets of selection.

High androgen receptor immunoexpression in human "Sertoli cell only" testis.

PubMed

Loukil, L Hadjkacem; Boudawara, T Sellami; Ayadi, I; Bahloul, A; Jlidi, R; Ayadi, H; Keskes, L Ammar

2005-01-01

Our purpose was to evaluate cellular androgen receptor (AR) distribution and intensity of immunostaining in the human azoospermic testis. Thirty six biopsy specimens from azoospermic men were immunostained, using a monoclonal antibody of human AR. The localization and the intensity of AR immunostaining was evaluated in Sertoli Cell Only (SCO) testis (G1, n = 21), in spermatogenesis arrest testis (G2, n = 11) and in histologically normal testis (G3, n = 4). We found an AR immunostaining in Sertoli, peritubular myoid and Leydig cells, but not in germ cells. The intensity of the immunostaining varied substantially between biopsy specimens of different patients. Sertoli and Leydig cells AR immunostaining (score and intensity) in SCO group was higher than in the other groups. For Sertoli cells, the score means of AR immunoreactivity were 20 +/- 2.36, 10.18 +/- 1.0 and 1 +/- 1, for G1, G2 and G3 groups, respectively. For Leydig cells, the score means were 10.24 +/- 1.37, 6 +/- 0.71 and 0, for G1, G2 and G3 groups, respectively. We found significant differences between G1 and G2 (p = 0.0008), between G1 and G3 (p = 1.54 10-7) and G2 and G3 (p = 0.00032). These results suggest that in the testis AR is located exclusively in somatic cells and its expression is higher in SCO syndrome than in normal and in arrest spermatogenesis testes.

HSP86 and HSP84 exhibit cellular specificity of expression and co-precipitate with an HSP70 family member in the murine testis

NASA Technical Reports Server (NTRS)

Gruppi, C. M.; Wolgemuth, D. J.

1993-01-01

This study extends to the protein level our previous observations, which had established the stage and cellular specificity of expression of hsp86 and hsp84 in the murine testis in the absence of exogenous stress. Immunoblot analysis was used to demonstrate that HSP86 protein was present throughout testicular development and that its levels increased with the appearance of differentiating germ cells. HSP86 was most abundant in the germ cell population and was present at significantly lower levels in the somatic cells. By contrast, the HSP84 protein was detected in the somatic cells of the testis rather than in germ cells. The steady-state levels of HSP86 and HSP84 paralleled the pattern of the expression of their respective mRNAs, suggesting that regulation at the level of translation was not a major mechanism controlling hsp90 gene expression in testicular cells. Immunoprecipitation analysis revealed that a 70-kDa protein coprecipitated with the HSP86/HSP84 proteins in testicular homogenates. This protein was identified as an HSP70 family member by immunoblot analysis, suggesting that HSP70 and HSP90 family members interact in testicular cells.

Characterization and expression of cyp19a gene in the Chinese giant salamander Andrias davidianus.

PubMed

Hu, Qiaomu; Xiao, Hanbing; Tian, HaiFeng; Meng, Yan

2016-02-01

We cloned the full length cyp19a of Chinese giant salamander Andrias davidianus, determined its distribution in tissues and developing gonads, and analyzed the CpG methylation pattern of the cyp19a promoter. The results revealed isoforms of 1706 bp (G arom) and 1698 bp (B arom) in length, differing in the 5' flanking region, both encoding 502 amino acids. The G arom gene was observed mainly in the ovary and kidney, with little in other investigated tissues, while B arom expression was high in the brain, ovary, testis, and pituitary, with low or undetected expression in other examined tissues. Total aromatase expression was high in the ovary; moderate in the kidney, brain, testis, and pituitary; and low in the remaining tissues. G arom expression was significantly higher in the ovary than in the testis and gradually decreased with maturation of the salamander. A single injection of methyltestosterone or letrozole resulted in ovarian G arom expression decreasing over a 12-96 h period. A 1366 bp sequence of the cyp19a promoter was cloned and shown to be conserved in selected species. CpG methylation level was negatively correlated with cyp19a expression in the examined tissues and developing ovaries. Five and three CpG methylation sites positively correlated with DNA methylation levels in tissues and developing ovary, suggesting that they play an important role in regulating cyp19a expression. The aromatase gene showed two isoforms with distinct expression patterns, and the promoter methylation level at specific CpG sites was associated with variation in expression profiles of tissues and developing ovaries. Copyright © 2015 Elsevier Inc. All rights reserved.

Cellular and Hormonal Disruption of Fetal Testis Development in Sheep Reared on Pasture Treated with Sewage Sludge

PubMed Central

Paul, Catriona; Rhind, Stewart M.; Kyle, Carol E.; Scott, Hayley; McKinnell, Chris; Sharpe, Richard M.

2005-01-01

The purpose of this study was to evaluate whether experimental exposure of pregnant sheep to a mixture of environmental chemicals added to pasture as sewage sludge (n = 9 treated animals) exerted effects on fetal testis development or function; application of sewage sludge was undertaken so as to maximize exposure of the ewes to its contents. Control ewes (n = 9) were reared on pasture treated with an equivalent amount of inorganic nitrogenous fertilizer. Treatment had no effect on body weight of ewes, but it reduced body weight by 12–15% in male (n = 12) and female (n = 8) fetuses on gestation day 110. In treated male fetuses (n = 11), testis weight was significantly reduced (32%), as were the numbers of Sertoli cells (34% reduction), Leydig cells (37% reduction), and gonocytes (44% reduction), compared with control fetuses (n = 8). Fetal blood levels of testosterone and inhibin A were also reduced (36% and 38%, respectively) in treated compared with control fetuses, whereas blood levels of luteinizing hormone and follicle-stimulating hormone were unchanged. Based on immunoexpression of anti-Müllerian hormone, cytochrome P450 side chain cleavage enzyme, and Leydig cell cytoplasmic volume, we conclude that the hormone changes in treated male fetuses probably result from the reduction in somatic cell numbers. This reduction could result from fetal growth restriction in male fetuses and/or from the lowered testosterone action; reduced immunoexpression of α-smooth muscle actin in peritubular cells and of androgen receptor in testes of treated animals supports the latter possibility. These findings indicate that exposure of the developing male sheep fetus to real-world mixtures of environmental chemicals can result in major attenuation of testicular development and hormonal function, which may have consequences in adulthood. PMID:16263515

[Expression and localization of transmembrane protein CMTM2 in human testis and sperm].

PubMed

Zhang, X W; Lan, K; Yang, W B; Li, Q; Zhao, Y P; Yin, H Q; Kite, B; Bai, W J; Xu, T

2017-08-18

To study the expression of transmembrane protein CMTM2 in the testis and sperm of adult males and to approach the potential function of the protein in the male reproductive system. The expression of CMTM2 in human testis and sperm was confirmed by Western blot. Immunohistochemical staining was used for detecting CMTM2 localization in the testis tissue, TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction, that is, immunofluorescent staining was used for detecting CMTM2 localization in both the testis and sperm before and after the acrosome reaction. CMTM2 was presented in both human testis and sperm. In the testis, CMTM2 immunoreactive particles were observed mainly in the membrane of the different stages of spermatogenic cells. In the human sperm, its immunoreactivity was restrictively localized to the posterior head where sperm-egg fusion occurred, and the CMTM2 localization was not affected by sperm acrosome reaction. CMTM2 was widely expressed in seminiferous tubules of the human testis, mainly in the cell membranes of spermatogenic cells, which was consistent with the previous reports. The immunofluorescence performed on frozen human testis slides showed similar findings with immunohistochemistry, which gave weight to the localization of CMTM2 in the cell membranes of spermatogenic cells at different stages. TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction. CMTM2 was localized at the posterior head of sperm before and after acrosome reaction. The localization and expression of CMTM2 were not affected by sperm acrosome reaction. Expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion. The expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion. However, it still remains to be further elucidated about the definite role of CMTM2 in male reproductive system and the process of spermatogenesis. And in vitro fertilization experiments are needed to confirm the role of CMTM2 in fertilization in future.

Sexual behavior and testis morphology in the BACHD rat model

PubMed Central

Novati, Arianna; Yu-Taeger, Libo; Gonzalez Menendez, Irene; Quintanilla Martinez, Leticia

2018-01-01

Background Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene, which results in brain neurodegeneration and peripheral pathology affecting different organs including testis. Patients with HD suffer from motor and cognitive impairment, and multiple psychiatric symptoms. Among behavioral abnormalities in HD, sexual disturbances have often been reported, but scarcely investigated in animal models. The BACHD rat model of HD carries the human full-length mutated HTT (mHTT) genomic sequence with 97 CAG-CAA repeats and displays HD-like alterations at neuropathological and behavioral level. Objective This study aims to phenotype the BACHD rats’ sexual behavior and performance as well as testis morphology because alterations in these aspects have been associated to HD. Methods Two rat cohorts at the age of 3 and 7 months were subjected to mating tests to assess different parameters of sexual behavior. Histological analyses for testis morphology were performed in different rat cohorts at 1.5, 7 and 12 months of age whereas immunohistochemical analyses were carried out at 7 and 12 months of age to visualize the presence of mHTT in testicular tissue. Furthermore, western blot analyses were used to assess HTT and mHTT expression levels in striatum and testis at three months of age. Results At 3 months, BACHD rats showed a decreased time exploring the female anogenital area (AGA), decreased latency to mount, increased number of intromissions and ejaculations and enhanced hit rate. At 7 months, all sexual parameters were comparable between genotypes with the exception that BACHD rats explored the AGA less than wild type rats. Testis analyses did not reveal any morphological alteration at any of the examined ages, but showed presence of mHTT limited to Sertoli cells in transgenic rats at both 7 and 12 months. BACHD rat HTT and mHTT expression levels in testis were lower than striatum at 3 months of age. Conclusions The testis phenotype in the BACHD rat model does not mimic the changes observed in human HD testis. The altered sexual behavior in BACHD rats at three months of age could be to a certain extent representative of and share common underlying pathways with some of the sexual disturbances in HD patients. Further investigating the biological causes of the sexual phenotype in BACHD rats may therefore contribute to clarifying the mechanisms at the base of sexual behavior changes in HD. PMID:29883458

Expression analysis of cancer-testis genes in prostate cancer reveals candidates for immunotherapy.

PubMed

Faramarzi, Sepideh; Ghafouri-Fard, Soudeh

2017-09-01

Prostate cancer is a prevalent disorder among men with a heterogeneous etiological background. Several molecular events and signaling perturbations have been found in this disorder. Among genes whose expressions have been altered during the prostate cancer development are cancer-testis antigens (CTAs). This group of antigens has limited expression in the normal adult tissues but aberrant expression in cancers. This property provides them the possibility to be used as cancer biomarkers and immunotherapeutic targets. Several CTAs have been shown to be immunogenic in prostate cancer patients and some of the have entered clinical trials. Based on the preliminary data obtained from these trials, it is expected that CTA-based therapeutic options are beneficial for at least a subset of prostate cancer patients.

Congenital intra-abdominal bilateral juvenile granulosa cell tumors of the testis associated with constitutional loss of material from chromosome 4.

PubMed

Yu, David C; Pathak, Bhavana; Vargas, Sara O; Javid, Patrick J; Hisama, Fuki M; Wilson, Jay M; Linden, Bradley C

2011-01-01

Juvenile granulosa cell tumor (JGCT) is an uncommon gonadal stromal tumor that occurs rarely in the testis. We report a newborn boy with bilateral intra-abdominal JGCT presenting with abdominal distention and respiratory distress at birth. He was taken to the operating room emergently, and 2 large masses connected by gubernacula to the inguinal canals were resected. Associated abnormalities included a constitutional chromosome 4 abnormality, polymicrogyria, and renal cysts. This report describes a rare presentation of JGCT with abdominal compression and expands the literature to include bilateral testicular involvement. Additionally, it is the 1st report of JGCT associated with a chromosome 4 abnormality, highlighting a genetic region that may be important in JGCT development.

Expression of the Wilms' tumor gene WT1 in the murine urogenital system.

PubMed

Pelletier, J; Schalling, M; Buckler, A J; Rogers, A; Haber, D A; Housman, D

1991-08-01

The Wilms' tumor gene WT1 is a recessive oncogene that encodes a putative transcription factor implicated in nephrogenesis during kidney development. In this report we analyze expression of WT1 in the murine urogenital system. WT1 is expressed in non-germ-cell components of the testis and ovaries in both young and adult mice. In situ mRNA hybridization studies demonstrate that WT1 is expressed in the granulosa and epithelial cells of ovaries, the Sertoli cells of the testis, and in the uterine wall. In addition to the 3.1-kb WT1 transcript detected by Northern blotting of RNA from kidney, uterus, and gonads, there is an approximately 2.5-kb WT1-related mRNA species in testis. The levels of WT1 mRNA in the gonads are among the highest observed, surpassing amounts detected in the embryonic kidney. During development, these levels are differentially regulated, depending on the sexual differentiation of the gonad. Expression of WT1 mRNA in the female reproductive system does not fluctuate significantly from days 4 to 40 postpartum. In contrast, WT1 mRNA levels in the tesis increase steadily after birth, reaching their highest expression levels at day 8 postpartum and decreasing slightly as the animal matures. Expression of WT1 in the gonads is detectable as early as 12.5 days postcoitum (p.c.). As an initial step toward exploring the tissue-specific expression of WT1, DNA elements upstream of WT1 were cloned and sequenced. Three putative transcription initiation sites, utilized in testis, ovaries, and uterus, were mapped by S1 nuclease protection assays. The sequences surrounding these sites have a high G + C content, and typical upstream CCAAT and TATAA boxes are not present. These studies allowed us to identify the translation initiation site for WT1 protein synthesis. We have also used an epitope-tagging protocol to demonstrate that WT1 is a nuclear protein, consistent with its role as a transcription factor. Our results demonstrate regulation of WT1 expression during development of the gonads, implicate WT1 in genitourinary development, and provide a molecular framework toward understanding genitourinary defects observed among hereditary cases of Wilms' tumor.

Unraveling the proteomic profile of mice testis during the initiation of meiosis.

PubMed

Shao, Binbin; Guo, Yueshuai; Wang, Lei; Zhou, Quan; Gao, Tingting; Zheng, Bo; Zheng, Haoyu; Zhou, Tao; Zhou, Zuomin; Guo, Xuejiang; Huang, Xiaoyan; Sha, Jiahao

2015-04-29

In mice, once primordial germ cells (PGCs) are generated, they continue to proliferate and migrate to eventually reach the future gonads. They initiate sexual differentiation after their colonization of the gonads. During this process, retinoic acid (RA) induces meiosis in the female germ cells, which proceeds to the diplotene stage of meiotic prophase I, whereas the male germ cells initiate growth arrest. After birth, meiosis is initiated in mice spermatogonia by their conversion to preleptotene spermatocytes. There are evidences showing the roles of RA in the regulation of spermatogonial differentiation and meiosis initiation. However, it is still not well known on what responds to RA and how RA signaling engages meiosis. Thus, we constructed a proteomic profile of proteins associated with meiosis onset during testis development in mouse and identified 104 differentially expressed proteins (≥1.5 folds). Bioinformatic analysis showed proteins functioning in specific cell processes. The expression patterns of five selected proteins were verified via Western blot, of which we found that Tfrc gene was RA responsive, with a RA responsive element, and could be up regulated by RA in spermatogonial stem cell (SSC) line. Taken together, the results provide an important reference profile for further functional study of meiosis initiation. Spermatogenesis involves mitosis of spermatogonia, meiosis of spermatocytes and spermiogenesis, in which meiosis is a unique event to germ cells, and not in the somatic cells. Till now, the detailed molecular mechanisms of the transition from mitosis to meiosis are still not elucidated. With high-throughput proteomic technology, it is now possible to systemically identify proteins possibly involved. With TMT-6plex based quantification, we identified 104 proteins differentially between testes without meiosis (day 8.5) and those that were meiosis initiated (day 10.5). And a well-known protein essential for meiosis initiation, stra8, was identified to be differentially expressed in the study. And bioinformatic analysis and functional studies revealed several proteins regulated by retinoic acid, a chemical known to regulate the meiosis initiation. Thus, this quantitative proteomic approach can identify meiosis initiation regulating proteins, and further functional studies of these proteins will help elucidate the mechanisms of meiosis initiation. Copyright © 2015. Published by Elsevier B.V.

De novo Transcriptome Analysis of Portunus trituberculatus Ovary and Testis by RNA-Seq: Identification of Genes Involved in Gonadal Development

PubMed Central

Meng, Xian-liang; Liu, Ping; Jia, Fu-long; Li, Jian; Gao, Bao-Quan

2015-01-01

The swimming crab Portunus trituberculatus is a commercially important crab species in East Asia countries. Gonadal development is a physiological process of great significance to the reproduction as well as commercial seed production for P. trituberculatus. However, little is currently known about the molecular mechanisms governing the developmental processes of gonads in this species. To open avenues of molecular research on P. trituberculatus gonadal development, Illumina paired-end sequencing technology was employed to develop deep-coverage transcriptome sequencing data for its gonads. Illumina sequencing generated 58,429,148 and 70,474,978 high-quality reads from the ovary and testis cDNA library, respectively. All these reads were assembled into 54,960 unigenes with an average sequence length of 879 bp, of which 12,340 unigenes (22.45% of the total) matched sequences in GenBank non-redundant database. Based on our transcriptome analysis as well as published literature, a number of candidate genes potentially involved in the regulation of gonadal development of P. trituberculatus were identified, such as FAOMeT, mPRγ, PGMRC1, PGDS, PGER4, 3β-HSD and 17β-HSDs. Differential expression analysis generated 5,919 differentially expressed genes between ovary and testis, among which many genes related to gametogenesis and several genes previously reported to be critical in differentiation and development of gonads were found, including Foxl2, Wnt4, Fst, Fem-1 and Sox9. Furthermore, 28,534 SSRs and 111,646 high-quality SNPs were identified in this transcriptome dataset. This work represents the first transcriptome analysis of P. trituberculatus gonads using the next generation sequencing technology and provides a valuable dataset for understanding molecular mechanisms controlling development of gonads and facilitating future investigation of reproductive biology in this species. The molecular markers obtained in this study will provide a fundamental basis for population genetics and functional genomics in P. trituberculatus and other closely related species. PMID:26042806

Gestational bisphenol A exposure and testis development.

PubMed

Williams, Cecilia; Bondesson, Maria; Krementsov, Dimitry N; Teuscher, Cory

Virtually all humans are exposed to bisphenol A (BPA). Since BPA can act as a ligand for estrogen receptors, potential hazardous effects of BPA should be evaluated in the context of endogenous estrogenic hormones. Because estrogen is metabolized in the placenta, developing fetuses are normally exposed to very low endogenous estrogen levels. BPA, on the other hand, passes through the placenta and might have distinct adverse consequences during the sensitive stages of fetal development. Testicular gametogenesis and steroidogenesis begin early during fetal development. These processes are sensitive to estrogens and play a role in determining the number of germ stem cells, sperm count, and male hormone levels in adulthood. Although studies have shown a correlation between BPA exposure and perturbed reproduction, a clear consensus has yet to be established as to whether current human gestational BPA exposure results in direct adverse effects on male genital development and reproduction. However, studies in animals and in vitro have provided direct evidence for the ability of BPA exposure to influence male reproductive development. This review discusses the current knowledge of potential effects of BPA exposure on male reproductive health and whether gestational exposure adversely affects testis development.

Effects of different levels of dietary selenium on the proliferation of spermatogonial stem cells and antioxidant status in testis of roosters.

PubMed

Shi, Lei; Zhao, Hui; Ren, Youshe; Yao, Xiaolei; Song, Ruigao; Yue, Wenbin

2014-10-01

The objective of this study was to investigate the different levels of dietary Se (from sodium selenite) on the proliferation of SSCs (spermatogonial stem cells) in testis of roosters. Also, the antioxidant status and Se content in blood plasma and testis were evaluated. A total of eighty 12-week-old Hy-Line Variety white roosters at an averaged body weight of 1.38 ± 0.2 kg were selected and randomly divided into four experimental groups. They were fed with the basal diet (0.044 mgSe/kg DM) supplemented with 0 (control), 0.5, 1.0 or 2.0 mgSe/kg DM (from sodium selenite). After the feeding experiment, blood and testis samples were collected for analysis of the antioxidant status and Se concentration. The testis samples were also used to examine the Thy-1 and β1-integrin mRNA expression by RT-PCR and detect the population of SSCs by immunofluorescence analysis. The results show that Se concentration in blood and testis of the animals was progressively increased with the increasing Se level in diet. The highest GSH-Px (glutathione peroxidase) activity and lowest MDA content in blood and testis was obtained in the treatment of 0.5mg/kg. RT-PCR analysis showed that mRNA expression of SSCs markers were significantly lower in the control and 1.0mg/kg groups when compared with that in the treatment of 0.5mg/kg. A similar trend was observed in the population of SSCs analyzed by immunofluorescence assay. These data suggest that dietary Se can influence the population of SSCs of roosters during spermatogenesis and that oxidative stress can modulate SSCs behavior through regulating some key factors during spermatogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

DEMONSTRATION IN VITRO OF ANAPHYLACTOID RESPONSE OF THE UTERUS AND ILEUM OF GUINEA PIGS INJECTED WITH TESTIS OR SPERM

PubMed Central

Katsh, Seymour

1958-01-01

Female guinea pigs were injected with the following materials: homogenates of guinea pig testis in saline or in adjuvant; suspensions of washed guinea pig sperm in saline or in adjuvant; homogenates of rabbit testis in adjuvant; guinea pig sperm and rabbit sperm in adjuvant. Control animals were not injected or were injected with adjuvant alone. At various times between 15 and 39 days after injection, the animals were sacrificed. Their ilea and uterine horns were removed and tested in vitro for reaction to washed epididymal sperm of the guinea pig, rabbit, or bull. It was found that the animals which were injected with homologous testis or sperm in adjuvant possessed organs which responded strongly to the challenge with homologous sperm. The response was a contracture which began 10 to 30 seconds after the sperm were injected into the bath and lasted for 5 minutes to 4 hours, the longest period of observation. Responses which lasted for periods of 5 minutes to 30 minutes were obtained with the uteri of the animals injected with guinea pig testis in saline or with guinea pig sperm in saline. Animals which were injected with rabbit testis and adjuvant responded to rabbit sperm, and animals injected with guinea pig sperm and rabbit sperm in adjuvant reacted to both gametes. A large proportion of the control animals possessed organs which reacted weakly to the challenge with homologous sperm. Retesting the organ which had contracted following exposure to sperm indicated that desensitization had occurred. Testing with heterologous sperm indicated a species selectivity. The evidence is interpreted to mean that injections of sperm or testis induce a hypersensitivity which is similar in some respects but differs from true anaphylaxis. The findings are discussed from the point of view of the nature of the response and the implications regarding natural immunity to sperm. PMID:13481258

A novel gene, RSD-3/HSD-3.1, encodes a meiotic-related protein expressed in rat and human testis.

PubMed

Zhang, Xiaodong; Liu, Huixian; Zhang, Yan; Qiao, Yuan; Miao, Shiying; Wang, Linfang; Zhang, Jianchao; Zong, Shudong; Koide, S S

2003-06-01

The expression of stage-specific genes during spermatogenesis was determined by isolating two segments of rat seminiferous tubule at different stages of the germinal epithelium cycle delineated by transillumination-delineated microdissection, combined with differential display polymerase chain reaction to identify the differential transcripts formed. A total of 22 cDNAs were identified and accepted by GenBank as new expressed sequence tags. One of the expressed sequence tags was radiolabeled and used as a probe to screen a rat testis cDNA library. A novel full-length cDNA composed of 2228 bp, designated as RSD-3 (rat sperm DNA no.3, GenBank accession no. AF094609) was isolated and characterized. The reading frame encodes a polypeptide consisting of 526 amino acid residues, containing a number of DNA binding motifs and phosphorylation sites for PKC, CK-II, and p34cdc2. Northern blot of mRNA prepared from various tissues of adult rats showed that RSD-3 is expressed only in the testis. The initial expression of the RSD-3 gene was detected in the testis on the 30th postnatal day and attained adult level on the 60th postnatal day. Immunolocalization of RSD-3 in germ cells of rat testis showed that its expression is restricted to primary spermatocytes, undergoing meiosis division I. A human testis homologue of RSD-3 cDNA, designated as HSD-3.1 (GenBank accession no. AF144487) was isolated by screening the Human Testis Rapid-Screen arrayed cDNA library panels by RT-PCR. The exon-intron boundaries of HSD-3.1 gene were determined by aligning the cDNA sequence with the corresponding genome sequence. The cDNA consisted of 12 exons that span approximately 52.8 kb of the genome sequence and was mapped to chromosome 14q31.3.

Optimizing a Male Reproductive Aging Mouse Model by d-Galactose Injection

PubMed Central

Liao, Chun-Hou; Chen, Bing-Huei; Chiang, Han-Sun; Chen, Chiu-Wei; Chen, Mei-Feng; Ke, Chih-Chun; Wang, Ya-Yun; Lin, Wei-Ning; Wang, Chi-Chung; Lin, Ying-Hung

2016-01-01

The d-galactose (d-gal)-injected animal model, which is typically established by administering consecutive subcutaneous d-gal injections to animals for approximately six or eight weeks, has been frequently used for aging research. In addition, this animal model has been demonstrated to accelerate aging in the brain, kidneys, liver and blood cells. However, studies on aging in male reproductive organs that have used this animal model remain few. Therefore, the current study aimed to optimize a model of male reproductive aging by administering d-gal injections to male mice and to determine the possible mechanism expediting senescence processes during spermatogenesis. In this study, C57Bl/6 mice were randomized into five groups (each containing 8–10 mice according to the daily intraperitoneal injection of vehicle control or 100 or 200 mg/kg dosages of d-gal for a period of six or eight weeks). First, mice subjected to d-gal injections for six or eight weeks demonstrated considerably decreased superoxide dismutase activity in the serum and testis lysates compared to those in the control group. The lipid peroxidation in testis also increased in the d-gal-injected groups. Furthermore, the d-gal-injected groups exhibited a decreased ratio of testis weight/body weight and sperm count compared to the control group. The percentages of both immotile sperm and abnormal sperm increased considerably in the d-gal-injected groups compared to those of the control group. To determine the genes influenced by the d-gal injection during murine spermatogenesis, a c-DNA microarray was conducted to compare testicular RNA samples between the treated groups and the control group. The d-gal-injected groups exhibited RNA transcripts of nine spermatogenesis-related genes (Cycl2, Hk1, Pltp, Utp3, Cabyr, Zpbp2, Speer2, Csnka2ip and Katnb1) that were up- or down-regulated by at least two-fold compared to the control group. Several of these genes are critical for forming sperm-head morphologies or maintaining nuclear integration (e.g., cylicin, basic protein of sperm head cytoskeleton 2 (Cylc2), casein kinase 2, alpha prime interacting protein (Csnka2ip) and katanin p80 (WD40-containing) subunit B1 (Katnb1)). These results indicate that d-gal-injected mice are suitable for investigating male reproductive aging. PMID:26771610

Sperm competition and maternal effects differentially influence testis and sperm size in Callosobruchus maculatus.

PubMed

Gay, L; Hosken, D J; Vasudev, R; Tregenza, T; Eady, P E

2009-05-01

The evolutionary factors affecting testis size are well documented, with sperm competition being of major importance. However, the factors affecting sperm length are not well understood; there are no clear theoretical predictions and the empirical evidence is inconsistent. Recently, maternal effects have been implicated in sperm length variation, a finding that may offer insights into its evolution. We investigated potential proximate and microevolutionary factors influencing testis and sperm size in the bruchid beetle Callosobruchus maculatus using a combined approach of an artificial evolution experiment over 90 generations and an environmental effects study. We found that while polyandry seems to select for larger testes, it had no detectable effect on sperm length. Furthermore, population density, a proximate indicator of sperm competition risk, was not significantly associated with sperm length or testis size variation. However, there were strong maternal effects influencing sperm length.

XX male sex reversal with genital abnormalities associated with a de novo SOX3 gene duplication.

PubMed

Moalem, Sharon; Babul-Hirji, Riyana; Stavropolous, Dmitri J; Wherrett, Diane; Bägli, Darius J; Thomas, Paul; Chitayat, David

2012-07-01

Differentiation of the bipotential gonad into testis is initiated by the Y chromosome-linked gene SRY (Sex-determining Region Y) through upregulation of its autosomal direct target gene SOX9 (Sry-related HMG box-containing gene 9). Sequence and chromosome homology studies have shown that SRY most probably evolved from SOX3, which in humans is located at Xq27.1. Mutations causing SOX3 loss-of-function do not affect the sex determination in mice or humans. However, transgenic mouse studies have shown that ectopic expression of Sox3 in the bipotential gonad results in upregulation of Sox9, resulting in testicular induction and XX male sex reversal. However, the mechanism by which these rearrangements cause sex reversal and the frequency with which they are associated with disorders of sex development remains unclear. Rearrangements of the SOX3 locus were identified recently in three cases of human XX male sex reversal. We report on a case of XX male sex reversal associated with a novel de novo duplication of the SOX3 gene. These data provide additional evidence that SOX3 gain-of-function in the XX bipotential gonad causes XX male sex reversal and further support the hypothesis that SOX3 is the evolutionary antecedent of SRY. Copyright © 2012 Wiley Periodicals, Inc.

Germline and reproductive tract effects intensify in male mice with successive generations of estrogenic exposure

PubMed Central

Horan, Tegan S.; Marre, Alyssa; Hassold, Terry; Lawson, Crystal; Hunt, Patricia A.

2017-01-01

The hypothesis that developmental estrogenic exposure induces a constellation of male reproductive tract abnormalities is supported by experimental and human evidence. Experimental data also suggest that some induced effects persist in descendants of exposed males. These multi- and transgenerational effects are assumed to result from epigenetic changes to the germline, but few studies have directly analyzed germ cells. Typically, studies of transgenerational effects have involved exposing one generation and monitoring effects in subsequent unexposed generations. This approach, however, has limited human relevance, since both the number and volume of estrogenic contaminants has increased steadily over time, intensifying rather than reducing or eliminating exposure. Using an outbred CD-1 mouse model, and a sensitive and quantitative marker of germline development, meiotic recombination, we tested the effect of successive generations of exposure on the testis. We targeted the germline during a narrow, perinatal window using oral exposure to the synthetic estrogen, ethinyl estradiol. A complex three generation exposure protocol allowed us to compare the effects of individual, paternal, and grandpaternal (ancestral) exposure. Our data indicate that multiple generations of exposure not only exacerbate germ cell exposure effects, but also increase the incidence and severity of reproductive tract abnormalities. Taken together, our data suggest that male sensitivity to environmental estrogens is increased by successive generations of exposure. PMID:28727826

Course of c-myc mRNA expression in the regenerating mouse testis determined by competitive reverse transcriptase polymerase chain reaction.

PubMed

Amendola, R

1994-11-01

The c-myc proto-oncogene is a reliable marker of the "G0-early G1" transition, and its down-regulation is believed to be necessary to obtain cellular differentiation. In murine spermatogenesis, the level of c-myc transcripts does not correlate with the rate of cellular division. Proliferation of supposed staminal spermatogonia to reproduce themselves is induced with a local 5 Gy X-ray dose in 90-day-old C57Bl/6 mice. c-myc quantification by a newly developed competitive reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to follow the expression course of this proto-oncogene. Damage and restoration of spermatogenesis were analyzed at days 3, 6, 9, 10, 13, 30, and 60 after injury by relative testes/body weight determination and histological examination. Proliferative status was determined by histone H3 Northern blot analysis. c-myc mRNA level was 10 times higher after 3 days in the irradiated animals compared to the controls. An increasing number of copies were noted up to 10 days, but promptly decreased to the base level found for irradiated mice from 13 to 60 days. Interestingly, the expression of histone H3 detected S phase only in testes at 60 days from damage.

Spermatogonial stem cells and progenitors are refractory to reprogramming to pluripotency by the transcription factors Oct3/4, c-Myc, Sox2 and Klf4

PubMed Central

Corbineau, Sébastien; Lassalle, Bruno; Givelet, Maelle; Souissi-Sarahoui, Inès; Firlej, Virginie; Romeo, Paul Henri; Allemand, Isabelle; Riou, Lydia; Fouchet, Pierre

2017-01-01

The male germinal lineage, which is defined as unipotent, produces sperm through spermatogenesis. However, embryonic primordial germ cells and postnatal spermatogonial stem cells (SSCs) can change their fate and convert to pluripotency in culture when they are not controlled by the testicular microenvironment. The mechanisms underlying these reprogramming processes are poorly understood. Testicular germ cell tumors, including teratoma, share some molecular characteristics with pluripotent cells, suggesting that cancer could result from an abnormal differentiation of primordial germ cells or from an abnormal conversion of SCCs to pluripotency in the testis. Here, we investigated whether the somatic reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM) could play a role in SSCs reprogramming and induce pluripotency using a doxycycline-inducible transgenic Col1a1-4F2A-OSKM mouse model. We showed that, in contrast to somatic cells, SSCs from adult mice are resistant to this reprogramming strategy, even in combination with small molecules, hypoxia, or p53 deficiency, which were previously described to favour the conversion of somatic cells to pluripotency. This finding suggests that adult SSCs have developed specific mechanisms to repress reprogramming by OSKM factors, contributing to circumvent testicular cancer initiation events. PMID:28052023

Transcript map of the Ovum mutant (Om) locus: isolation by exon trapping of new candidate genes for the DDK syndrome.

PubMed

Le Bras, Stéphanie; Cohen-Tannoudji, Michel; Guyot, Valérie; Vandormael-Pournin, Sandrine; Coumailleau, Franck; Babinet, Charles; Baldacci, Patricia

2002-08-21

The DDK syndrome is defined as the embryonic lethality of F1 mouse embryos from crosses between DDK females and males from other strains (named hereafter as non-DDK strains). Genetically controlled by the Ovum mutant (Om) locus, it is due to a deleterious interaction between a maternal factor present in DDK oocytes and the non-DDK paternal pronucleus. Therefore, the DDK syndrome constitutes a unique genetic tool to study the crucial interactions that take place between the parental genomes and the egg cytoplasm during mammalian development. In this paper, we present an extensive analysis performed by exon trapping on the Om region. Twenty-seven trapped sequences were from genes in the databases: beta-adaptin, CCT zeta2, DNA LigaseIII, Notchless, Rad51l3 and Scya1. Twenty-eight other sequences presented similarities with expressed sequence tags and genomic sequences whereas 57 did not. The pattern of expression of 37 of these markers was established. Importantly, five of them are expressed in DDK oocytes and are candidate genes for the maternal factor, and 20 are candidate genes for the paternal factor since they are expressed in testis. This data is an important step towards identifying the genes responsible for the DDK syndrome.

Undescended testis? How best to teach the physical examination.

PubMed

Zundel, S; Blumenstock, G; Herrmann-Werner, A; Trueck, M; Schmidt, A; Wiechers, S

2016-12-01

Undescended testis in boys is common. Guidelines recommend surgical treatment between the ages of 6 months and 2 years; nevertheless, orchidopexy is frequently performed at later ages. One reason is the belated diagnosis due to a perceived difficulty in the physical examination (PE) and correct localization of the testis. We aimed to find an effective method for teaching the physical examination of the testis in a child. An interdisciplinary team developed teaching sessions, including an educational video and a simulator. Medical students (n = 133) were randomized into three groups: self-study only, video, and video and simulator. The sessions were carried out and quantitative feedback was collected from the teachers and students. The learning achievements of the different groups were assessed with an objective structured clinical examination (OSCE). The differences in mean OSCE results between all three groups were tested using one-way analysis of variance (ANOVA). For multiple pairwise comparisons, a closed testing procedure was performed using unpaired t-tests. The self-study only group acquired the poorest results in the OSCE, with a mean score of 5.1 out of 10. The video-only-group reached a mean of 6.7, and the video-and-simulator group performed best with a mean score of 8.5. The differences between all three groups were found to be statistically significant, with P = 0.007. The attached figure illustrates this data. If analyzed in pairs, this difference was particularly apparent between the groups self-study only vs video and simulator, with P = 0.002. Qualitative feedback revealed doubtful effectiveness for educational videos, but positive reactions to training on a simulator. The poor results of the self-study-only group were in accordance with the literature, where textbook learning was found not to increase OSCE results. The effectiveness of video tutorials remains doubtful; studies focusing on this teaching method are divergent and the present students' feedback supports this data. The effective teaching with the simulator has been proven for other skills (i.e. ultrasound skills). The analyzed cohort for this study was small, and the study should be repeated at different institutions and with larger numbers of students to assure generalizability. Low-fidelity pediatric simulators with palpable testis are available and are able to improve examining skills in medical students. We hope the presented study inspires medical educators in their teaching of the PE of the pediatric testis. Copyright © 2016 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.

Connexin 43 reboots meiosis and reseals blood-testis barrier following toxicant-mediated aspermatogenesis and barrier disruption.

PubMed

Li, Nan; Mruk, Dolores D; Mok, Ka-Wai; Li, Michelle W M; Wong, Chris K C; Lee, Will M; Han, Daishu; Silvestrini, Bruno; Cheng, C Yan

2016-04-01

Earlier studies have shown that rats treated with an acute dose of 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (adjudin, a male contraceptive under development) causes permanent infertility due to irreversible blood-testis barrier (BTB) disruption even though the population of undifferentiated spermatogonia remains similar to normal rat testes, because spermatogonia fail to differentiate into spermatocytes to enter meiosis. Since other studies have illustrated the significance of connexin 43 (Cx43)-based gap junction in maintaining the homeostasis of BTB in the rat testis and the phenotypes of Sertoli cell-conditional Cx43 knockout mice share many of the similarities of the adjudin-treated rats, we sought to examine if overexpression of Cx43 in these adjudin-treated rats would reseal the disrupted BTB and reinitiate spermatogenesis. A full-length Cx43 cloned into mammalian expression vector pCI-neo was used to transfect testes of adjudin-treated ratsversusempty vector. It was found that overexpression of Cx43 indeed resealed the Sertoli cell tight junction-permeability barrier based on a functionalin vivoassay in tubules displaying signs of meiosis as noted by the presence of round spermatids. Thus, these findings suggest that overexpression of Cx43 reinitiated spermatogenesis at least through the steps of meiosis to generate round spermatids in testes of rats treated with an acute dose of adjudin that led to aspermatogenesis. It was also noted that the round spermatids underwent eventual degeneration with the formation of multinucleated cells following Cx43 overexpression due to the failure of spermiogenesis because no elongating/elongated spermatids were detected in any of the tubules examined. The mechanism by which overexpression of Cx43 reboots meiosis and rescues BTB function was also examined. In summary, overexpression of Cx43 in the testis with aspermatogenesis reboots meiosis and reseals toxicant-induced BTB disruption, even though it fails to support round spermatids to enter spermiogenesis.-Li, N., Mruk, D. D., Mok, K.-W., Li, M. W. M., Wong, C. K. C., Lee, W. M., Han, D., Silvestrini, B., Cheng, C. Y. Connexin 43 reboots meiosis and reseals blood-testis barrier following toxicant-mediated aspermatogenesis and barrier disruption. © FASEB.

Phthalate-Induced Pathology in the Foetal Testis Involves More Than Decreased Testosterone Production

EPA Science Inventory

Foetal exposure to phthalates is known to adversely impact male reproductive development and function. Developmental anomalies of reproductive tract have been attributed to impaired testosterone synthesis. However, species differences in the ability to produce testosterone have...

REPRODUCTIVE TOXICITY OF PHTHALATE ESTERS

EPA Science Inventory

Phthalate esters display several modes of toxicity in mammalian species. In the rat, in utero exposure at relatively low dosage levels disrupts development of the reproductive system of the male rat by altering fetal testis hormone production. This presentation is a review of t...

Transgenerational Effects of Di(2-ethylhexyl) Phthalate in the SD Male Rat

EPA Science Inventory

In the rat, some phthalates alter sexual differentiation at relatively low dosage levels by altering fetal Leydig cell development and hormone synthesis, thereby inducing abnormalities of the testis, gubernacular ligaments, epididymis and other androgen-dependent tissues. In ...

Protective effect of Urtica dioica L against nicotine-induced damage on sperm parameters, testosterone and testis tissue in mice.

PubMed

Jalili, Cyrus; Salahshoor, Mohammad Reza; Naseri, Ali

2014-06-01

Nicotine consumption can decrease fertility drive in males by inducing oxidative stress and DNA damage. Urtica dioica L (U.dioica) is a multipurpose herb in traditional medicine for which some anti-oxidative and anti-inflammatory properties have been identified. The main goal is to investigate whether the U.dioica could inhibit nicotine adverse effects on sperm cells viability, count, motility, and testis histology and testosterone hormone. In this study, hydro-alcoholic extract of U.dioica was prepared and various doses of U.dioica (0, 10, 20, and 50 mg/kg) and U.dioica plus nicotine (0, 10, 20, and 50 mg/kg) were administered intraperitoneally to 56 male mice for 28 consequent days. These mice were randomly assigned to 8 groups (n=7) and sperm parameters (sperm cells viability, count, motility, and morphology), testis and prostate weight, testis histology and testosterone hormone were analyzed and compared. The results indicated that nicotine administration (0.5 mg/kg) significantly decreased testosterone level, count and motility of sperm cells, and testis weight compared to control group (p=0.00). However, increasing the dose of U.dioica significantly boosted motility, count, normal morphology of sperm cells, seminiferous tubules diameter, and testosterone in all groups compared to control (p=0.00) and testis weight in 20 and 50 mg/kg doses in comparison with control group (p=0.00). It seems that U.dioica hydro-alcoholic extract administration could increase the quality of spermatozoa and inhibits nicotine-induced adverse effects on sperm parameters.

[Acceleration of Jingui Shenqi Pill on the testis telomerase activity in mice of Shen-yang deficiency].

PubMed

Xu, Cui-Ping; Zhu, Qing-Jun; Song, Jie; Li, Zhen; Zhang, Dan

2013-02-01

To explore the effects of Jingui Shenqi Pill (JSP) on the testis telomerase activity in mice of Shen-yang deficiency syndrome (SYDS). The SYDS model was prepared in 30 mice by over-fatigue and sexual overstrain. They were randomly divided into the model group and the JSP group, 15 in each group. Another 15 normal male mice were selected as the normal group. Mice in the normal group were fed routinely, with distilled water administered intragastrically at the daily dose of 0.1 mL/10 g. Mice in the model group were also administered intragastrically with distilled water at the daily dose of 0.1 mL/10 g while modeling establishment. Mice in the treatment group were administered intragastrically with JSP suspension at 0.1 mL/10 g (the concentration was 0.241 g/mL). The intervention lasted for 4 weeks. Four weeks later, the testis telomerase activity was detected in the three groups by ELISA. The SYDS model was replicated successfully by over-fatigue and sexual overstrain. JSP could improve the signs of mice of SYDS. Compared with the normal group, the activity of testis telomerase decreased in the model group (P < 0.01). Compared with the model group, the testis telomerase activity markedly increased in the treatment group (P < 0.01). The testis telomerase activity in mice of SYDS caused by over-fatigue and sexual overstrain obviously decreased, when compared with that in mice of the normal group. JSP could recover its activity.

Protective effect of Urtica dioica L against nicotine-induced damage on sperm parameters, testosterone and testis tissue in mice

PubMed Central

Jalili, Cyrus; Salahshoor, Mohammad Reza; Naseri, Ali

2014-01-01

Background: Nicotine consumption can decrease fertility drive in males by inducing oxidative stress and DNA damage. Urtica dioica L (U.dioica) is a multipurpose herb in traditional medicine for which some anti-oxidative and anti-inflammatory properties have been identified. Objective: The main goal is to investigate whether the U.dioica could inhibit nicotine adverse effects on sperm cells viability, count, motility, and testis histology and testosterone hormone. Materials and Methods: In this study, hydro-alcoholic extract of U.dioica was prepared and various doses of U.dioica (0, 10, 20, and 50 mg/kg) and U.dioica plus nicotine (0, 10, 20, and 50 mg/kg) were administered intraperitoneally to 56 male mice for 28 consequent days. These mice were randomly assigned to 8 groups (n=7) and sperm parameters (sperm cells viability, count, motility, and morphology), testis and prostate weight, testis histology and testosterone hormone were analyzed and compared. Results: The results indicated that nicotine administration (0.5 mg/kg) significantly decreased testosterone level, count and motility of sperm cells, and testis weight compared to control group (p=0.00). However, increasing the dose of U.dioica significantly boosted motility, count, normal morphology of sperm cells, seminiferous tubules diameter, and testosterone in all groups compared to control (p=0.00) and testis weight in 20 and 50 mg/kg doses in comparison with control group (p=0.00). Conclusion: It seems that U.dioica hydro-alcoholic extract administration could increase the quality of spermatozoa and inhibits nicotine-induced adverse effects on sperm parameters. PMID:25071848

Environmentally Induced Epigenetic Transgenerational Inheritance of Altered Sertoli Cell Transcriptome and Epigenome: Molecular Etiology of Male Infertility

PubMed Central

Guerrero-Bosagna, Carlos; Savenkova, Marina; Haque, Md. Muksitul; Nilsson, Eric; Skinner, Michael K.

2013-01-01

Environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of adult onset disease, including testis disease and male infertility. The current study was designed to determine the impact of an altered sperm epigenome on the subsequent development of an adult somatic cell (Sertoli cell) that influences the onset of a specific disease (male infertility). A gestating female rat (F0 generation) was exposed to the agriculture fungicide vinclozolin during gonadal sex determination and then the subsequent F3 generation progeny used for the isolation of Sertoli cells and assessment of testis disease. As previously observed, enhanced spermatogenic cell apoptosis was observed. The Sertoli cells provide the physical and nutritional support for the spermatogenic cells. Over 400 genes were differentially expressed in the F3 generation control versus vinclozolin lineage Sertoli cells. A number of specific cellular pathways were identified to be transgenerationally altered. One of the key metabolic processes affected was pyruvate/lactate production that is directly linked to spermatogenic cell viability. The Sertoli cell epigenome was also altered with over 100 promoter differential DNA methylation regions (DMR) modified. The genomic features and overlap with the sperm DMR were investigated. Observations demonstrate that the transgenerational sperm epigenetic alterations subsequently alters the development of a specific somatic cell (Sertoli cell) epigenome and transcriptome that correlates with adult onset disease (male infertility). The environmentally induced epigenetic transgenerational inheritance of testis disease appears to be a component of the molecular etiology of male infertility. PMID:23555832

The effects of pre- and postnatal exposure to the nonsteroidal antiandrogen flutamide on testis descent and morphology in the Albino Swiss rat

PubMed Central

KASSIM, NORMADIAH M.; McDONALD, S. W.; REID, O.; BENNETT, N. K.; GILMORE, D. P.; PAYNE, A. P.

1997-01-01

Exposure of male Albino Swiss rats to the nonsteroidal antiandrogen flutamide during the period from gestational day (d) 10 to birth resulted in feminisation of the external genitalia and the suppression of growth of the male reproductive tract. In adulthood, testes were found to be located in diverse positions. True cryptorchidism occurred in 10% of cases, whereas 50% of testes descended to the scrotum and 40% were located in a suprainguinal ectopic region. Varying degrees of tubule abnormality were seen in the testes of flutamide-treated animals, ranging from completely normal tubules with full spermatogenesis (and the expected frequency of the stages of spermatogenesis) to severely abnormal tubules lined with Sertoli cells only. For each individual testis, the overall severity of tubule damage was strongly correlated with its adult location, with intra-abdominal testes worst affected and scrotally-located testes least; only the latter contained normal tubules. Similarly, intra-abdominal testes were the smallest in weight and contained the least testosterone. By contrast, postnatal treatment of male rats with flutamide from birth to postnatal d 14 did not impair development of the external genitalia, the process of testicular descent or adult spermatogenesis. These findings confirm that androgen blockade during embryonic development interferes with testicular descent but also demonstrate that (1) prenatal flutamide treatment per se has a detrimental effect on adult testis morphology but (2) the degree of abnormality of the testes is strongly influenced by location. PMID:9183680