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Sample records for active germ cells

  1. Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

    SciTech Connect

    Yamano, Noriko; Kimura, Tohru; Watanabe-Kushima, Shoko; Shinohara, Takashi; Nakano, Toru

    2010-02-12

    Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.

  2. Testicular germ cell tumors.

    PubMed

    Looijenga, Leendert H J

    2014-02-01

    Human germ cell tumors are of interest because of their epidemiology, clinical behavior and pathobiology. Histologically, they are subdivided into various elements, with similarities to embryogenesis. Recent insights resulted in a division of five types of human germ cell tumors. In the context of male germ cells, three are relevant; Type I: teratomas and yolk sac tumors of neonates and infants; Type II: seminomas and nonseminomas of (predominantly) adolescents and adults; and Type III: spermatocytic seminomas of the elderly. Recent studies led to significant increases in understanding of the parameters involved in the earliest pathogenetic steps of human germ cells tumors, in particularly the seminomas and nonseminomas (Type II). In case of a disturbed gonadal physiology, either due to the germ cell itself, or the micro-environment, embryonic germ cells during a specific window of sensitization can be blocked in their maturation, resulting in carcinoma in situ or gonadoblastoma, the precursors of seminomas and nonseminomas. The level of testicularization of the gonad determines the histological composition of the precursor. These insights will allow better definition of individuals at risk to develop a germ cell malignancy, with putative preventive measurements, and allow better selection of scientific approaches to elucidate the pathogenesis. PMID:24683949

  3. [Retroperitoneal germ cell tumor].

    PubMed

    Borrell Palanca, A; García Garzón, J; Villamón Fort, R; Domenech Pérez, C; Martínez Lorente, A; Gunthner, S; García Sisamón, F

    1999-03-01

    We report a case of retroperitoneal extragonadal germ-cell tumor in an 17 years old patient who presented with aedema and pain in left inferior extremity asociated with hemopthysis caused by pulmonar metastasis, who was treated with chemotherapy and resection of residual mass and pulmonary nodes. Dyagnosis was stableshed by fine neadle aspiration biopsy of the wass. We comment on the difficult of stableshing differential dyagnosis between retroperitoneal extragonadal germ-cell tumor and metastasis of a testicular tumor. Dyagnosis is stableshed by the finding of a histologically malignant germ-cell tumor with normal testis. We considered physical examination and ecographyc exploration enough for a correct dyagnosis.

  4. Compliance of males with stage 1 testicular germ cell tumours on an active surveillance protocol.

    PubMed

    Honeyball, F; Murali-Ganesh, R; Hruby, G; Grimison, P

    2015-10-01

    The aim of this retrospective study was to determine the rate of compliance among 57 males with stage 1 testicular germ cell tumours on an active surveillance protocol at a single Australian centre. At median follow up of 24 months, 81% had adequate compliance with the follow-up regimen, 12% were lost to follow up, and 16% relapsed; none between protocol visits. Active surveillance is an acceptable alternative to adjuvant therapy for stage 1 testicular germ cell tumours, with reduced toxicity for most and equivalent survival, but requires efforts to maintain adequate compliance with follow up to avoid late detection of recurrence.

  5. Zebrafish Germ Cell Tumors.

    PubMed

    Sanchez, Angelica; Amatruda, James F

    2016-01-01

    Germ cell tumors (GCTs) are malignant cancers that arise from embryonic precursors known as Primordial Germ Cells. GCTs occur in neonates, children, adolescents and young adults and can occur in the testis, the ovary or extragonadal sites. Because GCTs arise from pluripotent cells, the tumors can exhibit a wide range of different histologies. Current cisplatin-based combination therapies cures most patients, however at the cost of significant toxicity to normal tissues. While GWAS studies and genomic analysis of human GCTs have uncovered somatic mutations and loci that might confer tumor susceptibility, little is still known about the exact mechanisms that drive tumor development, and animal models that faithfully recapitulate all the different GCT subtypes are lacking. Here, we summarize current understanding of germline development in humans and zebrafish, describe the biology of human germ cell tumors, and discuss progress and prospects for zebrafish GCT models that may contribute to better understanding of human GCTs. PMID:27165367

  6. Extragonadal Germ Cell Cancer (EGC)

    MedlinePlus

    ... Testicular Cancer Resource Center Extragonadal Germ Cell Cancer (EGC) 95% of all testicular tumors are germ cell ... seen in young adults. Patients with mediastinal nonseminomatous EGC are typically classed as poor risk patients because ...

  7. RNA Granules in Germ Cells

    PubMed Central

    Voronina, Ekaterina; Seydoux, Geraldine; Sassone-Corsi, Paolo; Nagamori, Ippei

    2011-01-01

    Germ granules” are cytoplasmic, nonmembrane-bound organelles unique to germline. Germ granules share components with the P bodies and stress granules of somatic cells, but also contain proteins and RNAs uniquely required for germ cell development. In this review, we focus on recent advances in our understanding of germ granule assembly, dynamics, and function. One hypothesis is that germ granules operate as hubs for the posttranscriptional control of gene expression, a function at the core of the germ cell differentiation program. PMID:21768607

  8. Ribosome Synthesis and MAPK Activity Modulate Ionizing Radiation-Induced Germ Cell Apoptosis in Caenorhabditis elegans

    PubMed Central

    Eberhard, Ralf; Stergiou, Lilli; Hofmann, E. Randal; Hofmann, Jen; Haenni, Simon; Teo, Youjin; Furger, André; Hengartner, Michael O.

    2013-01-01

    Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment. PMID:24278030

  9. [Mediastinal germ cell tumors].

    PubMed

    Bremmer, F; Ströbel, P

    2016-09-01

    The mediastinum is among the most frequent anatomic region in which germ cell tumors (GCT) arise, second only to the gonads. Mediastinal GCT (mGCT) account for 16 % of all mediastinal neoplasms. Although the morphology and (according to all available data) the molecular genetics of mediastinal and gonadal GCT are identical, a number of unique aspects exist. There is a highly relevant bi-modal age distribution. In pre-pubertal children of both sexes, mGCT consist exclusively of teratomas and yolk sac tumors. The prognosis is generally favorable with modern treatment. In post-pubertal adults, virtually all patients with malignant mGCT are males; the prognosis is more guarded and depends (among other factors) on the histological GCT components and is similar to GCT in other organs. So-called somatic type malignancies (i. e. clonally related, non-germ cell neoplasias arising in a GCT) are much more frequent in mGCT than in other organs, and the association between mediastinal yolk sac tumors and hematological malignancies, such as myelodysplasias and leukemias, is unique to mediastinal tumors. The prognosis of GCT with somatic type malignancies is generally dismal. PMID:27491549

  10. Specific deletion of AMP-activated protein kinase (α1AMPK) in mouse Sertoli cells modifies germ cell quality.

    PubMed

    Bertoldo, Michael J; Guibert, Edith; Faure, Melanie; Guillou, Florian; Ramé, Christelle; Nadal-Desbarats, Lydie; Foretz, Marc; Viollet, Benoit; Dupont, Joëlle; Froment, Pascal

    2016-03-01

    The AMP-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis which plays a role in fertility. Complete disruption of the AMPK catalytic subunit α1 gene (α1AMPK KO) in male mice results in a decrease in litter size which is associated with the production of altered sperm morphology and motility. Because of the importance of Sertoli cells in the formation of germ cells, we have chosen to selectively disrupt α1AMPK only in the Sertoli cells in mice (Sc-α1AMPK-KO mice). Specific deletion of the α1AMPK gene in Sertoli cells resulted in a 25% reduction in male fertility associated with abnormal spermatozoa with a thin head. No clear alterations in testis morphology or modification in the number of Sertoli cells in vivo were observed, but a dysregulation in energy metabolism in Sertoli cells occurred. We have reported an increase in lactate production, in lipid droplets, and a reduction in ATP production in Sc-α1AMPK-KO Sertoli cells. These perturbations were associated with lower expression of mitochondrial markers (cytochrome c and PGC1-α). In addition another metabolic sensor, the deacetylase SIRT1, had a reduction in expression which is correlated with a decline in deacetylase activity. Finally, expression and localization of junctions forming the blood-testis barrier between Sertoli cells themselves and with germ cells were deregulated in Sc-α1AMPK-KO. In conclusion, these results suggest that dysregulation of the energy sensing machinery exclusively through disruption of α1AMPK in Sertoli cells translates to a reduction in the quality of germ cells and fertility. PMID:26772142

  11. Anti-tumour activity of two novel compounds in cisplatin-resistant testicular germ cell cancer

    PubMed Central

    Nitzsche, B; Gloesenkamp, C; Schrader, M; Hoffmann, B; Zengerling, F; Balabanov, S; Honecker, F; Höpfner, M

    2012-01-01

    Background: Resistance to cisplatin-based chemotherapy is associated with poor prognosis in testicular germ cell cancer, emphasising the need for new therapeutic approaches. In this respect, the therapeutic concept of anti-angiogenesis is of particular interest. In a previous study, we presented two novel anti-angiogenic compounds, HP-2 and HP-14, blocking the tyrosine kinase activity of angiogenic growth factor receptors, such as vascular endothelial growth factor receptor-2 (VEGFR-2), and related signalling pathways in testicular cancer. In this study, we investigated the efficacy of these new compounds in platinum-resistant testicular germ cell tumours (TGCTs), in vitro and in vivo. Methods and results: Drug-induced changes in cell proliferation of the cisplatin-sensitive TGCT cell line 2102EP and its cisplatin-resistant counterpart 2102EP-R, both expressing the VEGFR-2, were evaluated by crystal violet staining. Both compounds inhibited the growth of cisplatin-resistant TGCT cells in a dose-dependent manner. In combination experiments with cisplatin, HP-14 revealed additive growth-inhibitory effects in TGCT cells, irrespective of the level of cisplatin resistance. Anti-angiogenic effects of HP compounds were confirmed by tube formation assays with freshly isolated human umbilical vein endothelial cells. Using TGCT cells inoculated onto the chorioallantoic membrane of fertilised chicken eggs (chicken chorioallantoic membrane assay), the anti-angiogenic and anti-proliferative potency of the novel compounds was also demonstrated in vivo. Gene expression profiling revealed changes in the expression pattern of genes related to DNA damage detection and repair, as well as in chaperone function after treatment with both cisplatin and HP-14, alone or in combination. This suggests that HP-14 can revert the lost effectiveness of cisplatin in the resistant cells by altering the expression of critical genes. Conclusion: The novel compound HP-14 effectively inhibits the

  12. Dissecting Germ Cell Metabolism through Network Modeling

    PubMed Central

    Whitmore, Leanne S.; Ye, Ping

    2015-01-01

    Metabolic pathways are increasingly postulated to be vital in programming cell fate, including stemness, differentiation, proliferation, and apoptosis. The commitment to meiosis is a critical fate decision for mammalian germ cells, and requires a metabolic derivative of vitamin A, retinoic acid (RA). Recent evidence showed that a pulse of RA is generated in the testis of male mice thereby triggering meiotic commitment. However, enzymes and reactions that regulate this RA pulse have yet to be identified. We developed a mouse germ cell-specific metabolic network with a curated vitamin A pathway. Using this network, we implemented flux balance analysis throughout the initial wave of spermatogenesis to elucidate important reactions and enzymes for the generation and degradation of RA. Our results indicate that primary RA sources in the germ cell include RA import from the extracellular region, release of RA from binding proteins, and metabolism of retinal to RA. Further, in silico knockouts of genes and reactions in the vitamin A pathway predict that deletion of Lipe, hormone-sensitive lipase, disrupts the RA pulse thereby causing spermatogenic defects. Examination of other metabolic pathways reveals that the citric acid cycle is the most active pathway. In addition, we discover that fatty acid synthesis/oxidation are the primary energy sources in the germ cell. In summary, this study predicts enzymes, reactions, and pathways important for germ cell commitment to meiosis. These findings enhance our understanding of the metabolic control of germ cell differentiation and will help guide future experiments to improve reproductive health. PMID:26367011

  13. Elucidating human male germ cell development by studying germ cell cancer.

    PubMed

    Nettersheim, Daniel; Jostes, Sina; Schneider, Simon; Schorle, Hubert

    2016-10-01

    Human germ cell development is regulated in a spatio-temporal manner by complex regulatory networks. Here, we summarize results obtained in germ cell tumors and respective cell lines and try to pinpoint similarities to normal germ cell development. This comparison allows speculating about the critical and error-prone mechanisms, which when disturbed, lead to the development of germ cell tumors. Short after specification, primordial germ cells express markers of pluripotency, which, in humans, persists up to the stage of fetal/infantile spermatogonia. Aside from the rare spermatocytic tumors, virtually all seminomas and embryonal carcinomas express markers of pluripotency and show signs of pluripotency or totipotency. Therefore, it appears that proper handling of the pluripotency program appears to be the most critical step in germ cell development in terms of tumor biology. Furthermore, data from mice reveal that germline cells display an epigenetic signature, which is highly similar to pluripotent cells. This signature (poised histone code, DNA hypomethylation) is required for the rapid induction of toti- and pluripotency upon fertilization. We propose that adult spermatogonial cells, when exposed to endocrine disruptors or epigenetic active substances, are prone to reinitiate the pluripotency program, giving rise to a germ cell tumor. The fact that pluripotent cells can be derived from adult murine and human testicular cells further corroborates this idea. PMID:27512122

  14. Palifosfamide in Treating Patients With Recurrent Germ Cell Tumors

    ClinicalTrials.gov

    2015-06-11

    Adult Central Nervous System Germ Cell Tumor; Adult Teratoma; Malignant Extragonadal Germ Cell Tumor; Malignant Extragonadal Non-Seminomatous Germ Cell Tumor; Extragonadal Seminoma; Recurrent Malignant Testicular Germ Cell Tumor; Recurrent Ovarian Germ Cell Tumor; Stage IV Extragonadal Non-Seminomatous Germ Cell Tumor; Stage IV Extragonadal Seminoma; Stage IV Ovarian Germ Cell Tumor

  15. NANOG alone induces germ cells in primed epiblast in vitro by activation of enhancers

    PubMed Central

    Zylicz, Jan J.; Tang, Walfred W. C.; Sengupta, Roopsha; Kobayashi, Toshihiro; Kim, Shinseog; Butler, Richard; Dietmann, Sabine; Surani, M. Azim

    2015-01-01

    Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGC) in mice1, where its precise role is yet unclear2-4. We investigated this in an in vitro model, where naïve pluripotent embryonic stem cells (ESCs) cultured in bFGF/ActivinA develop as epiblast-like cells (EpiLCs), and gain competence for PGC-like fate5. Consequently, bone morphogenetic protein (BMP4), or ectopic expression of key germline transcription factors Prdm1/ Prdm14/ Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ESCs6-8. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that following the dissolution of the naïve ESC pluripotency network during establishment of EpiLCs9,10, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG binding pattern between ESCs and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ESCs, they show contrasting roles in EpiLCs since Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development. PMID:26751055

  16. Pluripotent stem cells from germ cells.

    PubMed

    Kerr, Candace L; Shamblott, Michael J; Gearhart, John D

    2006-01-01

    To date, stem cells have been derived from three sources of germ cells. These include embryonic germ cells (EGCs), embryonal carcinoma cells (ECCs), and multipotent germ line stem cells (GSCs). EGCs are derived from primordial germ cells that arise in the late embryonic and early fetal period of development. ECCs are derived from adult testicular tumors whereas GSCs have been derived by culturing spermatogonial stem cells from mouse neonates and adults. For each of these lines, their pluripotency has been demonstrated by their ability to differentiate into cell types derived from the three germ layers in vitro and in vivo and in chimeric animals, including germ line transmission. These germ line-derived stem cells have been generated from many species including human, mice, porcine, and chicken albeit with only slight modifications. This chapter describes general considerations regarding critical aspects of their derivation compared with their counterpart, embryonic stem cells (ESCs). Detailed protocols for EGC derivation and maintenance from human and mouse primordial germ cells (PGCs) will be presented.

  17. General Information about Extragonadal Germ Cell Tumors

    MedlinePlus

    ... Germ Cell Tumors Treatment (PDQ®)–Patient Version General Information About Extragonadal Germ Cell Tumors Go to Health ... the PDQ Adult Treatment Editorial Board . Clinical Trial Information A clinical trial is a study to answer ...

  18. General Information about Ovarian Germ Cell Tumors

    MedlinePlus

    ... Germ Cell Tumors Treatment (PDQ®)–Patient Version General Information About Ovarian Germ Cell Tumors Go to Health ... the PDQ Adult Treatment Editorial Board . Clinical Trial Information A clinical trial is a study to answer ...

  19. HISTORY OF GERM CELL MUTAGENESIS

    EPA Science Inventory

    Much of the early work on germ cell mutation analysis was conducted with nonmammalian species, but this historical overview will begin with the rodent studies that provided quantitative data on induced mutations. The initial studies of mutation induction utilized the newly develo...

  20. Reprogramming of germ cells into pluripotency

    PubMed Central

    Sekita, Yoichi; Nakamura, Toshinobu; Kimura, Tohru

    2016-01-01

    Primordial germ cells (PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors.

  1. Reprogramming of germ cells into pluripotency.

    PubMed

    Sekita, Yoichi; Nakamura, Toshinobu; Kimura, Tohru

    2016-08-26

    Primordial germ cells (PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors. PMID:27621759

  2. Reprogramming of germ cells into pluripotency

    PubMed Central

    Sekita, Yoichi; Nakamura, Toshinobu; Kimura, Tohru

    2016-01-01

    Primordial germ cells (PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors. PMID:27621759

  3. Identification of Potential Germ-Cell Mutagens

    EPA Science Inventory

    The existence of agents that can induce germ-cell mutations in experimental systems has been recognized since 1927 with the discovery of the ability of X-rays to induce such mutations in Drosophila. Various rodent-based germ-cell mutation assays have been developed, and ~50 germ...

  4. Primordial Germ Cell Specification and Migration

    PubMed Central

    Marlow, Florence

    2015-01-01

    Primordial germ cells are the progenitor cells that give rise to the gametes. In some animals, the germline is induced by zygotic transcription factors, whereas in others, primordial germ cell specification occurs via inheritance of maternally provided gene products known as germ plasm. Once specified, the primordial germ cells of some animals must acquire motility and migrate to the gonad in order to survive. In all animals examined, perinuclear structures called germ granules form within germ cells. This review focuses on some of the recent studies, conducted by several groups using diverse systems, from invertebrates to vertebrates, which have provided mechanistic insight into the molecular regulation of germ cell specification and migration. PMID:26918157

  5. A Mechanism of Male Germ Cell Apoptosis Induced by Bisphenol-A and Nonylphenol Involving ADAM17 and p38 MAPK Activation

    PubMed Central

    Moreno, Ricardo D.

    2014-01-01

    Germ cell apoptosis regulation is pivotal in order to maintain proper daily sperm production. Several reports have shown that endocrine disruptors such as Bisphenol-A (BPA) and Nonylphenol (NP) induce germ cell apoptosis along with a decrease in sperm production. Given their ubiquitous distribution in plastic products used by humans it is important to clarify their mechanism of action. TACE/ADAM17 is a widely distributed extracellular metalloprotease and participates in the physiological apoptosis of germ cells during spermatogenesis. The aims of this work were: 1) to determine whether BPA and NP induce ADAM17 activation; and 2) to study whether ADAM17 and/or ADAM10 are involved in germ cell apoptosis induced by BPA and NP in the pubertal rat testis. A single dose of BPA or NP (50 mg/kg) induces germ cell apoptosis in 21-day-old male rats, which was prevented by a pharmacological inhibitor of ADAM17, but not by an inhibitor of ADAM10. In vitro, we showed that BPA and NP, at similar concentrations to those found in human samples, induce the shedding of exogenous and endogenous (TNF-α) ADAM17 substrates in primary rat Sertoli cell cultures and TM4 cell line. In addition, pharmacological inhibitors of metalloproteases and genetic silencing of ADAM17 prevent the shedding induced in vitro by BPA and NP. Finally, we showed that in vivo BPA and NP induced early activation (phosphorylation) of p38 MAPK and translocation of ADAM17 to the cell surface. Interestingly, the inhibition of p38 MAPK prevents germ cell apoptosis and translocation of ADAM17 to the cell surface. These results show for the first time that xenoestrogens can induce activation of ADAM17 at concentrations similar to those found in human samples, suggesting a mechanism by which they could imbalance para/juxtacrine cell-to-cell-communication and induce germ cell apoptosis. PMID:25474107

  6. Angiosarcoma associated with germ cell tumors

    SciTech Connect

    Ulbright, T.M.; Clark, S.A.; Einhorn, L.H.

    1985-03-01

    In two patients with malignant germ cell tumors angiosarcoma developed through two apparently different mechanisms. In one case the angiosarcoma probably developed as a complication of therapeutic radiation, since radiation changes were demonstrated in tissue adjacent to the neoplasm and since the angiosarcoma was not associated with elements of germ cell tumor. The absence of associated germ cell elements does not support the development of the angiosarcoma from a teratoma. In the second case, however, it is likely that the angiosarcoma developed as a result of malignant change within teratomatous foci, since angiosarcomatous elements were intermingled with teratomatous elements and the patient's primary germ cell tumor contained malignant and atypical teratomatous elements as well as prominent vascular proliferation. Malignant change within teratomatous components of germ cell tumors is a phenomenon of increasing importance in this era of effective chemotherapy for germ cell tumors. The development of angiosarcoma as a potential complication of testicular carcinoma has not been reported previously.

  7. A germ cell determinant reveals parallel pathways for germ line development in Caenorhabditis elegans.

    PubMed

    Mainpal, Rana; Nance, Jeremy; Yanowitz, Judith L

    2015-10-15

    Despite the central importance of germ cells for transmission of genetic material, our understanding of the molecular programs that control primordial germ cell (PGC) specification and differentiation are limited. Here, we present findings that X chromosome NonDisjunction factor-1 (XND-1), known for its role in regulating meiotic crossover formation, is an early determinant of germ cell fates in Caenorhabditis elegans. xnd-1 mutant embryos display a novel 'one PGC' phenotype as a result of G2 cell cycle arrest of the P4 blastomere. Larvae and adults display smaller germ lines and reduced brood size consistent with a role for XND-1 in germ cell proliferation. Maternal XND-1 proteins are found in the P4 lineage and are exclusively localized to the nucleus in PGCs, Z2 and Z3. Zygotic XND-1 turns on shortly thereafter, at the ∼300-cell stage, making XND-1 the earliest zygotically expressed gene in worm PGCs. Strikingly, a subset of xnd-1 mutants lack germ cells, a phenotype shared with nos-2, a member of the conserved Nanos family of germline determinants. We generated a nos-2 null allele and show that nos-2; xnd-1 double mutants display synthetic sterility. Further removal of nos-1 leads to almost complete sterility, with the vast majority of animals without germ cells. Sterility in xnd-1 mutants is correlated with an increase in transcriptional activation-associated histone modification and aberrant expression of somatic transgenes. Together, these data strongly suggest that xnd-1 defines a new branch for PGC development that functions redundantly with nos-2 and nos-1 to promote germline fates by maintaining transcriptional quiescence and regulating germ cell proliferation. PMID:26395476

  8. Treatment Option Overview (Extragonadal Germ Cell Tumors)

    MedlinePlus

    ... hCG and LDH may be at any level. Poor prognosis A nonseminoma extragonadal germ cell tumor is in the poor prognosis group if: the tumor is in the ... extragonadal germ cell tumor does not have a poor prognosis group. Treatment Option Overview Key Points There ...

  9. Specification of germ cell fate in mice.

    PubMed Central

    Saitou, Mitinori; Payer, Bernhard; Lange, Ulrike C; Erhardt, Sylvia; Barton, Sheila C; Surani, M Azim

    2003-01-01

    An early fundamental event during development is the segregation of germ cells from somatic cells. In many organisms, this is accomplished by the inheritance of preformed germ plasm, which apparently imposes transcriptional repression to prevent somatic cell fate. However, in mammals, pluripotent epiblast cells acquire germ cell fate in response to signalling molecules. We have used single cell analysis to study how epiblast cells acquire germ cell competence and undergo specification. Germ cell competent cells express Fragilis and initially progress towards a somatic mesodermal fate. However, a subset of these cells, the future primordial germ cells (PGCs), then shows rapid upregulation of Fragilis with concomitant transcriptional repression of a number of genes, including Hox and Smad genes. This repression may be a key event associated with germ cell specification. Furthermore, PGCs express Stella and other genes, such as Oct-4 that are associated with pluripotency. While these molecules are also detected in mature oocytes as maternally inherited factors, their early role is to regulate development and maintain pluripotency, and they do not serve the role of classical germline determinants. PMID:14511483

  10. Germ cell specification and regeneration in planarians.

    PubMed

    Newmark, P A; Wang, Y; Chong, T

    2008-01-01

    In metazoans, two apparently distinct mechanisms specify germ cell fate: Determinate specification (observed in animals including Drosophila, Caenorhabditis elegans, zebra fish, and Xenopus) uses cytoplasmic factors localized to specific regions of the egg, whereas epigenetic specification (observed in many basal metazoans, urodeles, and mammals) involves inductive interactions between cells. Much of our understanding of germ cell specification has emerged from studies of model organisms displaying determinate specification. In contrast, our understanding of epigenetic/inductive specification is less advanced and would benefit from studies of additional organisms. Freshwater planarians--widely known for their remarkable powers of regeneration--are well suited for studying the mechanisms by which germ cells can be induced. Classic experiments showed that planarians can regenerate germ cells from body fragments entirely lacking reproductive structures, suggesting that planarian germ cells could be specified by inductive signals. Furthermore, the availability of the genome sequence of the planarian Schmidtea mediterranea, coupled with the animal's susceptibility to systemic RNA interference (RNAi), facilitates functional genomic analyses of germ cell development and regeneration. Here, we describe recent progress in studies of planarian germ cells and frame some of the critical unresolved questions for future work.

  11. 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

  12. Specifying and protecting germ cell fate

    PubMed Central

    Strome, Susan; Updike, Dustin

    2015-01-01

    Germ cells are the special cells in the body that undergo meiosis to generate gametes and subsequently entire new organisms after fertilization, a process that continues generation after generation. Recent studies have expanded our understanding of the factors and mechanisms that specify germ cell fate, including the partitioning of maternally supplied ‘germ plasm’, inheritance of epigenetic memory and expression of transcription factors crucial for primordial germ cell (PGC) development. Even after PGCs are specified, germline fate is labile and thus requires protective mechanisms, such as global transcriptional repression, chromatin state alteration and translation of only germline-appropriate transcripts. Findings from diverse species continue to provide insights into the shared and divergent needs of these special reproductive cells. PMID:26122616

  13. Improvement of Chicken Primordial Germ Cell Maintenance In Vitro by Blockade of the Aryl Hydrocarbon Receptor Endogenous Activity.

    PubMed

    Pérez Sáez, Juan M; Bussmann, Leonardo E; Barañao, J Lino; Bussmann, Ursula A

    2016-06-01

    Primordial germ cells (PGCs) are the undifferentiated progenitors of gametes. Germline competent PGCs can be developed as a cell-based system for genetic modification in chickens, which provides a valuable tool for transgenic technology with both research and industrial applications. This implies manipulation of PGCs, which, in recent years, encouraged a lot of research focused on the study of PGCs and the way of improving their culture. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that besides mediating toxic responses to environmental contaminants plays pivotal physiological roles in various biological processes. Since a novel compound that acts as an antagonist of this receptor has been reported to promote expansion of hematopoietic stem cells, we conducted the present study with the aim of determining whether addition of an established AHR antagonist to the standard culture medium used nowadays for in vitro chicken PGCs culture improves ex vivo expansion. We have found that addition of α-naphthoflavone in culture medium promotes the amplification of undifferentiated cells and that this effect is exerted by the blockade of AHR action. Our results constitute the first report of the successful use of a readily available AHR antagonist to improve avian PGCs expansion, and they further extend the knowledge of the effects of AHR modulation in undifferentiated cells. PMID:27253627

  14. Improvement of Chicken Primordial Germ Cell Maintenance In Vitro by Blockade of the Aryl Hydrocarbon Receptor Endogenous Activity.

    PubMed

    Pérez Sáez, Juan M; Bussmann, Leonardo E; Barañao, J Lino; Bussmann, Ursula A

    2016-06-01

    Primordial germ cells (PGCs) are the undifferentiated progenitors of gametes. Germline competent PGCs can be developed as a cell-based system for genetic modification in chickens, which provides a valuable tool for transgenic technology with both research and industrial applications. This implies manipulation of PGCs, which, in recent years, encouraged a lot of research focused on the study of PGCs and the way of improving their culture. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that besides mediating toxic responses to environmental contaminants plays pivotal physiological roles in various biological processes. Since a novel compound that acts as an antagonist of this receptor has been reported to promote expansion of hematopoietic stem cells, we conducted the present study with the aim of determining whether addition of an established AHR antagonist to the standard culture medium used nowadays for in vitro chicken PGCs culture improves ex vivo expansion. We have found that addition of α-naphthoflavone in culture medium promotes the amplification of undifferentiated cells and that this effect is exerted by the blockade of AHR action. Our results constitute the first report of the successful use of a readily available AHR antagonist to improve avian PGCs expansion, and they further extend the knowledge of the effects of AHR modulation in undifferentiated cells.

  15. Functional Analysis of the Drosophila Embryonic Germ Cell Transcriptome by RNA Interference

    PubMed Central

    Bujna, Ágnes; Vilmos, Péter; Spirohn, Kerstin; Boutros, Michael; Erdélyi, Miklós

    2014-01-01

    In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general. PMID:24896584

  16. Wnt/β-catenin signaling pathway activation is required for proliferation of chicken primordial germ cells in vitro

    PubMed Central

    Lee, Hyung Chul; Lim, Sumi; Han, Jae Yong

    2016-01-01

    Here, we investigated the role of the Wnt/β-catenin signaling pathway in chicken primordial germ cells (PGCs) in vitro. We confirmed the expression of Wnt signaling pathway-related genes and the localization of β-catenin in the nucleus, revealing that this pathway is potentially activated in chicken PGCs. Then, using the single-cell pick-up assay, we examined the proliferative capacity of cultured PGCs in response to Wnt ligands, a β-catenin-mediated Wnt signaling activator (6-bromoindirubin-3′-oxime [BIO]) or inhibitor (JW74), in the presence or absence of basic fibroblast growth factor (bFGF). WNT1, WNT3A, and BIO promoted the proliferation of chicken PGCs similarly to bFGF, whereas JW74 inhibited this proliferation. Meanwhile, such treatments in combination with bFGF did not show a synergistic effect. bFGF treatment could not rescue PGC proliferation in the presence of JW74. In addition, we confirmed the translocation of β-catenin into the nucleus by the addition of bFGF after JW74 treatment. These results indicate that there is signaling crosstalk between FGF and Wnt, and that β-catenin acts on PGC proliferation downstream of bFGF. In conclusion, our study suggests that Wnt signaling enhances the proliferation of chicken PGCs via the stabilization of β-catenin and activation of its downstream genes. PMID:27687983

  17. Male germ cell transplantation in livestock.

    PubMed

    Hill, J R; Dobrinski, I

    2006-01-01

    Male germ cell transplantation is a powerful approach to study the control of spermatogenesis with the ultimate goal to enhance or suppress male fertility. In livestock animals, applications can be expanded to provide an alternative method of transgenesis and an alternative means of artificial insemination (AI). The transplantation technique uses testis stem cells, harvested from the donor animal. These donor stem cells are injected into seminiferous tubules, migrate from the lumen to relocate to the basement membrane and, amazingly, they can retain the capability to produce donor sperm in their new host. Adaptation of the mouse technique for livestock is progressing, with gradual gains in efficiency. Germ cell transfer in goats has produced offspring, but not yet in cattle and pigs. In goats and pigs, the applications of germ cell transplantation are mainly in facilitating transgenic animal production. In cattle, successful male germ cell transfer could create an alternative to AI in areas where it is impractical. Large-scale culture of testis stem cells would enhance the use of elite bulls by providing a renewable source of stem cells for transfer. Although still in a developmental state, germ cell transplantation is an emerging technology with the potential to create new opportunities in livestock production. PMID:16478598

  18. Comparison of methods for detecting mitomycin C- and ethyl nitrosourea-induced germ cell damage in mice: sperm enzyme activities, sperm motility, and testis weight

    SciTech Connect

    Ficsor, G.; Oldford, G.M.; Loughlin, K.R.; Panda, B.B.; Dubien, J.L.; Ginsberg, L.C.

    1984-01-01

    Testes weights, sperm motility and enzyme activities in single sperm were compared with respect to their ability to detect either developmental or mutational damage to germ cells. Male mice were injected i.p. with 2.5 mg/kg mitomycin C (MC) or 50 or 100 mg/kg ethylnitrosourea (ENU) or saline and were then killed at times such that sperm derived from treated vas sperm (SZ), spermatids (ST), preleptotene-late-spermatogonial cells (PLSG), spermatogonial cells (SG), or spermatogonial stem cells (SGS) could be evaluated. The authors conclude that testis weight, which is easily obtained, is a sensitive indicator of germ cell damage by these agents. Sperm from each animal were evaluated for sperm motility, acrosin activity, succinic dehydrogenase (SDH) activity with or without the competitive inhibitor malonate or after exposure to 60/sup 8/C for 10 min. The latter two assays were to detect sperm enzymes resistant to the inhibitor or heat. The presence of the acrosin protein was also detected immunologically. Of the sperm assays, acrosin activity proved to be the most sensitive indicator of germ cell damage and was the simplest to measure.

  19. Germ Cells Need Folate to Proliferate.

    PubMed

    Walker, Amy K

    2016-07-11

    In this issue of Developmental Cell, Chaudhari and colleagues (2016) use a novel method to create an in vitro proliferative cell line from tumorous C. elegans germ cells, and in the process discover that bacterial folates act as signals for proliferation, independent of their roles as vitamins. PMID:27404353

  20. Paediatric extracranial germ-cell tumours.

    PubMed

    Shaikh, Furqan; Murray, Matthew J; Amatruda, James F; Coleman, Nicholas; Nicholson, James C; Hale, Juliet P; Pashankar, Farzana; Stoneham, Sara J; Poynter, Jenny N; Olson, Thomas A; Billmire, Deborah F; Stark, Daniel; Rodriguez-Galindo, Carlos; Frazier, A Lindsay

    2016-04-01

    Management of paediatric extracranial germ-cell tumours carries a unique set of challenges. Germ-cell tumours are a heterogeneous group of neoplasms that present across a wide age range and vary in site, histology, and clinical behaviour. Patients with germ-cell tumours are managed by a diverse array of specialists. Thus, staging, risk stratification, and treatment approaches for germ-cell tumours have evolved disparately along several trajectories. Paediatric germ-cell tumours differ from the adolescent and adult disease in many ways, leading to complexities in applying age-appropriate, evidence-based care. Suboptimal outcomes remain for several groups of patients, including adolescents, and patients with extragonadal tumours, high tumour markers at diagnosis, or platinum-resistant disease. Survivors have significant long-term toxicities. The challenge moving forward will be to translate new insights from molecular studies and collaborative clinical data into improved patient outcomes. Future trials will be characterised by improved risk-stratification systems, biomarkers for response and toxic effects, rational reduction of therapy for low-risk patients and novel approaches for poor-risk patients, and improved international collaboration across paediatric and adult cooperative research groups. PMID:27300675

  1. Genomic Landscape of Developing Male Germ Cells

    PubMed Central

    Lee, Tin-Lap; Pang, Alan Lap-Yin; Rennert, Owen M.; Chan, Wai-Yee

    2010-01-01

    Spermatogenesis is a highly orchestrated developmental process by which spermatogonia develop into mature spermatozoa. This process involves many testis- or male germ cell-specific gene products whose expressions are strictly regulated. In the past decade the advent of high-throughput gene expression analytical techniques has made functional genomic studies of this process, particularly in model animals such as mice and rats, feasible and practical. These studies have just begun to reveal the complexity of the genomic landscape of the developing male germ cells. Over 50% of the mouse and rat genome are expressed during testicular development. Among transcripts present in germ cells, 40% – 60% are uncharacterized. A number of genes, and consequently their associated biological pathways, are differentially expressed at different stages of spermatogenesis. Developing male germ cells present a rich repertoire of genetic processes. Tissue-specific as well as spermatogenesis stage-specific alternative splicing of genes exemplifies the complexity of genome expression. In addition to this layer of control, discoveries of abundant presence of antisense transcripts, expressed psuedogenes, non-coding RNAs (ncRNA) including long ncRNAs, microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), and retrogenes all point to the presence of multiple layers of expression and functional regulation in male germ cells. It is anticipated that application of systems biology approaches will further our understanding of the regulatory mechanism of spermatogenesis.† PMID:19306351

  2. Surgery and Combination Chemotherapy in Treating Children With Extracranial Germ Cell Tumors

    ClinicalTrials.gov

    2016-05-06

    Childhood Embryonal Tumor; Childhood Extracranial Germ Cell Tumor; Childhood Extragonadal Germ Cell Tumor; Childhood Malignant Ovarian Germ Cell Tumor; Childhood Malignant Testicular Germ Cell Tumor; Childhood Teratoma; Ovarian Embryonal Carcinoma; Ovarian Yolk Sac Tumor; Stage II Malignant Testicular Germ Cell Tumor; Stage IIA Ovarian Germ Cell Tumor; Stage IIB Ovarian Germ Cell Tumor; Stage IIC Ovarian Germ Cell Tumor; Stage III Malignant Testicular Germ Cell Tumor; Stage IIIA Ovarian Germ Cell Tumor; Stage IIIB Ovarian Germ Cell Tumor; Stage IIIC Ovarian Germ Cell Tumor; Testicular Choriocarcinoma and Yolk Sac Tumor; Testicular Embryonal Carcinoma

  3. Use of DEAD-box polypeptide-4 (Ddx4) gene promoter-driven fluorescent reporter mice to identify mitotically active germ cells in post-natal mouse ovaries.

    PubMed

    Park, Eun-Sil; Tilly, Jonathan L

    2015-01-01

    Several laboratories have independently isolated mitotically active germ cells, termed female germline stem cells or oogonial stem cells (OSCs), from adult mouse ovaries. However, a recent study using Ddx4-Cre;Rosa26 reporter mice concluded that such germ cells do not exist. Given the disparity in conclusions drawn in this study compared with others, we felt it was important to re-assess the utility of Ddx4-Cre;Rosa26 reporter mice for identification of OSCs in adult mouse ovaries. Transgenic Ddx4-Cre mice were crossed with Rosa26(tdTm/tdTm) mice to drive restricted tomato red (tdTm) gene expression in cells in which the Ddx4 gene promoter has been activated. Crude dispersion of ovaries from recombined offspring generated cell fractions containing tdTm-positive immature oocytes, which are incapable of proliferation and thus probably represent the uncharacterized reporter-positive ovarian cells identified in the paper Zhang et al. (2012) as being mitotically inactive. Dispersed ovaries further subjected to fluorescence-activated cell sorting yielded a large population of non-germline tdTm-positive cells, indicative of promoter 'leakiness' in the Ddx4-Cre mouse line. Nonetheless, a small percentage of these tdTm-positive cells exhibited externalized (extracellular, ec) expression of Ddx4 protein (ecDdx4-positive), expressed markers of primitive germ cells but not of oocytes, and actively proliferated in culture, all of which are characteristic features of OSCs. Thus, crude dispersion of ovaries collected from Ddx4 gene promoter-driven reporter mice is not, by itself, a reliable approach to identify OSCs, whereas the same ovarian dispersates further subjected to cell sorting strategies yield purified OSCs that can be expanded in culture.

  4. Use of DEAD-box polypeptide-4 (Ddx4) gene promoter-driven fluorescent reporter mice to identify mitotically active germ cells in post-natal mouse ovaries.

    PubMed

    Park, Eun-Sil; Tilly, Jonathan L

    2015-01-01

    Several laboratories have independently isolated mitotically active germ cells, termed female germline stem cells or oogonial stem cells (OSCs), from adult mouse ovaries. However, a recent study using Ddx4-Cre;Rosa26 reporter mice concluded that such germ cells do not exist. Given the disparity in conclusions drawn in this study compared with others, we felt it was important to re-assess the utility of Ddx4-Cre;Rosa26 reporter mice for identification of OSCs in adult mouse ovaries. Transgenic Ddx4-Cre mice were crossed with Rosa26(tdTm/tdTm) mice to drive restricted tomato red (tdTm) gene expression in cells in which the Ddx4 gene promoter has been activated. Crude dispersion of ovaries from recombined offspring generated cell fractions containing tdTm-positive immature oocytes, which are incapable of proliferation and thus probably represent the uncharacterized reporter-positive ovarian cells identified in the paper Zhang et al. (2012) as being mitotically inactive. Dispersed ovaries further subjected to fluorescence-activated cell sorting yielded a large population of non-germline tdTm-positive cells, indicative of promoter 'leakiness' in the Ddx4-Cre mouse line. Nonetheless, a small percentage of these tdTm-positive cells exhibited externalized (extracellular, ec) expression of Ddx4 protein (ecDdx4-positive), expressed markers of primitive germ cells but not of oocytes, and actively proliferated in culture, all of which are characteristic features of OSCs. Thus, crude dispersion of ovaries collected from Ddx4 gene promoter-driven reporter mice is not, by itself, a reliable approach to identify OSCs, whereas the same ovarian dispersates further subjected to cell sorting strategies yield purified OSCs that can be expanded in culture. PMID:25147160

  5. Germ Smart: Children's Activities in Disease Prevention.

    ERIC Educational Resources Information Center

    Scheer, Judith K.

    This booklet is part of the "Children's Activity Series," a set of four supplemental teaching resources that promote awareness about health, family life, and cultural diversity for children in kindergarten through third grade. Nine activities are included in this booklet to help children be "germ smart" help children in kindergarten through third…

  6. The treatment of cranial germ cell tumours.

    PubMed

    Brandes, A A; Pasetto, L M; Monfardini, S

    2000-08-01

    Germ cell tumours of the central nervous system (CNS) include many subtypes whose response to treatment varies, even though the symptoms and radiological appearances are similar. Five-year survival rates are 96% for germinomas, 100% for mature teratomas, 67% for immature teratomas and 69% for immature teratomas mixed with germinomas; for beta-HCG secreting germinomas the rate is only 38%. Patients with choriocarcinoma, embryonal carcinoma, or yolk sac tumour have the lowest survival rates; patients with germinoma or mature teratoma have longer survival rates. Although a wider resection is associated with a higher rate of survival for patients with non-germinomatous germ cell (NGGC) tumours, to date an aggressive surgical approach has been advocated only for pineal region tumours, but not for hypothalamic/neurohypophyseal tumours. Beside the delayed injury induced by radiotherapy, the late injury induced by chemotherapy is becoming increasingly evident. Cisplatin is considered an indispensable drug, but it may cause renal damage, ototoxicity, peripheral neuropathy and sterility, while etoposide is associated with an excess frequency of second neoplasms. Taking into account all of the published literature, the following therapeutic options are suggested: in pure germinoma tumours (GT) radiotherapy alone will usually ensure adequate control of the disease, and the long-term sequelae may be limited by reducing the dose delivered, as was proposed for germ cell testicular tumours, to 30 Gy to limited fields plus 25-30 Gy to the spinal axis if there is disseminated disease. In cases of recurrence, which should be uncommon, patients may be rescued with both radiotherapy and chemotherapy. In NGGC tumours, the prognosis is more unfavourable and there is often dissemination to the spine at diagnosis; however, the tumour's high chemosensitivity suggests neoadjuvant treatment chemotherapy with cisplatin and etoposide for three cycles followed by consolidation radiotherapy with

  7. General Information about Childhood Extracranial Germ Cell Tumors

    MedlinePlus

    ... Germ Cell Tumors Treatment (PDQ®)–Patient Version General Information About Childhood Extracranial Germ Cell Tumors Go to ... the PDQ Pediatric Treatment Editorial Board . Clinical Trial Information A clinical trial is a study to answer ...

  8. piRNA pathway targets active LINE1 elements to establish the repressive H3K9me3 mark in germ cells.

    PubMed

    Pezic, Dubravka; Manakov, Sergei A; Sachidanandam, Ravi; Aravin, Alexei A

    2014-07-01

    Transposable elements (TEs) occupy a large fraction of metazoan genomes and pose a constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small noncoding piwi-interacting RNAs (piRNAs) recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments.

  9. Human GV oocytes generated by mitotically active germ cells obtained from follicular aspirates

    PubMed Central

    Ding, Xinbao; Liu, Guishu; Xu, Bo; Wu, Changqing; Hui, Ning; Ni, Xin; Wang, Jian; Du, Meirong; Teng, Xiaoming; Wu, Ji

    2016-01-01

    Human female germline stem cells (FGSCs) have opened new opportunities for understanding human oogenesis, delaying menopause, treating infertility, and providing a new strategy for preserving fertility. However, the shortage of adult human ovaries tissues available impedes their future investigations and clinical applications. Here, we have established FGSC lines from scarce ovarian cortical tissues that exist in follicular aspirates (faFGSCs), which are produced and discarded in in vitro fertilization centers worldwide. The faFGSCs have characteristics of germline stem cells involved in the gene expression profile, growth characteristics, and a normal karyotype consistent with that of FGSCs obtained from ovarian cortexes surgically removed from patients (srFGSCs). Furthermore, faFGSCs have developmental potentials including spontaneous differentiation into oocytes under feeder-free conditions, communicating with granulosa cells by gap junctions and paracrine factors, entering meiosis after RA induction, as well as forming follicles after injection into human ovarian cortical tissues xenografted into adult immunodeficient female mice. Lastly, we developed a strategy guiding FGSCs differentiated into germinal vesicle (GV) stage oocytes in vitro and revealed their developmental mechanisms. Our study not only provides a new approach to obtain human FGSCs for medical treatment, but also opens several avenues to investigate human oogenesis in vitro. PMID:27357640

  10. The association between institution at orchiectomy and outcomes on active surveillance for clinical stage I germ cell tumours

    PubMed Central

    Nayan, Madhur; Jewett, Michael A.S.; Anson-Cartwright, Lynn; Bedard, Philippe L.; Moore, Malcolm; Chung, Peter; Warde, Padraig; Sweet, Joan; O’Malley, Martin; Hamilton, Robert J.

    2016-01-01

    Introduction: Institutional experience has been associated with improved outcomes for various malignancies, including testicular cancer. The present study evaluated whether institution at orchiectomy was associated with outcomes on active surveillance (AS) for clinical stage (CS) I germ cell tumours (GCT). Methods: 815 patients with CSI GCT managed with AS at the Princess Margaret Cancer Centre were identified. Princess Margaret is a tertiary academic institution with a multidisciplinary testicular cancer clinic involving radiation oncologists, medical oncologists, and urologists, and has research experience in testicular cancer care. The association between institution of orchiectomy (Princess Margaret vs. Other) and time to progression on AS was analyzed using multivariable Cox proportional hazards models. Academic vs. non-academic institutions were compared in a sensitivity analysis. Results: Patients undergoing orchiectomy at Princess Margaret for non-seminoma GCT were significantly less likely to have pure embryonal carcinoma (EC) in orchiectomy pathology (odds ratio [OR] 0.33; p=0.008) and CSIB disease (OR 0.47; p=0.014). Seminoma characteristics did not differ significantly between institution groups. In non-seminoma GCT, median followup was 5.4 years, 27% progressed on AS, and institution of orchiectomy was not associated with time to progression in either univariate (hazard ratio [HR] 0.79; p=0.33) or multivariable analyses (HR 1.01; p=0.97). In seminoma, median followup was 4.7 years, 12% progressed on AS, and institution of orchiectomy was not associated with progression (univariate: HR 0.87; p=0.73; multivariable: HR 0.98; p=0.96). Sensitivity analyses demonstrated similar results. Conclusions: Among CSI GCT patients managed on AS at a specialized cancer centre, there appears to be no difference in oncologic outcomes based upon the institution where orchiectomy was performed.

  11. Tre1, a G Protein-Coupled Receptor, Directs Transepithelial Migration of Drosophila Germ Cells

    PubMed Central

    2003-01-01

    In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target. PMID:14691551

  12. Approaches for identifying germ cell mutagens: Report of the 2013 IWGT workshop on germ cell assays(☆).

    PubMed

    Yauk, Carole L; Aardema, Marilyn J; Benthem, Jan van; Bishop, Jack B; Dearfield, Kerry L; DeMarini, David M; Dubrova, Yuri E; Honma, Masamitsu; Lupski, James R; Marchetti, Francesco; Meistrich, Marvin L; Pacchierotti, Francesca; Stewart, Jane; Waters, Michael D; Douglas, George R

    2015-05-01

    This workshop reviewed the current science to inform and recommend the best evidence-based approaches on the use of germ cell genotoxicity tests. The workshop questions and key outcomes were as follows. (1) Do genotoxicity and mutagenicity assays in somatic cells predict germ cell effects? Limited data suggest that somatic cell tests detect most germ cell mutagens, but there are strong concerns that dictate caution in drawing conclusions. (2) Should germ cell tests be done, and when? If there is evidence that a chemical or its metabolite(s) will not reach target germ cells or gonadal tissue, it is not necessary to conduct germ cell tests, notwithstanding somatic outcomes. However, it was recommended that negative somatic cell mutagens with clear evidence for gonadal exposure and evidence of toxicity in germ cells could be considered for germ cell mutagenicity testing. For somatic mutagens that are known to reach the gonadal compartments and expose germ cells, the chemical could be assumed to be a germ cell mutagen without further testing. Nevertheless, germ cell mutagenicity testing would be needed for quantitative risk assessment. (3) What new assays should be implemented and how? There is an immediate need for research on the application of whole genome sequencing in heritable mutation analysis in humans and animals, and integration of germ cell assays with somatic cell genotoxicity tests. Focus should be on environmental exposures that can cause de novo mutations, particularly newly recognized types of genomic changes. Mutational events, which may occur by exposure of germ cells during embryonic development, should also be investigated. Finally, where there are indications of germ cell toxicity in repeat dose or reproductive toxicology tests, consideration should be given to leveraging those studies to inform of possible germ cell genotoxicity.

  13. H3K36 Trimethylation-Mediated Epigenetic Regulation is Activated by Bam and Promotes Germ Cell Differentiation During Early Oogenesis in Drosophila.

    PubMed

    Mukai, Masanori; Hira, Seiji; Nakamura, Katsuhiro; Nakamura, Shoichi; Kimura, Hiroshi; Sato, Masanao; Kobayashi, Satoru

    2015-01-01

    Epigenetic silencing is critical for maintaining germline stem cells in Drosophila ovaries. However, it remains unclear how the differentiation factor, Bag-of-marbles (Bam), counteracts transcriptional silencing. We found that the trimethylation of lysine 36 on histone H3 (H3K36me3), a modification that is associated with gene activation, is enhanced in Bam-expressing cells. H3K36me3 levels were reduced in flies deficient in Bam. Inactivation of the Set2 methyltransferase, which confers the H3K36me3 modification, in germline cells markedly reduced H3K36me3 and impaired differentiation. Genetic analyses revealed that Set2 acts downstream of Bam. Furthermore, orb expression, which is required for germ cell differentiation, was activated by Set2, probably through direct H3K36me3 modification of the orb locus. Our data indicate that H3K36me3-mediated epigenetic regulation is activated by bam, and that this modification facilitates germ cell differentiation, probably through transcriptional activation. This work provides a novel link between Bam and epigenetic transcriptional control. PMID:25572421

  14. Zebrafish germ cells: motility and guided migration.

    PubMed

    Paksa, Azadeh; Raz, Erez

    2015-10-01

    In the course of embryonic development, the process of cell migration is critical for establishment of the embryonic body plan, for morphogenesis and for organ function. Investigating the molecular mechanisms underlying cell migration is thus crucial for understanding developmental processes and clinical conditions resulting from abnormal cell migration such as cancer metastasis. The long-range migration of primordial germ cells toward the region at which the gonad develops occurs in embryos of various species and thus constitutes a useful in vivo model for single-cell migration. Recent studies employing zebrafish embryos have greatly contributed to the understanding of the mechanisms facilitating the migration of these cells en route to their target.

  15. Standard-Dose Combination Chemotherapy or High-Dose Combination Chemotherapy and Stem Cell Transplant in Treating Patients With Relapsed or Refractory Germ Cell Tumors

    ClinicalTrials.gov

    2016-07-26

    Germ Cell Tumor; Teratoma; Choriocarcinoma; Germinoma; Mixed Germ Cell Tumor; Yolk Sac Tumor; Childhood Teratoma; Malignant Germ Cell Neoplasm; Extragonadal Seminoma; Non-seminomatous Germ Cell Tumor; Seminoma

  16. Characterization of ribonuclease H activities present in two cell-free protein synthesizing systems, the wheat germ extract and the rabbit reticulocyte lysate.

    PubMed

    Cazenave, C; Frank, P; Büsen, W

    1993-01-01

    Experimental evidence accumulated to date by several research groups indicates that antisense oligodeoxynucleotides targeted against messenger RNA (mRNA) sequences located downstream of the initiation codon fail to inhibit the translation of this mRNA unless the hybrid is cleaved by RNase H. It has previously been shown that exogenous RNase H has to be added to rabbit reticulocyte lysate to obtain translational arrest (unless freshly prepared lysates are used). In contrast there is no need of exogenous RNase H by using wheat germ extract for translation because the level of endogenous RNase H is high enough to ensure cleavage of the hybrid formed between the antisense oligodeoxyribonucleotide and its complementary sequence on the mRNA. Surprisingly, we found that these two cell-free translation systems display similar amounts of RNase H activities when tested under standard conditions (extract diluted 500 times in the RNase H reaction mix). The RNase H activity of the rabbit reticulocyte lysate has a divalent cation requirement and sensitivity to inhibitors similar to class I ribonuclease H, whereas the activity of the wheat germ extract shows similarities to class II ribonuclease H. However, when these activities were assayed under conditions similar to those used for translation experiments, only highly reduced levels of activity were found in comparison to the standard assays. This reduction is due in part to sub-optimal ionic conditions for the endogenous RNase H activities in these extracts, and, for the other part, likely due to interactions with other proteins present in the lysates. In these conditions, however, the remaining activity found in the wheat germ extract was three times higher than the activity found in the rabbit reticulocyte lysate. Whether this difference can by itself explain the indicated differences in the two systems observed in hybrid-arrest of translation experiments remains open to discussion.

  17. Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon

    PubMed Central

    Wargelius, Anna; Leininger, Sven; Skaftnesmo, Kai Ove; Kleppe, Lene; Andersson, Eva; Taranger, Geir Lasse; Schulz, Rüdiger W; Edvardsen, Rolf B

    2016-01-01

    Introgression of farmed salmon escapees into wild stocks is a major threat to the genetic integrity of wild populations. Using germ cell-free fish in aquaculture may mitigate this problem. Our study investigated whether it is possible to produce germ cell-free salmon in F0 by using CRISPR-Cas9 to knock out dnd, a factor required for germ cell survival in vertebrates. To avoid studying mosaic animals, sgRNA targeting alb was simultaneously used as a visual tracer since the phenotype of alb KO is complete loss of pigmentation. Induced mutations for the tracer (alb) and the target (dnd) genes were highly correlated and produced germ cell-less fish lacking pigmentation, underlining the suitability of alb KO to serve as tracer for targeted double allelic mutations in F0 animals in species with prohibitively long generation times. This is also the first report describing dnd knockout in any fish species. Analyzing gene expression and histology of dnd KO fish revealed that sex differentiation of the somatic compartment does not depend on the presence of germ cells. However, the organization of the ovarian somatic compartment seems compromised in mutant fish. PMID:26888627

  18. A zebrafish homologue of the chemokine receptor Cxcr4 is a germ-cell guidance receptor

    NASA Astrophysics Data System (ADS)

    Knaut, Holger; Werz, Christian; Geisler, Robert; Tübingen 2000 Screen Consortium; Nüsslein-Volhard, Christiane

    2003-01-01

    Germ cells preserve an individual's genetic information and transmit it to the next generation. Early in development germ cells are set aside and undergo a specialized developmental programme, a hallmark of which is the migration from their site of origin to the future gonad. In Drosophila, several factors have been identified that control germ-cell migration to their target tissues; however, the germ-cell chemoattractant or its receptor have remained unknown. Here we apply genetics and in vivo imaging to show that odysseus, a zebrafish homologue of the G-protein-coupled chemokine receptor Cxcr4, is required specifically in germ cells for their chemotaxis. odysseus mutant germ cells are able to activate the migratory programme, but fail to undergo directed migration towards their target tissue, resulting in randomly dispersed germ cells. SDF-1, the presumptive cognate ligand for Cxcr4, shows a similar loss-of-function phenotype and can recruit germ cells to ectopic sites in the embryo, thus identifying a vertebrate ligand-receptor pair guiding migratory germ cells at all stages of migration towards their target.

  19. Sex Specification and Heterogeneity of Primordial Germ Cells in Mice

    PubMed Central

    Sakashita, Akihiko; Kawabata, Yukiko; Jincho, Yuko; Tajima, Shiun; Kumamoto, Soichiro; Kobayashi, Hisato; Matsui, Yasuhisa; Kono, Tomohiro

    2015-01-01

    In mice, primordial germ cells migrate into the genital ridges by embryonic day 13.5 (E13.5), where they are then subjected to a sex-specific fate with female and male primordial germ cells undergoing mitotic arrest and meiosis, respectively. However, the sex-specific basis of primordial germ cell differentiation is poorly understood. The aim of this study was to investigate the sex-specific features of mouse primordial germ cells. We performed RNA-sequencing (seq) of E13.5 female and male mouse primordial germ cells using next-generation sequencing. We identified 651 and 428 differentially expressed transcripts (>2-fold, P < 0.05) in female and male primordial germ cells, respectively. Of these, many transcription factors were identified. Gene ontology and network analysis revealed differing functions of the identified female- and male-specific genes that were associated with primordial germ cell acquisition of sex-specific properties required for differentiation into germ cells. Furthermore, DNA methylation and ChIP-seq analysis of histone modifications showed that hypomethylated gene promoter regions were bound with H3K4me3 and H3K27me3. Our global transcriptome data showed that in mice, primordial germ cells are decisively assigned to a sex-specific differentiation program by E13.5, which is necessary for the development of vital germ cells. PMID:26700643

  20. Combination Chemotherapy in Treating Young Patients With Recurrent or Resistant Malignant Germ Cell Tumors

    ClinicalTrials.gov

    2016-04-12

    Childhood Extracranial Germ Cell Tumor; Childhood Extragonadal Germ Cell Tumor; Childhood Malignant Ovarian Germ Cell Tumor; Childhood Malignant Testicular Germ Cell Tumor; Ovarian Choriocarcinoma; Ovarian Embryonal Carcinoma; Ovarian Yolk Sac Tumor; Recurrent Childhood Malignant Germ Cell Tumor; Recurrent Malignant Testicular Germ Cell Tumor; Recurrent Ovarian Germ Cell Tumor; Testicular Choriocarcinoma; Testicular Choriocarcinoma and Embryonal Carcinoma; Testicular Choriocarcinoma and Yolk Sac Tumor; Testicular Embryonal Carcinoma; Testicular Embryonal Carcinoma and Yolk Sac Tumor; Testicular Yolk Sac Tumor

  1. The proteasome inhibitor bortezomib induces testicular toxicity by upregulation of oxidative stress, AMP-activated protein kinase (AMPK) activation and deregulation of germ cell development in adult murine testis

    SciTech Connect

    Li, Wei; Fu, Jianfang; Zhang, Shun; Zhao, Jie; Xie, Nianlin; Cai, Guoqing

    2015-06-01

    Understanding how chemotherapeutic agents mediate testicular toxicity is crucial in light of compelling evidence that male infertility, one of the severe late side effects of intensive cancer treatment, occurs more often than they are expected to. Previous study demonstrated that bortezomib (BTZ), a 26S proteasome inhibitor used to treat refractory multiple myeloma (MM), exerts deleterious impacts on spermatogenesis in pubertal mice via unknown mechanisms. Here, we showed that intermittent treatment with BTZ resulted in fertility impairment in adult mice, evidenced by testicular atrophy, desquamation of immature germ cells and reduced caudal sperm storage. These deleterious effects may originate from the elevated apoptosis in distinct germ cells during the acute phase and the subsequent disruption of Sertoli–germ cell anchoring junctions (AJs) during the late recovery. Mechanistically, balance between AMP-activated protein kinase (AMPK) activation and Akt/ERK pathway appeared to be indispensable for AJ integrity during the late testicular recovery. Of particular interest, the upregulated testicular apoptosis and the following disturbance of Sertoli–germ cell interaction may both stem from the excessive oxidative stress elicited by BTZ exposure. We also provided the in vitro evidence that AMPK-dependent mechanisms counteract follicle-stimulating hormone (FSH) proliferative effects in BTZ-exposed Sertoli cells. Collectively, BTZ appeared to efficiently prevent germ cells from normal development via multiple mechanisms in adult mice. Employment of antioxidants and/or AMPK inhibitor may represent an attractive strategy of fertility preservation in male MM patients exposed to conventional BTZ therapy and warrants further investigation. - Highlights: • Intermittent treatment with BTZ caused fertility impairment in adult mice. • BTZ treatment elicited apoptosis during early phase of testicular recovery. • Up-regulation of oxidative stress by BTZ treatment

  2. Pathology of testicular germ cell tumors.

    PubMed

    Brodsky, G L

    1991-12-01

    The pathology report on a testicular germ cell tumor should include the following information: Tumor type: The histologic type of tumor present. If the tumor is of mixed type, the components should be listed, in order of relative abundance. The pathologist may endeavor to give a numeric estimate of the percentages of each element. Staging information: The size of the tumor should be listed. Local spread--into rete testis, tunica albuginea, epididymis, and spermatic cord--should be listed. If the cord is involved, possible involvement of its surgical resection margin should be assessed. Vascular/lymphatic invasion should be assessed for its presence or absence. Status of the remainder of the testis: Evidence of cryptorchidism or other dysgenetic features should be mentioned. Such features may imply a greater risk for the development of a contralateral tumor. Also, the presence of normal spermatogenesis elsewhere in the uninvolved testis should be reported. This finding may suggest a relatively decreased risk for contralateral tumor development and is a likely indicator of fertility should the patient consider sperm banking prior to retroperitoneal surgery and chemotherapy. The finding of mature sperm in the epididymis is an easy way to confirm spermatogenesis in the testis. Incidental findings: Lipomas or hydroceles of the cord, adrenal rests, and adnexal cysts may be found. The pathologist plays a crucial role in the diagnosis of germ cell tumors. In addition to elucidating tumor type, the pathologist is relied upon for precise local staging and for the classification of metastases, all of which have important implications in determining optimal therapy. As the clinical management of germ cell tumors evolves, the pathologist will continue to play a role in defining those features that have a bearing on patient outcome.

  3. Primordial Germ Cells: Current Knowledge and Perspectives

    PubMed Central

    Nikolic, Aleksandar; Volarevic, Vladislav; Armstrong, Lyle; Lako, Majlinda; Stojkovic, Miodrag

    2016-01-01

    Infertility is a condition that occurs very frequently and understanding what defines normal fertility is crucial to helping patients. Causes of infertility are numerous and the treatment often does not lead to desired pregnancy especially when there is a lack of functional gametes. In humans, the primordial germ cell (PGC) is the primary undifferentiated stem cell type that will differentiate towards gametes: spermatozoa or oocytes. With the development of stem cell biology and differentiation protocols, PGC can be obtained from pluripotent stem cells providing a new therapeutic possibility to treat infertile couples. Recent studies demonstrated that viable mouse pups could be obtained from in vitro differentiated stem cells suggesting that translation of these results to human is closer. Therefore, the aim of this review is to summarize current knowledge about PGC indicating the perspective of their use in both research and medical application for the treatment of infertility. PMID:26635880

  4. Late Relapse of Testicular Germ Cell Tumors.

    PubMed

    O'Shaughnessy, Matthew J; Feldman, Darren R; Carver, Brett S; Sheinfeld, Joel

    2015-08-01

    Germ cell tumors of the testis have an overall survival rate greater than 90% as a result of a successful multidisciplinary approach to management. Late relapse affects a subset of patients however, and tends to be chemorefractory and the overall prognosis is poor. Surgery is the mainstay in management of late relapse but salvage chemotherapy can be successful. In this review, the clinical presentation and detection of late relapse, clinical outcomes, and predictors of survival in late relapse and the importance of a multidisciplinary treatment approach for successful management of late relapse are discussed. PMID:26216823

  5. The proteasome inhibitor bortezomib induces testicular toxicity by upregulation of oxidative stress, AMP-activated protein kinase (AMPK) activation and deregulation of germ cell development in adult murine testis.

    PubMed

    Li, Wei; Fu, Jianfang; Zhang, Shun; Zhao, Jie; Xie, Nianlin; Cai, Guoqing

    2015-06-01

    Understanding how chemotherapeutic agents mediate testicular toxicity is crucial in light of compelling evidence that male infertility, one of the severe late side effects of intensive cancer treatment, occurs more often than they are expected to. Previous study demonstrated that bortezomib (BTZ), a 26S proteasome inhibitor used to treat refractory multiple myeloma (MM), exerts deleterious impacts on spermatogenesis in pubertal mice via unknown mechanisms. Here, we showed that intermittent treatment with BTZ resulted in fertility impairment in adult mice, evidenced by testicular atrophy, desquamation of immature germ cells and reduced caudal sperm storage. These deleterious effects may originate from the elevated apoptosis in distinct germ cells during the acute phase and the subsequent disruption of Sertoli-germ cell anchoring junctions (AJs) during the late recovery. Mechanistically, balance between AMP-activated protein kinase (AMPK) activation and Akt/ERK pathway appeared to be indispensable for AJ integrity during the late testicular recovery. Of particular interest, the upregulated testicular apoptosis and the following disturbance of Sertoli-germ cell interaction may both stem from the excessive oxidative stress elicited by BTZ exposure. We also provided the in vitro evidence that AMPK-dependent mechanisms counteract follicle-stimulating hormone (FSH) proliferative effects in BTZ-exposed Sertoli cells. Collectively, BTZ appeared to efficiently prevent germ cells from normal development via multiple mechanisms in adult mice. Employment of antioxidants and/or AMPK inhibitor may represent an attractive strategy of fertility preservation in male MM patients exposed to conventional BTZ therapy and warrants further investigation. PMID:25886977

  6. Germ line development: lessons learned from pluripotent stem cells.

    PubMed

    Martínez-Arroyo, Ana M; Medrano, Jose V; Remohí, José; Simón, Carlos

    2014-10-01

    Current knowledge about mammalian germ line development is mainly based on the mouse model and little is known about how this fundamental process occurs in humans. This review summarizes our current knowledge of genetic and epigenetic germ line development in mammals, mainly focusing on primordial germ cell (PGC) specification events, comparing the differences between mouse and human models. We also emphasize the knowledge derived from the most successful strategies used to generate germ cell-like cells in vitro in both models and major obstacles to obtaining bona fide in vitro-derived gametes are considered. PMID:25461452

  7. Environmentally induced transgenerational epigenetic reprogramming of primordial germ cells and the subsequent germ line.

    PubMed

    Skinner, Michael K; Guerrero-Bosagna, Carlos; Haque, M; Nilsson, Eric; Bhandari, Ramji; McCarrey, John R

    2013-01-01

    A number of environmental factors (e.g. toxicants) have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. Transgenerational inheritance requires the germline transmission of altered epigenetic information between generations in the absence of direct environmental exposures. The primary periods for epigenetic programming of the germ line are those associated with primordial germ cell development and subsequent fetal germline development. The current study examined the actions of an agricultural fungicide vinclozolin on gestating female (F0 generation) progeny in regards to the primordial germ cell (PGC) epigenetic reprogramming of the F3 generation (i.e. great-grandchildren). The F3 generation germline transcriptome and epigenome (DNA methylation) were altered transgenerationally. Interestingly, disruptions in DNA methylation patterns and altered transcriptomes were distinct between germ cells at the onset of gonadal sex determination at embryonic day 13 (E13) and after cord formation in the testis at embryonic day 16 (E16). A larger number of DNA methylation abnormalities (epimutations) and transcriptional alterations were observed in the E13 germ cells than in the E16 germ cells. These observations indicate that altered transgenerational epigenetic reprogramming and function of the male germline is a component of vinclozolin induced epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance are provided.

  8. Environmentally Induced Transgenerational Epigenetic Reprogramming of Primordial Germ Cells and the Subsequent Germ Line

    PubMed Central

    Skinner, Michael K.; Haque, Carlos Guerrero-Bosagna M.; Nilsson, Eric; Bhandari, Ramji; McCarrey, John R.

    2013-01-01

    A number of environmental factors (e.g. toxicants) have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. Transgenerational inheritance requires the germline transmission of altered epigenetic information between generations in the absence of direct environmental exposures. The primary periods for epigenetic programming of the germ line are those associated with primordial germ cell development and subsequent fetal germline development. The current study examined the actions of an agricultural fungicide vinclozolin on gestating female (F0 generation) progeny in regards to the primordial germ cell (PGC) epigenetic reprogramming of the F3 generation (i.e. great-grandchildren). The F3 generation germline transcriptome and epigenome (DNA methylation) were altered transgenerationally. Interestingly, disruptions in DNA methylation patterns and altered transcriptomes were distinct between germ cells at the onset of gonadal sex determination at embryonic day 13 (E13) and after cord formation in the testis at embryonic day 16 (E16). A larger number of DNA methylation abnormalities (epimutations) and transcriptional alterations were observed in the E13 germ cells than in the E16 germ cells. These observations indicate that altered transgenerational epigenetic reprogramming and function of the male germline is a component of vinclozolin induced epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance are provided. PMID:23869203

  9. Induction of Germ Cell-like Cells from Porcine Induced Pluripotent Stem Cells

    PubMed Central

    Wang, Hanning; Xiang, Jinzhu; Zhang, Wei; Li, Junhong; Wei, Qingqing; Zhong, Liang; Ouyang, Hongsheng; Han, Jianyong

    2016-01-01

    The ability to generate germ cells from pluripotent stem cells (PSCs) is valuable for human regenerative medicine and animal breeding. Germ cell-like cells (GCLCs) have been differentiated from mouse and human PSCs, but not from porcine PSCs, which are considered an ideal model for stem cell applications. Here, we developed a defined culture system for the induction of primordial germ cell-like cells (PGCLCs) from porcine induced PSCs (piPSCs). The identity of the PGCLCs was characterized by observing cell morphology, detecting germ cell marker gene expression and evaluating epigenetic properties. PGCLCs could further differentiate into spermatogonial stem cell-like cells (SSCLCs) in vitro. Importantly, meiosis occurred during SSCLC induction. Xenotransplantation of GCLCs into seminiferous tubules of infertile immunodeficient mice resulted in immunohistochemically identifiable germ cells in vivo. Overall, our study provides a feasible strategy for directing piPSCs to the germ cell fate and lays a foundation for exploring germ cell development mechanisms. PMID:27264660

  10. Paraneoplastic tumefactive demyelination with underlying combined germ cell cancer.

    PubMed

    Broadfoot, Jack R; Archer, Hilary A; Coulthard, Elizabeth; Appelman, Auke P A; Sutak, Judit; Braybrooke, Jeremy P; Love, Seth

    2015-12-01

    Paraneoplastic demyelination is a rare disorder of the central nervous system. We describe a 60-year-old man with tumefactive demyelination who had an underlying retroperitoneal germ cell cancer. He presented with visuospatial problems and memory loss and had a visual field defect. His MRI was interpreted as a glioma but stereotactic biopsy showed active demyelination. Investigation for multiple sclerosis was negative but CT imaging showed retroperitoneal lymphadenopathy, and nodal biopsy confirmed a combined germ cell cancer. He responded poorly to corticosteroid treatment, and his visual field defect progressed. However, 6 months after plasma exchange and successful chemotherapy, he has partially improved clinically and radiographically. Tumefactive demyelination is typically associated with multiple sclerosis but may be paraneoplastic. It is important to recognise paraneoplastic tumefactive demyelination early, as the neurological outcome relies on treating the associated malignancy. PMID:26088612

  11. Germ cell tumour: late recurrence after 43 years.

    PubMed

    Mukhtar, S; Beatty, J; Agrawal, S; Christmas, T J; Jameson, C; Huddart, R A

    2011-07-01

    We report the late relapse of a patient following 43 years of surveillance of a germ cell tumour, thought to be a pure seminoma, having undergone yolk sac differentiation. The longest previous recorded time to relapse was 32 years (malignant teratoma with adenocarcinoma de-differentiation).(1) This case report demonstrates a late relapse of a testicular germ cell tumour is possible whatever the initial stage. European Association of Urology guidelines state close and active follow-up is mandatory for at least five years' surveillance due to the high and often late rate of relapse. Furthermore, they also suggest continuing follow-up although it is unclear as to how long this should last.(7)

  12. DAZL Expression Explains Origin and Central Formation of Primordial Germ Cells in Chickens.

    PubMed

    Lee, Hyung Chul; Choi, Hee Jung; Lee, Hyo Gun; Lim, Jeong Mook; Ono, Tamao; Han, Jae Yong

    2016-01-01

    The timing and biological events associated with germ cell specification in chickens have not been determined yet. In this study, we report the origin of primordial germ cells (PGCs) and germ plasm dynamics through investigation of the expression of the chicken homolog of deleted in azoospermia-like (cDAZL) gene during germ cell specification. Asymmetric localization of germ plasm in the center of oocytes from preovulatory follicle stages leads to PGCs being formed in the center. During cleavage stages, DAZL expression pattern changes from a subcellular localization to a diffuse form before and after zygotic genome activation. Meanwhile, PGCs exhibit transcriptional active status during their specification. In addition, knockdown studies of cDAZL, which result in reduced proliferation, aberrant gene expression profiles, and PGC apoptosis in vitro, suggest its possible roles for PGC formation in chicken. In conclusion, DAZL expression reveals formation and initial positioning of PGCs in chickens.

  13. Absence of mDazl produces a final block on germ cell development at meiosis.

    PubMed

    Saunders, P T K; Turner, J M A; Ruggiu, M; Taggart, M; Burgoyne, P S; Elliott, D; Cooke, H J

    2003-11-01

    The autosomal gene DAZL is a member of a family of genes (DAZL, DAZ, BOULE), all of which contain a consensus RNA binding domain and are expressed in germ cells. Adult male and female mice null for Dazl lack gametes. In order to define more precisely the developmental stages in germ cells that require Dazl expression, the patterns of germ cell loss in immature male and female wild-type (+/+, WT) and Dazl -/- (DazlKO) mice were analysed. In females, loss of germ cells occurred during fetal life and was coincident with progression of cells through meiotic prophase. In males, testes were recovered from WT and DazlKO males obtained before and during the first wave of spermatogenesis (days 2-19). Mitotically active germ cells were present up to and including day 19. Functional differentiation of spermatogonia associated with detection of c-kit positive cells did not depend upon expression of Dazl. RBMY-positive cells (A, intermediate, B spermatogonia, zygotene and preleptotene spermatocytes) were reduced in DazlKO compared with WT testes. Staining of cell squashes from day 19 testes with anti-gamma-H2AX and anti-SCP3 antibodies showed that germ cells from DazlKO males were unable to progress beyond the leptotene stage of meiotic prophase I. It was concluded that in the absence of Dazl, germ cells can complete mitosis, and embark on functional differentiation but that, in both sexes, progression through meiotic prophase requires this RNA binding protein. PMID:14611631

  14. The Formation of Germ Cell for Organizational Learning

    ERIC Educational Resources Information Center

    Ivaldi, Silvia; Scaratti, Giuseppe

    2016-01-01

    Purpose: The aim of the paper is to analyze the process of "germ cell" formation by framing it as an opportunity for promoting organizational learning and transformation. The paper aims to specifically answer two research questions: Why does the "germ cell" have a pivotal role in organization's transformation? and Which…

  15. Is Tobacco Smoke a Germ-Cell Mutagen?

    EPA Science Inventory

    Although no international organization exists to declare whether an agent is a germ-cell mutagen, tobacco smoke may be a human germ-cell mutagen. In the mouse, tobacco smoke induces a significant increase in the mutation frequency at an expanded simple tandem repeat (ESTR) locus....

  16. DAZ Family Proteins, Key Players for Germ Cell Development

    PubMed Central

    Fu, Xia-Fei; Cheng, Shun-Feng; Wang, Lin-Qing; Yin, Shen; De Felici, Massimo; Shen, Wei

    2015-01-01

    DAZ family proteins are found almost exclusively in germ cells in distant animal species. Deletion or mutations of their encoding genes usually severely impair either oogenesis or spermatogenesis or both. The family includes Boule (or Boll), Dazl (or Dazla) and DAZ genes. Boule and Dazl are situated on autosomes while DAZ, exclusive of higher primates, is located on the Y chromosome. Deletion of DAZ gene is the most common causes of infertility in humans. These genes, encoding for RNA binding proteins, contain a highly conserved RNA recognition motif and at least one DAZ repeat encoding for a 24 amino acids sequence able to bind other mRNA binding proteins. Basically, Daz family proteins function as adaptors for target mRNA transport and activators of their translation. In some invertebrate species, BOULE protein play a pivotal role in germline specification and a conserved regulatory role in meiosis. Depending on the species, DAZL is expressed in primordial germ cells (PGCs) and/or pre-meiotic and meiotic germ cells of both sexes. Daz is found in fetal gonocytes, spermatogonia and spermatocytes of adult testes. Here we discuss DAZ family genes in a phylogenic perspective, focusing on the common and distinct features of these genes, and their pivotal roles during gametogenesis evolved during evolution. PMID:26327816

  17. A twist of fate: How a meiotic protein is providing new perspectives on germ cell development.

    PubMed

    Mainpal, Rana; Yanowitz, Judith L

    2016-01-01

    The molecular pathways that govern how germ line fate is acquired is an area of intense investigation that has major implications for the development of assisted reproductive technologies, infertility interventions, and treatment of germ cell cancers. Transcriptional repression has emerged as a primary mechanism to ensure suppression of somatic growth programs in primordial germ cells. In this commentary, we address how xnd-1 illuminates our understanding of transcriptional repression and how it is coordinated with the germ cell differentiation program. We recently identified xnd-1 as a novel, early determinant of germ cell fates in Caenorhabditis elegans. Our study revealed that XND-1 is maternally deposited into early embryos where it is selectively enriched in the germ lineage and then exclusively found on chromatin in the germ lineage throughout development and into adulthood when it dissociates from chromosomes in late pachytene. This localization is consistent with a range of interesting germ cell defects that suggest xnd-1 is a pivotal determinant of germ cell characteristics. Loss of xnd-1 results in a unique "one PGC (primordial germ cell)" phenotype due to G2 cell cycle arrest of the germline precursor blastomere, P4, which predisposes the animal and its progeny for reduced fecundity. The sterility in xnd-1 mutants is correlated with an increase in the transcriptional activation-associated histone modification, dimethylation of histone H3 lysine 4 (H3K4me2), and aberrant expression of somatic transgenes but overlapping roles with nos-2 and nos-1 suggest that transcriptional repression is achieved by multiple redundant mechanisms. PMID:27383565

  18. Hedgehog does not guide migrating Drosophila germ cells

    PubMed Central

    Renault, Andrew D.; Ricardo, Sara; Kunwar, Prabhat S.; Santos, Ana; Starz-Gaiano, Michelle; Stein, Jennifer; Lehmann, Ruth

    2009-01-01

    In many species, the germ cells, precursors of sperm and egg, migrate during embryogenesis. The signals that regulate this migration are thus essential for fertility. In flies, lipid signals have been shown to affect germ cell guidance. In particular, the synthesis of geranylgeranyl pyrophosphate through the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (Hmgcr) pathway is critical for attracting germ cells to their target tissue. In a genetic analysis of signaling pathways known to affect cell migration of other migratory cells, we failed to find a role for the Hedgehog (Hh) pathway in germ cell migration. However, previous reports had implicated Hh as a germ cell attractant in flies and suggested that Hh signaling is enhanced through the action of the Hmgcr pathway. We therefore repeated several critical experiments and carried out further experiments to test specifically whether Hh is a germ cell attractant in flies. In contrast to previously reported findings and consistent with findings in zebrafish our data do not support the notion that Hh has a direct role in the guidance of migrating germ cells in flies. PMID:19389345

  19. Alvocidib and Oxaliplatin With or Without Fluorouracil and Leucovorin Calcium in Treating Patients With Relapsed or Refractory Germ Cell Tumors

    ClinicalTrials.gov

    2015-05-11

    Recurrent Extragonadal Seminoma; Recurrent Malignant Extragonadal Germ Cell Tumor; Recurrent Malignant Extragonadal Non-Seminomatous Germ Cell Tumor; Recurrent Malignant Testicular Germ Cell Tumor; Recurrent Ovarian Germ Cell Tumor; Stage III Testicular Cancer; Stage IV Extragonadal Non-Seminomatous Germ Cell Tumor; Stage IV Extragonadal Seminoma; Stage IV Ovarian Germ Cell Tumor

  20. Use of Stirred Suspension Bioreactors for Male Germ Cell Enrichment.

    PubMed

    Sakib, Sadman; Dores, Camila; Rancourt, Derrick; Dobrinski, Ina

    2016-01-01

    Spermatogenesis is a stem cell based system. Both therapeutic and biomedical research applications of spermatogonial stem cells require a large number of cells. However, there are only few germ line stem cells in the testis, contained in the fraction of undifferentiated spermatogonia. The lack of specific markers makes it difficult to isolate these cells. The long term maintenance and proliferation of nonrodent germ cells in culture has so far been met with limited success, partially due to the lack of highly enriched starting populations. Differential plating, which depends on the differential adhesion properties of testicular somatic and germ cells to tissue culture dishes, has been the method of choice for germ cell enrichment, especially for nonrodent germ cells. However, for large animals, this process becomes labor intensive and increases variability due to the need for extensive handling. Here, we describe the use of stirred suspension bioreactors, as a novel system for enriching undifferentiated germ cells from 1-week-old pigs. This method capitalizes on the adherent properties of somatic cells within a controlled environment, thus promoting the enrichment of progenitor cells with minimal handling and variability.

  1. Germ cell transplantation and testis tissue xenografting in mice.

    PubMed

    Tang, Lin; Rodriguez-Sosa, Jose Rafael; Dobrinski, Ina

    2012-01-01

    Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 1994(1,2). These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal(2). The use of donor males carrying the bacterial β-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals(1). Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located(3). It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation(4,5), to study Sertoli cell-germ cell interaction(6,7), SSC homing and colonization(3,8), as well as SSC self-renewal and differentiation(9,10). Germ cell transplantation is also feasible in large species(11). In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the

  2. Tritium effects on germ cells and fertility

    SciTech Connect

    Dobson, R.L.; Kwan, T.C.; Straume, T.

    1982-11-19

    Primordial oocytes in juvenile mice show acute gamma-ray LD/sub 50/ as low as 6 rad. This provides opportunities for determining dose-response relations at low doses and chronic exposure in the intact animal - conditions of particular interest for hazard evaluation. Examined in this way, /sup 3/HOH in body water is found to kill murine oocytes exponentially with dose, the LD/sub 50/ level for chronic exposure being only 2..mu..Ci/ml (delivering 0.4 rad/day). At very low doses and dose rates, where comparisons between tritium and other radiations are of special significance for radiological protection, the RBE of tritium compared with /sup 60/Co gamma radiation reaches approximately 3. Effects on murine fertility from tritium-induced oocyte loss have been quantified by reproductive capacity measurements. Chronic low-level exposure has been examined also in three primate species - squirrel, rhesus, and bonnet monkeys. In squirrel monkeys the ovarian germ-cell supply is 99% destroyed by the time of birth from prenatal exposure to body-water levels of /sup 3/HOH (administered in maternal drinking water) of only 3 ..mu..Ci/ml, the LD/sub 50/ level being 0.5 ..mu..Ci/ml (giving 0.1 rad/day), one fourth that in mice. Though not completely ruled out, similar high sensitivity of female germ cells has not been found in macaques; and it probably does not occur in man. The exquisite radiosensitivity of primordial oocytes in mice is apparently due to vulnerability of the plasma membrane (or something of similar geometry and location), not DNA. Evidence for this comes from tritium data as well as neutron studies. Tritium administered as /sup 3/HOH, and therefore generally distributed, is much more effective in killing murine oocytes than is tritium administered as /sup 3/H-TdR, localized in the nucleus. This situation in the mouse may have implications for estimating radiation genetic risk in the human female.

  3. The Mouse Fetal Ovary Has Greater Sensitivity Than the Fetal Testis to Benzo[a]pyrene-Induced Germ Cell Death.

    PubMed

    Lim, Jinhwan; Kong, Weixi; Lu, Muzi; Luderer, Ulrike

    2016-08-01

    The polycyclic aromatic hydrocarbon pollutant benzo[a]pyrene (BaP) is a known developmental gonadotoxicant. However, the mechanism of BaP-induced germ cell death is unclear. We investigated whether exposure to BaP induces apoptotic germ cell death in the mouse fetal ovary or testis. Mouse fetal gonads were dissected at embryonic day 13.5 days postcoitum (dpc) and fixed immediately or cultured for 6, 24, 48, or 72 h with various concentrations of BaP (1-1000 ng/ml). Germ cells numbers, apoptosis, and proliferation were evaluated by immunostaining. Treatment of fetal ovaries with BaP for 72 h concentration-dependently depleted germ cells. Treatment with BaP elevated the expression of BAX protein at 6 h and activated downstream caspases-9 and -3 at 24 h in a concentration-dependent manner in germ cells of fetal ovaries. As a consequence, ovarian germ cell numbers were significantly and concentration-dependently decreased at 48 h. Pretreatment with z-VAD-fmk, a pan-caspase inhibitor, prior to exposure to 1000 ng/ml BaP prevented BaP-mediated ovarian germ cell death; there were no effects of BaP or z-VAD-fmk on germ cell proliferation. No significant effects of BaP exposure on caspase 3 activation or germ cell numbers were observed in fetal testes after 48 h of culture. Our findings show that BaP exposure increases caspase-dependent and BAX-associated germ cell apoptosis in the mouse fetal ovary, leading to germ cell depletion. In contrast, the cultured 13.5 dpc fetal testis is relatively resistant to BaP-induced germ cell death. This study provides a novel insight into molecular mechanisms by which BaP has direct gonadotoxicity in the mouse fetal ovary. PMID:27208085

  4. Signaling events during male germ cell differentiation: bases and perspectives.

    PubMed

    Berruti, G

    1998-11-01

    In all species, reproductive function depends on the ability of the individual to produce functional differentiated gametes. Spermatogenesis is a cyclic process in which diploid spermatogonia differentiate into mature haploid spermatozoa. Thus from a genetic point of view, spermatogenesis can be divided into two phases, namely the diploid and haploid phase. Indeed, this complex differentiation process is still more intriguing since primary spermatocytes, if genetically diploid, are functionally tetraploid, while elongating spermatids, the germ cells undergoing the most dramatic morphological changes, if genetically haploid, become functionally anucleate due to ongoing condensation of chromatin resulting in an inactive nuclear DNA. This multi-step differentiative pathway is dependent on a specific environment provided by the anatomical and cellular relationships that take place in the testis and more specifically within the seminiferous tubules. Already, early anatomists (mind comes to Enrico Sertoli and Gustaf Retzius) were fascinated by the mixed cellular composition of the testis correctly deciphered as a whole of interacting and interdependent cell types despite the fact these belong to two well-established and different cell lineages, i.e, the somatic and germinal line. Since their time (the XIX century) up to-day a conspicuous bulk of experimental work and a relative massive bibliographic documentation have been provided. From this it stands out : a) a sophisticated role played by the cyclic hormonal control elicited by the hypothalamic-pituitary axis; b) the structural membrane specializations of Sertoli-germ cell communications; c) the existence and action of a paracrine and autocrine testicular regulative secretion; d) a regulation of germ cell gene expression, highly specialized both at transcriptional, posttranscriptional, and translational level; e) an active participation of the haploid genome in the final steps of cell differentiation. Each of these

  5. Epigenetics: a way to understand the origin and biology of testicular germ cell tumors.

    PubMed

    Okamoto, Keisei

    2012-06-01

    Testicular germ cell tumors are neoplasms carrying two unique features. First, testicular germ cell tumors have a pluripotential nature and show protean histology ranging from that of germ cells to embryonal and differentiated somatic cells. Therefore, testicular germ cell tumors are interesting resources positioned at a crossroad in developmental and neoplastic processes. The second unique feature of testicular germ cell tumors is their exquisite sensitivity to cisplatin-based chemotherapy. This review summarizes recent research progress in the epigenetics of testicular germ cell tumors in an attempt to explain the abovementioned biological and clinical characteristics of testicular germ cell tumors.

  6. In vitro differentiation of germ cells from stem cells: a comparison between primordial germ cells and in vitro derived primordial germ cell-like cells

    PubMed Central

    Ge, W; Chen, C; De Felici, M; Shen, W

    2015-01-01

    Stem cells are unique cell types capable to proliferate, some of them indefinitely, while maintaining the ability to differentiate into a few or any cell lineages. In 2003, a group headed by Hans R. Schöler reported that oocyte-like cells could be produced from mouse embryonic stem (ES) cells in vitro. After more than 10 years, where have these researches reached? Which are the major successes achieved and the problems still remaining to be solved? Although during the last years, many reviews have been published about these topics, in the present work, we will focus on an aspect that has been little considered so far, namely a strict comparison between the in vitro and in vivo developmental capabilities of the primordial germ cells (PGCs) isolated from the embryo and the PGC-like cells (PGC-LCs) produced in vitro from different types of stem cells in the mouse, the species in which most investigation has been carried out. Actually, the formation and differentiation of PGCs are crucial for both male and female gametogenesis, and the faithful production of PGCs in vitro represents the basis for obtaining functional germ cells. PMID:26469955

  7. Topology of the germ plasm and development of primordial germ cells in inverted amphibian eggs

    NASA Technical Reports Server (NTRS)

    Wakahara, M.; Neff, A. W.; Malacinski, G. M.

    1984-01-01

    Inverted Xenopus eggs have reduced numbers of primordial germ cells (PGCs). The extent of the reduction varies from spawning to spawning. Histologic examination revealed that PGC counts were lowest in inverted eggs which displayed the greatest amount of shift in the vegetal mass of large yolk platelets, although the germ plasm itself always remained localized in the egg's original vegetal hemisphere. Even at blastulation the germ plasm continued to be localized in the egg's original vegetal hemisphere. In many cases, however, it was confined to the periphery of the embryo, which probably accounts for the reduced PGC number in some tadpoles. In other cases it may have been dispersed and therefore not detectable in histologic analyses. Although the altered site of involution in inverted embryos did not influence PGC development, subsequent cell movement patterns apparently did. Those embryos which displayed the largest degree of pattern reversal at the tail-bud stage also exhibited the most extreme reduction in PGC numbers. A brief cold shock (4 degrees C, 10 min) prior to first cleavage leads to a further reduction in PGC numbers in inverted embryos, probably as a result of the displacement of the germ plasm away from its original vegetal pole location.

  8. Germ Cell-Specific Excision of loxP-Flanked Transgenes in Rainbow Trout Oncorhynchus mykiss.

    PubMed

    Katayama, Naoto; Kume, Sachi; Hattori-Ihara, Shoko; Sadaie, Sakiko; Hayashi, Makoto; Yoshizaki, Goro

    2016-04-01

    Cre/loxP-mediated DNA excision in germ cell lineages could contribute substantially to the study of germ cell biology in salmonids, which are emerging as a model species in this field. However, a cell type-specific Cre/loxPsystem has not been successfully developed for any salmonid species. Therefore, we examined the feasibility of Cre/loxP-mediated, germ cell-specific gene excision and transgene activation in rainbow trout. Double-transgenic (wTg) progeny were obtained by mating a transgenic male carryingcrewith a transgenic female carrying thehsc-LRLGgene;crewas driven by rainbow troutvasaregulatory regions and thehsc-LRLGgene was made up of the rainbow troutheat-shock-cognate71promoter, theDsRedgene flanked by twoloxPsites, and theEgfpgene. PCR analysis, fluorescence imaging, and histological analysis revealed that excision of theloxP-flanked sequence and activation ofEgfpoccurred only in germ cells of wTg fish. However, progeny tests revealed that the excision efficiency ofloxP-flanked sequence in germ cells was low (≤3.27%). In contrast, the other wTg fish derived from two differentcre-transgenic males frequently excised theloxP-flanked sequence in germ cells (≤89.25%). Thus, we showed for the first time successful germ cell-specific transgene manipulation via the Cre/loxPsystem in rainbow trout. We anticipate that this technology will be suitable for studies of cell function through cell targeting, cell-linage tracing, and generating cell type-specific conditional gene knockouts and separately for developing sterile rainbow trout in aquaculture. PMID:26911430

  9. Germ Cell-Specific Excision of loxP-Flanked Transgenes in Rainbow Trout Oncorhynchus mykiss.

    PubMed

    Katayama, Naoto; Kume, Sachi; Hattori-Ihara, Shoko; Sadaie, Sakiko; Hayashi, Makoto; Yoshizaki, Goro

    2016-04-01

    Cre/loxP-mediated DNA excision in germ cell lineages could contribute substantially to the study of germ cell biology in salmonids, which are emerging as a model species in this field. However, a cell type-specific Cre/loxPsystem has not been successfully developed for any salmonid species. Therefore, we examined the feasibility of Cre/loxP-mediated, germ cell-specific gene excision and transgene activation in rainbow trout. Double-transgenic (wTg) progeny were obtained by mating a transgenic male carryingcrewith a transgenic female carrying thehsc-LRLGgene;crewas driven by rainbow troutvasaregulatory regions and thehsc-LRLGgene was made up of the rainbow troutheat-shock-cognate71promoter, theDsRedgene flanked by twoloxPsites, and theEgfpgene. PCR analysis, fluorescence imaging, and histological analysis revealed that excision of theloxP-flanked sequence and activation ofEgfpoccurred only in germ cells of wTg fish. However, progeny tests revealed that the excision efficiency ofloxP-flanked sequence in germ cells was low (≤3.27%). In contrast, the other wTg fish derived from two differentcre-transgenic males frequently excised theloxP-flanked sequence in germ cells (≤89.25%). Thus, we showed for the first time successful germ cell-specific transgene manipulation via the Cre/loxPsystem in rainbow trout. We anticipate that this technology will be suitable for studies of cell function through cell targeting, cell-linage tracing, and generating cell type-specific conditional gene knockouts and separately for developing sterile rainbow trout in aquaculture.

  10. The chemosensitivity of testicular germ cell tumors.

    PubMed

    Voutsadakis, Ioannis A

    2014-04-01

    Although rare cancers overall, testicular germ cell tumors (TGCTs) are the most common type of cancer in young males below 40 years of age. Both subtypes of TGCTs, i.e., seminomas and non-seminomas, are highly curable and the majority of even metastatic patients may expect to be cured. These high cure rates are not due to the indolent nature of these cancers, but rather to their sensitivity to chemotherapy (and for seminomas to radiotherapy). The delineation of the cause of chemosensitivity at the molecular level is of paramount importance, because it may provide insights into the minority of TGCTs that are chemo-resistant and, thereby, provide opportunities for specific therapeutic interventions aimed at reverting them to chemosensitivity. In addition, delineation of the molecular basis of TGCT chemo-sensitivity may be informative for the cause of chemo-resistance of other more common types of cancer and, thus, may create new therapeutic leads. p53, a frequently mutated tumor suppressor in cancers in general, is not mutated in TGCTs, a fact that has implications for their chemo-sensitivity. Oct4, an embryonic transcription factor, is uniformly expressed in the seminoma and embryonic carcinoma components of non-seminomas, and its interplay with p53 may be important in the chemotherapy response of these tumors. This interplay, together with other features of TGCTs such as the gain of genetic material from the short arm of chromosome 12 and the association with disorders of testicular development, will be discussed in this paper and integrated in a unifying hypothesis that may explain their chemo-sensitivity. PMID:24692098

  11. Selection of the Inducer for the Differentiation of Chicken Embryonic Stem Cells into Male Germ Cells In Vitro

    PubMed Central

    Zhang, Yani; Wang, Yingjie; Zuo, Qisheng; Wang, Xiaoyan; Li, Dong; Tang, Beibei; Li, Bichun

    2016-01-01

    Several inducers have been used to differentiate embryonic stem cells (ESCs) into male germ cells but the induction process has been inefficient. To solve the problem of low efficiency of inducer for ESCs differentiation into male germ cells, all-trans retinoic acid (ATRA), Am80(the retinoic acid receptor agonist), and estradiol (E2) was used to induce ESCs to differentiate into male germ cells in vitro. ESCs were cultured in media containing ATRA, Am80, or E2 respectively which can differentiate ESCs into a germ cell lineage. In process of ATRA and Am80 induction Group, germ cell-like cells can be observed in 10 days; but have no in E2 induction Group. The marker genes of germ cell: Dazl, Stra8, C-kit, Cvh, integrinα6, and integrinβ1 all showed a significant up-regulation in the expression level. The ATRA-induction group showed high expression of C-kit and Cvh around 4 days, and integrinα6 and integrinβ1 were activated on day 10, respectively, while the E2-,Am80- induction group showed a high expression of C-kit as early as 4 days immunocytochemistry results shown that, integrinα6 and integrinβ1 could be detected in the ATRA-, Am80-, and E2-induction group, Positive clones in the ATRA group were greater in number than those in the other two groups. we conclued that ATRA, Am80, and E2 can promote the expression of the corresponding genes of germ cells, and had different effect on the differentiation of ESCs into male germ cells. ATRA was the most effective inducer of germ cell differentiation. PMID:27741318

  12. Mechanisms and chemical induction of aneuploidy in rodent germ cells

    SciTech Connect

    Mailhes, J B; Marchetti, F

    2004-10-15

    The objective of this review is to suggest that the advances being made in our understanding of the molecular events surrounding chromosome segregation in non-mammalian and somatic cell models be considered when designing experiments for studying aneuploidy in mammalian germ cells. Accurate chromosome segregation requires the temporal control and unique interactions among a vast array of proteins and cellular organelles. Abnormal function and temporal disarray among these, and others to be inidentified, biochemical reactions and cellular organelles have the potential for predisposing cells to aneuploidy. Although numerous studies have demonstrated that certain chemicals (mainly those that alter microtubule function) can induce aneuploidy in mammalian germ cells, it seems relevant to point out that such data can be influenced by gender, meiotic stage, and time of cell-fixation post-treatment. Additionally, a consensus has not been reached regarding which of several germ cell aneuploidy assays most accurately reflects the human condition. More recent studies have shown that certain kinase, phosphatase, proteasome, and topoisomerase inhibitors can also induce aneuploidy in rodent germ cells. We suggest that molecular approaches be prudently incorporated into mammalian germ cell aneuploidy research in order to eventually understand the causes and mechanisms of human aneuploidy. Such an enormous undertaking would benefit from collaboration among scientists representing several disciplines.

  13. In Vitro Ectopic Behavior of Porcine Spermatogonial Germ Cells and Testicular Somatic Cells.

    PubMed

    Lee, Kyung Hoon; Lee, Won Young; Do, Jung Tae; Park, Chan Kyu; Kim, Nam Hyung; Kim, Jin Hoi; Chung, Hak Jae; Kim, Dong Woon; Song, Hyuk

    2016-08-01

    Embryonic body-like colony formation is a unique pattern in male germ cell cultures, including spermatogonial stem cells. However, detailed information of the colony formation has not yet been sufficiently reported in male germ cell culture. To elucidate the formation of germ cell-derived colony (GDC), glial cell-derived neurotrophic factor receptor alpha-1 (GFRα-1)-positive pig germ cells were isolated using an immunomagnetic cell isolation method and labeled with red- or green-fluorescent dye. In GDC culture, red-fluorescent-labeled germ cells were evenly distributed in the wells from day 1 to 4, and they clustered together at the time of GDC formation on day 6. Interestingly, feeder cells migrated to the site of colony formation as spermatogonia carriers. Furthermore, when freshly prepared green-labeled GFRα-1-positive germ cells were added, mixed-fluorescent dye (red and green) colonies were observed. On bromodeoxyuridine (BrdU) treatment, 58% ± 3.13% of germ cells were positive to protein gene product 9.5 but negative to BrdU cells. Immunocytochemistry and reverse transcription-polymerase chain reaction results showed that cultured GDC cells were positive to stem cell- and pig germ cell-specific marker genes. In conclusion, in vitro formation of GDCs is mainly dependent on the aggregation of single germ cells as well as on the slow proliferation of germ cells.

  14. In Vitro Ectopic Behavior of Porcine Spermatogonial Germ Cells and Testicular Somatic Cells.

    PubMed

    Lee, Kyung Hoon; Lee, Won Young; Do, Jung Tae; Park, Chan Kyu; Kim, Nam Hyung; Kim, Jin Hoi; Chung, Hak Jae; Kim, Dong Woon; Song, Hyuk

    2016-08-01

    Embryonic body-like colony formation is a unique pattern in male germ cell cultures, including spermatogonial stem cells. However, detailed information of the colony formation has not yet been sufficiently reported in male germ cell culture. To elucidate the formation of germ cell-derived colony (GDC), glial cell-derived neurotrophic factor receptor alpha-1 (GFRα-1)-positive pig germ cells were isolated using an immunomagnetic cell isolation method and labeled with red- or green-fluorescent dye. In GDC culture, red-fluorescent-labeled germ cells were evenly distributed in the wells from day 1 to 4, and they clustered together at the time of GDC formation on day 6. Interestingly, feeder cells migrated to the site of colony formation as spermatogonia carriers. Furthermore, when freshly prepared green-labeled GFRα-1-positive germ cells were added, mixed-fluorescent dye (red and green) colonies were observed. On bromodeoxyuridine (BrdU) treatment, 58% ± 3.13% of germ cells were positive to protein gene product 9.5 but negative to BrdU cells. Immunocytochemistry and reverse transcription-polymerase chain reaction results showed that cultured GDC cells were positive to stem cell- and pig germ cell-specific marker genes. In conclusion, in vitro formation of GDCs is mainly dependent on the aggregation of single germ cells as well as on the slow proliferation of germ cells. PMID:27328332

  15. Primordial germ cells: the first cell lineage or the last cells standing?

    PubMed Central

    Johnson, Andrew D.; Alberio, Ramiro

    2015-01-01

    Embryos of many animal models express germ line determinants that suppress transcription and mediate early germ line commitment, which occurs before the somatic cell lineages are established. However, not all animals segregate their germ line in this manner. The ‘last cell standing’ model describes primordial germ cell (PGC) development in axolotls, in which PGCs are maintained by an extracellular signalling niche, and germ line commitment occurs after gastrulation. Here, we propose that this ‘stochastic’ mode of PGC specification is conserved in vertebrates, including non-rodent mammals. We postulate that early germ line segregation liberates genetic regulatory networks for somatic development to evolve, and that it therefore emerged repeatedly in the animal kingdom in response to natural selection. PMID:26286941

  16. Germ Cell Nuclear Factor Regulates Gametogenesis in Developing Gonads

    PubMed Central

    Sabour, Davood; Xu, Xueping; Chung, Arthur C. K.; Le Menuet, Damien; Ko, Kinarm; Tapia, Natalia; Araúzo-Bravo, Marcos J.; Gentile, Luca; Greber, Boris; Hübner, Karin; Sebastiano, Vittorio; Wu, Guangming; Schöler, Hans R.; Cooney, Austin J.

    2014-01-01

    Expression of germ cell nuclear factor (GCNF; Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU-domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads. PMID:25140725

  17. Loss of Sertoli-germ cell adhesion determines the rapid germ cell elimination during the seasonal regression of the seminiferous epithelium of the large hairy armadillo Chaetophractus villosus.

    PubMed

    Luaces, Juan Pablo; Rossi, Luis Francisco; Sciurano, Roberta Beatriz; Rebuzzini, Paola; Merico, Valeria; Zuccotti, Maurizio; Merani, Maria Susana; Garagna, Silvia

    2014-03-01

    The armadillo Chaetophractus villosus is a seasonal breeder whose seminiferous epithelium undergoes rapid regression with massive germ cell loss, leaving the tubules with only Sertoli cells and spermatogonia. Here, we addressed the question of whether this regression entails 1) the disassembly of cell junctions (immunolocalization of nectin-3, Cadm1, N-cadherin, and beta-catenin, and transmission electron microscopy [TEM]); 2) apoptosis (immunolocalization of cytochrome c and caspase 3; TUNEL assay); and 3) the involvement of Sertoli cells in germ cell phagocytosis (TEM). We showed a dramatic reduction in the extension of vimentin filaments associated with desmosomelike junctions at the interface between Sertoli and germ cells, and an increased diffusion of the immunosignals of nectin-3, Cadm1, N-cadherin, and beta-catenin. Together, these results suggest loss of Sertoli-germ cell adhesion, which in turn might determine postmeiotic cell sloughing at the beginning of epithelium regression. Then, loss of Sertoli-germ cell adhesion triggers cell death. Cytochrome c is released from mitochondria, but although postmeiotic cells were negative for late apoptotic markers, at advanced regression spermatocytes were positive for all apoptotic markers. Transmission electron microscopy analysis showed cytoplasmic engulfment of cell debris and lipid droplets within Sertoli cells, a sign of their phagocytic activity, which contributes to the elimination of the residual meiocytes still present in the latest regression phases. These findings are novel and add new players to the mechanisms of seminiferous epithelium regression occurring in seasonal breeders, and they introduce the armadillo as an interesting model for studying seasonal spermatogenesis. PMID:24451984

  18. Lactate Regulates Rat Male Germ Cell Function through Reactive Oxygen Species

    PubMed Central

    Galardo, María Noel; Regueira, Mariana; Riera, María Fernanda; Pellizzari, Eliana Herminia; Cigorraga, Selva Beatriz; Meroni, Silvina Beatriz

    2014-01-01

    Besides giving structural support, Sertoli cells regulate the fate of germ cells by supplying a variety of factors. These factors include hormones, several pro- and anti-apoptotic agents and also energetic substrates. Lactate is one of the compounds produced by Sertoli cells, which is utilized as an energetic substrate by germ cells, particularly spermatocytes and spermatids. Beyond its function as an energy source, some studies have proposed a role of lactate in the regulation of gene expression not strictly related to the energetic state of the cells. The general hypothesis that motivated this investigation was that lactate affects male germ cell function, far beyond its well-known role as energetic substrate. To evaluate this hypothesis we investigated: 1) if lactate was able to regulate germ cell gene expression and if reactive oxygen species (ROS) participated in this regulation, 2) if different signal transduction pathways were modified by the production of ROS in response to lactate and 3) possible mechanisms that may be involved in lactate stimulation of ROS production. In order to achieve these goals, cultures of germ cells obtained from male 30-day old rats were exposed to 10 or 20 mM lactate. Increases in lactate dehydrogenase (LDH) C and monocarboxylate transporter (MCT)2 expression, in Akt and p38-MAPK phosphorylation levels and in ROS production were observed. These effects were impaired in the presence of a ROS scavenger. Lactate stimulated ROS production was also inhibited by a LDH inhibitor or a NAD(P)H oxidase (NOX) inhibitor. NOX4 expression was identified in male germ cells. The results obtained herein are consistent with a scenario where lactate, taken up by germ cells, becomes oxidized to pyruvate with the resultant increase in NADH, which is a substrate for NOX4. ROS, products of NOX4 activity, may act as second messengers regulating signal transduction pathways and gene expression. PMID:24498241

  19. Vanadium induced ultrastructural changes and apoptosis in male germ cells.

    PubMed

    Aragón, M A; Ayala, M E; Fortoul, T I; Bizarro, P; Altamirano-Lozano, M

    2005-01-01

    Vanadium is a transition metal that is emitted to the atmosphere during combustion of fossil fuels. In the environment, vanadium occurs in the (V) oxidized form, but in the body it is found exclusively in the (IV) oxidized form. Vanadium tetraoxide is an inorganic chemical species in the (IV) oxidized form that has been shown to induce toxic effects in vitro and in vivo. The reproductive toxicity of vanadium in males was studied through monitoring germ cell apoptosis during spermatogenesis. We analyzed ultrastructural damage, and testosterone and progesterone concentrations following vanadium tetraoxide administered to male mice for 60 days. Spermatogenesis stages I-III and X-XII frequently showed apoptotic germ cells in control and treated animals; vanadium tetraoxide treatment induced an increase in the number of germ cell apoptosis in stages I-III and XII at 9.4 and 18.8 mg/kg, respectively. Although spermatogenesis is regulated by testosterone, in our study this hormone level was not modified by vanadium administration; thus, germ cell death was not related with testosterone concentration. At the ultrastructural level, we observed inclusion structures that varied as to location and content in the Sertoli and germ cells. PMID:15808796

  20. Toll-like receptor 11-initiated innate immune response in male mouse germ cells.

    PubMed

    Chen, Qiaoyuan; Zhu, Weiwei; Liu, Zhenghui; Yan, Keqin; Zhao, Shutao; Han, Daishu

    2014-02-01

    Toxoplasma gondii and uropathogenic Escherichia coli (UPEC) may infect the testis and impair testicular function. Mechanisms underlying testicular innate immune response to these two pathogens remain to be clarified. The present study examined the function of TLR11, which can be recognized by T. gondii-derived profilin and UPEC, in initiating innate immune response in male mouse germ cells. TLR11 is predominantly expressed in spermatids. Profilin and UPEC induced the expressions of different inflammatory cytokine profiles in the germ cells. In particular, profilin induced the expressions of macrophage chemotactic protein 1 (MCP1), interleukin 12 (IL12), and interferon gamma (IFNG) through nuclear factor KB (NFKB) activation. UPEC induced the expressions of MCP1, IL12, and IFNG, as well as tumor necrosis factor alpha (TNFA), IL6, and IFNB, through the activation of NFKB, IFN regulatory factor 3, and mitogen-activated protein kinases. Evidence showed that profilin induced the innate response in male germ cells through TLR11 signaling, and UPEC triggered the response through TLR11 and other TLR-signaling pathways. We also provided evidence that local injection of profilin or UPEC induces the innate immune response in the germ cells. Data describe TLR11-mediated innate immune function of male germ cells in response to T. gondii profilin and UPEC stimulations. This system may play a role in testicular defense against T. gondii and UPEC infections in mice.

  1. Crucial Genes and Pathways in Chicken Germ Stem Cell Differentiation

    PubMed Central

    Zhang, Zhentao; Elsayed, Ahmed Kamel; Shi, Qingqing; Zhang, Yani; Zuo, Qisheng; Li, Dong; Lian, Chao; Tang, Beibei; Xiao, Tianrong; Xu, Qi; Chang, Guobin; Chen, Guohong; Zhang, Lei; Wang, Kehua; Wang, Yingjie; Jin, Kai; Wang, Yilin; Song, Jiuzhou; Cui, Hengmi; Li, Bichun

    2015-01-01

    Male germ cell differentiation is a subtle and complex regulatory process. Currently, its regulatory mechanism is still not fully understood. In our experiment, we performed the first comprehensive genome and transcriptome-wide analyses of the crucial genes and signaling pathways in three kinds of crucial cells (embryonic stem cells, primordial germ cell, and spermatogonial stem cells) that are associated with the male germ cell differentiation. We identified thousands of differentially expressed genes in this process, and from these we chose 173 candidate genes, of which 98 genes were involved in cell differentiation, 19 were involved in the metabolic process, and 56 were involved in the differentiation and metabolic processes, like GAL9, AMH, PLK1, and PSMD7 and so on. In addition, we found that 18 key signaling pathways were involved mainly in cell proliferation, differentiation, and signal transduction processes like TGF-β, Notch, and Jak-STAT. Further exploration found that the candidate gene expression patterns were the same between in vitro induction experiments and transcriptome results. Our results yield clues to the mechanistic basis of male germ cell differentiation and provide an important reference for further studies. PMID:25847247

  2. Salvage Therapy for Patients With Germ Cell Tumor.

    PubMed

    Rashdan, Sawsan; Einhorn, Lawrence H

    2016-05-01

    The introduction of cisplatin combination chemotherapy, 40 years ago, transformed metastatic testicular germ cell tumors from an almost uniformly fatal disease into a model for a curable neoplasm. Before the era of platinum combination chemotherapy, the 5-year survival rate among men with metastatic testicular germ cell tumors was 5% to 10%. Currently, the 5-year survival rate is 80% for patients with metastatic disease and 95% overall. Despite the substantial advances in the treatment of germ cell tumors, 20% to 30% of patients will relapse after first-line chemotherapy and will require additional salvage therapies. Standard-dose or high-dose chemotherapy can cure ≤ 50% of these patients. Relapses after high-dose chemotherapy generally carry a poor prognosis; however, cure is still possible in a small percentage of patients by using further salvage chemotherapy or salvage surgery. PMID:27170693

  3. Giant Mediastinal Germ Cell Tumour: An Enigma of Surgical Consideration

    PubMed Central

    Ali, Nurayub Mohd; Azizan, Nornazirah; Zakaria, Andee Dzulkarnaen; Rahman, Mohd Ramzisham Abdul

    2016-01-01

    We present a case of 16-year-old male, who was referred from private centre for dyspnoea, fatigue, and orthopnea. The chest radiograph revealed complete opacification of left chest which was confirmed by computed tomography as a large left mediastinal mass measuring 14 × 15 × 18 cm. The diagnostic needle core biopsy revealed mixed germ cell tumour with possible combination of embryonal carcinoma, yolk sac, and teratoma. After 4 cycles of neoadjuvant BEP regime, there was initial response of tumour markers but not tumour bulk. Instead of classic median sternotomy or clamshell incision, posterolateral approach with piecemeal manner was chosen. Histology confirmed mixed germ cell tumour with residual teratomatous component without yolk sac or embryonal carcinoma component. Weighing 3.5 kg, it is one of the largest mediastinal germ cell tumours ever reported. We describe this rare and gigantic intrathoracic tumour and discuss the spectrum of surgical approach and treatment of this exceptional tumour. PMID:27807495

  4. A functional genomic screen in planarians identifies novel regulators of germ cell development

    PubMed Central

    Wang, Yuying; Stary, Joel M.; Wilhelm, James E.; Newmark, Phillip A.

    2010-01-01

    Germ cells serve as intriguing examples of differentiated cells that retain the capacity to generate all cell types of an organism. Here we used functional genomic approaches in planarians to identify genes required for proper germ cell development. We conducted microarray analyses and in situ hybridization to discover and validate germ cell-enriched transcripts, and then used RNAi to screen for genes required for discrete stages of germ cell development. The majority of genes we identified encode conserved RNA-binding proteins, several of which have not been implicated previously in germ cell development. We also show that a germ cell-specific subunit of the conserved transcription factor CCAAT-binding protein/nuclear factor-Y is required for maintaining spermatogonial stem cells. Our results demonstrate that conserved transcriptional and post-transcriptional mechanisms regulate germ cell development in planarians. These findings suggest that studies of planarians will inform our understanding of germ cell biology in higher organisms. PMID:20844018

  5. Germ-cell malignant tumours in father and son.

    PubMed Central

    Musa, M. B.

    1975-01-01

    Germ-cell malignant tumours occurred in a man and his son. The father, who had a teratoma of the right testicle removed 24 years ago, is presently alive and well. The son, who had a choriocarcinoma presenting as an abdominal mass, possibly originating in the testicle, died within 7 months of the diagnosis with metastases in the lungs, liver and retroperitoneum. This report documents the third such case of germ-cell neoplasms occurring in father and son. Images FIG. 1 FIG. 2 FIG. 3 PMID:1168534

  6. Expression of steroidogenesis-related genes in murine male germ cells.

    PubMed

    Culty, Martine; Liu, Ying; Manku, Gurpreet; Chan, Wai-Yee; Papadopoulos, Vassilios

    2015-11-01

    For decades, only few tissues and cell types were defined as steroidogenic, capable of de novo steroid synthesis from cholesterol. However, with the refinement of detection methods, several tissues have now been added to the list of steroidogenic tissues. Besides their critical role as long-range acting hormones, steroids are also playing more discreet roles as local mediators and signaling molecules within the tissues they are produced. In testis, steroidogenesis is carried out by the Leydig cells through a broad network of proteins, mediating cholesterol delivery to CYP11A1, the first cytochrome of the steroidogenic cascade, and the sequential action of enzymes insuring the production of active steroids, the main one being testosterone. The knowledge that male germ cells can be directly regulated by steroids and that they express several steroidogenesis-related proteins led us to hypothesize that germ cells could produce steroids, acting as autocrine, intracrine and juxtacrine modulators, as a way to insure synchronized progression within spermatogenic cycles, and preventing inappropriate cell behaviors between neighboring cells. Gene expression and protein analyses of mouse and rat germ cells from neonatal gonocytes to spermatozoa showed that most steroidogenesis-associated genes are expressed in germ cells, showing cell type-, spermatogenic cycle-, and age-specific expression profiles. Highly expressed genes included genes involved in steroidogenesis and other cell functions, such as Acbd1 and 3, Tspo and Vdac1-3, and genes involved in fatty acids metabolism or synthesis, including Hsb17b4 10 and 12, implying broader roles than steroid synthesis in germ cells. These results support the possibility of an additional level of regulation of spermatogenesis exerted between adjacent germ cells.

  7. Premeiotic germ cell defect in seminiferous tubules of Atm-null testis

    SciTech Connect

    Takubo, Keiyo . E-mail: keiyot@gmail.com; Hirao, Atsushi; Ohmura, Masako; Azuma, Masaki; Arai, Fumio; Nagamatsu, Go; Suda, Toshio . E-mail: sudato@sc.itc.keio.ac.jp

    2006-12-29

    Lifelong spermatogenesis is maintained by coordinated sequential processes including self-renewal of stem cells, proliferation of spermatogonial cells, meiotic division, and spermiogenesis. It has been shown that ataxia telangiectasia-mutated (ATM) is required for meiotic division of the seminiferous tubules. Here, we show that, in addition to its role in meiosis, ATM has a pivotal role in premeiotic germ cell maintenance. ATM is activated in premeiotic spermatogonial cells and the Atm-null testis shows progressive degeneration. In Atm-null testicular cells, differing from bone marrow cells of Atm-null mice, reactive oxygen species-mediated p16{sup Ink4a} activation does not occur in Atm-null premeiotic germ cells, which suggests the involvement of different signaling pathways from bone marrow defects. Although Atm-null bone marrow undergoes p16{sup Ink4a}-mediated cellular senescence program, Atm-null premeiotic germ cells exhibited cell cycle arrest and apoptotic elimination of premeiotic germ cells, which is different from p16{sup Ink4a}-mediated senescence.

  8. Ascorbic acid protects against cadmium-induced endoplasmic reticulum stress and germ cell apoptosis in testes.

    PubMed

    Ji, Yan-Li; Wang, Zhen; Wang, Hua; Zhang, Cheng; Zhang, Ying; Zhao, Mei; Chen, Yuan-Hua; Meng, Xiu-Hong; Xu, De-Xiang

    2012-11-01

    Cadmium (Cd) is a testicular toxicant which induces endoplasmic reticulum (ER) stress and germ cell apoptosis in testes. This study investigated the effects of ascorbic acid on Cd-evoked ER stress and germ cell apoptosis in testes. Male mice were intraperitoneally injected with CdCl(2) (2.0 mg/kg). As expected, a single dose of Cd induced testicular germ cell apoptosis. Interestingly, Cd-triggered testicular germ cell apoptosis was almost completely inhibited in mice treated with ascorbic acid. Interestingly, ascorbic acid significantly attenuated Cd-induced upregulation of GRP78 in testes. In addition, ascorbic acid significantly attenuated Cd-triggered testicular IRE1α and eIF2α phosphorylation and XBP-1 activation, indicating that this antioxidant counteracts Cd-induced unfolded protein response (UPR) in testes. Finally, ascorbic acid significantly attenuated Cd-evoked upregulation of CHOP and JNK phosphorylation, two components in ER stress-mediated apoptotic pathway. In conclusion, ascorbic acid protects mice from Cd-triggered germ cell apoptosis via inhibiting ER stress and UPR in testes. PMID:22569276

  9. Electron microscopy of the germ cells and the ovarian wall in Xiphinema (Nematoda).

    PubMed

    Van de Velde, M C; Coomans, A

    1988-01-01

    The ovary of Xiphinema theresiae is studied ultrastructurally. It consists of two cell types, the ovarian epithelial cells and the germ cells. The ovarian epithelial cells form a thin layer around the germ cells. Their nuclei are located in between the germ cells. At some sites, processes of the ovarian epithelial cells migrate inward and form a central cytoplasmic mass. The germ cells have a large lobated nucleus, with an eccentric nucleolus, and are considered to represent young previtellogenic oocytes. In contact with the central cytoplasmic mass, the germ cells develop two membrane derived features, the villi and the small coated bulges, which most probably play a role in transport.

  10. New evidence for the origin of intracranial germ cell tumours from primordial germ cells: expression of pluripotency and cell differentiation markers.

    PubMed

    Hoei-Hansen, C E; Sehested, A; Juhler, M; Lau, Y-F C; Skakkebaek, N E; Laursen, H; Rajpert-de Meyts, E

    2006-05-01

    Primary intracranial germ cell tumours are rare neoplasms that occur in children and adolescents. This study examined both the biology and the origin of these tumours, as it has been hypothesized that they originate from a totipotent primordial germ cell. We applied recent knowledge from gonadal germ cell tumours and analysed expression of a wide panel of stem cell-related proteins (C-KIT, OCT-3/4 (POU5F1), AP-2gamma (TFAP2C), and NANOG) and developmentally regulated germ cell-specific proteins (including MAGE-A4, NY-ESO-1, and TSPY). Expression at the protein level was analysed in 21 children and young adults with intracranial germinomas and non-germinomas, contributing to a careful description of these unusual tumours and adding to the understanding of pathogenesis. Stem cell related proteins were highly expressed in intracranial germ cell tumours, and many similarities were detected with their gonadal equivalents, including a close similarity with primordial germ cells. A notable difference was the sex-specific expression of TSPY, a gene previously implicated in the origin of gonadoblastoma. TSPY was only detected in germ cell tumours in the central nervous system (CNS) from males, suggesting that it is not required for the initiation of malignant germ cell transformation. The expression of genes associated with embryonic stem cell pluripotency in CNS germ cell tumours strongly suggests that these tumours are derived from cells that retain, at least partially, an embryonic stem cell-like phenotype, which is a hallmark of primordial germ cells. PMID:16456896

  11. Testicular structure and germ cells morphology in salamanders

    PubMed Central

    Uribe, Mari Carmen; Mejía-Roa, Víctor

    2014-01-01

    Testes of salamanders or urodeles are paired elongated organs that are attached to the dorsal wall of the body by a mesorchium. The testes are composed of one or several lobes. Each lobe is morphologically and functionally a similar testicular unit. The lobes of the testis are joined by cords covered by a single peritoneal epithelium and subjacent connective tissue. The cords contain spermatogonia. Spermatogonia associate with Sertoli cells to form spermatocysts or cysts. The spermatogenic cells in a cyst undergo their development through spermatogenesis synchronously. The distribution of cysts displays the cephalo-caudal gradient in respect to the stage of spermatogenesis. The formation of cysts at cephalic end of the testis causes their migration along the lobules to the caudal end. Consequently, the disposition in cephalo-caudal regions of spermatogenesis can be observed in longitudinal sections of the testis. The germ cells are spermatogonia, diploid cells with mitotic activity; primary and second spermatocytes characterized by meiotic divisions that develop haploid spermatids; during spermiogenesis the spermatids differentiate to spermatozoa. During spermiation the cysts open and spermatozoa leave the testicular lobules. After spermiation occurs the development of Leydig cells into glandular tissue. This glandular tissue regressed at the end of the reproductive cycle. PMID:26413406

  12. Declaring the Existence of Human Germ-Cell Mutagens

    EPA Science Inventory

    After more than 80 years of searching for human germ-cell mutagens, I think that sufficient evidence already exists for a number of agents to be so considered, and definitive confirmation seems imminent due to the application ofrecently developed genomic techniques. In preparatio...

  13. Genome-wide analysis of germ cell proliferation in C. elegans identifies VRK-1 as a key regulator of CEP-1/p53

    PubMed Central

    Waters, Katherine; Yang, Alison Z.; Reinke, Valerie

    2012-01-01

    Proliferating germ cells in Caenorhabditis elegans provide a useful model system for deciphering fundamental mechanisms underlying the balance between proliferation and differentiation. Using gene expression profiling, we identified approximately 200 genes upregulated in the proliferating germ cells of C. elegans. Functional characterization using RNA-mediated interference demonstrated that over forty of these factors are required for normal germline proliferation and development. Detailed analysis of two of these factors defined an important regulatory relationship controlling germ cell proliferation. We established that the kinase VRK-1 is required for normal germ cell proliferation, and that it acts in part to regulate CEP-1(p53) activity. Loss of cep-1 significantly rescued the proliferation defects of vrk-1 mutants. We suggest that VRK-1 prevents CEP-1 from triggering an inappropriate cell cycle arrest, thereby promoting germ cell proliferation. This finding reveals a previously unsuspected mechanism for negative regulation of p53 activity in germ cells to control proliferation. PMID:20599896

  14. DNA Analysis in Samples From Younger Patients With Germ Cell Tumors and Their Parents or Siblings

    ClinicalTrials.gov

    2016-10-05

    Childhood Malignant Ovarian Germ Cell Tumor; Childhood Malignant Testicular Germ Cell Tumor; Ovarian Choriocarcinoma; Ovarian Embryonal Carcinoma; Ovarian Mixed Germ Cell Tumor; Ovarian Teratoma; Ovarian Yolk Sac Tumor; Testicular Choriocarcinoma; Testicular Embryonal Carcinoma; Testicular Seminoma; Testicular Teratoma; Testicular Yolk Sac Tumor

  15. Primordial germ cells in the embryos of Medaka fish.

    PubMed

    Ijiri, K; Narita, T; Mizuno, R

    1996-10-01

    In the second International Microgravity Laboratory (IML-2) mission in 1994, four small Japanese killifish (Medaka, Oryzias latipes) made a space travel of 15 days aboard a space shuttle. These four adult Medaka fish successfully mated in space for the first time among vertebrate animals. Moreover, the eggs they laid developed normally, at least in their external appearance, hatching as fry (baby fish) in space. Fish mated and laid eggs every day during the first week. Near the end of the mission most of the eggs had a well-developed body with two pigmented eyes. In total, 43 eggs were laid (detected), out of which 8 fry hatched in space, as truly 'space-originated' babies. A further 30 fry hatched within 3 days after landing. This is the normal hatching rate, compared with the ground-based data. Among the 8 space-originated fry, four were killed for histological sections, and germ cells at the gonadal region were counted for each fry. Their numbers were in the range of the germ cells of the normal control fry (ground-kept samples). Thus, as embryos developed normally in their external appearance, inside the embryos the formation of primordial germ cells took place normally in space, and their migration to the genital ridges was not hindered by microgravity. The two of the remaining space-originated fry have grown up and been creating their offspring in the laboratory. This proved that the primordial germ cells formed in space were also normal from a functional point of view. The four space-travelled adult fish re-started mating and laying eggs on the 7th day after landing and continued to do so every day afterward. Fertilization rate and hatchability of these eggs were as high as the eggs laid by the laboratory-kept fish. This fact implies that in gametogenesis of adult fish, there are no specific stages of germ cells extremely susceptible to microgravity.

  16. Protective effect of quercetin on cadmium-induced oxidative toxicity on germ cells in male mice.

    PubMed

    Bu, Tongliang; Mi, Yuling; Zeng, Weidong; Zhang, Caiqiao

    2011-03-01

    Cadmium is a toxic heavy metal that is widely distributed in the environment. As a critical process, oxidative toxicity mediates the morphological and functional damages in germ cells after cadmium exposure. In this study, the protective effect of quercetin on cadmium-induced oxidative toxicity was investigated in mouse testicular germ cells. After oral administration of cadmium chloride at 4 mg/kg body weight for 2 weeks, damages in spermatozoa occurred in the early stage of spermatogenesis. Cadmium treatment significantly decreased the testicular antioxidant system, including decreases in the glutathione (GSH) level, superoxide dismutase (SOD), and GSH peroxidase (GSH-Px) activities. Moreover, exposure to cadmium resulted in an increase of hydrogen peroxide production and lipid peroxidation in testes. In addition, cadmium provoked germ cell apoptosis by upregulating expression of the proapoptotic proteins Bax and caspase-3 and downregulating expression of the antiapoptotic protein Bcl-XL. However, combined administration of a common flavonoid quercetin at 75 mg/kg body weight significantly attenuated cadmium-induced germ cell apoptosis by suppressing the hydrogen peroxide production and lipid peroxidation in testicular tissue. Simultaneous supplementation of quercetin markedly restored the decrease in GSH level and SOD and GSH-Px activities elicited by cadmium treatment. Additionally, quercetin protected germ cells from cadmium-induced apoptosis by downregulating the expression of Bax and caspase-3 and upregulating Bcl-XL expression. These results indicate that quercetin, due to its antioxidative and antiapoptotic characters, may manifest effective protective action against cadmium-induced oxidative toxicity in mouse testicular germ cells. PMID:21337715

  17. Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile.

    PubMed

    Sugawa, Fumihiro; Araúzo-Bravo, Marcos J; Yoon, Juyong; Kim, Kee-Pyo; Aramaki, Shinya; Wu, Guangming; Stehling, Martin; Psathaki, Olympia E; Hübner, Karin; Schöler, Hans R

    2015-04-15

    Primordial germ cells (PGCs) develop only into sperm and oocytes in vivo. The molecular mechanisms underlying human PGC specification are poorly understood due to inaccessibility of cell materials and lack of in vitro models for tracking the earliest stages of germ cell development. Here, we describe a defined and stepwise differentiation system for inducing pre-migratory PGC-like cells (PGCLCs) from human pluripotent stem cells (PSCs). In response to cytokines, PSCs differentiate first into a heterogeneous mesoderm-like cell population and then into PGCLCs, which exhibit minimal PRDM14 expression. PGC specification in humans is similar to the murine process, with the sequential activation of mesodermal and PGC genes, and the suppression of neural induction and of de novo DNA methylation, suggesting that human PGC formation is induced via epigenesis, the process of germ cell specification via inductive signals from surrounding somatic cells. This study demonstrates that PGC commitment in humans shares key features with that of the mouse, but also highlights key differences, including transcriptional regulation during the early stage of human PGC development (3-6 weeks). A more comprehensive understanding of human germ cell development may lead to methodology for successfully generating PSC-derived gametes for reproductive medicine.

  18. Melatonin alleviates cadmium-induced cellular stress and germ cell apoptosis in testes.

    PubMed

    Ji, Yan-Li; Wang, Hua; Meng, Can; Zhao, Xian-Feng; Zhang, Cheng; Zhang, Ying; Zhao, Mei; Chen, Yuan-Hua; Meng, Xiu-Hong; Xu, De-Xiang

    2012-01-01

    Increasing evidence demonstrates that melatonin has an anti-apoptotic effect in somatic cells. However, whether melatonin can protect against germ cell apoptosis remains obscure. Cadmium (Cd) is a testicular toxicant and induces germ cell apoptosis. In this study, we investigated the effects of melatonin on Cd-evoked germ cell apoptosis in testes. Male ICR mice were intraperitoneally (i.p.) injected with melatonin (5 mg/kg) every 8 hr, beginning at 8 hr before CdCl(2) (2.0 mg/kg, i.p.). As expected, acute Cd exposure resulted in germ cell apoptosis in testes, as determined by terminal dUTP nick-end labeling (TUNEL) staining. Melatonin significantly alleviated Cd-induced testicular germ cell apoptosis. An additional experiment showed that spliced form of XBP-1, the target of the IRE-1 pathway, was significantly increased in testes of mice injected with CdCl(2). GRP78, an endoplasmic reticulum (ER) chaperone, and CHOP, a downstream target of the PERK pathway, were upregulated in testes of Cd-treated mice. In addition, acute Cd exposure significantly increased testicular eIF2α and JNK phosphorylation, indicating that the unfolded protein response (UPR) pathway was activated by CdCl(2). Interestingly, melatonin almost completely inhibited Cd-induced ER stress and the UPR in testes. In addition, melatonin obviously attenuated Cd-induced heme oxygenase (HO)-1 expression and protein nitration in testes. Taken together, these results suggest that melatonin alleviates Cd-induced cellular stress and germ cell apoptosis in testes. Melatonin may be useful as pharmacological agents to protect against Cd-induced testicular toxicity. PMID:21793897

  19. Ghrelin modulates testicular germ cells apoptosis and proliferation in adult normal rats

    SciTech Connect

    Kheradmand, Arash; Dezfoulian, Omid; Alirezaei, Masoud; Rasoulian, Bahram

    2012-03-09

    , ghrelin balanced Bax/Bcl-2 ratio toward at increase of Bax level in the spermatocytes and therefore may stimulate apoptosis in these germ cells. In contrast, ghrelin administration significantly suppressed proliferation-associated peptide PCNA in the spermatocytes as well as spermatogonia (P < 0.05). Whereas, caspase-3 activity did not show any marked alteration during the experiment in both groups (P > 0.05). Upstream of Bax substance parallel to down-regulation of PCNA demonstrate that ghrelin may prevent massive accumulation of germ cells during normal spermatogenesis. These observations also indicate that ghrelin may be considered as a modulator of spermatogenesis in normal adult rats and could be potentially implicated for abnormal spermatogenesis in some testicular germ cell tumors.

  20. Formation and cultivation of medaka primordial germ cells.

    PubMed

    Li, Zhendong; Li, Mingyou; Hong, Ni; Yi, Meisheng; Hong, Yunhan

    2014-07-01

    Primordial germ cell (PGC) formation is pivotal for fertility. Mammalian PGCs are epigenetically induced without the need for maternal factors and can also be derived in culture from pluripotent stem cells. In egg-laying animals such as Drosophila and zebrafish, PGCs are specified by maternal germ plasm factors without the need for inducing factors. In these organisms, PGC formation and cultivation in vitro from indeterminate embryonic cells have not been possible. Here, we report PGC formation and cultivation in vitro from blastomeres dissociated from midblastula embryos (MBEs) of the fish medaka (Oryzias latipes). PGCs were identified by using germ-cell-specific green fluorescent protein (GFP) expression from a transgene under the control of the vasa promoter. Embryo perturbation was exploited to study PGC formation in vivo, and dissociated MBE cells were cultivated under various conditions to study PGC formation in vitro. Perturbation of somatic development did not prevent PGC formation in live embryos. Dissociated MBE blastomeres formed PGCs in the absence of normal somatic structures and of known inducing factors. Most importantly, under culture conditions conducive to stem cell derivation, some dissociated MBE blastomeres produced GFP-positive PGC-like cells. These GFP-positive cells contained genuine PGCs, as they expressed PGC markers and migrated into the embryonic gonad to generate germline chimeras. Our data thus provide evidence for PGC preformation in medaka and demonstrate, for the first time, that PGC formation and derivation can be obtained in culture from early embryos of medaka as a lower vertebrate model.

  1. Are testicular mast cells involved in the regulation of germ cells in man?

    PubMed

    Windschüttl, S; Nettersheim, D; Schlatt, S; Huber, A; Welter, H; Schwarzer, J U; Köhn, F M; Schorle, H; Mayerhofer, A

    2014-07-01

    Protease activated receptor-2 (PAR-2) is the receptor for the prototype mast cell product tryptase. PAR-2 expression by cells of the human germinal epithelium was reported, but the exact cellular sites of testicular expression remained unknown. That became of interest, because mast cells, expressing tryptase, were found in the walls of seminiferous tubules of patients suffering from sub- and infertility. This location suggested that mast cells via tryptase might be able to influence PAR-2-expressing cells in the germinal epithelium. To explore these points, we used testicular paraffin-embedded sections for immunohistochemistry. PAR-2-positive cells were mostly basally located cells of the seminiferous epithelium, namely spermatogonia. Some stained for the receptor for GDNF (GFRalpha-1), and possibly represent spermatogonial stem cells (SSCs). As true human SSCs could not be examined, we turned to TCam-2 seminoma cells, expressing PAR-2 and stem cell markers, including GFRalpha-1. TCam-2 cells robustly responded to stimulation with a specific PAR-2 agonist (SLIGKV) by increased intracellular Ca(2+) levels. Recombinant tryptase and trypsin, but not a control peptide (VKGILS) evoked this response, implying functional PAR-2. Video imaging and caspase 3/7 assays showed that SLIGKV and tryptase prevented spontaneous apoptosis and increased proliferation of TCam-2 cells. The expression of the marker of pluripotency OCT3/4 was unchanged upon activation of PAR-2, suggesting that the stem cell-like character is not changed. Furthermore, human germ cell cancers were examined. A subset of seminoma and carcinoma in situ samples expressed PAR-2, indicating that yet unknown subgroups exist. Collectively, the descriptive data obtained in human testicular sections, in germ cell cancers and the functional results in TCam-2 cells imply a trophic role of mast cell-derived tryptase for human germ cells. This may be relevant for subtypes of human germ cell cancers, and possibly SSCs. It

  2. Regulative germ cell specification in axolotl embryos: a primitive trait conserved in the mammalian lineage.

    PubMed

    Johnson, Andrew D; Crother, Brian; White, Mary E; Patient, Roger; Bachvarova, Rosemary F; Drum, Matthew; Masi, Thomas

    2003-08-29

    How germ cells are specified in the embryos of animals has been a mystery for decades. Unlike most developmental processes, which are highly conserved, embryos specify germ cells in very different ways. Curiously, in mouse embryos germ cells are specified by extracellular signals; they are not autonomously specified by maternal germ cell determinants (germ plasm), as are the germ cells in most animal model systems. We have developed the axolotl (Ambystoma mexicanum), a salamander, as an experimental system, because classic experiments have shown that the germ cells in this species are induced by extracellular signals in the absence of germ plasm. Here, we provide evidence that the germ cells in axolotls arise from naive mesoderm in response to simple inducing agents. In addition, by analysing the sequences of axolotl germ-cell-specific genes, we provide evidence that mice and urodele amphibians share a common mechanism of germ cell development that is ancestral to tetrapods. Our results imply that germ plasm, as found in species such as frogs and teleosts, is the result of convergent evolution. We discuss the evolutionary implications of our findings. PMID:14511484

  3. The Magea gene cluster regulates male germ cell apoptosis without affecting the fertility in mice.

    PubMed

    Hou, Siyuan; Xian, Li; Shi, Peiliang; Li, Chaojun; Lin, Zhaoyu; Gao, Xiang

    2016-01-01

    While apoptosis is essential for male germ cell development, improper activation of apoptosis in the testis can affect spermatogenesis and cause reproduction defects. Members of the MAGE-A (melanoma antigen family A) gene family are frequently clustered in mammalian genomes and are exclusively expressed in the testes of normal animals but abnormally activated in a wide variety of cancers. We investigated the potential roles of these genes in spermatogenesis by generating a mouse model with a 210-kb genomic deletion encompassing six members of the Magea gene cluster (Magea1, Magea2, Magea3, Magea5, Magea6 and Magea8). Male mice carrying the deletion displayed smaller testes from 2 months old with a marked increase in apoptotic germ cells in the first wave of spermatogenesis. Furthermore, we found that Magea genes prevented stress-induced spermatogenic apoptosis after N-ethyl-N-nitrosourea (ENU) treatment during the adult stage. Mechanistically, deletion of the Magea gene cluster resulted in a dramatic increase in apoptotic germ cells, predominantly spermatocytes, with activation of p53 and induction of Bax in the testes. These observations demonstrate that the Magea genes are crucial in maintaining normal testicular size and protecting germ cells from excessive apoptosis under genotoxic stress. PMID:27226137

  4. The Magea gene cluster regulates male germ cell apoptosis without affecting the fertility in mice

    PubMed Central

    Hou, Siyuan; Xian, Li; Shi, Peiliang; Li, Chaojun; Lin, Zhaoyu; Gao, Xiang

    2016-01-01

    While apoptosis is essential for male germ cell development, improper activation of apoptosis in the testis can affect spermatogenesis and cause reproduction defects. Members of the MAGE-A (melanoma antigen family A) gene family are frequently clustered in mammalian genomes and are exclusively expressed in the testes of normal animals but abnormally activated in a wide variety of cancers. We investigated the potential roles of these genes in spermatogenesis by generating a mouse model with a 210-kb genomic deletion encompassing six members of the Magea gene cluster (Magea1, Magea2, Magea3, Magea5, Magea6 and Magea8). Male mice carrying the deletion displayed smaller testes from 2 months old with a marked increase in apoptotic germ cells in the first wave of spermatogenesis. Furthermore, we found that Magea genes prevented stress-induced spermatogenic apoptosis after N-ethyl-N-nitrosourea (ENU) treatment during the adult stage. Mechanistically, deletion of the Magea gene cluster resulted in a dramatic increase in apoptotic germ cells, predominantly spermatocytes, with activation of p53 and induction of Bax in the testes. These observations demonstrate that the Magea genes are crucial in maintaining normal testicular size and protecting germ cells from excessive apoptosis under genotoxic stress. PMID:27226137

  5. A conserved chromatin architecture marks and maintains the restricted germ cell lineage in worms and flies.

    PubMed

    Schaner, Christine E; Deshpande, Girish; Schedl, Paul D; Kelly, William G

    2003-11-01

    In C. elegans, mRNA production is initially repressed in the embryonic germline by a protein unique to C. elegans germ cells, PIE-1. PIE-1 is degraded upon the birth of the germ cell precursors, Z2 and Z3. We have identified a chromatin-based mechanism that succeeds PIE-1 repression in these cells. A subset of nucleosomal histone modifications, methylated lysine 4 on histone H3 (H3meK4) and acetylated lysine 8 on histone H4 (H4acetylK8), are globally lost and the DNA appears more condensed. This coincides with PIE-1 degradation and requires that germline identity is not disrupted. Drosophila pole cell chromatin also lacks H3meK4, indicating that a unique chromatin architecture is a conserved feature of embryonic germ cells. Regulation of the germline-specific chromatin architecture requires functional nanos activity in both organisms. These results indicate that genome-wide repression via a nanos-regulated, germ cell-specific chromatin organization is a conserved feature of germline maintenance during embryogenesis.

  6. Positive mRNA Translational Control in Germ Cells by Initiation Factor Selectivity

    PubMed Central

    Friday, Andrew J.; Keiper, Brett D.

    2015-01-01

    Ultimately, the production of new proteins in undetermined cells pushes them to new fates. Other proteins hold a stem cell in a mode of self-renewal. In germ cells, these decision-making proteins are produced largely from translational control of preexisting mRNAs. To date, all of the regulation has been attributed to RNA binding proteins (RBPs) that repress mRNAs in many models of germ cell development (Drosophila, mouse, C. elegans, and Xenopus). In this review, we focus on the selective, positive function of translation initiation factors eIF4E and eIF4G, which recruit mRNAs to ribosomes upon derepression. Evidence now shows that the two events are not separate but rather are coordinated through composite complexes of repressors and germ cell isoforms of eIF4 factors. Strikingly, the initiation factor isoforms are themselves mRNA selective. The mRNP complexes of translation factors and RBPs are built on specific populations of mRNAs to prime them for subsequent translation initiation. Simple rearrangement of the partners causes a dormant mRNP to become synthetically active in germ cells when and where they are required to support gametogenesis. PMID:26357652

  7. Ectopic Expression of Testis Germ Cell Proteins in Cancer and Its Potential Role in Genomic Instability.

    PubMed

    Nielsen, Aaraby Yoheswaran; Gjerstorff, Morten Frier

    2016-01-01

    Genomic instability is a hallmark of human cancer and an enabling factor for the genetic alterations that drive cancer development. The processes involved in genomic instability resemble those of meiosis, where genetic material is interchanged between homologous chromosomes. In most types of human cancer, epigenetic changes, including hypomethylation of gene promoters, lead to the ectopic expression of a large number of proteins normally restricted to the germ cells of the testis. Due to the similarities between meiosis and genomic instability, it has been proposed that activation of meiotic programs may drive genomic instability in cancer cells. Some germ cell proteins with ectopic expression in cancer cells indeed seem to promote genomic instability, while others reduce polyploidy and maintain mitotic fidelity. Furthermore, oncogenic germ cell proteins may indirectly contribute to genomic instability through induction of replication stress, similar to classic oncogenes. Thus, current evidence suggests that testis germ cell proteins are implicated in cancer development by regulating genomic instability during tumorigenesis, and these proteins therefore represent promising targets for novel therapeutic strategies.

  8. Cryopreservation of specialized chicken lines using cultured primordial germ cells.

    PubMed

    Nandi, S; Whyte, J; Taylor, L; Sherman, A; Nair, V; Kaiser, P; McGrew, M J

    2016-08-01

    Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells ( PGCS: ). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- ( MHC-: ) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs. PMID:27099306

  9. PGC-Enriched miRNAs Control Germ Cell Development

    PubMed Central

    Bhin, Jinhyuk; Jeong, Hoe-Su; Kim, Jong Soo; Shin, Jeong Oh; Hong, Ki Sung; Jung, Han-Sung; Kim, Changhoon; Hwang, Daehee; Kim, Kye-Seong

    2015-01-01

    Non-coding microRNAs (miRNAs) regulate the translation of target messenger RNAs (mRNAs) involved in the growth and development of a variety of cells, including primordial germ cells (PGCs) which play an essential role in germ cell development. However, the target mRNAs and the regulatory networks influenced by miRNAs in PGCs remain unclear. Here, we demonstrate a novel miRNAs control PGC development through targeting mRNAs involved in various cellular pathways. We reveal the PGC-enriched expression patterns of nine miRNAs, including miR-10b, -18a, -93, -106b, -126-3p, -127, -181a, -181b, and -301, using miRNA expression analysis along with mRNA microarray analysis in PGCs, embryonic gonads, and postnatal testes. These miRNAs are highly expressed in PGCs, as demonstrated by Northern blotting, miRNA in situ hybridization assay, and miRNA qPCR analysis. This integrative study utilizing mRNA microarray analysis and miRNA target prediction demonstrates the regulatory networks through which these miRNAs regulate their potential target genes during PGC development. The elucidated networks of miRNAs disclose a coordinated molecular mechanism by which these miRNAs regulate distinct cellular pathways in PGCs that determine germ cell development. PMID:26442865

  10. NUT protein immunoreactivity in ovarian germ cell tumours.

    PubMed

    Iacobelli, J F; Charles, A K; Crook, M; Stewart, C J R

    2015-02-01

    The aim of this study was to investigate NUT (nuclear protein in the testis) expression in ovarian germ cell tumours (GCTs). Immunostaining for NUT protein was performed in 10 mature cystic teratomas and in 49 malignant ovarian GCTs including 15 pure dysgerminomas, six dysgerminomas associated with gonadoblastoma, nine yolk sac tumours, 12 immature teratomas, and seven mixed malignant tumours. Only nuclear staining was considered a positive finding although cytoplasmic staining was noted when present. Thirty-seven (76%) malignant GCTs were NUT positive but staining was usually of weak to moderate intensity and observed in a relatively small proportion of neoplastic cells. Staining in immature teratomas and yolk sac tumours was restricted to foci of hepatoid and intestinal/glandular differentiation, where both nuclear and cytoplasmic reactivity were observed. In dysgerminoma associated with gonadoblastoma only the in situ and invasive germ cell elements were NUT positive. Nuclear staining was not seen in benign teratomas. Most malignant ovarian GCTs express NUT protein, albeit focally, and this should be considered when evaluating immunostaining in the differential diagnosis of poorly differentiated malignancies, particularly NUT midline carcinoma. Since NUT protein appears to play a role in normal germ cell maturation it may influence intestinal or hepatoid differentiation within malignant GCTs.

  11. Cryopreservation of specialized chicken lines using cultured primordial germ cells

    PubMed Central

    Nandi, S.; Whyte, J.; Taylor, L.; Sherman, A.; Nair, V.; Kaiser, P.; McGrew, M. J.

    2016-01-01

    Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells (PGCs). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- (MHC-) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs. PMID:27099306

  12. FGF, Insulin, and SMAD Signaling Cooperate for Avian Primordial Germ Cell Self-Renewal

    PubMed Central

    Whyte, Jemima; Glover, James D.; Woodcock, Mark; Brzeszczynska, Joanna; Taylor, Lorna; Sherman, Adrian; Kaiser, Pete; McGrew, Michael J.

    2015-01-01

    Summary Precise self-renewal of the germ cell lineage is fundamental to fertility and reproductive success. The early precursors for the germ lineage, primordial germ cells (PGCs), survive and proliferate in several embryonic locations during their migration to the embryonic gonad. By elucidating the active signaling pathways in migratory PGCs in vivo, we were able to create culture conditions that recapitulate this embryonic germ cell environment. In defined medium conditions without feeder cells, the growth factors FGF2, insulin, and Activin A, signaling through their cognate-signaling pathways, were sufficient for self-renewal of germline-competent PGCs. Forced expression of constitutively active MEK1, AKT, and SMAD3 proteins could replace their respective upstream growth factors. Unexpectedly, we found that BMP4 could replace Activin A in non-clonal growth conditions. These defined medium conditions identify the key molecular pathways required for PGC self-renewal and will facilitate efforts in biobanking of chicken genetic resources and genome editing. PMID:26677769

  13. Radiation-induced bystander signaling from somatic cells to germ cells in Caenorhabditis elegans.

    PubMed

    Guo, Xiaoying; Sun, Jie; Bian, Po; Chen, Lianyun; Zhan, Furu; Wang, Jun; Xu, An; Wang, Yugang; Hei, Tom K; Wu, Lijun

    2013-09-01

    Recently, radiation-induced bystander effects (RIBE) have been studied in mouse models in vivo, which clearly demonstrated bystander effects among somatic cells. However, there is currently no evidence for RIBE between somatic cells and germ cells in animal models in vivo. In the current study, the model animal Caenorhabditis elegans was used to investigate the bystander signaling from somatic cells to germ cells, as well as underlying mechanisms. C. elegans body size allows for precise microbeam irradiation and the abundant mutant strains for genetic dissection relative to currently adopted mouse models make it ideal for such analysis. Our results showed that irradiation of posterior pharynx bulbs and tails of C. elegans enhanced the level of germ cell apoptosis in bystander gonads. The irradiation of posterior pharynx bulbs also increased the level of DNA damage in bystander germ cells and genomic instability in the F1 progeny of irradiated worms, suggesting a potential carcinogenic risk in progeny even only somatic cells of parents are exposed to ionizing radiation (IR). It was also shown that DNA damage-induced germ cell death machinery and MAPK signaling pathways were both involved in the induction of germ cell apoptosis by microbeam induced bystander signaling, indicating a complex cooperation among multiple signaling pathways for bystander effects from somatic cells to germ cells.

  14. Lifetime stress experience: transgenerational epigenetics and germ cell programming.

    PubMed

    Bale, Tracy L

    2014-09-01

    The transgenerational epigenetic programming involved in the passage of environmental exposures to stressful periods from one generation to the next has been examined in human populations, and mechanistically in animal models. Epidemiological studies suggest that gestational exposures to environmental factors including stress are strongly associated with an increased risk of neurodevelopmental disorders, including attention deficit-hyperactivity disorder, schizophrenia, and autism spectrum disorders. Both maternal and paternal life experiences with stress can be passed on to offspring directly during pregnancy or through epigenetic marks in the germ cell. Animal models of parental stress have examined relevant offspring phenotypes and transgenerational outcomes, and provided unique insight into the germ cell epigenetic changes associated with disruptions in neurodevelopment. Understanding germline susceptibility to exogenous signals during stress exposure and the identification of the types of epigenetic marks is critical for defining mechanisms underlying disease risk.

  15. Mediastinal germ cell tumors: a radiologic-pathologic review.

    PubMed

    Drevelegas, A; Palladas, P; Scordalaki, A

    2001-01-01

    Germ cell tumors of the mediastinum are histologically identical to those found in the testes and ovaries. Early diagnosis and treatment improve the survival rate. Imaging studies of teratoma demonstrate a rounded, often lobulated heterogeneous mass containing soft tissue elements with fluid and fat attenuation. Calcification is present in 20-43% of cases. Seminomas are large masses of homogeneous soft tissue attenuation. Malignant nonseminomatous germ cell tumors are heterogeneous tumors with irregular borders due to invasion of adjacent structures. CT shows the location and extent of the tumors as well as intrinsic elements including soft tissue, fat, fluid, and calcification. CT is the modality of choice for the diagnostic evaluation of these tumors. MRI reveals masses of heterogeneous signal intensity, is more sensitive in depicting infiltration of the adjacent structures by fat plane obliteration, and is performed as an ancillary study.

  16. Lifetime stress experience: transgenerational epigenetics and germ cell programming

    PubMed Central

    Bale, Tracy L.

    2014-01-01

    The transgenerational epigenetic programming involved in the passage of environmental exposures to stressful periods from one generation to the next has been examined in human populations, and mechanistically in animal models. Epidemiological studies suggest that gestational exposures to environmental factors including stress are strongly associated with an increased risk of neurodevelopmental disorders, including attention deficit-hyperactivity disorder, schizophrenia, and autism spectrum disorders. Both maternal and paternal life experiences with stress can be passed on to offspring directly during pregnancy or through epigenetic marks in the germ cell. Animal models of parental stress have examined relevant offspring phenotypes and transgenerational outcomes, and provided unique insight into the germ cell epigenetic changes associated with disruptions in neurodevelopment. Understanding germline susceptibility to exogenous signals during stress exposure and the identification of the types of epigenetic marks is critical for defining mechanisms underlying disease risk. PMID:25364281

  17. Germ cell transplantation: a potential treatment of severe testicular failure.

    PubMed

    Cozzolino, D J; Lamb, D J

    2000-12-01

    Although the process of spermatogenesis is relatively efficient and resistant to damage, male infertility can result from exposure to toxic agents such as chemotherapeutic regimes, radiation, or occupational exposures to chemicals. Other types of infertility may result from migratory defects or poor survival of primordial germ cells during development, abnormal repopulation of the tubules by spermatogonia during development, or low cellularity of the testis (hypospermatogenesis). Presently, there are no effective therapies available to treat these patients. Recent studies in animal models have demonstrated that isolated testicular germ cells collected from testes may be transplanted into sterile recipient mice to regenerate spermatogenesis. This technology will have widespread applications in efforts to manipulate the genome and produce transgenic offspring, to improve agricultural species, to enhance sperm production in endangered species, to improve our understanding of the control mechanisms regulating spermatogenesis, and to treat male infertility.

  18. Intensive chemotherapy as salvage treatment for solid tumors: focus on germ cell cancer

    PubMed Central

    Selle, F.; Gligorov, J.; Richard, S.; Khalil, A.; Alexandre, I.; Avenin, D.; Provent, S.; Soares, D.G.; Lotz, J.P.

    2014-01-01

    Germ cell tumors present contrasting biological and molecular features compared to many solid tumors, which may partially explain their unusual sensitivity to chemotherapy. Reduced DNA repair capacity and enhanced induction of apoptosis appear to be key factors in the sensitivity of germ cell tumors to cisplatin. Despite substantial cure rates, some patients relapse and subsequently die of their disease. Intensive doses of chemotherapy are used to counter mechanisms of drug resistance. So far, high-dose chemotherapy with hematopoietic stem cell support for solid tumors is used only in the setting of testicular germ cell tumors. In that indication, high-dose chemotherapy is given as the first or late salvage treatment for patients with either relapsed or progressive tumors after initial conventional salvage chemotherapy. High-dose chemotherapy is usually given as two or three sequential cycles using carboplatin and etoposide with or without ifosfamide. The administration of intensive therapy carries significant side effects and can only be efficiently and safely conducted in specialized referral centers to assure optimum patient care outcomes. In breast and ovarian cancer, most studies have demonstrated improvement in progression-free survival (PFS), but overall survival remained unchanged. Therefore, most of these approaches have been dropped. In germ cell tumors, clinical trials are currently investigating novel therapeutic combinations and active treatments. In particular, the integration of targeted therapies constitutes an important area of research for patients with a poor prognosis. PMID:25493378

  19. DAZL limits pluripotency, differentiation, and apoptosis in developing primordial germ cells.

    PubMed

    Chen, Hsu-Hsin; Welling, Maaike; Bloch, Donald B; Muñoz, Javier; Mientjes, Edwin; Chen, Xinjie; Tramp, Cody; Wu, Jie; Yabuuchi, Akiko; Chou, Yu-Fen; Buecker, Christa; Krainer, Adrian; Willemsen, Rob; Heck, Albert J; Geijsen, Niels

    2014-11-11

    The scarcity of primordial germ cells (PGCs) in the developing mammalian embryo hampers robust biochemical analysis of the processes that underlie early germ cell formation. Here, we demonstrate that DAZL, a germ cell-specific RNA binding protein, is a robust PGC marker during in vitro germ cell development. Using Dazl-GFP reporter ESCs, we demonstrate that DAZL plays a central role in a large mRNA/protein interactive network that blocks the translation of core pluripotency factors, including Sox2 and Sall4, as well as of Suz12, a polycomb family member required for differentiation of pluripotent cells. Thus, DAZL limits both pluripotency and somatic differentiation in nascent PGCs. In addition, we observed that DAZL associates with mRNAs of key Caspases and similarly inhibits their translation. This elegant fail-safe mechanism ensures that, whereas loss of DAZL results in prolonged expression of pluripotency factors, teratoma formation is avoided due to the concomitant activation of the apoptotic cascade. PMID:25418731

  20. Endobronchial metastasis of mixed germ cell tumors: two cases.

    PubMed

    Öztürk, Ayperi; Aktaş, Zafer; Yılmaz, Aydın

    2016-06-01

    Lung metastases from extrapulmonary malignancies are common however endobronchial metastases (EBM) from nonpulmonary neoplasms are rare. A variety of extrathoracic tumors have a tendency to EBM especially breast, colon, and renal carcinomas are most frequent reported tumors however EBM of germ cell tumors are extremely rare. A 39-year-old and a 27-year-old male patient were admitted to our hospital with hemoptysis and dyspnea at different times. Both of them had a history of left orchiectomy due to mixed germ cell tumor two years and one year ago, respectively. On chest X-Ray and thorax computed tomography, first had a right upper lobe atelectasis and second had right total atelectasis. Fiberoptic bronchoscopy (FOB) performed and a vascularized endobronchial lesion (EBL) which tended to bleed was seen in the orifis of right upper lobe in the first case and right main bronchus was totally obstructed by EBL also in the second. Interventional bronchoscopy was performed via rigid bronchoscopy for biopsy and palliative treatment (argon plasma coagulation and debulking) in both two patients because of tendency to bleeding. A partial aperture was achieved at right upper lobe bronchus in the first case and total atelectasis resolved in the second case. Immunohistochemically, histopathological examinations of both patients biopsies confirmed EBM of mixed germ cell tumors. In conclusion, EBM of the germ cell tumors especially with total or partial atelectasis are extremely rare. We want to present these cases to emphasize the importance of distinguishing EBM from primary lung carcinoma which treatment and survival could be different. PMID:27481085

  1. Detection of phase specificity of in vivo germ cell mutagens in an in vitro germ cell system.

    PubMed

    Habas, Khaled; Anderson, Diana; Brinkworth, Martin

    2016-04-15

    In vivo tests for male reproductive genotoxicity are time consuming, resource-intensive and their use should be minimised according to the principles of the 3Rs. Accordingly, we investigated the effects in vitro, of a variety of known, phase-specific germ cell mutagens, i.e., pre-meiotic, meiotic, and post-meiotic genotoxins, on rat spermatogenic cell types separated using Staput unit-gravity velocity sedimentation, evaluating DNA damage using the Comet assay. N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU) (spermatogenic phase), 6-mercaptopurine (6-MP) and 5-bromo-2'-deoxy-uridine (5-BrdU) (meiotic phase), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS) (post-meiotic phase) were selected for use as they are potent male rodent, germ cell mutagens in vivo. DNA damage was detected directly using the Comet assay and indirectly using the TUNEL assay. Treatment of the isolated cells with ENU and MNU produced the greatest concentration-related increase in DNA damage in spermatogonia. Spermatocytes were most sensitive to 6-MP and 5-BrdU while spermatids were particularly susceptible to MMS and EMS. Increases were found when measuring both Olive tail moment (OTM) and% tail DNA, but the greatest changes were in OTM. Parallel results were found with the TUNEL assay, which showed highly significant, concentration dependent effects of all these genotoxins on spermatogonia, spermatocytes and spermatids in the same way as for DNA damage. The specific effects of these chemicals on different germ cell types matches those produced in vivo. This approach therefore shows potential for use in the detection of male germ cell genotoxicity and could contribute to the reduction of the use of animals in such toxicity assays. PMID:27059372

  2. The diversity of nanos expression in echinoderm embryos supports different mechanisms in germ cell specification.

    PubMed

    Fresques, Tara; Swartz, Steven Zachary; Juliano, Celina; Morino, Yoshiaki; Kikuchi, Mani; Akasaka, Koji; Wada, Hiroshi; Yajima, Mamiko; Wessel, Gary M

    2016-07-01

    Specification of the germ cell lineage is required for sexual reproduction in all animals. However, the timing and mechanisms of germ cell specification is remarkably diverse in animal development. Echinoderms, such as sea urchins and sea stars, are excellent model systems to study the molecular and cellular mechanisms that contribute to germ cell specification. In several echinoderm embryos tested, the germ cell factor Vasa accumulates broadly during early development and is restricted after gastrulation to cells that contribute to the germ cell lineage. In the sea urchin, however, the germ cell factor Vasa is restricted to a specific lineage by the 32-cell stage. We therefore hypothesized that the germ cell specification program in the sea urchin/Euechinoid lineage has evolved to an earlier developmental time point. To test this hypothesis we determined the expression pattern of a second germ cell factor, Nanos, in four out of five extant echinoderm clades. Here we find that Nanos mRNA does not accumulate until the blastula stage or later during the development of all other echinoderm embryos except those that belong to the Echinoid lineage. Instead, Nanos is expressed in a restricted domain at the 32-128 cell stage in Echinoid embryos. Our results support the model that the germ cell specification program underwent a heterochronic shift in the Echinoid lineage. A comparison of Echinoid and non-Echinoid germ cell specification mechanisms will contribute to our understanding of how these mechanisms have changed during animal evolution. PMID:27402572

  3. Development of germ cell neoplasia in situ in chinchilla rabbits.

    PubMed

    Vigueras-Villaseñor, Rosa María; Montelongo Solís, Paola; Chávez-Saldaña, Margarita; Gutiérrez-Pérez, Oscar; Cortés Trujillo, Lucero; Rojas-Castañeda, Julio César

    2016-05-01

    The present study was designed to describe the development of germ cell neoplasia in situ in Chinchilla rabbit by administration of estradiol. The study was performed in rabbits distributed into two groups: control and 17 β-estradiol. The determination of histological alterations and POU5F1 and c-kit proteins employed as biomarkers for the diagnosis of this neoplasia was carried out. Testicular descent and complete spermatogenesis were observed in the control group. The protein biomarkers were negative. However, in the rabbits treated with estradiol, the testes remained undescended with the gonocytes undifferentiated to spermatogonia. There were histological lesions owing to germ cell neoplasia in situ and positive to POU5F1 and c-kit proteins. These findings indicate that the chinchilla rabbit is an ideal model to study this neoplasia in which the histological characteristics and biomarkers of the disease could be clearly observed. Using this model we suggested that the persisting gonocytes could be responsible for the development of germ cell neoplasia in situ. PMID:26617392

  4. POMB/ACE chemotherapy for mediastinal germ cell tumours.

    PubMed

    Bower, M; Brock, C; Holden, L; Nelstrop, A; Makey, A R; Rustin, G J; Newlands, E S

    1997-05-01

    Mediastinal germ cell tumours (MGCT) are rare and most published series reflect the experiences of individual institutions over many years. Since 1979, we have treated 16 men (12 non-seminomatous germ cell tumours and 4 seminomas) with newly diagnosed primary MGCT with POMB/ACE chemotherapy and elective surgical resection of residual masses. This approach yielded complete remissions in 15/16 (94%) patients. The median follow-up was 6.0 years and no relapses occurred more than 2 years after treatment. The 5 year overall survival in the non-seminomatous germ cell tumours (NSGCT) is 73% (95% confidence interval 43-90%). One patient with NSGCT developed drug-resistant disease and died without achieving remission and 2 patients died of relapsed disease. In addition, 4 patients with bulky and/or metastatic seminoma were treated with POMB/ACE. One died of treatment-related neutropenic sepsis in complete remission and one died of relapsed disease. Finally, 4 patients (2 NSGCT and 2 seminomas) referred at relapse were treated with POMB/ACE and one was successfully salvaged. The combination of POMB/ACE chemotherapy and surgery is effective management for MGCT producing high long-term survival rates.

  5. Gonadal development and germ cell tumors in mouse and humans.

    PubMed

    Dolci, Susanna; Campolo, Federica; De Felici, Massimo

    2015-09-01

    In multicellular organisms, proper development of gonads and germ cells is essential for the transmission of genetic information to the next generations and eventually for the survival of the species. For this reason, germline development is finely regulated to control germ cell proliferation, survival and differentiation. Disruption of such controls can lead to infertility or germ cell tumors (GCTs). GCTs are particularly hideous pathologies since they occur mainly in neonates, infants, and children, rarely in the adults. They arise primarily in the testes and ovaries, though they can also develop in extragonadal sites along the midline of the body and the brain. Many similarities exist between most types of GCTs of the ovary and testis, including a morphological resemblance (often constituting a caricature of normal embryogenesis) and a similar pattern of chromosomal alterations. Furthermore, families with both ovarian and testicular GCTs have been reported, suggesting a possible common genetic etiology. This review focuses on the cellular processes, differentiation events and molecular mechanisms occurring during gonad development in mice and humans whose disturbance can be implicated in GCT formation.

  6. Germ cell formation from embryonic stem cells and the use of somatic cell nuclei in oocytes.

    PubMed

    Pelosi, Emanuele; Forabosco, Antonino; Schlessinger, David

    2011-03-01

    Embryonic stem cells (ESCs) have remarkable properties of pluripotency and self-renewal, along with the retention of chromosomal integrity. Germ cells function as a kind of "transgenerational stem cells," transmitting genetic information from one generation to the next. The formation of putative primordial germ cells (PGCs) and germ cells from mouse and human ESCs (hESCs) has, in fact, been shown, and the apparent derivation of functional mouse male gametes has also been described. Additionally, investigators have successfully reprogrammed somatic nuclei into a pluripotent state by inserting them into ESCs or oocytes. This would enable the generation of ESCs genetically identical to the somatic cell donor and their use in cell therapy. However, these methodologies are still inefficient and their mechanisms poorly understood. Until full comprehension of these processes is obtained, clinical applications remain remote. Nevertheless, they represent promising tools in the future, enhancing methods of therapeutic cloning and infertility treatment.

  7. Generation of viable fish from cryopreserved primordial germ cells.

    PubMed

    Kobayashi, Terumasa; Takeuchi, Yutaka; Takeuchi, Toshio; Yoshizaki, Goro

    2007-02-01

    An increasing number of wild fish species are in danger of extinction, often as a result of human activities. The cryopreservation of gametes and embryos has great potential for maintaining and restoring threatened species. The conservation of both paternal and maternal genetic information is essential. However, although this technique has been successfully applied to the spermatozoa of many fish species, reliable methods are lacking for the long-term preservation of fish eggs and embryos. Here, we describe a protocol for use with rainbow trout (Oncorhynchus mykiss) primordial germ cells (PGCs) and document the restoration of live fish from gametes derived from these cryopreserved progenitors. Genital ridges (GRs), which are embryonic tissues containing PGCs, were successfully cryopreserved in a medium containing 1.8 M ethylene glycol (EG). The thawed PGCs that were transplanted into the peritoneal cavities of allogenic trout hatchlings differentiated into mature spermatozoa and eggs in the recipient gonads. Furthermore, the fertilization of eggs derived from cryopreserved PGCs by cryopreserved spermatozoa resulted in the development of fertile F1 fish. This PGC cryopreservation technique represents a promising tool in efforts to save threatened fish species. Moreover, this approach has significant potential for maintaining domesticated fish strains carrying commercially valuable traits for aquaculture purposes.

  8. Prepubertal male rats with high rates of germ-cell apoptosis present exacerbated rates of germ-cell apoptosis after serotonin depletion.

    PubMed

    Méndez Palacios, Néstor; Escobar, María Elena Ayala; Mendoza, Maximino Méndez; Crispín, Rubén Huerta; Andrade, Octavio Guerrero; Melández, Javier Hernández; Martínez, Andrés Aragón

    2016-04-01

    Male germ-cell apoptosis occurs naturally and can be increased by exposure to drugs and toxic chemicals. Individuals may have different rates of apoptosis and are likely to also exhibit differential sensitivity to outside influences. Previously, we reported that p-chloroamphetamine (pCA), a substance that inhibits serotonin synthesis, induced germ-cell apoptosis in prepubertal male rats. Here, we identified prepubertal rats with naturally high or low rates of germ-cell apoptosis and evaluated gene expression in both groups. Bax and Shbg mRNA levels were higher in rats with high rates of germ-cell apoptosis. Rats were then treated with pCA and the neuro-hormonal response and gene expression were evaluated. Treatment with pCA induced a reduction in serotonin concentrations but levels of sex hormones and gonadotrophins were not changed. Rats with initially high rates of germ-cell apoptosis had even higher rates of germ-cell apoptosis after treatment with pCA. In rats with high rates of germ-cell apoptosis Bax mRNA expression remained high after treatment with pCA. On the basis of category, an inverse relationship between mRNA expression of Bax and Bcl2, Bax and AR and Bax and Hsd3b2 was found. Here we provide evidence that innate levels of germ-cell apoptosis could be explained by the level of mRNA expression of genes involved with apoptosis and spermatogenesis.

  9. Mechanisms guiding primordial germ cell migration: strategies from different organisms

    PubMed Central

    Richardson, Brian E.; Lehmann, Ruth

    2015-01-01

    Preface The regulated migration of cells is essential for development and tissue homeostasis, and aberrant cell migration can lead to an impaired immune response and the progression of cancer. Primordial germ cells (PGCs), precursors to sperm and eggs, have to migrate across the embryo to reach somatic gonadal precursors (SGPs) and fulfill their function. Studies of model organisms have revealed that, despite important differences, several features of PGC migration are conserved. PGCs require both an intrinsic motility program and external guidance cues to survive and successfully migrate. Proper guidance involves both attractive and repulsive cues mediated by protein and lipid signalling. PMID:20027186

  10. Phytochemical profile, antioxidant and cytotoxic activities of the carob tree (Ceratonia siliqua L.) germ flour extracts.

    PubMed

    Custódio, Luísa; Escapa, Ana Luísa; Fernandes, Eliana; Fajardo, Alba; Aligué, Rosa; Alberício, Fernando; Neng, Nuno; Nogueira, José Manuel Florêncio; Romano, Anabela

    2011-03-01

    This work aimed to evaluate the phytochemical content and to determine the antioxidant and cytotoxic activities of methanol extracts of the carob tree (Ceratonia siliqua L.) germ flour. The extracts were rich in phenolic compounds, had considerable antioxidant activity, and reduced the viability of cervical (HeLa) cancer cells. The chemical content and the biological activities of the extracts were significantly affected by gender and cultivar. Female cultivar Galhosa had the highest levels of phenolic compounds, and the highest antioxidant activity. Extracts from the hermaphrodite trees and from the female cultivars Galhosa and Costela/Canela exhibited the highest cytotoxic activity. The most abundant compound was theophylline. The phenolic content was correlated to both antioxidant and cytotoxic activities. Our findings provide new knowledge about the health implications of consuming food supplemented with carob germ flour.

  11. A mutation of cdc-25.1 causes defects in germ cells but not in somatic tissues in C. elegans.

    PubMed

    Kim, Jiyoung; Lee, Ah-Reum; Kawasaki, Ichiro; Strome, Susan; Shim, Yhong-Hee

    2009-07-31

    By screening C. elegans mutants for severe defects in germline proliferation, we isolated a new loss-of-function allele of cdc-25.1, bn115. bn115 and another previously identified loss-of-function allele nr2036 do not exhibit noticeable cell division defects in the somatic tissues but have reduced numbers of germ cells and are sterile, indicating that cdc-25.1 functions predominantly in the germ line during postembryonic development, and that cdc-25.1 activity is probably not required in somatic lineages during larval development. We analyzed cell division of germ cells and somatic tissues in bn115 homozygotes with germline-specific anti-PGL-1 immunofluorescence and GFP transgenes that express in intestinal cells, in distal tip cells, and in gonadal sheath cells, respectively. We also analyzed the expression pattern of cdc-25.1 with conventional and quantitative RT-PCR. In the presence of three other family members of cdc-25 in C. elegans defects are observed only in the germ line but not in the somatic tissues in cdc-25.1 single mutants, and cdc-25.1 is expressed predominantly, if not exclusively, in the germ line during postembryonic stages. Our findings indicate that the function of cdc-25.1 is unique in the germ line but likely redundant with other members in the soma.

  12. Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells

    PubMed Central

    Panula, Sarita; Medrano, Jose V.; Kee, Kehkooi; Bergström, Rosita; Nguyen, Ha Nam; Byers, Blake; Wilson, Kitchener D.; Wu, Joseph C.; Simon, Carlos; Hovatta, Outi; Reijo Pera, Renee A.

    2011-01-01

    Historically, our understanding of molecular genetic aspects of human germ cell development has been limited, at least in part due to inaccessibility of early stages of human development to experimentation. However, the derivation of pluripotent stem cells may provide the necessary human genetic system to study germ cell development. In this study, we compared the potential of human induced pluripotent stem cells (iPSCs), derived from adult and fetal somatic cells to form primordial and meiotic germ cells, relative to human embryonic stem cells. We found that ∼5% of human iPSCs differentiated to primordial germ cells (PGCs) following induction with bone morphogenetic proteins. Furthermore, we observed that PGCs expressed green fluorescent protein from a germ cell-specific reporter and were enriched for the expression of endogenous germ cell-specific proteins and mRNAs. In response to the overexpression of intrinsic regulators, we also observed that iPSCs formed meiotic cells with extensive synaptonemal complexes and post-meiotic haploid cells with a similar pattern of ACROSIN staining as observed in human spermatids. These results indicate that human iPSCs derived from reprogramming of adult somatic cells can form germline cells. This system may provide a useful model for molecular genetic studies of human germline formation and pathology and a novel platform for clinical studies and potential therapeutical applications. PMID:21131292

  13. Primary Malignant Mixed Germ Cell Tumour with Squamous Cell Carcinoma of the Mandible; A Rare Entity

    PubMed Central

    Paul, Arun; Parmar, Harshad; Chacko, Rabin

    2015-01-01

    Germ cell Tumours (GCT) are neoplasm derived from germ cells. GCT usually occurs inside the gonads. Extragonadal GCT’s are rare. Most common GCT associated with head and neck region are the teratomas. Of the few teratomas found in the head and neck, malignant transformation of a teratomatous element is very uncommon, and primary bone involvement within the head and neck is even rare. We present a case of primary malignant mixed germ cell Tumour involving the mandible, the present case presented malignant transformation of the epithelial component showing foci of squamous cell carcinoma within the GCT. PMID:26266228

  14. Mouse TEX14 is required for embryonic germ cell intercellular bridges but not female fertility.

    PubMed

    Greenbaum, Michael P; Iwamori, Naoki; Agno, Julio E; Matzuk, Martin M

    2009-03-01

    A conserved feature of germ cell cytokinesis is the formation of stable intercellular bridges between daughter cells. These intercellular bridges are seen in diverse species from Drosophila melanogaster to Homo sapiens and have been shown to have roles in communication of large numbers of germ cells. In testis expressed gene 14 (Tex14) knockout mice, intercellular bridges do not form during spermatogenesis, and male mice are sterile, demonstrating an essential role for intercellular bridges in postnatal spermatogenesis in mammals. Intercellular bridges also form between dividing germ cells in both male and female embryos. However, little is known about the formation or role of the embryonic intercellular bridges in mammals. In females, embryonic intercellular bridges have been proposed to have a role in development of the presumptive oocyte. Herein, we show that TEX14 is an essential component of male and female embryonic intercellular bridges. In addition, we demonstrate that mitotic kinesin-like protein 1 (MKLP1, official symbol KIF23), which we have discovered is a component of intercellular bridges during spermatogenesis, is also a component of male and female embryonic intercellular bridges. Germ cell intercellular bridges are readily identified by KIF23 immunofluorescence between the gonocytes and oogonia of control mice but are absent between germ cells of Tex14-null mice. Furthermore, by electron microscopy, intercellular bridges are present in all control newborn ovaries but are absent in the Tex14 knockout ovaries. Despite the absence of embryonic intercellular bridges in the Tex14-null mice, male mice initiate spermatogenesis, and female mice are fertile. Although fewer oocytes were present in Tex14-null neonatal ovaries, folliculogenesis was still active at 1 yr of age. Thus, while TEX14 and intercellular bridges have an essential role in postnatal spermatogenesis, they are not required in the embryo.

  15. Generation of exogenous germ cells in the ovaries of sterile NANOS3-null beef cattle

    PubMed Central

    Ideta, Atsushi; Yamashita, Shiro; Seki-Soma, Marie; Yamaguchi, Ryosaku; Chiba, Shiori; Komaki, Haruna; Ito, Tetsuya; Konishi, Masato; Aoyagi, Yoshito; Sendai, Yutaka

    2016-01-01

    Blastocyst complementation (BC) systems have enabled in vivo generation of organs from allogeneic pluripotent cells, compensating for an empty germ cell niche in gene knockout (KO) animals. Here, we succeeded in producing chimeric beef cattle (Wagyu) by transferring allogenic germ cells into ovaries using somatic cell nuclear transfer and BC technology. The KO of NANOS3 (NANOS3−/−) in Wagyu bovine ovaries produced a complete loss of germ cells. Holstein blastomeres (NANOS3+/+) were injected into NANOS3−/− Wagyu embryos. Subsequently, exogenous germ cells (NANOS3+/+) were identified in the NANOS3−/− ovary. These results clearly indicate that allogeneic germ cells can be generated in recipient germ cell-free gonads using cloning and BC technologies. PMID:27117862

  16. Generation of exogenous germ cells in the ovaries of sterile NANOS3-null beef cattle.

    PubMed

    Ideta, Atsushi; Yamashita, Shiro; Seki-Soma, Marie; Yamaguchi, Ryosaku; Chiba, Shiori; Komaki, Haruna; Ito, Tetsuya; Konishi, Masato; Aoyagi, Yoshito; Sendai, Yutaka

    2016-01-01

    Blastocyst complementation (BC) systems have enabled in vivo generation of organs from allogeneic pluripotent cells, compensating for an empty germ cell niche in gene knockout (KO) animals. Here, we succeeded in producing chimeric beef cattle (Wagyu) by transferring allogenic germ cells into ovaries using somatic cell nuclear transfer and BC technology. The KO of NANOS3 (NANOS3(-/-)) in Wagyu bovine ovaries produced a complete loss of germ cells. Holstein blastomeres (NANOS3(+/+)) were injected into NANOS3(-/-) Wagyu embryos. Subsequently, exogenous germ cells (NANOS3(+/+)) were identified in the NANOS3(-/-) ovary. These results clearly indicate that allogeneic germ cells can be generated in recipient germ cell-free gonads using cloning and BC technologies. PMID:27117862

  17. LINEing germ and embryonic stem cells' silencing of retrotransposons.

    PubMed

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena

    2014-07-01

    Almost half of our genome is occupied by transposable elements. Although most of them are inactive, one type of non-long terminal repeat (LTR) retrotransposon, long interspersed nuclear element 1 (LINE1), is capable of retrotransposition. Two studies in this issue, Pezic and colleagues (pp. 1410-1428) and Castro-Diaz and colleagues (pp. 1397-1409), provide novel insight into the regulation of LINE1s in human embryonic stem cells and mouse germ cells and shed new light on the conservation of complex mechanisms to ensure silencing of transposable elements in mammals.

  18. Human somatic cells subjected to genetic induction with six germ line-related factors display meiotic germ cell-like features

    PubMed Central

    Medrano, Jose V.; Martínez-Arroyo, Ana M.; Míguez, Jose M.; Moreno, Inmaculada; Martínez, Sebastián; Quiñonero, Alicia; Díaz-Gimeno, Patricia; Marqués-Marí, Ana I.; Pellicer, Antonio; Remohí, Jose; Simón, Carlos

    2016-01-01

    The in vitro derivation of human germ cells has attracted interest in the last years, but their direct conversion from human somatic cells has not yet been reported. Here we tested the ability of human male somatic cells to directly convert into a meiotic germ cell-like phenotype by inducing them with a combination of selected key germ cell developmental factors. We started with a pool of 12 candidates that were reduced to 6, demonstrating that ectopic expression of the germ line-related genes PRDM1, PRDM14, LIN28A, DAZL, VASA and SYCP3 induced direct conversion of somatic cells (hFSK (46, XY), and hMSC (46, XY)) into a germ cell-like phenotype in vitro. Induced germ cell-like cells showed a marked switch in their transcriptomic profile and expressed several post-meiotic germ line related markers, showed meiotic progression, evidence of epigenetic reprogramming, and approximately 1% were able to complete meiosis as demonstrated by their haploid status and the expression of several post-meiotic markers. Furthermore, xenotransplantation assays demonstrated that a subset of induced cells properly colonize the spermatogonial niche. Knowledge obtained from this work can be used to create in vitro models to study gamete-related diseases in humans. PMID:27112843

  19. The role of MAPK and FAS death receptor pathways in testicular germ cell apoptosis induced by lead.

    PubMed

    Dong, Shuying; Liang, Duoping; An, Na; Jia, Li; Shan, Yujuan; Chen, Chao; Sun, Kuo; Niu, Fei; Li, Huiyan; Fu, Songbin

    2009-09-01

    The aim of the present study is to investigate gene expression involved in the signal pathway of MAPK and death signal receptor pathway of FAS in lead-induced apoptosis of testicular germ cells. First, cell viabilities were determined by MTT assay. Second, using single cell gel-electrophoresis test (comet assay) and TUNEL staining technique, apoptotic rate and cell apoptosis localization of testicular germ cells were measured in mice treated with 0.15%, 0.3%, and 0.6% lead, respectively. Third, the immunolocalization of K-ras, c-fos, Fas, and active caspase-3 proteins was determined by immunohistochemistry. Finally, changes in the translational levels of K-ras, c-fos, Fas, and active caspase-3 were further detected by western blot analysis. Our results showed that lead could significantly induce testicular germ cell apoptosis in a dose-dependent manner (P<0.01). The mechanisms were closely related to the increased expressions of K-ras, c-fos, Fas, and active caspase-3 in apoptotic germ cells. In conclusion, K-ras/c-fos and Fas/caspase-3 death signaling receptor pathways were involved in the lead-induced apoptosis of the testicular germ cells in mice. PMID:19727529

  20. Insights into female germ cell biology: from in vivo development to in vitro derivations

    PubMed Central

    Jung, Dajung; Kee, Kehkooi

    2015-01-01

    Understanding the mechanisms of human germ cell biology is important for developing infertility treatments. However, little is known about the mechanisms that regulate human gametogenesis due to the difficulties in collecting samples, especially germ cells during fetal development. In contrast to the mitotic arrest of spermatogonia stem cells in the fetal testis, female germ cells proceed into meiosis and began folliculogenesis in fetal ovaries. Regulations of these developmental events, including the initiation of meiosis and the endowment of primordial follicles, remain an enigma. Studying the molecular mechanisms of female germ cell biology in the human ovary has been mostly limited to spatiotemporal characterizations of genes or proteins. Recent efforts in utilizing in vitro differentiation system of stem cells to derive germ cells have allowed researchers to begin studying molecular mechanisms during human germ cell development. Meanwhile, the possibility of isolating female germline stem cells in adult ovaries also excites researchers and generates many debates. This review will mainly focus on presenting and discussing recent in vivo and in vitro studies on female germ cell biology in human. The topics will highlight the progress made in understanding the three main stages of germ cell developments: namely, primordial germ cell formation, meiotic initiation, and folliculogenesis. PMID:25652637

  1. Insights into female germ cell biology: from in vivo development to in vitro derivations.

    PubMed

    Jung, Dajung; Kee, Kehkooi

    2015-01-01

    Understanding the mechanisms of human germ cell biology is important for developing infertility treatments. However, little is known about the mechanisms that regulate human gametogenesis due to the difficulties in collecting samples, especially germ cells during fetal development. In contrast to the mitotic arrest of spermatogonia stem cells in the fetal testis, female germ cells proceed into meiosis and began folliculogenesis in fetal ovaries. Regulations of these developmental events, including the initiation of meiosis and the endowment of primordial follicles, remain an enigma. Studying the molecular mechanisms of female germ cell biology in the human ovary has been mostly limited to spatiotemporal characterizations of genes or proteins. Recent efforts in utilizing in vitro differentiation system of stem cells to derive germ cells have allowed researchers to begin studying molecular mechanisms during human germ cell development. Meanwhile, the possibility of isolating female germline stem cells in adult ovaries also excites researchers and generates many debates. This review will mainly focus on presenting and discussing recent in vivo and in vitro studies on female germ cell biology in human. The topics will highlight the progress made in understanding the three main stages of germ cell developments: namely, primordial germ cell formation, meiotic initiation, and folliculogenesis.

  2. Methods to study maternal regulation of germ cell specification in zebrafish.

    PubMed

    Kaufman, O H; Marlow, F L

    2016-01-01

    The process by which the germ line is specified in the zebrafish embryo is under the control of maternal gene products that were produced during oogenesis. Zebrafish are highly amenable to microscopic observation of the processes governing maternal germ cell specification because early embryos are transparent, and the germ line is specified rapidly (within 4-5h post fertilization). Advantages of zebrafish over other models used to study vertebrate germ cell formation include their genetic tractability, the large numbers of progeny, and the easily manipulable genome, all of which make zebrafish an ideal system for studying the genetic regulators and cellular basis of germ cell formation and maintenance. Classical molecular biology techniques, including expression analysis through in situ hybridization and forward genetic screens, have laid the foundation for our understanding of germ cell development in zebrafish. In this chapter, we discuss some of these classic techniques, as well as recent cutting-edge methodologies that have improved our ability to visualize the process of germ cell specification and differentiation, and the tracking of specific molecules involved in these processes. Additionally, we discuss traditional and novel technologies for manipulating the zebrafish genome to identify new components through loss-of-function studies of putative germ cell regulators. Together with the numerous aforementioned advantages of zebrafish as a genetic model for studying development, we believe these new techniques will continue to advance zebrafish to the forefront for investigation of the molecular regulators of germ cell specification and germ line biology. PMID:27312489

  3. Methods to study maternal regulation of germ cell specification in zebrafish

    PubMed Central

    Kaufman, O.H.; Marlow, F.L.

    2016-01-01

    The process by which the germ line is specified in the zebrafish embryo is under the control of maternal gene products that were produced during oogenesis. Zebrafish are highly amenable to microscopic observation of the processes governing maternal germ cell specification because early embryos are transparent, and the germ line is specified rapidly (within 4–5 h post fertilization). Advantages of zebrafish over other models used to study vertebrate germ cell formation include their genetic tractability, the large numbers of progeny, and the easily manipulable genome, all of which make zebrafish an ideal system for studying the genetic regulators and cellular basis of germ cell formation and maintenance. Classical molecular biology techniques, including expression analysis through in situ hybridization and forward genetic screens, have laid the foundation for our understanding of germ cell development in zebrafish. In this chapter, we discuss some of these classic techniques, as well as recent cutting-edge methodologies that have improved our ability to visualize the process of germ cell specification and differentiation, and the tracking of specific molecules involved in these processes. Additionally, we discuss traditional and novel technologies for manipulating the zebrafish genome to identify new components through loss-of-function studies of putative germ cell regulators. Together with the numerous aforementioned advantages of zebrafish as a genetic model for studying development, we believe these new techniques will continue to advance zebrafish to the forefront for investigation of the molecular regulators of germ cell specification and germ line biology. PMID:27312489

  4. Metabolomic Response of Human Embryonic Stem Cell Derived Germ-like Cells after Exposure to Steroid Hormones

    EPA Science Inventory

    To assess the potential risks of human exposure to endocrine active compounds (EACs), the mechanisms of toxicity must first be identified and characterized. Currently, there are no robust in vitro models for identifying the mechanisms of toxicity in germ cells resulting from EAC ...

  5. Testicular germ cell tumors and related research from a historical point of view.

    PubMed

    Damjanov, Ivan; Wewer-Albrechtsen, Nicolai

    2013-01-01

    In this brief overview of the history of testicular germ cell tumors, we touch upon the key events and personalities that have contributed to our current understanding of germ cell tumors in general, and those of the testis in particular. The intricacies of human germ cell tumor pathology and histogenesis have been elucidated in part by contributions in the field of experimental pathology and developmental biology. Correlation between clinical oncologic findings, pathology and experimental studies of germ cell tumors and related topics ushered the era of cellular and genetic engineering that have revolutionized contemporary cell and molecular biology.

  6. Germ Cell Tumors in Adolescents and Young Adults.

    PubMed

    Calaminus, Gabriele; Joffe, Jonathan

    2016-01-01

    Germ cell tumors (GCTs) represent a group of biologically complex malignancies that affect patients at different sites within the body and at different ages. The varying nature of these tumors reflects their cell of origin which is the primordial germ cell, which normally gives rise to ovarian and testicular egg and sperm producing cells. These cells retain an ability to give rise to all types of human tissues, and this is illustrated by the different kinds of GCTs that occur. In adolescent and young adult (AYA) patients, GCTs predominantly present as testicular, ovarian or mediastinal primary GCTs, and represent some of the most complex therapeutic challenges within any AYA practice. The varying types of GCTs, defined by primary site and/or age at presentation, can look very similar microscopically. However, there is growing evidence that they may have different molecular characteristics, different biology and different requirements for curative treatments. Whilst in adult testicular GCTs there is evidence for an environmental cause during fetal development and a genetic component, these causative factors are much less well understood in other GCTs. GCTs are some of the most curable cancers in adults, but some patients exhibit resistance to standard treatments. Because of this, today's clinical research is directed at understanding how to best utilize toxic therapies and promote healthy survivorship. This chapter explores the biology, behavior and treatment of GCTs and discusses how the AYA group of GCTs may hold some of the keys to understanding fundamental unanswered questions of biological variance and curability in GCTs. PMID:27595361

  7. Spectrum of germ cell tumors: from head to toe.

    PubMed

    Ueno, Teruko; Tanaka, Yumiko Oishi; Nagata, Michio; Tsunoda, Hajime; Anno, Izumi; Ishikawa, Shigemi; Kawai, Koji; Itai, Yuji

    2004-01-01

    Germ cell tumors (GCTs) occur most frequently in the gonads and are relatively rare in other sites, such as the pineal gland, neurohypophysis, mediastinum, and retroperitoneum. GCTs are thought to originate from primordial germ cells, which migrate to the primitive gonadal glands in the urogenital ridge. Extragonadal GCTs might also originate from these cells when the cells are sequestered during their migration. Pathologic subtypes of GCTs vary, and the prevalence of mixed tumors is high. These factors produce a diversity of radiologic findings and make prospective radiologic diagnosis difficult in many cases. However, similar radiologic findings have been observed in pathologically equivalent tumors in varying sites. Seminomas appear as uniformly solid, lobulated masses with fibrovascular septa that enhance intensely. Nonseminomatous GCTs appear as heterogeneous masses with areas of necrosis, hemorrhage, or cystic degeneration. Fat and calcifications are hallmarks of teratomas, most of which are benign. In immature teratomas, scattered fat and calcification within larger solid components are occasionally seen. These imaging characteristics reflect the pathologic features of each tumor, and histologically similar GCTs at varying sites have similar radiologic features. Knowledge of the pathologic appearances of GCTs and their corresponding radiologic appearances will allow radiologists to diagnose these tumors correctly. PMID:15026588

  8. Hypoxia Inducible Factor 1 Alpha Is Expressed in Germ Cells throughout the Murine Life Cycle

    PubMed Central

    Gardner, Lauren H.; Mathews, Juanita; Yamazaki, Yuki; Allsopp, Richard C.

    2016-01-01

    Pluripotent stem cells of the early embryo, and germ line cells, are essential to ensure uncompromised development to adulthood as well as species propagation, respectively. Recently, the transcription factor hypoxia inducible factor 1 alpha (Hif1α) has been shown to have important roles in embryonic stem cells; in particular, regulation of conversion to glycolytic metabolism and, as we have shown, maintenance of functional levels of telomerase. In the present study, we sought to assess whether Hif1α was also expressed in the primitive cells of the murine embryo. We observed expression of Hif1α in pre-implantation embryos, specifically the 2-cell stage, morula, and blastocyst. Robust Hif1α expression was also observed in male and female primordial germ cells. We subsequently assessed whether Hif1α was expressed in adult male and female germ cells. In the testis, Hif1α was robustly expressed in spermatogonial cells, in both juvenile (6-week old) and adult (3-month old) males. In the ovaries, Hif1α was expressed in mature oocytes from adult females, as assessed both in situ and in individual oocytes flushed from super-ovulated females. Analysis of Hif1α transcript levels indicates a mechanism of regulation during early development that involves stockpiling of Hif1α protein in mature oocytes, presumably to provide protection from hypoxic stress until the gene is re-activated at the blastocyst stage. Together, these observations show that Hif1α is expressed throughout the life-cycle, including both the male and female germ line, and point to an important role for Hif1α in early progenitor cells. PMID:27148974

  9. Refractory sacrococcygeal germ cell tumor in Schinzel-Giedion syndrome.

    PubMed

    Kishimoto, Kenji; Kobayashi, Ryoji; Yonemaru, Nozomi; Yamamoto, Hiroshi; Tsujioka, Takao; Sano, Hirozumi; Suzuki, Daisuke; Yasuda, Kazue; Suzuki, Masahiko; Ando, Akiko; Tonoki, Hidefumi; Iizuka, Susumu; Uetake, Kimiaki; Kobayashi, Kunihiko

    2015-05-01

    We describe a boy with Schinzel-Giedion syndrome who developed refractory sacrococcygeal germ cell tumor with elements of embryonal carcinoma and immature teratoma. He developed local recurrence soon after tumor resection. The tumor was highly resistant to platinum-based combination chemotherapy, local irradiation, and salvage chemotherapy. Frequent infections resulted in a delay in treatment, although apparent fragility had not been observed clinically. He died from tumor progression at 32 months of age. Intensification of chemotherapy does not seem to be feasible for tumors in patients with Schinzel-Giedion syndrome. PMID:25171454

  10. BMP signaling and the maintenance of primordial germ cell identity in Drosophila embryos.

    PubMed

    Deshpande, Girish; Willis, Elinor; Chatterjee, Sandip; Fernandez, Robert; Dias, Kristen; Schedl, Paul

    2014-01-01

    The specification of primordial germ cells (PGCs) and subsequent maintenance of germ-line identity in Drosophila embryos has long been thought to occur solely under the control of cell-autonomous factors deposited in the posterior pole plasm during oogenesis. However, here we document a novel role for somatic BMP signaling in the maintenance of PGC fate during the period leading up to embryonic gonad coalescence. We find that PGCs fail to maintain their germline identity when BMP signaling is compromised. They initiate but are unable to properly assemble the germline stem cell-specific organelle, the spectrosome, and they lose expression of the germline-specific gene Vasa. BMP signaling must, however, be finely tuned as there are deleterious consequences to PGCs when the pathway is excessively active. We show that one mechanism used to calibrate the effects of BMP signals is dependent on the Ubc9 homolog Lesswright (Lwr).

  11. BMP Signaling and the Maintenance of Primordial Germ Cell Identity in Drosophila Embryos

    PubMed Central

    Deshpande, Girish; Willis, Elinor; Chatterjee, Sandip; Fernandez, Robert; Dias, Kristen; Schedl, Paul

    2014-01-01

    The specification of primordial germ cells (PGCs) and subsequent maintenance of germ-line identity in Drosophila embryos has long been thought to occur solely under the control of cell-autonomous factors deposited in the posterior pole plasm during oogenesis. However, here we document a novel role for somatic BMP signaling in the maintenance of PGC fate during the period leading up to embryonic gonad coalescence. We find that PGCs fail to maintain their germline identity when BMP signaling is compromised. They initiate but are unable to properly assemble the germline stem cell-specific organelle, the spectrosome, and they lose expression of the germline-specific gene Vasa. BMP signaling must, however, be finely tuned as there are deleterious consequences to PGCs when the pathway is excessively active. We show that one mechanism used to calibrate the effects of BMP signals is dependent on the Ubc9 homolog Lesswright (Lwr). PMID:24551179

  12. Repression of Pumilio Protein Expression by Rbfox1 Promotes Germ Cell Differentiation.

    PubMed

    Carreira-Rosario, Arnaldo; Bhargava, Varsha; Hillebrand, Jens; Kollipara, Rahul K; Ramaswami, Mani; Buszczak, Michael

    2016-03-01

    RNA-binding Fox (Rbfox) proteins have well-established roles in regulating alternative splicing, but specific Rbfox isoforms lack nuclear localization signals and accumulate in the cytoplasm. The potential splicing-independent functions of these proteins remain unknown. Here we demonstrate that cytoplasmic Drosophila Rbfox1 regulates germ cell development and represses the translation of mRNAs containing (U)GCAUG elements within their 3'UTRs. During germline cyst differentiation, Rbfox1 targets pumilio mRNA for destabilization and translational silencing, thereby promoting germ cell development. Mis-expression of pumilio results in the formation of germline tumors, which contain cysts that break down and dedifferentiate back to single, mitotically active cells. Together, these results reveal that cytoplasmic Rbfox family members regulate the translation of specific target mRNAs. In the Drosophila ovary, this activity provides a genetic barrier that prevents germ cells from reverting back to an earlier developmental state. The finding that Rbfox proteins regulate mRNA translation has implications for Rbfox-related diseases. PMID:26954550

  13. The Ter Mutation In The Dead End Gene Causes Germ Cell Loss And Testicular Germ Cell Tumours

    SciTech Connect

    Youngren, Kirsten K.; Coveney, Douglas; Peng, Xiaoning; Bhattacharya, Chitralekha; Schmidt, Laura S.; Nickerson, Michael L.; Lamb, Bruce T.; Deng Jian Min; Behringer, Richard R.; Capel, Blanche; Rubin, Edward M.; Nadeau, Joseph H.; Matin, Angabin

    2005-01-01

    In mice, the Ter mutation causes primordial germ cell (PGC) loss in all genetic backgrounds1. Ter is also a potent modifier of spontaneous testicular germ cell tumour (TGCT) susceptibility in the 129 family of inbred strains, and markedly increases TGCT incidence in 129-Ter/Ter males2 4. In 129-Ter/Ter mice, some of the remaining PGCs transform into undifferentiated pluripotent embryonal carcinoma cells2 6, and after birth differentiate into various cells and tissues that compose TGCTs. Here, we report the positional cloning of Ter, revealing a point mutation that introduces a termination codon in the mouse orthologue (Dnd1) of the zebrafish dead end (dnd) gene. PGC deficiency is corrected both with bacterial artificial chromosomes that contain Dnd1 and with a Dnd1-encoding transgene. Dnd1 is expressed in fetal gonads during the critical period when TGCTs originate. DND1 has an RNA recognition motif and is most similar to the apobec complementation factor, a component of the cytidine t o uridine RNA-editing complex. These results suggest that Ter may adversely affect essential aspects of RNA biology during PGC development. DND1 is the first protein known to have an RNA recognition motif directly implicated as a heritable cause of spontaneous tumorigenesis. TGCT development in the 129-Ter mouse strain models paediatric TGCT in humans. This work will have important implications for our understanding of the genetic control of TGCT pathogenesis and PGC biology.

  14. Benign Leydig cell tumour and germ cell carcinoma in situ in a young man with gynaecomastia.

    PubMed Central

    Fink, R. S.; Mann, M. S.; Hopewell, J. P.; Ginsburg, J.

    1984-01-01

    A 21-year-old man presented with a 16-year history of recurrent pyrexial episodes and a 5-year history of gynaecomastia. Blood and urinary oestrogen levels were elevated and a mass was found in the upper pole of a retractile right testis. After orchidectomy, oestrogen levels fell, gynaecomastia regressed and the pyrexial episodes ceased. Histological examination of the right testis showed a benign Leydig cell tumour in the upper pole and a germinal cell carcinoma in situ in the remaining part of the testis. Thus a potentially lethal condition was detected at an early pre-malignant phase by virtue of a benign, endocrinologically active tumour. This would seem to be the first report of the co-existence of a Leydig cell tumour and germ cell carcinoma in the same testis. Images Fig. 1 Fig. 2 Fig. 3 PMID:6694953

  15. Bmi1 expression in long-term germ stem cells

    PubMed Central

    Komai, Yoshihiro; Tanaka, Toshihiro; Tokuyama, Yoko; Yanai, Hirotsugu; Ohe, Shuichi; Omachi, Taichi; Atsumi, Naho; Yoshida, Naoko; Kumano, Keiki; Hisha, Hiroko; Matsuda, Tadashi; Ueno, Hiroo

    2014-01-01

    Asingle cells in undifferentiated spermatogonia are considered to be the most primitive forms of germ stem cells (GSCs). Although GFRα1 is thought to be a marker of Asingle cells, we found that Bmi1High is more specific than GFRα1 for Asingle cells. Bmi1High expression in Asingle cells is correlated with seminiferous stages, and its expression was followed by the proliferative stage of Asingle GSCs. In contrast, GFRα1 expression was seminiferous stage-independent. Fate analyses of EdU-positive Bmi1High-positive cell-derived Asingle cells revealed that these cells self-renewed or generated transient amplifying Apaired cells. Bmi1High-positive cells were resistant to irradiation-induced injury, after which they regenerated. Elimination of Bmi1High-positive cells from seminiferous tubules resulted in the appearance of tubules with seminiferous stage mismatches. Thus, in this study, we found that Bmi1High is a seminiferous stage-dependent marker for long-term GSCs and that Bmi1High-positive cells play important roles in maintaining GSCs and in regenerating spermatogenic progenitors after injury. PMID:25146451

  16. MAPK15 upregulation promotes cell proliferation and prevents DNA damage in male germ cell tumors.

    PubMed

    Rossi, Matteo; Colecchia, David; Ilardi, Gennaro; Acunzo, Mario; Nigita, Giovanni; Sasdelli, Federica; Celetti, Angela; Strambi, Angela; Staibano, Stefania; Croce, Carlo Maria; Chiariello, Mario

    2016-04-12

    Germ cell tumors (GCT) are the most common malignancies in males between 15 and 35 years of age. Despite the high cure rate, achieved through chemotherapy and/or surgery, the molecular basis of GCT etiology is still largely obscure. Here, we show a positive correlation between MAPK15 (ERK8; ERK7) expression and specific GCT subtypes, with the highest levels found in the aggressive embryonal carcinomas (EC). Indeed, in corresponding cellular models for EC, MAPK15 enhanced tumorigenicity in vivo and promoted cell proliferation in vitro, supporting a role for this kinase in human GCT. At molecular level, we demonstrated that endogenous MAPK15 is necessary to sustain cell cycle progression of EC cells, by limiting p53 activation and preventing the triggering of p53-dependent mechanisms resulting in cell cycle arrest.To understand MAPK15-dependent mechanisms impinging on p53 activation, we demonstrate that this kinase efficiently protects cells from DNA damage. Moreover, we show that the ability of MAPK15 to control the autophagic process is necessary for basal management of DNA damage and for tumor formation controlled by the kinase.In conclusion, our findings suggest that MAPK15 overexpression may contribute to the malignant transformation of germ cells by controlling a "stress support" autophagic pathway, able to prevent DNA damage and the consequent activation of the p53 tumor suppressor. Moreover, in light of these results, MAPK15-specific inhibitors might represent new tools to enhance the therapeutic index of cytotoxic therapy in GCT treatment, and to increase the sensitivity to DNA-damaging drugs in other chemotherapy-resistant human tumors. PMID:26988910

  17. MAPK15 upregulation promotes cell proliferation and prevents DNA damage in male germ cell tumors

    PubMed Central

    Ilardi, Gennaro; Acunzo, Mario; Nigita, Giovanni; Sasdelli, Federica; Celetti, Angela; Strambi, Angela; Staibano, Stefania; Croce, Carlo Maria; Chiariello, Mario

    2016-01-01

    Germ cell tumors (GCT) are the most common malignancies in males between 15 and 35 years of age. Despite the high cure rate, achieved through chemotherapy and/or surgery, the molecular basis of GCT etiology is still largely obscure. Here, we show a positive correlation between MAPK15 (ERK8; ERK7) expression and specific GCT subtypes, with the highest levels found in the aggressive embryonal carcinomas (EC). Indeed, in corresponding cellular models for EC, MAPK15 enhanced tumorigenicity in vivo and promoted cell proliferation in vitro, supporting a role for this kinase in human GCT. At molecular level, we demonstrated that endogenous MAPK15 is necessary to sustain cell cycle progression of EC cells, by limiting p53 activation and preventing the triggering of p53-dependent mechanisms resulting in cell cycle arrest. To understand MAPK15-dependent mechanisms impinging on p53 activation, we demonstrate that this kinase efficiently protects cells from DNA damage. Moreover, we show that the ability of MAPK15 to control the autophagic process is necessary for basal management of DNA damage and for tumor formation controlled by the kinase. In conclusion, our findings suggest that MAPK15 overexpression may contribute to the malignant transformation of germ cells by controlling a “stress support” autophagic pathway, able to prevent DNA damage and the consequent activation of the p53 tumor suppressor. Moreover, in light of these results, MAPK15-specific inhibitors might represent new tools to enhance the therapeutic index of cytotoxic therapy in GCT treatment, and to increase the sensitivity to DNA-damaging drugs in other chemotherapy-resistant human tumors. PMID:26988910

  18. Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment.

    PubMed

    Sekinaka, Tamotsu; Hayashi, Yohei; Noce, Toshiaki; Niwa, Hitoshi; Matsui, Yasuhisa

    2016-09-09

    Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs.

  19. Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment

    PubMed Central

    Sekinaka, Tamotsu; Hayashi, Yohei; Noce, Toshiaki; Niwa, Hitoshi; Matsui, Yasuhisa

    2016-01-01

    Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs. PMID:27608931

  20. Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment

    NASA Astrophysics Data System (ADS)

    Sekinaka, Tamotsu; Hayashi, Yohei; Noce, Toshiaki; Niwa, Hitoshi; Matsui, Yasuhisa

    2016-09-01

    Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs.

  1. Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment.

    PubMed

    Sekinaka, Tamotsu; Hayashi, Yohei; Noce, Toshiaki; Niwa, Hitoshi; Matsui, Yasuhisa

    2016-01-01

    Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs. PMID:27608931

  2. Modeling cell elongation during germ band retraction: cell autonomy versus applied anisotropic stress

    NASA Astrophysics Data System (ADS)

    Lynch, Holley E.; Veldhuis, Jim; Brodland, G. Wayne; Hutson, M. Shane

    2014-05-01

    The morphogenetic process of germ band retraction in Drosophila embryos involves coordinated movements of two epithelial tissues—germ band and amnioserosa. The germ band shortens along its rostral-caudal or head-to-tail axis, widens along its perpendicular dorsal-ventral axis, and uncurls from an initial ‘U’ shape. The amnioserosa mechanically assists this process by pulling on the crook of the U-shaped germ band. The amnioserosa may also provide biochemical signals that drive germ band cells to change shape in a mechanically autonomous fashion. Here, we use a finite-element model to investigate how these two contributions reshape the germ band. We do so by modeling the response to laser-induced wounds in each of the germ band’s spatially distinct segments (T1-T3, A1-A9) during the middle of retraction when segments T1-A3 form the ventral arm of the ‘U’, A4-A7 form its crook, and A8-A9 complete the dorsal arm. We explore these responses under a range of externally applied stresses and internal anisotropy of cell edge tensions—akin to a planar cell polarity that can drive elongation of cells in a direction parallel to the minimum edge tension—and identify regions of parameter space (edge-tension anisotropy versus stress anisotropy) that best match previous experiments for each germ band segment. All but three germ band segments are best fit when the applied stress anisotropy and the edge-tension anisotropy work against one another—i.e., when the isolated effects would elongate cells in perpendicular directions. Segments in the crook of the germ band (A4-A7) have cells that elongate in the direction of maximum external stress, i.e., external stress anisotropy is dominant. In most other segments, the dominant factor is internal edge-tension anisotropy. These results are consistent with models in which the amnioserosa pulls on the crook of the germ band to mechanically assist retraction. In addition, they suggest a mechanical cue for edge

  3. Modeling cell elongation during germ band retraction: cell autonomy versus applied anisotropic stress

    PubMed Central

    Lynch, Holley E.; Veldhuis, Jim; Brodland, G. Wayne; Hutson, M. Shane

    2014-01-01

    The morphogenetic process of germ band retraction in Drosophila embryos involves coordinated movements of two epithelial tissues – germ band and amnioserosa. The germ band shortens along its rostral-caudal or head-to-tail axis, widens along its perpendicular dorsal-ventral axis, and uncurls from an initial ‘U’ shape. The amnioserosa mechanically assists this process by pulling on the crook of the U-shaped germ band. The amnioserosa may also provide biochemical signals that drive germ band cells to change shape in a mechanically autonomous fashion. Here, we use a finite-element model to investigate how these two contributions reshape the germ band. We do so by modeling the response to laser-induced wounds in each of the germ band’s spatially distinct segments (T1-T3, A1-A9) during the middle of retraction when segments T1-A3 form the ventral arm of the ‘U’, A4-A7 form its crook, and A8-A9 complete the dorsal arm. We explore these responses under a range of externally applied stresses and internal anisotropy of cell edge tensions – akin to a planar cell polarity that can drive elongation of cells in a direction parallel to the minimum edge tension – and identify regions of parameter space (edge-tension anisotropy versus stress anisotropy) that best match previous experiments for each germ band segment. All but three germ band segments are best fit when the applied stress anisotropy and the edge-tension anisotropy work against one another – i.e., when the isolated effects would elongate cells in perpendicular directions. Segments in the crook of the germ band (A4-A7) have cells that elongate in the direction of maximum external stress, i.e., external stress anisotropy is dominant. In most other segments, the dominant factor is internal edge-tension anisotropy. These results are consistent with models in which the amnioserosa pulls on the crook of the germ band to mechanically assist retraction. In addition, they suggest a mechanical cue for edge

  4. Human iPS Cell-Derived Germ Cells: Current Status and Clinical Potential

    PubMed Central

    Ishii, Tetsuya

    2014-01-01

    Recently, fertile spermatozoa and oocytes were generated from mouse induced pluripotent (iPS) cells using a combined in vitro and in vivo induction system. With regard to germ cell induction from human iPS cells, progress has been made particularly in the male germline, demonstrating in vitro generation of haploid, round spermatids. Although iPS-derived germ cells are expected to be developed to yield a form of assisted reproductive technology (ART) that can address unmet reproductive needs, genetic and/or epigenetic instabilities abound in iPS cell generation and germ cell induction. In addition, there is still room to improve the induction protocol in the female germline. However, rapid advances in stem cell research are likely to make such obstacles surmountable, potentially translating induced germ cells into the clinical setting in the immediate future. This review examines the current status of the induction of germ cells from human iPS cells and discusses the clinical potential, as well as future directions. PMID:26237592

  5. Regulatory mechanism of protein metabolic pathway during the differentiation process of chicken male germ cell.

    PubMed

    Li, Dong; Zuo, Qisheng; Lian, Chao; Zhang, Lei; Shi, Qingqing; Zhang, Zhentao; Wang, Yingjie; Ahmed, Mahmoud F; Tang, Beibei; Xiao, Tianrong; Zhang, Yani; Li, Bichun

    2015-08-01

    We explored the regulatory mechanism of protein metabolism during the differentiation process of chicken male germ cells and provide a basis for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro. We sequenced the transcriptome of embryonic stem cells, primordial germ cells, and spermatogonial stem cells with RNA sequencing (RNA-Seq), bioinformatics analysis methods, and detection of the key genes by quantitative reverse transcription PCR (qRT-PCR). Finally, we found 16 amino acid metabolic pathways enriched in the biological metabolism during the differentiation process of embryonic stem cells to primordial germ cells and 15 amino acid metabolic pathways enriched in the differentiation stage of primordial germ cells to spermatogonial stem cells. We found three pathways, arginine-proline metabolic pathway, tyrosine metabolic pathway, and tryptophan metabolic pathway, significantly enriched in the whole differentiation process of embryonic stem cells to spermatogonial stem cells. Moreover, for these three pathways, we screened key genes such as NOS2, ADC, FAH, and IDO. qRT-PCR results showed that the expression trend of these genes were the same to RNA-Seq. Our findings showed that the three pathways and these key genes play an important role in the differentiation process of embryonic stem cells to male germ cells. These results provide basic information for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro.

  6. A novel somatic MAPK1 mutation in primary ovarian mixed germ cell tumors.

    PubMed

    Zou, Yang; Deng, Wei; Wang, Feng; Yu, Xiao-Hong; Liu, Fa-Ying; Yang, Bi-Cheng; Huang, Mei-Zhen; Guo, Jiu-Bai; Xie, Qiu-Hua; He, Ming; Huang, Ou-Ping

    2016-02-01

    A recent exome-sequencing study revealed prevalent mitogen-activated protein kinase 1 (MAPK1) p.E322K mutation in cervical carcinoma. It remains largely unknown whether ovarian carcinomas also harbor MAPK1 mutations. As paralogous gene mutations co‑occur frequently in human malignancies, we analyzed here a total of 263 ovarian carcinomas for the presence of MAPK1 and paralogous MAPK3 mutations by DNA sequencing. A previously unreported MAPK1 p.D321N somatic mutation was identified in 2 out of 18 (11.1%) ovarian mixed germ cell tumors, while no other MAPK1 or MAPK3 mutation was detected in our samples. Of note, OCC‑115, the MAPK1‑mutated sample with bilateral cancerous ovaries affected, harbored MAPK1 mutation in the right ovary while retained the left ovary intact, implicating that the genetic alterations underlying ovarian mixed germ cell tumor may be different, even in patients with similar genetic backgrounds and tumor microenvironments. The results of evolutionary conservation and protein structure modeling analysis implicated that MAPK1 p.D321N mutation may be pathogenic. Additionally, mutations in protein phosphatase 2 regulatory subunit α (PPP2R1A), ring finger protein 43 (RNF43), DNA directed polymerase ε (POLE1), ribonuclease type III (DICER1), CCCTC‑binding factor (CTCF), ribosomal protein L22 (RPL22), DNA methyltransferase 3α (DNMT3A), transformation/transcription domain‑associated protein (TRRAP), isocitrate dehydrogenase (IDH)1 and IDH2 were not detected in ovarian mixed germ cell tumors, implicating these genetic alterations may be not associated with MAPK1 mutation in the development of this malignancy. The present study identified a previously unreported MAPK1 mutation in ovarian mixed germ cell tumors for the first time, and this mutation may be actively involved in the tumorigenesis of this disease.

  7. The role of sex chromosomes in mammalian germ cell differentiation: can the germ cells carrying X and Y chromosomes differentiate into fertile oocytes?

    PubMed

    Taketo, Teruko

    2015-01-01

    The sexual differentiation of germ cells into spermatozoa or oocytes is strictly regulated by their gonadal environment, testis or ovary, which is determined by the presence or absence of the Y chromosome, respectively. Hence, in normal mammalian development, male germ cells differentiate in the presence of X and Y chromosomes, and female germ cells do so in the presence of two X chromosomes. However, gonadal sex reversal occurs in humans as well as in other mammalian species, and the resultant XX males and XY females can lead healthy lives, except for a complete or partial loss of fertility. Germ cells carrying an abnormal set of sex chromosomes are efficiently eliminated by multilayered surveillance mechanisms in the testis, and also, though more variably, in the ovary. Studying the molecular basis for sex-specific responses to a set of sex chromosomes during gametogenesis will promote our understanding of meiotic processes contributing to the evolution of sex determining mechanisms. This review discusses the fate of germ cells carrying various sex chromosomal compositions in mouse models, the limitation of which may be overcome by recent successes in the differentiation of functional germ cells from embryonic stem cells under experimental conditions.

  8. The role of sex chromosomes in mammalian germ cell differentiation: can the germ cells carrying X and Y chromosomes differentiate into fertile oocytes?

    PubMed Central

    Taketo, Teruko

    2015-01-01

    The sexual differentiation of germ cells into spermatozoa or oocytes is strictly regulated by their gonadal environment, testis or ovary, which is determined by the presence or absence of the Y chromosome, respectively. Hence, in normal mammalian development, male germ cells differentiate in the presence of X and Y chromosomes, and female germ cells do so in the presence of two X chromosomes. However, gonadal sex reversal occurs in humans as well as in other mammalian species, and the resultant XX males and XY females can lead healthy lives, except for a complete or partial loss of fertility. Germ cells carrying an abnormal set of sex chromosomes are efficiently eliminated by multilayered surveillance mechanisms in the testis, and also, though more variably, in the ovary. Studying the molecular basis for sex-specific responses to a set of sex chromosomes during gametogenesis will promote our understanding of meiotic processes contributing to the evolution of sex determining mechanisms. This review discusses the fate of germ cells carrying various sex chromosomal compositions in mouse models, the limitation of which may be overcome by recent successes in the differentiation of functional germ cells from embryonic stem cells under experimental conditions. PMID:25578929

  9. A novel gene, Pog, is necessary for primordial germ cell proliferation in the mouse and underlies the germ cell deficient mutation, gcd.

    PubMed

    Agoulnik, Alexander I; Lu, Baisong; Zhu, Qichao; Truong, Cavatina; Ty, Maria T; Arango, Nelson; Chada, Kiran K; Bishop, Colin E

    2002-11-15

    Primordial germ cells (PGCs) are the precursor of the germ cells in adult gonads. They arise extra-gonadally and migrate through somatic tissues to the presumptive genital ridges, where they proliferate and differentiate into oogonia or spermatogonia cells. Abnormalities in this developmental process can cause embryonic depletion of germ cells leading to infertility in the adult. We report here that the mouse gcd (germ cell deficient) mutant phenotype, characterized by reduced numbers of PGCs and adult sterility, is due to reduced PGC proliferation rather than aberrant migration and is caused by the partial deletion of a single novel gene, Pog (proliferation of germ cells). Pog is critical for normal PGC proliferation, starting between 9.5 and 10.25 dpc when germ cells begin to migrate to the developing genital ridge. Deletion of Pog is also accompanied by reduced embryonic body weight and, on some genetic backgrounds, embryonic lethality. Thus, in addition to being necessary for PGC proliferation, Pog may have a wider significance in early embryonic development.

  10. The relevance of spontaneous- and chemically-induced alterations in testicular germ cell apoptosis to toxicology.

    PubMed

    Richburg, J H

    2000-03-15

    Elimination of germ cells via apoptosis occurs spontaneously under normal physiologic conditions and is often heightened after chemical-induced testicular injury. Though many different apoptosis-related elements have been identified in the testis, the molecular and cellular mechanisms that regulate germ cell apoptosis are not thoroughly understood. In this review, the role of germ cell apoptosis in spermatogenesis and possible key regulators of apoptosis is described. The involvement of the Fas-signaling pathway between Sertoli cells and germ cells is highlighted as a crucial paracrine-signaling mechanism that responds to both physiologic- or toxicant-induced declines in the supportive capacity of the testis and reduces the germ cell population accordingly.

  11. Single exposure to heat induces stage-specific germ cell apoptosis in rats: role of intratesticular testosterone on stage specificity.

    PubMed

    Lue, Y H; Hikim, A P; Swerdloff, R S; Im, P; Taing, K S; Bui, T; Leung, A; Wang, C

    1999-04-01

    Short term exposure of the testis to heat causes degeneration of germ cells. However, the mechanisms underlying this process are poorly understood. The major objectives of this study were to determine whether the heat-induced loss of germ cells in the adult rat occurs via apoptosis, to document its stage-specific and cell-specific distribution, and to examine whether intratesticular testosterone (T) plays any role in the stage specificity of heat-induced germ cell death. Testes of adult male Sprague-Dawley rats were exposed to 22 C (control) or 43 C for 15 min. Animals were killed on days 1, 2, 9, and 56 after heat exposure. Germ cell apoptosis was characterized by DNA gel electrophoresis and in situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling assay. The incidence of germ cell apoptosis [apoptotic index (AI)] was quite low in control rats (AI = 0.04-0.1). Mild hyperthermia within 1 or 2 days resulted in a marked activation (AI = 4.7-5.6) of germ cell apoptosis predominantly at early (I-IV) and late (XII-XIV) stages. Stages V-VI and VII-VIII were relatively protected from heat-induced apoptosis. Spermatocytes, including pachytenes at stages I-IV and IX-XII, diplotene and dividing spermatocytes at stages XIII-XIV, and early (steps 1-4) spermatids, were most susceptible to heat. On day 9, the majority of the tubules were severely damaged and displayed only a few remaining apoptotic germ cells. By day 56, spermatogenesis was completely recovered, and the incidence of germ cell apoptosis was compatible with the control levels. To determine whether intratesticular T plays a role in protecting germ cells at stages VII-VIII against heat-induced cell death, adult rats were exposed to local testicular heating on day 2 or were given a daily sc injection of GnRH antagonist (GnRH-A) for 4 days with and without a single exposure of testes to heat applied on day 2. By day 4, the incidence of increased germ cell apoptosis at stages other than VII

  12. Are There Human Germ-Cell Mutagens? We May Know Soon

    EPA Science Inventory

    The existence of agents that can induce germ-cell mutations in experimental systems has been recognized since 1927 with the discovery of the ability of X-rays to induce such mutations in Drosophila. Since then, various rodent-based assays have been used to identify ~50 germ-cell...

  13. Germ cell tumor located in the midline of the anterior neck.

    PubMed

    Pirdopska, Tatyana; Terziev, Ivan; Hristova, Sv; Mladenovsky, W; Petkov, R

    2011-01-01

    Primary germ cell tumors involving midline of the anterior neck are extremely rare. Here we report a 68-year-old male who was operated due to a mass lesion in the anterior neck with infiltration of the isthmus of the thyroid gland. Histopathological examination revealed a germ cell tumor with extragonadal localization in the anterior neck infiltrating the isthmus of the thyroid gland.

  14. Development of interspecies testicular germ-cell transplantation in flatfish.

    PubMed

    Pacchiarini, Tiziana; Sarasquete, Carmen; Cabrita, Elsa

    2014-06-01

    Interspecific testicular germ cell (TGC) transplantation was investigated in two commercial flatfish species. Testes from donor species (Senegalese sole) were evaluated using classical histological techniques (haematoxylin-eosin staining and haematoxylin-light green-orange G-acid fuchsine staining), in situ hybridisation and immunohistochemical analysis. Both Ssvasa1-2 mRNAs and SsVasa protein allowed the characterisation of TGCs, confirming the usefulness of the vasa gene in the detection of Senegalese sole TGCs. Xenogenic transplants were carried out using TGCs from one-year-old Senegalese sole into turbot larvae. Propidium iodide-SYBR-14 and 4',6'-diamidino-2-phenylindole (DAPI) staining showed that 87.98% of the extracted testicular cells were viable for microinjection and that 15.63% of the total recovered cells were spermatogonia. The vasa gene was characterised in turbot recipients using cDNA cloning. Smvasa mRNA was confirmed as a germ cell-specific molecular marker in this species. Smvasa expression analysis during turbot ontogeny was carried out before Senegalese sole TGC transplants into turbot larvae. Turbot larvae at 18 days after hatching (DAH) proved to be susceptible to manipulation procedures. High survival rates (83.75±15.90-100%) were obtained for turbot larvae at 27, 34 and 42 DAH. These data highlight the huge potential of this species for transplantation studies. Quantitative PCR was employed to detect Senegalese sole vasa mRNAs (Ssvasa1-2) in the recipient turbot larvae. The Ssvasa mRNAs showed a significant increase in relative expression in 42-DAH microinjected larvae three weeks after treatment, showing the proliferation of Senegalese sole spermatogonia in transplanted turbot larvae.

  15. Human germ cell formation in xenotransplants of induced pluripotent stem cells carrying X chromosome aneuploidies.

    PubMed

    Dominguez, Antonia A; Chiang, H Rosaria; Sukhwani, Meena; Orwig, Kyle E; Reijo Pera, Renee A

    2014-09-22

    Turner syndrome is caused by complete or partial loss of the second sex chromosome and is characterized by spontaneous fetal loss in >90% of conceptions. Survivors possess an array of somatic and germline clinical characteristics. Induced pluripotent stem cells (iPSCs) offer an opportunity for insight into genetic requirements of the X chromosome linked to Turner syndrome. We derived iPSCs from Turner syndrome and control individuals and examined germ cell development as a function of X chromosome composition. We demonstrate that two X chromosomes are not necessary for reprogramming or maintenance of pluripotency and that there are minimal differences in gene expression, at the single cell level, linked to X chromosome aneuploidies. Formation of germ cells, as assessed in vivo through a murine xenotransplantation model, indicated that undifferentiated iPSCs, independent of X chromosome composition, are capable of forming germ-cell-like cells (GCLCs) in vivo. In combination with clinical data regarding infertility in women with X chromosome aneuploidies, results suggest that two intact X chromosomes are not required for human germ cell formation, qualitatively or quantitatively, but rather are likely to be required for maintenance of human germ cells to adulthood.

  16. Alternative germ cell death pathway in Drosophila involves HtrA2/Omi, lysosomes, and a caspase-9 counterpart.

    PubMed

    Yacobi-Sharon, Keren; Namdar, Yuval; Arama, Eli

    2013-04-15

    In both flies and mammals, almost one-third of the newly emerging male germ cells are spontaneously eliminated before entering meiosis. Here, we show that in Drosophila, germ cell death (GCD) involves the initiator caspase Dronc independently of the apoptosome and the main executioner caspases. Electron microscopy of dying germ cells revealed mixed morphologies of apoptosis and necrosis. We further show that the lysosomes and their catabolic enzymes, but not macroautophagy, are involved in the execution of GCD. We then identified, in a screen, the Parkinson's disease-associated mitochondrial protease, HtrA2/Omi, as an important mediator of GCD, acting mainly through its catalytic activity rather than by antagonizing inhibitor of apoptosis proteins. Concomitantly, other mitochondrial-associated factors were also implicated in GCD, including Pink1 (but not Parkin), the Bcl-2-related proteins, and endonuclease G, which establish the mitochondria as central mediators of GCD. These findings uncover an alternative developmental cell death pathway in metazoans.

  17. Exogenous supplementation of Activin A enhances germ cell differentiation of human embryonic stem cells.

    PubMed

    Duggal, Galbha; Heindryckx, Björn; Warrier, Sharat; Taelman, Jasin; Van der Jeught, Margot; Deforce, Dieter; Chuva de Sousa Lopes, Susana; De Sutter, Petra

    2015-05-01

    Human embryonic stem cells (hESCs) derived in the presence of Activin A (ActA) demonstrate an increased differentiation propensity toward the germ cell lineage. In addition, mouse epiblast stem cells and mouse epiblast-like cells are poised toward germ cell differentiation and are derived in the presence of ActA. We therefore investigated whether supplementation with ActA enhances in vitro hESC differentiation toward germ cell lineage. ActA up-regulated early primordial germ cell (PGC) genes STELLA/DPPA3 (developmental pluripotency associated 3) and tyrosine kinase receptor cKIT in both ActA-derived and standard-derived hESCs indicating its role in priming hESCs toward the PGC lineage. Indeed, ActA plus bone morphogenic protein 4 (BMP4) strongly increased germ cell differentiation potential of hESCs based on the high expression of late PGC markers DAZL (deleted in azoospermia-like) and VASA/DDX4 (DEAD-box polypeptide 4) at mRNA and protein level. Hence, the combination of ActA with BMP4 provides an additional boost for hESCs to develop into postmigratory germ cells. Together with increased VASA expression in the presence of ActA and BMP4, we also observed up-regulation of endoderm-specific genes GATA4 (GATA binding protein 4) and GATA6. Finally, we were able to further mature these in vitro-derived PGC-like cells (PGCLCs) by culturing them in in vitro maturation (IVM) medium, resulting in the formation of germ cell-like clusters and induction of meiotic gene expression. In conclusion, we demonstrate for the first time a synergism between ActA and BMP4 in facilitating germ cell-directed differentiation of hESCs, which is enhanced by extended culture in IVM medium, as shown by cytoplasmic VASA-expressing PGCLCs. We propose a novel relationship between the endoderm and germ cell lineage during hESC differentiation.

  18. Murine somatic cell nuclear transfer using reprogrammed donor cells expressing male germ cell-specific genes.

    PubMed

    Kang, Hoin; Park, Jong Im; Roh, Sangho

    2016-01-01

    In vivo-matured mouse oocytes were enucleated, and a single murine embryonic fibroblast (control or reprogrammed by introducing extracts from murine testis tissue, which showed expression of male germ cell-specific genes) was injected into the cytoplasm of the oocytes. The rate of blastocyst development and expression levels of Oct-4, Eomes and Cdx-2 were not significantly different in both experimental groups. However, the expression levels of Nanog, Sox9 and Glut-1 were significantly increased when reprogrammed cells were used as donor nuclei. Increased expression of Nanog can be supportive of complete reprogramming of somatic cell nuclear transfer murine embryos. The present study suggested that donor cells expressing male germ cell-specific genes can be reconstructed and can develop into embryos with normal high expression of developmentally essential genes. PMID:26369430

  19. Crosstalk between endoplasmic reticulum stress and mitochondrial pathway mediates cadmium-induced germ cell apoptosis in testes.

    PubMed

    Ji, Yan-Li; Wang, Hua; Zhao, Xian-Feng; Wang, Qun; Zhang, Cheng; Zhang, Ying; Zhao, Mei; Chen, Yuan-Hua; Meng, Xiu-Hong; Xu, De-Xiang

    2011-12-01

    Cadmium (Cd) is associated with male infertility and poor semen quality in humans. Increasing evidence demonstrates that Cd induces testicular germ cell apoptosis in rodent animals. However, the molecular mechanisms of Cd-induced testicular germ cell apoptosis remain poorly understood. In the present study, we investigated the role of endoplasmic reticulum (ER) stress on Cd-evoked germ cell apoptosis in testes. We show that spliced form of XBP-1, the target of the IRE1 pathway, was significantly increased in testes of mice injected with CdCl(2). GRP78, an ER chaperone, and CHOP, a downstream target of the PERK pathway, were upregulated in testes of Cd-treated mice. In addition, acute Cd exposure significantly caused eIF2α and JNK phosphorylation in testes, indicating that the unfolded protein response pathway in testes was activated by Cd. Interestingly, phenylbutyric acid (PBA), an ER chemical chaperone, attenuated Cd-induced ER stress and protected against germ cell apoptosis in testes. In addition, PBA significantly attenuated Cd-evoked release of cytochrome c from mitochondria to cytoplasm in testes. Taken together, these results suggest that crosstalk between ER stress signaling and mitochondrial pathway mediates Cd-induced testicular germ cell apoptosis. PMID:21908765

  20. Endocrine disrupters, microRNAs, and primordial germ cells: a dangerous cocktail.

    PubMed

    Brieño-Enríquez, Miguel Angel; Larriba, Eduardo; Del Mazo, Jesús

    2016-09-15

    Endocrine-disrupting chemicals (EDCs) are environmental pollutants that may change the homeostasis of the endocrine system, altering the differentiation of germ cells with consequences for reproduction. In mammals, germ cell differentiation begins with primordial germ cells (PGCs) during embryogenesis. Primordial germ cell development and gametogenesis are genetically regulated processes, in which the posttranscriptional gene regulation could be mediated by small noncoding RNAs (sncRNAs) such as microRNAs (miRNAs). Here, we review the deleterious effects of exposure during fetal life to EDCs mediated by deregulation of ncRNAs, and specifically miRNAs on PGC differentiation. Moreover, the environmental stress induced by exposure to some EDCs during the embryonic window of development could trigger reproductive dysfunctions transgenerationally transmitted by epigenetic mechanisms with the involvement of miRNAs expressed in germ line cells. PMID:27521771

  1. Novel strong tissue specific promoter for gene expression in human germ cells

    PubMed Central

    2010-01-01

    Background Tissue specific promoters may be utilized for a variety of applications, including programmed gene expression in cell types, tissues and organs of interest, for developing different cell culture models or for use in gene therapy. We report a novel, tissue-specific promoter that was identified and engineered from the native upstream regulatory region of the human gene NDUFV1 containing an endogenous retroviral sequence. Results Among seven established human cell lines and five primary cultures, this modified NDUFV1 upstream sequence (mNUS) was active only in human undifferentiated germ-derived cells (lines Tera-1 and EP2102), where it demonstrated high promoter activity (~twice greater than that of the SV40 early promoter, and comparable to the routinely used cytomegaloviral promoter). To investigate the potential applicability of the mNUS promoter for biotechnological needs, a construct carrying a recombinant cytosine deaminase (RCD) suicide gene under the control of mNUS was tested in cell lines of different tissue origin. High cytotoxic effect of RCD with a cell-death rate ~60% was observed only in germ-derived cells (Tera-1), whereas no effect was seen in a somatic, kidney-derived control cell line (HEK293). In further experiments, we tested mNUS-driven expression of a hyperactive Sleeping Beauty transposase (SB100X). The mNUS-SB100X construct mediated stable transgene insertions exclusively in germ-derived cells, thereby providing further evidence of tissue-specificity of the mNUS promoter. Conclusions We conclude that mNUS may be used as an efficient promoter for tissue-specific gene expression in human germ-derived cells in many applications. Our data also suggest that the 91 bp-long sequence located exactly upstream NDUFV1 transcriptional start site plays a crucial role in the activity of this gene promoter in vitro in the majority of tested cell types (10/12), and an important role - in the rest two cell lines. PMID:20716342

  2. Migratory mechanisms of chick primordial germ cells toward gonadal anlage.

    PubMed

    Kuwana, T; Rogulska, T

    1999-07-01

    After appearing at the germinal crescent region, chick primordial germ cells (PGCs) migrate toward the presumptive gonads (pG) till stage 19 (Hamburger and Hamilton, 1951). This study seeks to elucidate the roles of passive and active factors in the PGC-migration, physical trapping of circulating PGCs by the capillary network and PGC attraction by chemotactic factor from presumptive gonads. Firstly, latex beads/pollens (the same size or larger than PGCs) were injected into the embryonic bloodstream at stage 13-19 (when PGCs are in the migrating and settlement phase to the presumptive gonad) in ovo in order to determine whether the PGCs passively reach pG. Most of such particles accumulated in the head region (60%), whereas the remainder did the same in the gonadal region (23% at the peak) at stage 16 when both the head and gonadal regions are rich in capillary plexus. After 3 days, most particles in the gonadal region were located at the angles of dorsal mesentery near the developing gonads where many extra-gonadal PGCs had been located, and a few particles were detected close to the gonad. These results suggest that one of the mechanisms of PGC-migration to the developing gonads is an autonomous trapping of PGCs by the capillary network quite close to the germinal epithelium (GE) and passive translocation by morphogenetic movement. Secondly, the attraction for PGCs by the gonadal anlage proper was examined in ovo using chick and quail embryos. Grafts of quail gonadal anlage containing gonadal epithelium and neighbouring mesenchymal tissue were excised from the quail embryo at stages 12 to 16 (staging by Zacchei, 1961). With the aims of eliminating the influence of surrounding tissue, the quail graft was ectopically transplanted into the posterior to the optic vesicle of 8 to 17 somite chick embryo from the point of a posterior region to the auditory vesicle by a fine tungsten needle under the illumination by the method of Hara (1971). Then the region posterior to

  3. Production of loach (Misgurnus anguillicaudatus) germ-line chimera using transplantation of primordial germ cells isolated from cryopreserved blastomeres.

    PubMed

    Yasui, G S; Fujimoto, T; Sakao, S; Yamaha, E; Arai, K

    2011-08-01

    An efficient procedure for the cryopreservation of fish blastomeres followed by restoration through germ-line chimera formation was established. Blastomeres of the loach (Misgurnus anguillicaudatus) were cryopreserved in 250-µL straws in Eagle's minimum essential medium with various concentrations of dimethyl-sulfoxide (0, 5, 10, 15, and 20%), and the best concentration was combined with glycerol (1, 2, and 4%) and external cryoprotectants (1 or 2% sucrose; 2, 5, or 10% fetal bovine serum; 1 or 2% BSA). Postthaw viability of the blastomeres was used to optimize cryopreservation conditions. Donor blastomeres were injected with zebrafish green fluorescence protein-nos1 3' untranslated region mRNA and biotin dextran before cryopreservation in the optimal freeze medium. Host embryos were injected with zebrafish DsRed-nos1 3' untranslated region mRNA and reared to the blastula stage. Donor blastomeres were thawed at 25 °C for 10 s and transplanted to the host embryos either immediately or after incubation for 16 h at 20 °C. Donor and host primordial germ cell migration was visualized with fluorescent imaging during the early stages of embryogenesis, and also by histology in 4-d-old embryos. Transplantation of blastomeres immediately after thawing gave decreased hatching rates (approximately 3%) and generated a smaller percentage of germ-line chimeras (approximately 1.1%). In contrast, incubation of a cryopreserved sample for 16 h followed by transplantation of the green fluorescence protein-positive blastomeres improved the hatching rate to 90%, and successfully produced presumable germ-line chimeras at a rate of 16.5%. The improved survival rates and germ-line chimerism may be an effective method for gene banking and subsequent reconstitution of endangered fish genotypes.

  4. Implications of Sertoli cell induced germ cell apoptosis to testicular pathology

    PubMed Central

    Murphy, Caitlin J; Richburg, John H

    2014-01-01

    After exposure to toxicants, degenerating germ cells represents the most common testicular histopathological alteration, regardless of the mechanism of toxicity. Therefore, deciphering the primary toxicant cellular target and mechanism of action can be extremely difficult. However, most testicular toxicants display a cell-specific and a stage-specific pattern of damage, which is the best evidence for identifying the primary cellular target (i.e. germ cell, Sertoli cell, peritubular myoid cell, or Leydig cell). Some toxicant-induced Sertoli cell injury presents with germ cell apoptosis occurring primarily in spermatocytes in rats in stages XI-XIV, I and II. Although some toxicants result in spermatid degeneration and apoptosis, it is still unclear if spermatid apoptosis is a result of Sertoli cell-selective apoptosis or a direct effect of toxicants on spermatids, therefore if this is seen as the earliest change, one cannot infer the mechanism of apoptosis. This review summarizes some of the distinguishing features of Sertoli cell-induced germ cell apoptosis and the associated mechanisms of cell death to provide the toxicologist observing similar cell death, with evidence about a potential mode of action. PMID:26413394

  5. HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse

    PubMed Central

    Lim, Shu Ly; Geoghegan, Joel; Hempfling, Anna-Lena; Bergmann, Martin; Goodnow, Christopher C.; Ormandy, Christopher J.; Wong, Lee; Mann, Jeff; Scott, Hamish S.; Jamsai, Duangporn; Adelson, David L.

    2015-01-01

    piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2’ O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program. PMID:26496356

  6. HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse.

    PubMed

    Lim, Shu Ly; Qu, Zhi Peng; Kortschak, R Daniel; Lawrence, David M; Geoghegan, Joel; Hempfling, Anna-Lena; Bergmann, Martin; Goodnow, Christopher C; Ormandy, Christopher J; Wong, Lee; Mann, Jeff; Scott, Hamish S; Jamsai, Duangporn; Adelson, David L; O'Bryan, Moira K

    2015-10-01

    piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2' O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program. PMID:26496356

  7. Germ cell dynamics during the annual reproductive cycle of Dendropsophus minutus (Anura: Hylidae).

    PubMed

    Santos, Lia Raquel de Souza; Franco-Belussi, Lilian; de Oliveira, Classius

    2011-11-01

    Thirty male specimens of Dendropsophus minutus Peters, 1972, were collected from April 2004 to March 2005 in the region de Sao José do Rio Preto/SP, to conduct a histological study during the seasonal and annual cycles. Testicular activity was inferred based on the volume occupied by each type of cellular cyst present in the seminiferous tubules, as well as the quantity of germ cells in the final development stage, the spermatozoids. All data analyzed were correlated with climatic variables (temperature, rainfall and photoperiod) registered in the region where specimens were collected. A significant variation was verified in the quantity of spermatozoids as well as in the volume occupied by spermatids and spermatozoids throughout the year and between the cold/dry and hot/ humid seasons. It has also been reported that environmental conditions are important factors closely related to species reproduction and that production of germ cells and volume occupied by germ cysts is independent of anatomical aspect of the gonads. Thus, it was possible to verify that although the species reproduces throughout the year, individuals exhibit a preferential reproduction season, resulting in a reproductive (October to the end of February) and a post-reproductive period. PMID:22035307

  8. Melphalan, Carboplatin, Mannitol, and Sodium Thiosulfate in Treating Patients With Recurrent or Progressive CNS Embryonal or Germ Cell Tumors

    ClinicalTrials.gov

    2016-04-28

    Adult Central Nervous System Germ Cell Tumor; Adult Ependymoblastoma; Adult Medulloblastoma; Adult Pineoblastoma; Adult Supratentorial Primitive Neuroectodermal Tumor; Childhood Atypical Teratoid/Rhabdoid Tumor; Childhood Central Nervous System Germ Cell Tumor; Childhood Ependymoblastoma; Medulloepithelioma; Ototoxicity; Recurrent Adult Brain Neoplasm; Recurrent Childhood Central Nervous System Embryonal Neoplasm; Recurrent Childhood Malignant Germ Cell Tumor; Recurrent Childhood Medulloblastoma; Recurrent Childhood Pineoblastoma; Recurrent Childhood Supratentorial Primitive Neuroectodermal Tumor

  9. Mechano-logical model of C. elegans germ line suggests feedback on the cell cycle

    PubMed Central

    Atwell, Kathryn; Qin, Zhao; Gavaghan, David; Kugler, Hillel; Hubbard, E. Jane Albert; Osborne, James M.

    2015-01-01

    The Caenorhabditis elegans germ line is an outstanding model system in which to study the control of cell division and differentiation. Although many of the molecules that regulate germ cell proliferation and fate decisions have been identified, how these signals interact with cellular dynamics and physical forces within the gonad remains poorly understood. We therefore developed a dynamic, 3D in silico model of the C. elegans germ line, incorporating both the mechanical interactions between cells and the decision-making processes within cells. Our model successfully reproduces key features of the germ line during development and adulthood, including a reasonable ovulation rate, correct sperm count, and appropriate organization of the germ line into stably maintained zones. The model highlights a previously overlooked way in which germ cell pressure may influence gonadogenesis, and also predicts that adult germ cells might be subject to mechanical feedback on the cell cycle akin to contact inhibition. We provide experimental data consistent with the latter hypothesis. Finally, we present cell trajectories and ancestry recorded over the course of a simulation. The novel approaches and software described here link mechanics and cellular decision-making, and are applicable to modeling other developmental and stem cell systems. PMID:26428008

  10. N-acetylcysteine protects against cadmium-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in testes.

    PubMed

    Ji, Yan-Li; Wang, Hua; Zhang, Cheng; Zhang, Ying; Zhao, Mei; Chen, Yuan-Hua; Xu, De-Xiang

    2013-03-01

    Cadmium (Cd) is a reproductive toxicant that induces germ cell apoptosis in the testes. Previous studies have demonstrated that endoplasmic reticulum (ER) stress is involved in Cd-induced germ cell apoptosis. The aim of the present study was to investigate the effects of N-acetylcysteine (NAC), an antioxidant, on Cd-induced ER stress and germ cell apoptosis in the testes. Male CD-1 mice were intraperitoneally injected with CdCl2 (2.0 mg kg(-1)). As expected, acute Cd exposure induced germ cell apoptosis in the testes, as determined by terminal dUTP nick-end labelling (TUNEL). However, the administration of NAC alleviated Cd-induced germ cell apoptosis in the testes. Further analysis showed that NAC attenuated the Cd-induced upregulation of testicular glucose-regulated protein 78 (GRP78), an important ER molecular chaperone. Moreover, NAC inhibited the Cd-induced phosphorylation of testicular eukaryotic translation initiation factor 2α (eIF2α), a downstream target of the double-stranded RNA-activated kinase-like ER kinase (PERK) pathway. In addition, NAC blocked the Cd-induced activation of testicular X binding protein (XBP)-1, indicating that NAC attenuates the Cd-induced ER stress and the unfolded protein response (UPR). Interestingly, NAC almost completely prevented the Cd-induced elevation of C/EBP homologous protein (CHOP) and phosphorylation of c-Jun N-terminal kinase (JNK), two components of the ER stress-mediated apoptotic pathway. In conclusion, NAC protects against Cd-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in the testes. PMID:23353715

  11. Control over the morphology and segregation of Zebrafish germ cell granules during embryonic development

    PubMed Central

    Strasser, Markus J; Mackenzie, Natalia C; Dumstrei, Karin; Nakkrasae, La-Iad; Stebler, Jürg; Raz, Erez

    2008-01-01

    Background Zebrafish germ cells contain granular-like structures, organized around the cell nucleus. These structures share common features with polar granules in Drosophila, germinal granules in Xenopus and chromatoid bodies in mice germ cells, such as the localization of the zebrafish Vasa, Piwi and Nanos proteins, among others. Little is known about the structure of these granules as well as their segregation in mitosis during early germ-cell development. Results Using transgenic fish expressing a fluorescently labeled novel component of Zebrafish germ cell granules termed Granulito, we followed the morphology and distribution of the granules. We show that whereas these granules initially exhibit a wide size variation, by the end of the first day of development they become a homogeneous population of medium size granules. We investigated this resizing event and demonstrated the role of microtubules and the minus-end microtubule dependent motor protein Dynein in the process. Last, we show that the function of the germ cell granule resident protein the Tudor domain containing protein-7 (Tdrd7) is required for determination of granule morphology and number. Conclusion Our results suggest that Zebrafish germ cell granules undergo a transformation process, which involves germ cell specific proteins as well as the microtubular network. PMID:18507824

  12. Essential role of brc-2 in chromosome integrity of germ cells in C. elegans.

    PubMed

    Ko, Eunkyong; Lee, Junho; Lee, Hyunsook

    2008-12-31

    brc-2, an ortholog of BRCA2 in Caenorhabditis elegans, is essential in the maintenance of genetic integrity. In C. elegans, cellular location correlates with meiotic progression, and transgene-induced cosuppression is observed in the germ line but not in somatic cells. We used these unique features to dissect the role of brc-2 in the germ line from that in somatic cells. In situ hybridization of wild type animals revealed that brc-2 gene expression was higher in oocytes than in other germline cells, and was barely detectable in mitotic cells. In contrast, germ cells containing multicopies of the brc-2 transgene showed no significant in situ hybridization signal at any oogenesis stage, confirming that brc-2 expression was functionally cosuppressed in the transgenic germ line. RAD-51 foci formation in response to DNA damage was abrogated in brc-2-cosuppressed germ cells, whereas wild-type germ cells showed strong RAD-51 foci formation. These germ cells exhibited massive chromosome fragmentation and decompaction instead of six bivalent chromosomes in diakinesis. Accordingly, lethality was observed after the early stage of germline development. These results suggest that brc-2 plays essential roles in chromosome integrity in early prophase, and therefore is crucial in meiotic progression and embryonic survival.

  13. Apoptotic extinction of germ cells in testes of Cyp26b1 knockout mice.

    PubMed

    MacLean, Glenn; Li, Hui; Metzger, Daniel; Chambon, Pierre; Petkovich, Martin

    2007-10-01

    Cyp26b1 encodes a retinoic acid (RA) metabolizing cytochrome P450 enzyme that is expressed in embryonic tissues undergoing morphogenesis, including the testes. We have generated transgenic mice lacking Cyp26b1 and have observed increased RA levels in embryonic testes. Cyp26b1(-/-) germ cells prematurely enter meiosis at embryonic d 13.5 and appear to arrest at pachytene stage. Furthermore, after embryonic d 13.5, a rapid increase in apoptosis is observed in male germ cells derived from Cyp26b1(-/-) embryos; germ cells are essentially absent in mutant male neonates. In contrast, testicular somatic cells appear to develop normally in the absence of Cyp26b1. Moreover, ovarian germ and somatic cells appear unaffected by the lack of CYP26B1. We also show that the synthetic retinoid Am580, which is resistant to CYP26 metabolism, induces meiosis of male germ cells in cultured gonads, suggesting that abnormal development of germ cells in the Cyp26b1(-/-) testes results from excess RA rather than the absence of CYP26B1-generated metabolites of RA. These results provide evidence that CYP26B1 maintains low levels of RA in the developing testes that blocks entry into meiosis and acts as a survival factor to prevent apoptosis of male germ cells.

  14. Effect of Antioxidants and Apoptosis Inhibitors on Cryopreservation of Murine Germ Cells Enriched for Spermatogonial Stem Cells.

    PubMed

    Ha, Seung-Jung; Kim, Byung-Gak; Lee, Yong-An; Kim, Yong-Hee; Kim, Bang-Jin; Jung, Sang-Eun; Pang, Myeong-Geol; Ryu, Buom-Yong

    2016-01-01

    Spermatogonial stem cells (SSCs) are germline stem cells that serve as the foundation of spermatogenesis to maintain fertility throughout a male's lifetime. To treat male infertility using stem cell banking systems and transplantation, it is important to be able to preserve SSCs for long periods of time. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using antioxidants and apoptosis inhibitors in freezing medium. No differences were observed compared to controls when SSCs were cryopreserved in the presence of apoptosis inhibitors by themselves. However, mouse germ cells cryopreserved in basal medium containing the antioxidant hypotaurine (14 mM) resulted in significantly greater proliferation potential and mitochondrial activity. Furthermore, treatment groups with combinations containing 200 mM trehalose and 14 mM hypotaurine showed higher proliferation rates compared to controls. In addition, several serum free conditions were evaluated for SSC cryopreservation. Treatment media containing 10% or 20% knockout serum replacement resulted in similar cryopreservation results compared to media containing FBS. SSC transplantation was also performed to confirm the functionality of SSCs frozen in 14 mM hypotaurine. Donor SSCs formed normal spermatogenic colonies and sperm in the recipient testis. These data indicate that inclusion of 14 mM hypotaurine in cryopreservation media is an effective way to efficiently cryopreserve germ cells enriched for SSCs and that knockout serum replacement can replace FBS in germ cell cryopreservation media. PMID:27548381

  15. Effect of Antioxidants and Apoptosis Inhibitors on Cryopreservation of Murine Germ Cells Enriched for Spermatogonial Stem Cells

    PubMed Central

    Lee, Yong-An; Kim, Yong-Hee; Kim, Bang-Jin; Jung, Sang-Eun; Pang, Myeong-Geol; Ryu, Buom-Yong

    2016-01-01

    Spermatogonial stem cells (SSCs) are germline stem cells that serve as the foundation of spermatogenesis to maintain fertility throughout a male’s lifetime. To treat male infertility using stem cell banking systems and transplantation, it is important to be able to preserve SSCs for long periods of time. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using antioxidants and apoptosis inhibitors in freezing medium. No differences were observed compared to controls when SSCs were cryopreserved in the presence of apoptosis inhibitors by themselves. However, mouse germ cells cryopreserved in basal medium containing the antioxidant hypotaurine (14 mM) resulted in significantly greater proliferation potential and mitochondrial activity. Furthermore, treatment groups with combinations containing 200 mM trehalose and 14 mM hypotaurine showed higher proliferation rates compared to controls. In addition, several serum free conditions were evaluated for SSC cryopreservation. Treatment media containing 10% or 20% knockout serum replacement resulted in similar cryopreservation results compared to media containing FBS. SSC transplantation was also performed to confirm the functionality of SSCs frozen in 14 mM hypotaurine. Donor SSCs formed normal spermatogenic colonies and sperm in the recipient testis. These data indicate that inclusion of 14 mM hypotaurine in cryopreservation media is an effective way to efficiently cryopreserve germ cells enriched for SSCs and that knockout serum replacement can replace FBS in germ cell cryopreservation media. PMID:27548381

  16. Innate mechanisms for Bifidobacterium lactis to activate transient pro-inflammatory host responses in intestinal epithelial cells after the colonization of germ-free rats.

    PubMed

    Ruiz, Pedro A; Hoffmann, Micha; Szcesny, Silke; Blaut, Michael; Haller, Dirk

    2005-08-01

    Bifidobacteria comprise a dominant microbial population group in the human intestinal tract with purported beneficial health effects on the host. In this study, we characterized the molecular mechanisms for the initial interaction of probiotic Bifidobacterium lactis strain BB12 with native and intestinal epithelial cell (IEC) lines. We showed that B. lactis-monoassociated Fisher F344 rats transiently induce phosphorylation/activation of the NF-kappaB transcriptionally active subunit RelA and the mitogen-activated protein kinase (MAPK) p38 in native IEC at day 5 after initial bacterial colonization. In addition, Interleukin 6 (IL-6) gene expression was significantly increased at day 5, demonstrating the physiological relevance of transient transcription factor activation in IEC. In contrast, Bacteroides vulgatus-monoassociated Fisher rats revealed RelA but not p38 MAPK phosphorylation and failed to trigger significant IL-6 gene expression in native IEC. Moreover, we demonstrated that B. lactis triggers NF-kappaB RelA and p38 MAPK phosphorylation in IEC lines. Adenoviral delivery of mutant IKK-beta (Ad5dnIKKbeta) and inhibition of the p38 MAPK pathway through the pharmacological inhibitor SB203580 significantly blocked B. lactis-induced IL-6 gene expression in IEC, suggesting that B. lactis triggers NF-kappaB and MAPK signaling to induce gene expression in the intestinal epithelium. Regarding the mechanisms of bacteria epithelial cell cross-talk, B. lactis-induced IL-6 gene expression was completely inhibited in TLR2 deficient mouse embryogenic fibroblasts (MEF TLR2-/-) as well as TLR2DeltaTIR transfected Mode-K cells. In conclusion, we demonstrated that probiotic bacteria transiently trigger innate signal transduction and pro-inflammatory gene expression in the intestinal epithelium at early stages of bacterial colonization.

  17. Regulating the balance between differentiation and apoptosis: role of CREM in the male germ cells.

    PubMed

    Sassone-Corsi, P

    1998-01-01

    Various endocrine and neuronal functions are governed by the cAMP-dependent signaling pathway. In eukaryotes, transcriptional regulation upon stimulation of the adenylyl cyclase signaling pathway is mediated by a family of cAMP-responsive nuclear factors. This family consists of a large number of members which may act as activators or repressors. These factors contain the basic domain/leucine zipper motifs and bind as dimers to cAMP-response elements (CRE). The function of CRE-binding proteins (CREBs) is modulated by phosphorylation by several kinases. Direct activation of gene expression by CREBs requires phosphorylation by the cAMP-dependent protein kinase A to the serine-133 residue. The gene CREM encodes various transcription factors which play key physiological and developmental roles within the hypothalamic-pituitary-gonadal axis. We have previously shown that the transcriptional activator CREMtau is highly expressed in postmeiotic cells. Spermiogenesis is a complex process by which postmeiotic male germ cells differentiate into mature spermatozoa. This process involves remarkable structural and biochemical changes which are under the hormonal control of the hypothalamic-pituitary axis. We have addressed the specific role of CREM in spermiogenesis using CREM-mutant mice generated by homologous recombination. Analysis of the seminiferous epithelium from mutant male mice reveals that spermatogenesis stops at the first step of spermiogenesis. Late spermatids are completely absent while there is a significant increase in apoptotic germ cells. A series of postmeiotic germ cell-specific genes are not expressed. Mutant male mice completely lack spermatozoa. This phenotype is reminiscent of cases of human infertility.

  18. DNA replication licensing in somatic and germ cells.

    PubMed

    Eward, Kathryn Leigh; Obermann, Ellen C; Shreeram, S; Loddo, Marco; Fanshawe, Thomas; Williams, Craig; Jung, Hyo-Il; Prevost, A Toby; Blow, J Julian; Stoeber, Kai; Williams, Gareth H

    2004-11-15

    The DNA replication (or origin) licensing system ensures precise duplication of the genome in each cell cycle and is a powerful regulator of cell proliferation in metazoa. Studies in yeast, Drosophila melanogaster and Xenopus laevis have characterised the molecular machinery that constitutes the licensing system, but it remains to be determined how this important evolutionary conserved pathway is regulated in Homo sapiens. We have investigated regulation of the origin licensing factors Cdc6, Cdt1, Mcm2 and Geminin in human somatic and germ cells. Cdc6 and Cdt1 play an essential role in DNA replication initiation by loading the Mcm2-7 complex, which is required for unwinding the DNA helix, onto chromosomal origins. Geminin is a repressor of origin licensing that blocks Mcm2-7 loading onto origins. Our studies demonstrate that Cdc6, Cdt1 and Mcm2 play a central role in coordinating growth during the proliferation-differentiation switch in somatic self-renewing systems and that Cdc6 expression is rate-limiting for acquisition of replication competence in primary oocytes. In striking contrast, we show that proliferation control during male gametogenesis is not linked to Cdc6 or Mcm2, but appears to be coordinated by the negative regulator Geminin with Cdt1 becoming rate-limiting in late prophase. Our data demonstrate a striking sexual dimorphism in the mechanisms repressing origin licensing and preventing untimely DNA synthesis during meiosis I, implicating a pivotal role for Geminin in maintaining integrity of the male germline genome.

  19. The fog-3 gene and regulation of cell fate in the germ line of Caenorhabditis elegans

    SciTech Connect

    Ellis, R.; Kimble, J.

    1995-02-01

    In the nematode Caenorhabditis elegans, germ cells normally adopt one of three fates: mitosis, spermatogenesis or oogenesis. We have identified and characterized the gene fog-3, which is required for germ cells to differentiate as sperm rather than as oocytes. Analysis of double mutants suggests that fog-3 is absolutely required for spermatogenesis and acts at the end of the regulatory hierarchy controlling sex determination for the germ line. By contrast, mutations in fog-3 do not alter the sexual identity of other tissues. We also have characterized the null phenotype of fog-1, another gene required for spermatogenesis; we demonstrate that it too controls the sexual identity of germ cells but not of other tissues. Finally, we have studied the same interaction of these two fog genes with gld-1, a gene required for germ cells to undergo oogenesis rather than mitosis. On the basis of these results, we propose that germ-cell fate might be controlled by a set of inhibitory interactions among genes that specify one of three fates: mitosis, spermatogenesis or oogenesis. Such a regulatory network would link the adoption of one germ-cell fate to the suppression of the other two. 68 refs., 7 figs., 6 tabs.

  20. Genome-wide analysis of the maternal-to-zygotic transition in Drosophila primordial germ cells

    PubMed Central

    2012-01-01

    Background During the maternal-to-zygotic transition (MZT) vast changes in the embryonic transcriptome are produced by a combination of two processes: elimination of maternally provided mRNAs and synthesis of new transcripts from the zygotic genome. Previous genome-wide analyses of the MZT have been restricted to whole embryos. Here we report the first such analysis for primordial germ cells (PGCs), the progenitors of the germ-line stem cells. Results We purified PGCs from Drosophila embryos, defined their proteome and transcriptome, and assessed the content, scale and dynamics of their MZT. Transcripts encoding proteins that implement particular types of biological functions group into nine distinct expression profiles, reflecting coordinate control at the transcriptional and posttranscriptional levels. mRNAs encoding germ-plasm components and cell-cell signaling molecules are rapidly degraded while new transcription produces mRNAs encoding the core transcriptional and protein synthetic machineries. The RNA-binding protein Smaug is essential for the PGC MZT, clearing transcripts encoding proteins that regulate stem cell behavior, transcriptional and posttranscriptional processes. Computational analyses suggest that Smaug and AU-rich element binding proteins function independently to control transcript elimination. Conclusions The scale of the MZT is similar in the soma and PGCs. However, the timing and content of their MZTs differ, reflecting the distinct developmental imperatives of these cell types. The PGC MZT is delayed relative to that in the soma, likely because relief of PGC-specific transcriptional silencing is required for zygotic genome activation as well as for efficient maternal transcript clearance. PMID:22348290

  1. Chemotherapy for Good-Risk Nonseminomatous Germ Cell Tumors: Current Concepts and Controversies.

    PubMed

    In, Gino; Dorff, Tanya

    2015-08-01

    The rate of diagnosis of germ cell tumors has remained fairly constant. By the International Germ Cell Cancer Consensus Classification, roughly 60% of all metastatic germ cell tumors are classified as good risk. This group of patients has an excellent prognosis, with greater than 90% expectation of cure. Treatment standards have not changed much in recent years. This article focuses on key concepts in the development of the currently accepted first-line regimens and addresses some evolving areas of interest, if not controversy. PMID:26216822

  2. Atypical presentation of pediatric mixed germ cell tumors in the sellar-suprasellar region.

    PubMed

    Furtado, Sunil V; Thakar, Sumit; Ghosal, Nandita; Hegde, Alangar S

    2012-01-01

    Intracranial germ cell tumors constitute a unique group of tumors, more often reported from the Asian region. Amongst them, the non-germinomatous variety occurs with a lesser frequency than the germinomatous variety. We report two children with mixed germ cell tumors with unusual clinical presentations: Central diabetes insipidus and recent-onset oculomotor palsy mimicking pituitary apoplexy. Unlike in adults, suprasellar lesions with a pituitary apoplexy-like picture in the pediatric age group may suggest a possibility of a mixed germ cell tumor. PMID:22406789

  3. Gonadogenesis and slow proliferation of germ cells in juveniles of cultured yellowfin tuna, Thunnus albacares.

    PubMed

    Kobayashi, Toru; Honryo, Tomoki; Agawa, Yasuo; Sawada, Yoshifumi; Tapia, Ileana; Macìas, Karla A; Cano, Amado; Scholey, Vernon P; Margulies, Daniel; Yagishita, Naoki

    2015-06-01

    To develop techniques for seedling production of yellowfin tuna, the behavior of primordial germ cells (PGCs) and gonadogenesis were examined at 1-30 days post hatching (dph) using morphometric analysis, histological examination, and in situ hybridization. Immediately after hatching, PGCs were located on the dorsal side of the posterior end of the rectum under the peritoneum of the larvae, and at 3 dph they came into contact with stromal cells. PGCs and stromal cells gradually moved forward from the anus prior to 5 dph. At 7-10 dph, germ cells were surrounded by stromal cells and the gonadal primordia were formed. In individuals collected at 12 dph, PGCs were detected by in situ hybridization using a vasa mRNA probe that is a germ-cell-specific detection marker. The proliferation of germ cells in the gonadal primordia began at 7-10 dph. We observed double the number of germ cells at 30 dph (22 ± 3.2 cells), compared to that at 1 dph (11 ± 2.1 cells). Therefore, based on our data and previous reports, the initial germ cell proliferation of yellowfin tuna is relatively slower than that of other fish species.

  4. Gonadogenesis and slow proliferation of germ cells in juveniles of cultured yellowfin tuna, Thunnus albacares.

    PubMed

    Kobayashi, Toru; Honryo, Tomoki; Agawa, Yasuo; Sawada, Yoshifumi; Tapia, Ileana; Macìas, Karla A; Cano, Amado; Scholey, Vernon P; Margulies, Daniel; Yagishita, Naoki

    2015-06-01

    To develop techniques for seedling production of yellowfin tuna, the behavior of primordial germ cells (PGCs) and gonadogenesis were examined at 1-30 days post hatching (dph) using morphometric analysis, histological examination, and in situ hybridization. Immediately after hatching, PGCs were located on the dorsal side of the posterior end of the rectum under the peritoneum of the larvae, and at 3 dph they came into contact with stromal cells. PGCs and stromal cells gradually moved forward from the anus prior to 5 dph. At 7-10 dph, germ cells were surrounded by stromal cells and the gonadal primordia were formed. In individuals collected at 12 dph, PGCs were detected by in situ hybridization using a vasa mRNA probe that is a germ-cell-specific detection marker. The proliferation of germ cells in the gonadal primordia began at 7-10 dph. We observed double the number of germ cells at 30 dph (22 ± 3.2 cells), compared to that at 1 dph (11 ± 2.1 cells). Therefore, based on our data and previous reports, the initial germ cell proliferation of yellowfin tuna is relatively slower than that of other fish species. PMID:26051459

  5. Mouse oocytes differentiate through organelle enrichment from sister cyst germ cells.

    PubMed

    Lei, Lei; Spradling, Allan C

    2016-04-01

    Oocytes differentiate in diverse species by receiving organelles and cytoplasm from sister germ cells while joined in germline cysts or syncytia. Mouse primordial germ cells form germline cysts, but the role of cysts in oogenesis is unknown. We find that mouse germ cells receive organelles from neighboring cyst cells and build a Balbiani body to become oocytes, whereas nurselike germ cells die. Organelle movement, Balbiani body formation, and oocyte fate determination are selectively blocked by low levels of microtubule-dependent transport inhibitors. Membrane breakdown within the cyst and an apoptosis-like process are associated with organelle transfer into the oocyte, events reminiscent of nurse cell dumping in Drosophila We propose that cytoplasmic and organelle transport plays an evolutionarily conserved and functionally important role in mammalian oocyte differentiation.

  6. [Current progress and future direction in the biology of ovarian germ stem cells in mammals].

    PubMed

    Li, Chao-Hui; Guo, Kun; Zheng, Ping

    2012-12-01

    Whether or not oogenesis continues after birth in mammalian ovaries remains controversial. Since the 1950's, it has been generally accepted that oogenesis takes place during embryogenesis in mammals and ceases at birth. At birth, germ cells in mammalian ovaries have progressed to the diplotene stage of meiotic prophase and have formed primordial follicles with surrounding somatic cells. These primordial follicles represent follicle reserves of the reproductive life. However, this view has been recently challenged by a growing body of evidence showing the isolation and propagation of germ stem cells from mouse and human ovaries. These ovarian germ stem cells are capable of regenerating functional oocytes when transplanted back into recipient ovaries. Despite the discovery of the potential germ stem cells in mammalian ovaries, it remains uncertain whether these cells exist and function in ovaries under physiological conditions. Herein we review the current progress and future direction in this infant area.

  7. Mouse oocytes differentiate through organelle enrichment from sister cyst germ cells.

    PubMed

    Lei, Lei; Spradling, Allan C

    2016-04-01

    Oocytes differentiate in diverse species by receiving organelles and cytoplasm from sister germ cells while joined in germline cysts or syncytia. Mouse primordial germ cells form germline cysts, but the role of cysts in oogenesis is unknown. We find that mouse germ cells receive organelles from neighboring cyst cells and build a Balbiani body to become oocytes, whereas nurselike germ cells die. Organelle movement, Balbiani body formation, and oocyte fate determination are selectively blocked by low levels of microtubule-dependent transport inhibitors. Membrane breakdown within the cyst and an apoptosis-like process are associated with organelle transfer into the oocyte, events reminiscent of nurse cell dumping in Drosophila We propose that cytoplasmic and organelle transport plays an evolutionarily conserved and functionally important role in mammalian oocyte differentiation. PMID:26917595

  8. Identification of novel fusion genes in testicular germ cell tumors

    PubMed Central

    Hoff, Andreas M.; Alagaratnam, Sharmini; Zhao, Sen; Bruun, Jarle; Andrews, Peter W.; Lothe, Ragnhild A.; Skotheim, Rolf I.

    2015-01-01

    Testicular germ cell tumors (TGCT) are the most frequently diagnosed solid tumors in young men ages 15 to 44 years. Embryonal carcinomas (EC) comprise a subset of TGCTs that exhibit pluripotent characteristics similar to embryonic stem (ES) cells, but the genetic drivers underlying malignant transformation of ECs are unknown. To elucidate the abnormal genetic events potentially contributing to TGCT malignancy, such as the existence of fusion genes or aberrant fusion transcript expression, we performed RNA sequencing of EC cell lines and their non-malignant ES cell line counterparts. We identified eight novel fusion transcripts and one gene with alternative promoter usage, ETV6. Four out of nine transcripts were found recurrently expressed in an extended panel of primary TGCTs and additional EC cell lines, but not in normal parenchyma of the testis, implying tumor-specific expression. Two of the recurrent transcripts involved an intrachromosomal fusion between RCC1 and HENMT1 located 80 Mbp apart and an interchromosomal fusion between RCC1 and ABHD12B. RCC1-ABHD12B and the ETV6 transcript variant were found to be preferentially expressed in the more undifferentiated TGCT subtypes. In vitro differentiation of the NTERA2 EC cell line resulted in significantly reduced expression of both fusion transcripts involving RCC1 and the ETV6 transcript variant, indicating that they are markers of pluripotency in a malignant setting. In conclusion, we identified eight novel fusion transcripts that, to our knowledge, are the first fusion genes described in TGCT and may therefore potentially serve as genomic biomarkers of malignant progression. PMID:26659575

  9. The Origin And Migration Of Primordial Germ Cells In Sturgeons

    PubMed Central

    Saito, Taiju; Pšenička, Martin; Goto, Rie; Adachi, Shinji; Inoue, Kunio; Arai, Katsutoshi; Yamaha, Etsuro

    2014-01-01

    Primordial germ cells (PGCs) arise elsewhere in the embryo and migrate into developing gonadal ridges during embryonic development. In several model animals, formation and migration patterns of PGCs have been studied, and it is known that these patterns vary. Sturgeons (genus Acipenser) have great potential for comparative and evolutionary studies of development. Sturgeons belong to the super class Actinoptergii, and their developmental pattern is similar to that of amphibians, although their phylogenetic position is an out-group to teleost fishes. Here, we reveal an injection technique for sturgeon eggs allowing visualization of germplasm and PGCs. Using this technique, we demonstrate that the PGCs are generated at the vegetal pole of the egg and they migrate on the yolky cell mass toward the gonadal ridge. We also provide evidence showing that PGCs are specified by inheritance of maternally supplied germplasm. Furthermore, we demonstrate that the migratory mechanism is well-conserved between sturgeon and other remotely related teleosts, such as goldfish, by a single PGCs transplantation (SPT) assay. The mode of PGCs specification in sturgeon is similar to that of anurans, but the migration pattern resembles that of teleosts. PMID:24505272

  10. Poultry genetic resource conservation using primordial germ cells

    PubMed Central

    NAKAMURA, Yoshiaki

    2016-01-01

    The majority of poultry genetic resources are maintained in situ in living populations. However, in situ conservation of poultry genetic resources always carries the risk of loss owing to pathogen outbreaks, genetic problems, breeding cessation, or natural disasters. Cryobanking of germplasm in birds has been limited to the use of semen, preventing conservation of the W chromosome and mitochondrial DNA. A further challenge is posed by the structure of avian eggs, which restricts the cryopreservation of ova and fertilized embryos, a technique widely used for mammalian species. By using a unique biological property and accessibility of avian primordial germ cells (PGCs), precursor cells for gametes, which temporally circulate in the vasculature during early development, an avian PGC transplantation technique has been established. To date, several techniques for PGC manipulation including purification, cryopreservation, depletion, and long-term culture have been developed in chickens. PGC transplantation combined with recent advanced PGC manipulation techniques have enabled ex situ conservation of poultry genetic resources in their complete form. Here, the updated technologies for avian PGC manipulation are introduced, and then the concept of a poultry PGC-bank is proposed by considering the biological properties of avian PGCs. PMID:27210834

  11. Expression of Genes Related to Germ Cell Lineage and Pluripotency in Single Cells and Colonies of Human Adult Germ Stem Cells

    PubMed Central

    Conrad, Sabine; Azizi, Hossein; Hatami, Maryam; Kubista, Mikael; Bonin, Michael; Hennenlotter, Jörg; Sievert, Karl-Dietrich; Skutella, Thomas

    2016-01-01

    The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen−/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profile in vitro, related but not identical to true pluripotent stem cells. Under ES-cell conditions haGSC colonies could be selected and maintained in a partial pluripotent state at the molecular level, which may be related to their cell plasticity and potential to differentiate into cells of all germ layers. PMID:26649052

  12. Cyclic AMP and c-KIT Signaling in Familial Testicular Germ Cell Tumor Predisposition

    PubMed Central

    Azevedo, Monalisa F.; Horvath, Anelia; Bornstein, Ethan R.; Almeida, Madson Q.; Xekouki, Paraskevi; Faucz, Fabio R.; Gourgari, Evgenia; Nadella, Kiran; Remmers, Elaine F.; Quezado, Martha; de Alexandre, Rodrigo Bertollo; Kratz, Christian P.; Nesterova, Maria; Greene, Mark H.

    2013-01-01

    Background: Familial testicular germ cell tumors (FTGCTs) are hypothesized to result from the combined interaction of multiple low-penetrance genes. We reported inactivating germline mutations of the cAMP-binding phosphodiesterase 11A (PDE11A) as modifiers of FTGCT risk. Recent genome-wide association studies have identified single-nucleotide polymorphisms in the KITLG gene, the ligand for the cKIT tyrosine kinase receptor, as strong modifiers of susceptibility to both familial and sporadic testicular germ cell tumors. Design: We studied 94 patients with FTGCTs and 50 at-risk male relatives from 63 unrelated kindreds, in whom the PDE11A gene had been sequenced by investigating the association between KITLG genome-wide association study single-nucleotide polymorphisms rs3782179 and rs4474514 and FTGCT risk in these patients and in 692 controls. We also examined cAMP and c-KIT signaling in testicular tissues and cell lines and extended the studies to 2 sporadic cases, one with a PDE11A defect and one without, as a comparison. Results: We found a higher frequency of the KITLG risk alleles in FTGCT patients who also had a PDE11A sequence variant, compared with those with a wild-type PDE11A sequence. In NTERA-2 and Tcam-2 cells transfected with the mutated forms of PDE11A (R52T, F258Y, Y727C, R804H, V820M, R867G, and M878V), cAMP levels were significantly higher, and the relative phosphodiesterase activity was lower than in the wild-type cells. KITLG expression was consistently increased in the presence of PDE11A-inactivating defects, both at the RNA and protein levels, in familial testicular germ cell tumors. The 2 sporadic cases that were studied, one with a PDE11A defect and another without, agreed with the data in FTGTCT and in the cell lines. Conclusions: Patients with FTGCT and PDE11A defects also carry KITLG risk alleles more frequently. There may be an interaction between cAMP and c-KIT signaling in predisposition to testicular germ cell tumors. PMID:23771924

  13. Germ-cell deficient (gcd), an insertional mutation manifested as infertility in transgenic mice.

    PubMed

    Pellas, T C; Ramachandran, B; Duncan, M; Pan, S S; Marone, M; Chada, K

    1991-10-01

    A genetic analysis is necessary to gain a greater understanding of the complex developmental processes in mammals. Toward this end, an insertional transgenic mouse mutant has been isolated that results in abnormal germ-cell development. This recessive mutation manifests as infertility in both males and females and is specific for the reproductive organs, since all other tissues examined were histologically normal. A developmental analysis of the gonadal tissues demonstrated that the germ cells were specifically depleted as early as day 11.5 of embryonic development, while the various somatic cells were apparently unaffected. Therefore, the mutated locus must play a critical role in the migration/proliferation of primordial germ cells to the genital ridges of developing embryos. In addition, females homozygous for the mutation could potentially be a valuable animal model of a human syndrome, premature ovarian failure. This mutation has been named germ-cell deficient, gcd.

  14. In vitro production of haploid sperm cells from male germ cells of foetal cattle.

    PubMed

    Dong, Wu-Zi; Hua, Jin-Lian; Shen, Wen-Zheng; Dou, Zhong-Ying

    2010-04-01

    The purpose of this study was to isolate the foetal cattle male germ cells (mGCs) and then induce them into sperm cells. The mGCs were purified and enriched by a two-step plating method based on the different adherence velocities of mGCs and somatic cells. The percentage of the vasa and the c-kit positive cells were 95.34+/-2.25% and 53.3+/-1.03% by using flow cytometry analysis (FCA), respectively. In feeder-free culture system, the half-suspending cells appeared and formed a 16-cell rosary in medium after the mGCs were cultured for 6-8 days. On immunocytochemical staining during the second passage, some single cells adhering to the plate appeared to be both Oct-4 and alpha6-integrin positive. During the third passage, the mGCs were induced for 48 h by retinol acid (RA) on Sertoli cell-feeder layer, followed by 5-7 days culture in an RA-free medium. Some elongated sperm-like cells appeared in the medium at this stage. We found that the most effective concentration of RA for the inducement was 10(-7)moll(-1) (P<0.01). The haploid cells in suspension were identified by FCA. The elongated sperm-like cells showed proacrosome-like structure and the flagellum with fibre construct under electron microscopy. The mRNA of outer dense fibre-3 (ODF-3) and transcription protein-1 (TP-1) could be detected in the suspended cells by using reverse transcription polymerase chain reaction (RT-PCR). About 23.1% bovine oocytes could be activated to perform cleavage by intracytoplasmic injection with the sperm-like cells, but embryos did not further develop. Our investigation further demonstrated that foetal cattle mGCs could be induced in vitro into haploid sperm in the short term. PMID:19632794

  15. In vitro production of haploid sperm cells from male germ cells of foetal cattle.

    PubMed

    Dong, Wu-Zi; Hua, Jin-Lian; Shen, Wen-Zheng; Dou, Zhong-Ying

    2010-04-01

    The purpose of this study was to isolate the foetal cattle male germ cells (mGCs) and then induce them into sperm cells. The mGCs were purified and enriched by a two-step plating method based on the different adherence velocities of mGCs and somatic cells. The percentage of the vasa and the c-kit positive cells were 95.34+/-2.25% and 53.3+/-1.03% by using flow cytometry analysis (FCA), respectively. In feeder-free culture system, the half-suspending cells appeared and formed a 16-cell rosary in medium after the mGCs were cultured for 6-8 days. On immunocytochemical staining during the second passage, some single cells adhering to the plate appeared to be both Oct-4 and alpha6-integrin positive. During the third passage, the mGCs were induced for 48 h by retinol acid (RA) on Sertoli cell-feeder layer, followed by 5-7 days culture in an RA-free medium. Some elongated sperm-like cells appeared in the medium at this stage. We found that the most effective concentration of RA for the inducement was 10(-7)moll(-1) (P<0.01). The haploid cells in suspension were identified by FCA. The elongated sperm-like cells showed proacrosome-like structure and the flagellum with fibre construct under electron microscopy. The mRNA of outer dense fibre-3 (ODF-3) and transcription protein-1 (TP-1) could be detected in the suspended cells by using reverse transcription polymerase chain reaction (RT-PCR). About 23.1% bovine oocytes could be activated to perform cleavage by intracytoplasmic injection with the sperm-like cells, but embryos did not further develop. Our investigation further demonstrated that foetal cattle mGCs could be induced in vitro into haploid sperm in the short term.

  16. Progesterone regulates chicken embryonic germ cell meiotic initiation independent of retinoic acid signaling.

    PubMed

    Mi, Yuling; He, Bin; Li, Jian; Zhang, Caiqiao

    2014-07-15

    The signaling molecule retinoic acid (RA) is known to trigger germ cells to enter meiosis. However, RA may not be the only secreted inducer of meiosis. Our previous data indicate that luteinizing hormone also promotes germ cell meiotic initiation by upregulating 3βHSDII transcription. Here, using chicken embryos, we investigate the role of progesterone (P4) in regulating germ cell meiotic initiation. Progesterone treatment at embryonic Day 9.5 accelerated germ cell meiosis entry in the female chicken embryos. However, P4 treatment in vivo did no influence on testicular germ cells but triggered their meiotic initiation in the cultured testes. As treatment with an RA receptor (RAR) inhibitor did not block the stimulatory effect of P4 on germ cell meiotic initiation, this P4 stimulatory effect seems to be independent of RAR-mediated signaling. The abundance of RA metabolism-related enzymes and RAR (RARβ) mRNAs did not differ significantly between P4-treated and control individuals. The RA concentration in the ovaries remained unchanged by P4 treatment in vivo. Because no inhibition by the P4 receptor (PR) nuclear receptor antagonist mifepristone on P4 effect was observed in either in vitro or in vivo experiments, the effect of P4 on germ cell meiotic initiation is probably mediated by membrane PRs (mPR). The mPRα, mPRβ, and mPRγ mRNAs were all expressed in the embryonic ovaries. The expression of mPRα and mPRβ was higher than that of mPRγ. Immunohistochemical results showed that mPRα-positive cells were mainly scattered in the ovarian cortex area where most germ cells were distributed. The mPRβ-positive cells were widely distributed in the ovaries, and positive cells were clustered with a similar morphology to that of germ cell clusters. In conclusion, P4 may regulate embryonic germ cell meiotic initiation independent of RA signaling through the membrane PRs. This study provides a new insight into the mechanisms of germ cell meiotic initiation in the chicken

  17. CREM, a master-switch of the transcriptional cascade in male germ cells.

    PubMed

    De Cesare, D; Fimia, G M; Sassone-Corsi, P

    2000-10-01

    In eukaryotes, transcriptional regulation upon stimulation of the adenylyl cyclase signalling pathway is mediated by a family of cAMP-responsive nuclear factors. The CREB and CREM transcription factors are activated by phosphorylation of a key serine residue by kinase stimulated by cyclic AMP, calcium, growth factors and stress signals. Phosphorylation allows recruitment of CBP (CREB Binding Protein), a large co-activator that contacts the general transcriptional machinery. The CREM gene plays a key physiological and developmental role within the hypothalamic-pituitary-gonadal axis. CREM is highly expressed in postmeiotic cells upon a striking developmental switch regulated by the pituitary hormone FSH. CREM-mutant mice generated by homologous recombination reveal that spermatogenesis stops at the first step of spermiogenesis. Late spermatids are completely absent while there is a significant increase in apoptotic germ cells. Mutant male mice completely lack spermatozoa, a phenotype reminiscent of cases of human infertility. Interestingly, in male germ cells, CREM is not phosphorylated but associates with ACT, a member of the LIM-only class of proteins that has intrinsic transcriptional activity. Thus, in some circumstance, CREM can bypass the classical requirement for phosphorylation and association with CBP.

  18. Human endogenous retrovirus rec interferes with germ cell development in mice and may cause carcinoma in situ, the predecessor lesion of germ cell tumors.

    PubMed

    Galli, Uwe M; Sauter, Marlies; Lecher, Bernd; Maurer, Simone; Herbst, Hermann; Roemer, Klaus; Mueller-Lantzsch, Nikolaus

    2005-04-28

    Germ cell tumors (GCTs) are among the most common malignancies in young men. We have previously documented that patients with GCT frequently produce serum antibodies directed against proteins encoded by human endogenous retrovirus (HERV) type K sequences. Transcripts originating from the env gene of HERV-K, including the rec-relative of human immunodeficiency virus rev, are highly expressed in GCTs. We report here that mice that inducibly express HERV-K rec show a disturbed germ cell development and may exhibit, by 19 months of age, changes reminiscent of carcinoma in situ, the predecessor lesion of classic seminoma in humans. This provides the first direct evidence that the expression of a human endogenous retroviral gene previously established as a marker in human germ cell tumors may contribute to organ-specific tumorigenesis in a transgenic mouse model.

  19. The PUF binding landscape in metazoan germ cells

    PubMed Central

    Prasad, Aman; Porter, Douglas F.; Kroll-Conner, Peggy L.; Mohanty, Ipsita; Ryan, Anne R.; Crittenden, Sarah L.; Wickens, Marvin; Kimble, Judith

    2016-01-01

    PUF (Pumilio/FBF) proteins are RNA-binding proteins and conserved stem cell regulators. The Caenorhabditis elegans PUF proteins FBF-1 and FBF-2 (collectively FBF) regulate mRNAs in germ cells. Without FBF, adult germlines lose all stem cells. A major gap in our understanding of PUF proteins, including FBF, is a global view of their binding sites in their native context (i.e., their “binding landscape”). To understand the interactions underlying FBF function, we used iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) to determine binding landscapes of C. elegans FBF-1 and FBF-2 in the germline tissue of intact animals. Multiple iCLIP peak-calling methods were compared to maximize identification of both established FBF binding sites and positive control target mRNAs in our iCLIP data. We discovered that FBF-1 and FBF-2 bind to RNAs through canonical as well as alternate motifs. We also analyzed crosslinking-induced mutations to map binding sites precisely and to identify key nucleotides that may be critical for FBF–RNA interactions. FBF-1 and FBF-2 can bind sites in the 5′UTR, coding region, or 3′UTR, but have a strong bias for the 3′ end of transcripts. FBF-1 and FBF-2 have strongly overlapping target profiles, including mRNAs and noncoding RNAs. From a statistically robust list of 1404 common FBF targets, 847 were previously unknown, 154 were related to cell cycle regulation, three were lincRNAs, and 335 were shared with the human PUF protein PUM2. PMID:27165521

  20. Testicular cell-conditioned medium supports embryonic stem cell differentiation toward germ lineage and to spermatocyte- and oocyte-like cells.

    PubMed

    Shah, Syed M; Saini, Neha; Singh, Manoj K; Manik, Radheysham; Singla, Suresh K; Palta, Prabhat; Chauhan, Manmohan S

    2016-08-01

    Testicular cells are believed to secrete various growth factors that activate signaling pathways finally leading to gametogenesis. In vitro gametogenesis is an obscure but paramountly important task primarily because of paucity of the precursor cells and first trimester gonadal tissues. To overcome these limitations for development of in vitro gametes, the present study was designed to induce differentiation of buffalo embryonic stem (ES) cells into germ lineage cells on stimulation by testicular cell-conditioned medium (TCM), on the basis of the assumption that ES cells have the intrinsic property to differentiate into any cell type and TCM would provide the necessary growth factors for differentiation toward germ cell lineage. For this purpose, buffalo ES cells were differentiated as embryoid bodies (EB) in floating cultures and as monolayer adherent cultures in different doses (10%, 20%, and 40%) of TCM for different culture intervals (4, 8, and 14 days), to identify the optimum dose-and-time period. We observed that 40% TCM dose induces highest expression of primordial germ cell-specific (DAZL, VASA, and PLZF), meiotic (SYCP3, MLH1, TNP1/2, and PRM2), spermatocyte-specific (BOULE and TEKT1), and oocyte-specific genes (GDF9 and ZP2/3) for a culture period of 14 days under both floating and adherent differentiation. Immunocytochemical analysis of EBs and adherent cultures revealed presence of primordial germ cell markers (c-KIT, DAZL, and VASA), meiotic markers (SYCP3, MLH1 and PROTAMINE1), spermatocyte markers (ACROSIN and HAPRIN), and oocyte markers (GDF9 and ZP4), indicating progression into post-meiotic gametogenesis. The detection of germ cell-specific proteins in Day 14 EBs like VASA, GDF9, and ZP4 by Western blotting further confirmed germ lineage differentiation. The significantly lower (P < 0.05) concentration of 5-methyl-2-deoxycytidine in optimally differentiated EBs is suggestive of the process of methylation erasure. Oocyte-like structures

  1. Germ cell toxicity: significance in genetic and fertility effects of radiation and chemicals

    SciTech Connect

    Oakberg, E.F.

    1983-01-01

    The response of the male and female to radiation and chemicals is different. Any loss of oocytes in the female cannot be replaced, and if severe enough, will result in a shortening of the reproductive span. In the male, a temporary sterile period may be induced owing to destruction of the differentiating spermatogonia, but the stem cells are the most resistant spermatogonial type, are capable of repopulating the seminiferous epithelium, and fertility usually returns. The response of both the male and female changes with development of the embryonic to the adult gonad, and with differentiation and maturation in the adult. The primordial germ cells, early oocytes, and differentiating spermatogonia of the adult male are unusually sensitive to the cytotoxic action of noxious agents, but each agent elicits a specific response owing to the intricate biochemical and physiological changes associated with development and maturation of the gametes. The relationship of germ cell killing to fertility is direct, and long-term fertility effects can be predicted from histological analysis of the gonads. The relationship to genetic effects, on the other hand, is indirect, and acts primarily by limiting the cell stages available for testing, by affecting the distribution of mitotically active stem cells among the different stages of the mitotic cycle, and thereby, changing both the type and frequency of genetic effects observed. 100 references, 38 figures, 7 tables.

  2. Germ cells of the centipede Strigamia maritima are specified early in embryonic development.

    PubMed

    Green, Jack E; Akam, Michael

    2014-08-15

    We provide the first systematic description of germ cell development with molecular markers in a myriapod, the centipede Strigamia maritima. By examining the expression of Strigamia vasa and nanos orthologues, we find that the primordial germ cells are specified from at least the blastoderm stage. This is a much earlier embryonic stage than previously described for centipedes, or any other member of the Myriapoda. Using these genes as markers, and taking advantage of the developmental synchrony of Strigamia embryos within single clutches, we are able to track the development of the germ cells throughout embryogenesis. We find that the germ cells accumulate at the blastopore; that the cells do not internalize through the hindgut, but rather through the closing blastopore; and that the cells undergo a long-range migration to the embryonic gonad. This is the first evidence for primordial germ cells displaying these behaviours in any myriapod. The myriapods are a phylogenetically important group in the arthropod radiation for which relatively little developmental data is currently available. Our study provides valuable comparative data that complements the growing number of studies in insects, crustaceans and chelicerates, and is important for the correct reconstruction of ancestral states and a fuller understanding of how germ cell development has evolved in different arthropod lineages.

  3. Germ cells of the centipede Strigamia maritima are specified early in embryonic development.

    PubMed

    Green, Jack E; Akam, Michael

    2014-08-15

    We provide the first systematic description of germ cell development with molecular markers in a myriapod, the centipede Strigamia maritima. By examining the expression of Strigamia vasa and nanos orthologues, we find that the primordial germ cells are specified from at least the blastoderm stage. This is a much earlier embryonic stage than previously described for centipedes, or any other member of the Myriapoda. Using these genes as markers, and taking advantage of the developmental synchrony of Strigamia embryos within single clutches, we are able to track the development of the germ cells throughout embryogenesis. We find that the germ cells accumulate at the blastopore; that the cells do not internalize through the hindgut, but rather through the closing blastopore; and that the cells undergo a long-range migration to the embryonic gonad. This is the first evidence for primordial germ cells displaying these behaviours in any myriapod. The myriapods are a phylogenetically important group in the arthropod radiation for which relatively little developmental data is currently available. Our study provides valuable comparative data that complements the growing number of studies in insects, crustaceans and chelicerates, and is important for the correct reconstruction of ancestral states and a fuller understanding of how germ cell development has evolved in different arthropod lineages. PMID:24930702

  4. [Clinical evaluation of nonseminomatous testicular germ cell tumors].

    PubMed

    Nukui, M; Nakao, M; Nakagawa, S; Takada, H; Ebisui, K; Sugimoto, K; Watanabe, H; Maekawa, M

    1991-10-01

    We treated 26 patients with nonseminomatous germ cell tumors (NSGCT) between January 1976 and March 1989. Histologically, 7 were embryonal carcinoma (27%), 4 were teratoma (15%), 2 were yolk sac tumor (8%), 10 were teratocarcinoma (38%) and 3 were other mixed tumors. As regards staging, 18 belonged to stage I (69%), 1 to stage II A (4%), 1 to stage IIB (4%), 1 to stage IIIA, 2 to stage III B1 (8%) and 3 to stage III B2 (12%). Patients in stage I were treated by orchidectomy with lymphadenectomy and occasionally chemotherapy before 1984, resulting in a 100% 5-year survival. However, after 1985, 5 cases in stage I were treated by orchidectomy alone according to a watch-and-see policy. Two cases among them relapsed within two years and both of them contained immature teratoma elements. Six patients with metastatic tumor were treated with PVB therapy of which response rate was 66.7%. The total 5-year survival rate of patients in stage I, II and III was 100%, 50%, 50%, respectively and that in overall cases was 84.6%.

  5. Familial testicular germ cell tumor: no associated syndromic pattern identified

    PubMed Central

    2014-01-01

    Background Testicular germ cell tumor (TGCT) is the most common malignancy in young men. Familial clustering, epidemiologic evidence of increased risk with family or personal history, and the association of TGCT with genitourinary (GU) tract anomalies have suggested an underlying genetic predisposition. Linkage data have not identified a rare, highly-penetrant, single gene in familial TGCT (FTGCT) cases. Based on its association with congenital GU tract anomalies and suggestions that there is an intrauterine origin to TGCT, we hypothesized the existence of unrecognized dysmorphic features in FTGCT. Methods We evaluated 38 FTGCT individuals and 41 first-degree relatives from 22 multiple-case families with detailed dysmorphology examinations, physician-based medical history and physical examination, laboratory testing, and genitourinary imaging studies. Results The prevalence of major abnormalities and minor variants did not significantly differ between either FTGCT individuals or their first-degree relatives when compared with normal population controls, except for tall stature, macrocephaly, flat midface, and retro-/micrognathia. However, these four traits were not manifest as a constellation of features in any one individual or family. We did detect an excess prevalence of the genitourinary anomalies cryptorchidism and congenital inguinal hernia in our population, as previously described in sporadic TGCT, but no congenital renal, retroperitoneal or mediastinal anomalies were detected. Conclusions Overall, our study did not identify a constellation of dysmorphic features in FTGCT individuals, which is consistent with results of genetic studies suggesting that multiple low-penetrance genes are likely responsible for FTGCT susceptibility. PMID:24559313

  6. Analysis of the gene expression profile of mouse male meiotic germ cells.

    PubMed

    Rossi, Pellegrino; Dolci, Susanna; Sette, Claudio; Capolunghi, Federica; Pellegrini, Manuela; Loiarro, Maria; Di Agostino, Silvia; Paronetto, Maria Paola; Grimaldi, Paola; Merico, Daniele; Martegani, Enzo; Geremia, Raffaele

    2004-05-01

    Wide genome analysis of difference in gene expression between spermatogonial populations from 7-day-old mice and pachytene spermatocytes from 18-day-old mice was performed using Affymetrix gene chips representing approximately 12,500 mouse known genes or EST sequences, spanning approximately 1/3rd of the mouse genome. To delineate differences in the profile of gene expression between mitotic and meiotic stages of male germ cell differentiation, expressed genes were grouped in functional clusters. The analysis confirmed the previously described pre-meiotic or meiotic expression for several genes, in particular for those involved in the regulation of the mitotic and meiotic cell cycle, and for those whose transcripts are accumulated during the meiotic stages to be translated later in post-meiotic stages. Differential expression of several additional genes was discovered. In few cases (pro-apoptotic factors Bak, Bad and Bax), data were in conflict with the previously published stage-dependent expression of genes already known to be expressed in male germ cells. Northern blot analysis of selected genes confirmed the results obtained with the microarray chips. Six of these were novel genes specifically expressed in pachytene spermatocytes: a chromatin remodeling factor (chrac1/YCL1), a homeobox gene (hmx1), a novel G-coupled receptor for an unknown ligand (Gpr19), a glycoprotein of the intestinal epithelium (mucin 3), a novel RAS activator (Ranbp9), and the A630056B21Rik gene (predicted to encode a novel zinc finger protein). These studies will help to delineate the global patterns of gene expression characterizing male germ cell differentiation for a better understanding of regulation of spermatogenesis in mammals.

  7. Mammalian male germ cells are fertile ground for expression profiling of sexual reproduction.

    PubMed

    Wrobel, Gunnar; Primig, Michael

    2005-01-01

    Recent large-scale transcriptional profiling experiments of mammalian spermatogenesis using rodent model systems and different types of microarrays have yielded insight into the expression program of male germ cells. These studies revealed that an astonishingly large number of loci are differentially expressed during spermatogenesis. Among them are several hundred transcripts that appear to be specific for meiotic and post-meiotic germ cells. This group includes many genes that were previously implicated in spermatogenesis and/or fertility and others that are as yet poorly characterized. Profiling experiments thus reveal candidates for regulation of spermatogenesis and fertility as well as targets for innovative contraceptives that act on gene products absent in somatic tissues. In this review, consolidated high density oligonucleotide microarray data from rodent total testis and purified germ cell samples are analyzed and their impact on our understanding of the transcriptional program governing male germ cell differentiation is discussed. PMID:15615893

  8. Automatic classification of fish germ cells through optimum-path forest.

    PubMed

    Papa, João P; Gutierrez, Mario E M; Nakamura, Rodrigo Y M; Papa, Luciene P; Vicentini, Irene B F; Vicentini, Carlos A

    2011-01-01

    The spermatogenesis is crucial to the species reproduction, and its monitoring may shed light over some important information of such process. Thus, the germ cells quantification can provide useful tools to improve the reproduction cycle. In this paper, we present the first work that address this problem in fishes with machine learning techniques. We show here how to obtain high recognition accuracies in order to identify fish germ cells with several state-of-the-art supervised pattern recognition techniques.

  9. Wnt signaling-mediated redox regulation maintains the germ line stem cell differentiation niche

    PubMed Central

    Wang, Su; Gao, Yuan; Song, Xiaoqing; Ma, Xing; Zhu, Xiujuan; Mao, Ying; Yang, Zhihao; Ni, Jianquan; Li, Hua; Malanowski, Kathryn E; Anoja, Perera; Park, Jungeun; Haug, Jeff; Xie, Ting

    2015-01-01

    Adult stem cells continuously undergo self-renewal and generate differentiated cells. In the Drosophila ovary, two separate niches control germ line stem cell (GSC) self-renewal and differentiation processes. Compared to the self-renewing niche, relatively little is known about the maintenance and function of the differentiation niche. In this study, we show that the cellular redox state regulated by Wnt signaling is critical for the maintenance and function of the differentiation niche to promote GSC progeny differentiation. Defective Wnt signaling causes the loss of the differentiation niche and the upregulated BMP signaling in differentiated GSC progeny, thereby disrupting germ cell differentiation. Mechanistically, Wnt signaling controls the expression of multiple glutathione-S-transferase family genes and the cellular redox state. Finally, Wnt2 and Wnt4 function redundantly to maintain active Wnt signaling in the differentiation niche. Therefore, this study has revealed a novel strategy for Wnt signaling in regulating the cellular redox state and maintaining the differentiation niche. DOI: http://dx.doi.org/10.7554/eLife.08174.001 PMID:26452202

  10. Identification and characterization of a haploid germ cell-specific nuclear protein kinase (Haspin) in spermatid nuclei and its effects on somatic cells.

    PubMed

    Tanaka, H; Yoshimura, Y; Nozaki, M; Yomogida, K; Tsuchida, J; Tosaka, Y; Habu, T; Nakanishi, T; Okada, M; Nojima, H; Nishimune, Y

    1999-06-11

    We have cloned the entire coding region of a mouse germ cell-specific cDNA encoding a unique protein kinase whose catalytic domain contains only three consensus subdomains (I-III) instead of the normal 12. The protein possesses intrinsic Ser/Thr kinase activity and is exclusively expressed in haploid germ cells, localizing only in their nuclei, and was thus named Haspin (for haploid germ cell-specific nuclear protein kinase). Western blot analysis showed that specific antibodies recognized a protein of Mr 83,000 in the testis. Ectopically expressed Haspin was detected exclusively in the nuclei of cultured somatic cells. Even in the absence of kinase activity, however, Haspin caused cell cycle arrest at G1, resulting in growth arrest of the transfected somatic cells. In a DNA binding experiment, approximately one-half of wild-type Haspin was able to bind to a DNA-cellulose column, whereas the other half was not. In contrast, all of the deletion mutant Haspin that lacked autophosphorylation bound to the DNA column. Thus, the DNA-binding activity of Haspin may, in some way, be associated with its kinase activity. These observations suggest that Haspin has some critical roles in cell cycle cessation and differentiation of haploid germ cells. PMID:10358056

  11. Expression and Localization of Opioid Receptors in Male Germ Cells and the Implication for Mouse Spermatogenesis

    PubMed Central

    Gianzo, Marta; Urizar-Arenaza, Itziar; Casis, Luis; Irazusta, Jon; Subirán, Nerea

    2016-01-01

    The presence of endogenous opioid peptides in different testicular cell types has been extensively characterized and provides evidence for the participation of the opioid system in the regulation of testicular function. However, the exact role of the opioid system during the spermatogenesis has remained controversial since the presence of the mu-, delta- and kappa-opioid receptors in spermatogenic cells was yet to be demonstrated. Through a combination of quantitative real-time PCR, immunofluorescence, immunohistochemistry and flow cytometry approaches, we report for the first time the presence of active mu-, delta- and kappa-opioid receptors in mouse male germ cells. They show an exposition time-dependent response to opioid agonist, hence suggesting their active involvement in spermatogenesis. Our results contribute to understanding the role of the opioid receptors in the spermatogenesis and could help to develop new strategies to employ the opioid system as a biochemical tool for the diagnosis and treatment of male infertility. PMID:27031701

  12. Recovery from Choriocarcinoma Syndrome Associated with a Metastatic Extragonadal Germ Cell Tumor Hemorrhage

    PubMed Central

    Komori, Koji; Takahari, Daisuke; Kimura, Kenya; Kinoshita, Takashi; Ito, Seiji; Abe, Tetsuya; Senda, Yoshiki; Misawa, Kazunari; Ito, Yuichi; Uemura, Norihisa; Natsume, Seiji; Kawakami, Jiro; Iwata, Yoshinori; Tsutsuyama, Masayuki; Shigeyoshi, Itaru; Akazawa, Tomoyuki; Hayashi, Daisuke; Ouchi, Akira; Shimizu, Yasuhiro

    2016-01-01

    A germ cell tumor is the most common form of malignancy in early male life, and can be classified as either seminomatous or nonseminomatous. Choriocarcinoma, comprised of nonseminomatous germ cells, is the most aggressive type of germ cell tumor and characteristically metastasizes to the retroperitoneal lymph nodes and less frequently to the lungs, liver, bone or brain [Shibuya et al., 2009;48: 551–554]. A 56-year-old man was admitted to another hospital complaining of abdominal distension. Symptoms included anorexia, vomiting, and diarrhea. The patient was diagnosed with an extragonadal germ cell tumor and referred to our hospital to receive chemotherapy. The day after admission, the patient's abdominal distension gradually worsened. An emergency operation revealed venous hemorrhage from the surface of a metastatic extragonadal germ cell tumor between the ligament of Treitz and the inferior mesenteric vein in a horizontal position. Hemostatic treatment was performed with 4-0 proline thread attached to a medicated cotton sponge, rather than using a simple proline thread, and the closure area was manually compressed. Chemotherapy was initiated on postoperative day 10. A metastatic extragonadal germ cell tumor that causes massive hemorrhage and gastrointestinal hemorrhage is very rare, and represents a life-threatening emergency. If the patient's condition carries a substantial risk of bleeding to death, it may be worthwhile to attempt abdominal operations. PMID:27403124

  13. Recovery from Choriocarcinoma Syndrome Associated with a Metastatic Extragonadal Germ Cell Tumor Hemorrhage.

    PubMed

    Komori, Koji; Takahari, Daisuke; Kimura, Kenya; Kinoshita, Takashi; Ito, Seiji; Abe, Tetsuya; Senda, Yoshiki; Misawa, Kazunari; Ito, Yuichi; Uemura, Norihisa; Natsume, Seiji; Kawakami, Jiro; Iwata, Yoshinori; Tsutsuyama, Masayuki; Shigeyoshi, Itaru; Akazawa, Tomoyuki; Hayashi, Daisuke; Ouchi, Akira; Shimizu, Yasuhiro

    2016-01-01

    A germ cell tumor is the most common form of malignancy in early male life, and can be classified as either seminomatous or nonseminomatous. Choriocarcinoma, comprised of nonseminomatous germ cells, is the most aggressive type of germ cell tumor and characteristically metastasizes to the retroperitoneal lymph nodes and less frequently to the lungs, liver, bone or brain [Shibuya et al., 2009;48: 551-554]. A 56-year-old man was admitted to another hospital complaining of abdominal distension. Symptoms included anorexia, vomiting, and diarrhea. The patient was diagnosed with an extragonadal germ cell tumor and referred to our hospital to receive chemotherapy. The day after admission, the patient's abdominal distension gradually worsened. An emergency operation revealed venous hemorrhage from the surface of a metastatic extragonadal germ cell tumor between the ligament of Treitz and the inferior mesenteric vein in a horizontal position. Hemostatic treatment was performed with 4-0 proline thread attached to a medicated cotton sponge, rather than using a simple proline thread, and the closure area was manually compressed. Chemotherapy was initiated on postoperative day 10. A metastatic extragonadal germ cell tumor that causes massive hemorrhage and gastrointestinal hemorrhage is very rare, and represents a life-threatening emergency. If the patient's condition carries a substantial risk of bleeding to death, it may be worthwhile to attempt abdominal operations. PMID:27403124

  14. Dearth and Delayed Maturation of Testicular Germ Cells in Fanconi Anemia E Mutant Male Mice

    PubMed Central

    Fu, Chun; Begum, Khurshida; Jordan, Philip W.; He, Yan; Overbeek, Paul A.

    2016-01-01

    After using a self-inactivating lentivirus for non-targeted insertional mutagenesis in mice, we identified a transgenic family with a recessive mutation that resulted in reduced fertility in homozygous transgenic mice. The lentiviral integration site was amplified by inverse PCR. Sequencing revealed that integration had occurred in intron 8 of the mouse Fance gene, which encodes the Fanconi anemia E (Fance) protein. Fanconi anemia (FA) proteins play pivotal roles in cellular responses to DNA damage and Fance acts as a molecular bridge between the FA core complex and Fancd2. To investigate the reduced fertility in the mutant males, we analyzed postnatal development of testicular germ cells. At one week after birth, most tubules in the mutant testes contained few or no germ cells. Over the next 2–3 weeks, germ cells accumulated in a limited number of tubules, so that some tubules contained germ cells around the full periphery of the tubule. Once sufficient numbers of germ cells had accumulated, they began to undergo the later stages of spermatogenesis. Immunoassays revealed that the Fancd2 protein accumulated around the periphery of the nucleus in normal developing spermatocytes, but we did not detect a similar localization of Fancd2 in the Fance mutant testes. Our assays indicate that although Fance mutant males are germ cell deficient at birth, the extant germ cells can proliferate and, if they reach a threshold density, can differentiate into mature sperm. Analogous to previous studies of FA genes in mice, our results show that the Fance protein plays an important, but not absolutely essential, role in the initial developmental expansion of the male germ line. PMID:27486799

  15. EphrinB/EphB Signaling Controls Embryonic Germ Layer Separation by Contact-Induced Cell Detachment

    PubMed Central

    Rohani, Nazanin; Canty, Laura; Luu, Olivia

    2011-01-01

    Background The primordial organization of the metazoan body is achieved during gastrulation by the establishment of the germ layers. Adhesion differences between ectoderm, mesoderm, and endoderm cells could in principle be sufficient to maintain germ layer integrity and prevent intermixing. However, in organisms as diverse as fly, fish, or amphibian, the ectoderm-mesoderm boundary not only keeps these germ layers separated, but the ectoderm also serves as substratum for mesoderm migration, and the boundary must be compatible with repeated cell attachment and detachment. Principal Findings We show that localized detachment resulting from contact-induced signals at the boundary is at the core of ectoderm-mesoderm segregation. Cells alternate between adhesion and detachment, and detachment requires ephrinB/EphB signaling. Multiple ephrinB ligands and EphB receptors are expressed on each side of the boundary, and tissue separation depends on forward signaling across the boundary in both directions, involving partially redundant ligands and receptors and activation of Rac and RhoA. Conclusion This mechanism differs from a simple differential adhesion process of germ layer formation. Instead, it involves localized responses to signals exchanged at the tissue boundary and an attachment/detachment cycle which allows for cell migration across a cellular substratum. PMID:21390298

  16. Endogenous DNA Damage and Risk of Testicular Germ Cell Tumors

    SciTech Connect

    Cook, M B; Sigurdson, A J; Jones, I M; Thomas, C B; Graubard, B I; Korde, L; Greene, M H; McGlynn, K A

    2008-01-18

    Testicular germ cell tumors (TGCT) are comprised of two histologic groups, seminomas and nonseminomas. We postulated that the possible divergent pathogeneses of these histologies may be partially explained by variable endogenous DNA damage. To assess our hypothesis, we conducted a case-case analysis of seminomas and nonseminomas using the alkaline comet assay to quantify single-strand DNA breaks and alkali-labile sites. The Familial Testicular Cancer study and the U.S. Radiologic Technologists cohort provided 112 TGCT cases (51 seminomas & 61 nonseminomas). A lymphoblastoid cell line was cultured for each patient and the alkaline comet assay was used to determine four parameters: tail DNA, tail length, comet distributed moment (CDM) and Olive tail moment (OTM). Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated using logistic regression. Values for tail length, tail DNA, CDM and OTM were modeled as categorical variables using the 50th and 75th percentiles of the seminoma group. Tail DNA was significantly associated with nonseminoma compared to seminoma (OR{sub 50th percentile} = 3.31, 95%CI: 1.00, 10.98; OR{sub 75th percentile} = 3.71, 95%CI: 1.04, 13.20; p for trend=0.039). OTM exhibited similar, albeit statistically non-significant, risk estimates (OR{sub 50th percentile} = 2.27, 95%CI: 0.75, 6.87; OR{sub 75th percentile} = 2.40, 95%CI: 0.75, 7.71; p for trend=0.12) whereas tail length and CDM showed no association. In conclusion, the results for tail DNA and OTM indicate that endogenous DNA damage levels are higher in patients who develop nonseminoma compared with seminoma. This may partly explain the more aggressive biology and younger age-of-onset of this histologic subgroup compared with the relatively less aggressive, later-onset seminoma.

  17. Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras.

    PubMed

    Keighren, Margaret A; Flockhart, Jean H; West, John D

    2016-05-15

    The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1(-/-) null mouse embryos die but a previous study showed that some homozygous Gpi1(-/-) null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1(-/-)↔Gpi1(c/c) chimaera with functional Gpi1(-/-) null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1(-/-) null cells in adult Gpi1(-/-)↔Gpi1(c/c) chimaeras and determine if Gpi1(-/-) null germ cells are functional. Analysis of adult Gpi1(-/-)↔Gpi1(c/c) chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1(-/-) null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1(-/-) null oocytes in one female Gpi1(-/-)↔Gpi1(c/c) chimaera were functional and provided preliminary evidence that one male putative Gpi1(-/-)↔Gpi1(c/c) chimaera produced functional spermatozoa from homozygous Gpi1(-/-) null germ cells. Although the male chimaera was almost certainly Gpi1(-/-)↔Gpi1(c/c), this part of the study is considered preliminary because only blood was typed for GPI. Gpi1(-/-) null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1(-/-) null germ cells, it successfully identified functional Gpi1(-/-) null oocytes and revealed that some Gpi1(-/-) null cells could survive in many adult tissues.

  18. Regulation of Caenorhabditis elegans p53/CEP-1–Dependent Germ Cell Apoptosis by Ras/MAPK Signaling

    PubMed Central

    Rutkowski, Rachael; Dickinson, Robin; Stewart, Graeme; Craig, Ashley; Schimpl, Marianne; Keyse, Stephen M.; Gartner, Anton

    2011-01-01

    Maintaining genome stability in the germline is thought to be an evolutionarily ancient role of the p53 family. The sole Caenorhabditis elegans p53 family member CEP-1 is required for apoptosis induction in meiotic, late-stage pachytene germ cells in response to DNA damage and meiotic recombination failure. In an unbiased genetic screen for negative regulators of CEP-1, we found that increased activation of the C. elegans ERK orthologue MPK-1, resulting from either loss of the lip-1 phosphatase or activation of let-60 Ras, results in enhanced cep-1–dependent DNA damage induced apoptosis. We further show that MPK-1 is required for DNA damage–induced germ cell apoptosis. We provide evidence that MPK-1 signaling regulates the apoptotic competency of germ cells by restricting CEP-1 protein expression to cells in late pachytene. Restricting CEP-1 expression to cells in late pachytene is thought to ensure that apoptosis doesn't occur in earlier-stage cells where meiotic recombination occurs. MPK-1 signaling regulates CEP-1 expression in part by regulating the levels of GLD-1, a translational repressor of CEP-1, but also via a GLD-1–independent mechanism. In addition, we show that MPK-1 is phosphorylated and activated upon ionising radiation (IR) in late pachytene germ cells and that MPK-1–dependent CEP-1 activation may be in part direct, as these two proteins interact in a yeast two-hybrid assay. In summary, we report our novel finding that MAP kinase signaling controls CEP-1–dependent apoptosis by several different pathways that converge on CEP-1. Since apoptosis is also restricted to pachytene stage cells in mammalian germlines, analogous mechanisms regulating p53 family members are likely to be conserved throughout evolution. PMID:21901106

  19. Mouse Tafazzin Is Required for Male Germ Cell Meiosis and Spermatogenesis

    PubMed Central

    Cadalbert, Laurence C.; Ghaffar, Farah Naz; Stevenson, David; Bryson, Sheila; Vaz, Frédéric M.; Gottlieb, Eyal; Strathdee, Douglas

    2015-01-01

    Barth syndrome is an X-linked mitochondrial disease, symptoms of which include neutropenia and cardiac myopathy. These symptoms are the most significant clinical consequences of a disease, which is increasingly recognised to have a variable presentation. Mutation in the Taz gene in Xq28 is thought to be responsible for the condition, by altering mitochondrial lipid content and mitochondrial function. Male chimeras carrying a targeted mutation of Taz on their X-chromosome were infertile. Testes from the Taz knockout chimeras were smaller than their control counterparts and this was associated with a disruption of the progression of spermatocytes through meiosis to spermiogenesis. Taz knockout ES cells also showed a defect when differentiated to germ cells in vitro. Mutant spermatocytes failed to progress past the pachytene stage of meiosis and had higher levels of DNA double strand damage and increased levels of endogenous retrotransposon activity. Altogether these data revealed a novel role for Taz in helping to maintain genome integrity in meiosis and facilitating germ cell differentiation. We have unravelled a novel function for the Taz protein, which should contribute to an understanding of how a disruption of the Taz gene results in the complex symptoms underlying Barth Syndrome. PMID:26114544

  20. Mouse Tafazzin Is Required for Male Germ Cell Meiosis and Spermatogenesis.

    PubMed

    Cadalbert, Laurence C; Ghaffar, Farah Naz; Stevenson, David; Bryson, Sheila; Vaz, Frédéric M; Gottlieb, Eyal; Strathdee, Douglas

    2015-01-01

    Barth syndrome is an X-linked mitochondrial disease, symptoms of which include neutropenia and cardiac myopathy. These symptoms are the most significant clinical consequences of a disease, which is increasingly recognised to have a variable presentation. Mutation in the Taz gene in Xq28 is thought to be responsible for the condition, by altering mitochondrial lipid content and mitochondrial function. Male chimeras carrying a targeted mutation of Taz on their X-chromosome were infertile. Testes from the Taz knockout chimeras were smaller than their control counterparts and this was associated with a disruption of the progression of spermatocytes through meiosis to spermiogenesis. Taz knockout ES cells also showed a defect when differentiated to germ cells in vitro. Mutant spermatocytes failed to progress past the pachytene stage of meiosis and had higher levels of DNA double strand damage and increased levels of endogenous retrotransposon activity. Altogether these data revealed a novel role for Taz in helping to maintain genome integrity in meiosis and facilitating germ cell differentiation. We have unravelled a novel function for the Taz protein, which should contribute to an understanding of how a disruption of the Taz gene results in the complex symptoms underlying Barth Syndrome.

  1. The transcriptional repressor Blimp-1 acts downstream of BMP signaling to generate primordial germ cells in the cricket Gryllus bimaculatus.

    PubMed

    Nakamura, Taro; Extavour, Cassandra G

    2016-01-15

    Segregation of the germ line from the soma is an essential event for transmission of genetic information across generations in all sexually reproducing animals. Although some well-studied systems such as Drosophila and Xenopus use maternally inherited germ determinants to specify germ cells, most animals, including mice, appear to utilize zygotic inductive cell signals to specify germ cells during later embryogenesis. Such inductive germ cell specification is thought to be an ancestral trait of Bilateria, but major questions remain as to the nature of an ancestral mechanism to induce germ cells, and how that mechanism evolved. We previously reported that BMP signaling-based germ cell induction is conserved in both the mouse Mus musculus and the cricket Gryllus bimaculatus, which is an emerging model organism for functional studies of induction-based germ cell formation. In order to gain further insight into the functional evolution of germ cell specification, here we examined the Gryllus ortholog of the transcription factor Blimp-1 (also known as Prdm1), which is a widely conserved bilaterian gene known to play a crucial role in the specification of germ cells in mice. Our functional analyses of the Gryllus Blimp-1 ortholog revealed that it is essential for Gryllus primordial germ cell development, and is regulated by upstream input from the BMP signaling pathway. This functional conservation of the epistatic relationship between BMP signaling and Blimp-1 in inductive germ cell specification between mouse and cricket supports the hypothesis that this molecular mechanism regulated primordial germ cell specification in a last common bilaterian ancestor.

  2. Time series analysis supporting the hypothesis that enhanced cosmic radiation during germ cell formation can increase breast cancer mortality in germ cell cohorts

    NASA Astrophysics Data System (ADS)

    Juckett, D. A.; Rosenberg, Barnett

    Techniques from cancer epidemiology and time series analysis were used to explore the hypothesis that cosmic radiation can induce germ cell changes leading to increases in future breast cancer mortality. A birth cohort time series for female breast cancer mortality was obtained using a model-independent, age-period-cohort analysis on age-specific mortality data for 1940-1990. The birth cohort series contained several oscillatory components, which were isolated and compared to the corresponding frequency components of a cosmic ray surrogate time series - Greenland ice-core 10Be concentrations. A technique, referred to as component wave-train alignment, was used to show that the breast cancer and cosmic ray oscillations were phase-locked approx. 25 years before the time of birth. This is consistent with the time of germ cell formation, which occurs during the fetal development stage of the preceding generation. Evidence is presented that the observable oscillations in the birth cohort series were residues of oscillations of much larger amplitude in the germ cell cohort, which were attenuated by the effect of the broad maternal age distribution. It is predicted that a minimum of 50% of breast cancer risk is associated with germ cell damage by cosmic radiation (priming event), which leads to the development of individuals with a higher risk of breast cancer. It is proposed that the priming event, by preceding other steps of carcinogenesis, works in concert with risk factor exposure during life. The priming event is consistent with epigenetic changes such as imprinting.

  3. Role of the ABC transporter Mdr49 in Hedgehog signaling and germ cell migration.

    PubMed

    Deshpande, Girish; Manry, Diane; Jourjine, Nicholas; Mogila, Vladic; Mozes, Henny; Bialistoky, Tzofia; Gerlitz, Offer; Schedl, Paul

    2016-06-15

    Coalescence of the embryonic gonad in Drosophila melanogaster requires directed migration of primordial germ cells (PGCs) towards somatic gonadal precursor cells (SGPs). It was recently proposed that the ATP-binding cassette (ABC) transporter Mdr49 functions in the embryonic mesoderm to facilitate the transmission of the PGC attractant from the SGPs; however, the precise molecular identity of the Mdr49-dependent guidance signal remained elusive. Employing the loss- and gain-of-function strategies, we show that Mdr49 is a component of the Hedgehog (hh) pathway and it potentiates the signaling activity. This function is direct because in Mdr49 mutant embryos the Hh ligand is inappropriately sequestered in the hh-expressing cells. Our data also suggest that the role of Mdr49 is to provide cholesterol for the correct processing of the Hh precursor protein. Supporting this conclusion, PGC migration defects in Mdr49 embryos are substantially ameliorated by a cholesterol-rich diet. PMID:27122170

  4. Pathogenesis of germ cell neoplasia in testicular dysgenesis and disorders of sex development.

    PubMed

    Jørgensen, Anne; Lindhardt Johansen, Marie; Juul, Anders; Skakkebaek, Niels E; Main, Katharina M; Rajpert-De Meyts, Ewa

    2015-09-01

    Development of human gonads is a sex-dimorphic process which evolved to produce sex-specific types of germ cells. The process of gonadal sex differentiation is directed by the action of the somatic cells and ultimately results in germ cells differentiating to become functional gametes through spermatogenesis or oogenesis. This tightly controlled process depends on the proper sequential expression of many genes and signalling pathways. Disturbances of this process can be manifested as a large spectrum of disorders, ranging from severe disorders of sex development (DSD) to - in the genetic male - mild reproductive problems within the testicular dysgenesis syndrome (TDS), with large overlap between the syndromes. These disorders carry an increased but variable risk of germ cell neoplasia. In this review, we discuss the pathogenesis of germ cell neoplasia associated with gonadal dysgenesis, especially in individuals with 46,XY DSD. We summarise knowledge concerning development and sex differentiation of human gonads, with focus on sex-dimorphic steps of germ cell maturation, including meiosis. We also briefly outline the histopathology of germ cell neoplasia in situ (GCNIS) and gonadoblastoma (GDB), which are essentially the same precursor lesion but with different morphological structure dependent upon the masculinisation of the somatic niche. To assess the risk of germ cell neoplasia in different types of DSD, we have performed a PubMed search and provide here a synthesis of the evidence from studies published since 2006. We present a model for pathogenesis of GCNIS/GDB in TDS/DSD, with the risk of malignancy determined by the presence of the testis-inducing Y chromosome and the degree of masculinisation. The associations between phenotype and the risk of neoplasia are likely further modulated in each individual by the constellation of the gene polymorphisms and environmental factors.

  5. Germ cell degeneration in high-temperature treated pufferfish, Takifugu rubripes.

    PubMed

    Lee, K H; Yamaguchi, A; Rashid, H; Kadomura, K; Yasumoto, S; Matsuyama, M

    2009-01-01

    Exogenous factors such as temperature, social behavior, and salinity play a crucial role during the critical sensitive period of sex differentiation in many vertebrates. In fishes, amphibians, and reptiles temperature treatment is known to induce all-male (or female) individuals, and genes related to sex differentiation have been studied. The Japanese pufferfish, Takifugu rubripes, possesses the most compact genome among vertebrates and has immense potential for studies focusing on comparative genome analysis. In this study, we describe gonadal morphology and vasa (germ cell marker) and dmrt1 (Sertoli cell marker) expression on a molecular level in relation to the development of temperature-treated pufferfish. To investigate the relationship between temperature and gonadal development, pufferfish were exposed to high-temperature conditions (32 degrees C) during early gonadal development. Morphological observations showed that this high-temperature treatment did not influence sexual differentiation as determined by ovarian cavity characteristics; however, high-temperature treatment induces gonadal degeneration that is devoid of germ cells. RT-PCR results revealed no vasa expression within germ cell-degenerated gonads. In situ hybridization results showed that dmrt1 was expressed in somatic cells of germ cell-degenerated ovaries. These results suggest that high-temperature treatment during early gonadal development induces germ cell degeneration and masculinization of ovarian somatic cells in pufferfish.

  6. The degenerative fate of germ cells not conforming to stage in the pubertal golden hamster testis.

    PubMed

    Miething, A

    1998-11-01

    In the golden hamster (Mesocricetus auratus), pubertal establishment of spermatogenesis includes a defined period (d 26-30 of life) during which elongation of spermatids is selectively arrested. The resulting appearance of germ cell associations not conforming to stage and the phenomenon of desynchronisation-related germ cell degeneration are analysed both quantitatively and qualitatively by means of light and 'retrospective' electron microscopy. From d 26 onwards, the portion of tubules containing non-stage conforming germ cell associations gradually increases up to 37.5% of sectioned tubules on d 32. Concomitantly, the degree of desynchronisation rises to a maturational gap between spermatids and associated younger germ cells of 7 stages of the seminiferous epithelium cycle, i.e. of fully half a cycle. Beyond d 32, the frequency of desynchronised tubule segments decreases again. Some of the arrested round spermatids and, eventually, all belatedly elongating spermatids degenerate and are lost from the epithelium. Thus a regular maturation of advanced spermatids does not succeed under non-stage conforming conditions. Possibly it is not the desynchronisation between the associated germ cell generations and the spermatids by itself that impedes normal further development of the latter cells. Instead this may be due to the maturational delay of the stage-aberrant cells by several stages compared to the seminiferous epithelium as a whole and, especially, in relation to the stage-conditioned functional state of the neighbouring Sertoli cells.

  7. Stochastic specification of primordial germ cells from mesoderm precursors in axolotl embryos.

    PubMed

    Chatfield, Jodie; O'Reilly, Marie-Anne; Bachvarova, Rosemary F; Ferjentsik, Zoltan; Redwood, Catherine; Walmsley, Maggie; Patient, Roger; Loose, Mathew; Johnson, Andrew D

    2014-06-01

    A common feature of development in most vertebrate models is the early segregation of the germ line from the soma. For example, in Xenopus and zebrafish embryos primordial germ cells (PGCs) are specified by germ plasm that is inherited from the egg; in mice, Blimp1 expression in the epiblast mediates the commitment of cells to the germ line. How these disparate mechanisms of PGC specification evolved is unknown. Here, in order to identify the ancestral mechanism of PGC specification in vertebrates, we studied PGC specification in embryos from the axolotl (Mexican salamander), a model for the tetrapod ancestor. In the axolotl, PGCs develop within mesoderm, and classic studies have reported their induction from primitive ectoderm (animal cap). We used an axolotl animal cap system to demonstrate that signalling through FGF and BMP4 induces PGCs. The role of FGF was then confirmed in vivo. We also showed PGC induction by Brachyury, in the presence of BMP4. These conditions induced pluripotent mesodermal precursors that give rise to a variety of somatic cell types, in addition to PGCs. Irreversible restriction of the germ line did not occur until the mid-tailbud stage, days after the somatic germ layers are established. Before this, germline potential was maintained by MAP kinase signalling. We propose that this stochastic mechanism of PGC specification, from mesodermal precursors, is conserved in vertebrates.

  8. Zebrafish vasa is required for germ-cell differentiation and maintenance.

    PubMed

    Hartung, Odelya; Forbes, Meredyth M; Marlow, Florence L

    2014-10-01

    Vasa is a universal marker of the germ line in animals, yet mutations disrupting vasa cause sexually dimorphic infertility, with impaired development of the ovary in some animals and the testis in others. The basis for this sexually dimorphic requirement for Vasa is not clear; in most animals examined, both the male and female gonad express vasa throughout the life of the germ line. Here we characterized a loss-of-function mutation disrupting zebrafish vasa. We show that maternally provided Vasa is stable through the first ten days of development in zebrafish, and thus likely fulfills any early roles for Vasa during germ-line specification, migration, survival, and maintenance. Although zygotic Vasa is not essential for the development of juvenile gonads, vasa mutants develop exclusively as sterile males. Furthermore, phenotypes of vasa;p53 compound mutants are indistinguishable from those of vasa mutants, therefore the failure of vasa mutants to differentiate as females and to support germ-cell development in the testis is not due to p53-mediated apoptosis. Instead, we found that failure to progress beyond the pachytene stage of meiosis causes the loss of germ-line stem cells, leaving empty somatic tubules. Our studies provide insight into the function of zebrafish vasa during female meiosis, differentiation, and maintenance of germ-line stem cells.

  9. Stochastic specification of primordial germ cells from mesoderm precursors in axolotl embryos

    PubMed Central

    Chatfield, Jodie; O'Reilly, Marie-Anne; Bachvarova, Rosemary F.; Ferjentsik, Zoltan; Redwood, Catherine; Walmsley, Maggie; Patient, Roger; Loose, Mathew; Johnson, Andrew D.

    2014-01-01

    A common feature of development in most vertebrate models is the early segregation of the germ line from the soma. For example, in Xenopus and zebrafish embryos primordial germ cells (PGCs) are specified by germ plasm that is inherited from the egg; in mice, Blimp1 expression in the epiblast mediates the commitment of cells to the germ line. How these disparate mechanisms of PGC specification evolved is unknown. Here, in order to identify the ancestral mechanism of PGC specification in vertebrates, we studied PGC specification in embryos from the axolotl (Mexican salamander), a model for the tetrapod ancestor. In the axolotl, PGCs develop within mesoderm, and classic studies have reported their induction from primitive ectoderm (animal cap). We used an axolotl animal cap system to demonstrate that signalling through FGF and BMP4 induces PGCs. The role of FGF was then confirmed in vivo. We also showed PGC induction by Brachyury, in the presence of BMP4. These conditions induced pluripotent mesodermal precursors that give rise to a variety of somatic cell types, in addition to PGCs. Irreversible restriction of the germ line did not occur until the mid-tailbud stage, days after the somatic germ layers are established. Before this, germline potential was maintained by MAP kinase signalling. We propose that this stochastic mechanism of PGC specification, from mesodermal precursors, is conserved in vertebrates. PMID:24917499

  10. Stochastic specification of primordial germ cells from mesoderm precursors in axolotl embryos.

    PubMed

    Chatfield, Jodie; O'Reilly, Marie-Anne; Bachvarova, Rosemary F; Ferjentsik, Zoltan; Redwood, Catherine; Walmsley, Maggie; Patient, Roger; Loose, Mathew; Johnson, Andrew D

    2014-06-01

    A common feature of development in most vertebrate models is the early segregation of the germ line from the soma. For example, in Xenopus and zebrafish embryos primordial germ cells (PGCs) are specified by germ plasm that is inherited from the egg; in mice, Blimp1 expression in the epiblast mediates the commitment of cells to the germ line. How these disparate mechanisms of PGC specification evolved is unknown. Here, in order to identify the ancestral mechanism of PGC specification in vertebrates, we studied PGC specification in embryos from the axolotl (Mexican salamander), a model for the tetrapod ancestor. In the axolotl, PGCs develop within mesoderm, and classic studies have reported their induction from primitive ectoderm (animal cap). We used an axolotl animal cap system to demonstrate that signalling through FGF and BMP4 induces PGCs. The role of FGF was then confirmed in vivo. We also showed PGC induction by Brachyury, in the presence of BMP4. These conditions induced pluripotent mesodermal precursors that give rise to a variety of somatic cell types, in addition to PGCs. Irreversible restriction of the germ line did not occur until the mid-tailbud stage, days after the somatic germ layers are established. Before this, germline potential was maintained by MAP kinase signalling. We propose that this stochastic mechanism of PGC specification, from mesodermal precursors, is conserved in vertebrates. PMID:24917499

  11. A Small RNA-Based Immune System Defends Germ Cells against Mobile Genetic Elements

    PubMed Central

    2016-01-01

    Transposons are mobile genetic elements that threaten the survival of species by destabilizing the germline genomes. Limiting the spread of these selfish elements is imperative. Germ cells employ specialized small regulatory RNA pathways to restrain transposon activity. PIWI proteins and Piwi-interacting RNAs (piRNAs) silence transposons at the transcriptional and posttranscriptional level with loss-of-function mutant animals universally exhibiting sterility often associated with germ cell defects. This short review aims to illustrate basic strategies of piRNA-guided defense against transposons. Mechanisms of piRNA silencing are most readily studied in Drosophila melanogaster, which serves as a model to delineate molecular concepts and as a reference for mammalian piRNA systems. PiRNA pathways utilize two major strategies to handle the challenges of transposon control: (1) the hard-wired molecular memory of prior transpositions enables recognition of mobile genetic elements and discriminates transposons from host genes; (2) a feed-forward adaptation mechanism shapes piRNA populations to selectively combat the immediate threat of transposon transcripts. In flies, maternally contributed PIWI-piRNA complexes bolster both of these lines of defense and ensure transgenerational immunity. While recent studies have provided a conceptual framework of what could be viewed as an ancient immune system, we are just beginning to appreciate its many molecular innovations. PMID:26681955

  12. Indirect Immunofluorescence of Proteins in Oogenic Germ Cells of Caenorhabditis elegans.

    PubMed

    Brenner, John L; Schedl, Tim

    2016-01-01

    Formation of full-grown oocytes requires the control and coordination of a number of processes (e.g., oocyte growth) through multiple stages, where disruption at any one step can result in infertility. Numerous proteins are required for the regulation and execution of the various oogenic processes as well as functioning as maternal products needed for embryogenesis. Immunofluorescence microscopy combined with staining using antibodies against specific proteins, or their posttranslationally modified forms, is a standard approach to determine the temporal and spatial location of gene products that function in oocyte development. The simple linear organization of the germline in the model organism Caenorhabditis elegans allows easy correlation of protein localization and germ cell developmental stage, thus aiding in our understanding of protein function during gametogenesis. Here we outline co-immunofluorescence staining for two major regulators of C. elegans germline development, the translational repressor GLD-1 and activated form of MPK-1 (dpMPK-1) ERK MAP kinase in dissected gonads from adult C. elegans. Worms are first dissected and the extruded gonads are fixed and permeabilized before being bathed in primary antibodies against GLD-1 and dpMPK-1. Secondary antibodies conjugated to fluorophore dyes and that target the IgG domains of the primary antibody reagents are then used to provide a fluorescent signal that corresponds to the position of GLD-1 and dpMPK-1. The outlined procedure is amenable to many other proteins expressed in C. elegans germ cells. PMID:27557570

  13. Suppression of meiosis of male germ cells by an antagonist of luteinizing hormone-releasing hormone.

    PubMed Central

    Szende, B; Redding, T W; Schally, A V

    1990-01-01

    Male nude mice were implanted with osmotic minipumps releasing 50 micrograms of a potent antagonist of luteinizing hormone-releasing hormone (LH-RH) per day [N-Ac-[D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-RH] (SB-75) [Nal(2), 3-(2-naphthyl)alanine; Phe(pCl), 4-chlorophenylalanine; Pal(3), 3-(3-pyridyl)alanine; Cit, citrulline], or they were treated with s.c. injections of SB-75 (25 micrograms twice a day). Another group of nude mice received an injection of microcapsules of the agonist [D-Trp6]LH-RH liberating 25 micrograms/day. One month after the initiation of treatment, the testicular weights were significantly reduced and the blood testosterone values were at castration levels in all treated groups. Histologically, only the testicles of the mice treated with SB-75 released from minipumps showed a significant decrease of meiosis. The most advanced forms of germ cells were spermatogonia in 26%, spermatocytes in 17%, and round spermatids in 35% of the seminiferous tubules. Only 22% of the tubules contained elongated spermatids. The suppression of meiotic activity by this modern LH-RH antagonist can possibly be used for the development of methods for male contraception and for the protection of germ cells against the damage caused by cytotoxic drugs and x-radiation. Images PMID:2405399

  14. Production of reproductively sterile fish: A mini-review of germ cell elimination technologies.

    PubMed

    Wong, Ten-Tsao; Zohar, Yonathan

    2015-09-15

    As seafood consumption shifts from fisheries harvests to artificially propagated aquatic species, the increase of aquaculture activities poses a biological threat to our environment. Selectively bred, non-native and (eventually) genetically engineered farmed fish may escape from aquaculture operations, propagate and/or interbreed with wild stocks and subsequently alter the genetic makeup of populations in the environment. Thus, an effective strategy for bio-containment of farmed fish is critically needed. Farming reproductively sterile fish is the most environmentally sustainable approach to ensure complete bio-containment in large-scale aquaculture operations. Chromosome set manipulations to produce sterile fish, including polyploidy and hybridization, are currently the most common practices in the aquaculture industry. However, they do not always result in 100% sterility of the treated fish. Moreover, triploid fish typically do not perform as well as the non-manipulated diploids under commercial culture conditions. In the last half decade, several genetic engineering methods have been developed to produce sterile fish. In this review, we will address the latest technologies that use transgenic approaches to eliminate germ cells, resulting in the production of sterile fish. These latest advances also led us to the development of egg/embryo immersion methodologies to deliver and screen compounds that can be used to eliminate primordial germ cells and produce sterile fish. This emerging non-transgenic strategy for the production of reproductively sterile fish in aquaculture will also be discussed.

  15. HMGA2 expression distinguishes between different types of postpubertal testicular germ cell tumour.

    PubMed

    Kloth, Lars; Gottlieb, Andrea; Helmke, Burkhard; Wosniok, Werner; Löning, Thomas; Burchardt, Käte; Belge, Gazanfer; Günther, Kathrin; Bullerdiek, Jörn

    2015-10-01

    The group of postpubertal testicular germ cell tumours encompasses lesions with highly diverse differentiation - seminomas, embryonal carcinomas, yolk sac tumours, teratomas and choriocarcinomas. Heterogeneous differentiation is often present within individual tumours and the correct identification of the components is of clinical relevance. HMGA2 re-expression has been reported in many tumours, including testicular germ cell tumours. This is the first study investigating HMGA2 expression in a representative group of testicular germ cell tumours with the highly sensitive method of quantitative real-time PCR as well as with immunohistochemistry. The expression of HMGA2 and HPRT was measured using quantitative real-time PCR in 59 postpubertal testicular germ cell tumours. Thirty specimens contained only one type of tumour and 29 were mixed neoplasms. With the exception of choriocarcinomas, at least two pure specimens from each subgroup of testicular germ cell tumour were included. In order to validate the quantitative real-time PCR data and gather information about the localisation of the protein, additional immunohistochemical analysis with an antibody specific for HMGA2 was performed in 23 cases. Expression of HMGA2 in testicular germ cell tumours depended on the histological differentiation. Seminomas and embryonal carcinomas showed no or very little expression, whereas yolk sac tumours strongly expressed HMGA2 at the transcriptome as well as the protein level. In teratomas, the expression varied and in choriocarcinomas the expression was moderate. In part, these results contradict data from previous studies but HMGA2 seems to represent a novel marker to assist pathological subtyping of testicular germ cell tumours. The results indicate a critical role in yolk sac tumours and some forms of teratoma. PMID:27499908

  16. Interaction of Insulin-like Growth Factor-binding Protein-3 and BAX in Mitochondria Promotes Male Germ Cell Apoptosis

    PubMed Central

    Jia, Yue; Lee, Kuk-Wha; Swerdloff, Ronald; Hwang, David; Cobb, Laura J.; Sinha Hikim, Amiya; Lue, Yan He; Cohen, Pinchas; Wang, Christina

    2010-01-01

    Germ cell apoptosis is crucial for spermatogenesis and can be triggered by various stimuli, including intratesticular hormone deprivation. This study proposes a role for insulin-like growth factor binding protein-3 (IGFBP-3) in male germ cell apoptosis. Groups of adult Sprague-Dawley male rats received one of the following treatments for 5 days: (i) daily intratesticular (IT) injections with saline (control); (ii) a single subcutaneous injection of the gonadotropin-releasing hormone antagonist (GnRH-A), acyline, on day 1 and a daily IT injection of saline; (iii) daily IT injection of IGFBP-3; and (iv) a GnRH-A injection on day 1 and a daily IT injection of IGFBP-3. Germ cell apoptosis increased significantly after IGFBP-3 or GnRH-A treatment which was further enhanced by the combined treatment. After co-immunoprecipitation with BAX antibody, IGFBP-3 association with BAX was demonstrated in total and mitochondrial fractions but not in the cytosol of testis extracts. BAX-associated IGFBP-3 expression was increased in mitochondria after treatment compared with control, which was confirmed by an IGFBP-3 enzyme-linked immunosorbent assay. Dot blot studies further validated the BAX-IGFBP-3 binding in vitro. IGFBP-3 as well as BAX induced release of cytochrome c and DIABLO from isolated testicular mitochondria in vitro. IGFBP-3, when combined with an ineffective dose of BAX, triggered release of these proteins from isolated mitochondria at a 4-fold lower dose than IGFBP-3 alone. Our data demonstrate that the IGFBP-3 and BAX interaction activates germ cell apoptosis via the mitochondria-dependent pathway. This represents a novel pathway regulating germ call homeostasis that may have significance for male fertility and testicular disease. PMID:19887447

  17. Circulating tumor cells in germ cell tumors: are those biomarkers of real prognostic value? A review

    PubMed Central

    CEBOTARU, CRISTINA LIGIA; OLTEANU, ELENA DIANA; ANTONE, NICOLETA ZENOVIA; BUIGA, RARES; NAGY, VIORICA

    2016-01-01

    Analysis of circulating tumor cells from patients with different types of cancer is nowadays a fascinating new tool of research and their number is proven to be useful as a prognostic factor in metastatic breast, colon and prostate cancer patients. Studies are going beyond enumeration, exploring the circulating tumor cells to better understand the mechanisms of tumorigenesis, invasion and metastasis and their value for characterization, prognosis and tailoring of treatment. Few studies investigated the prognostic significance of circulating tumor cells in germ cell tumors. In this review, we examine the possible significance of the detection of circulating tumor cells in this setting. PMID:27152069

  18. Reproductive stage-dependent effects of additional cryoprotectant agents for the cryopreservation of stallion germ cells.

    PubMed

    Jung, Heejun; Kim, Namyoung; Yoon, Minjung

    2016-10-01

    The main objective of this study was to evaluate the efficacy of an additional cryoprotectant in 10% dimethyl sulfoxide (DMSO) on cryopreserving germ cells from stallions at different reproductive stages. Testicular samples were obtained from pre-pubertal (1-1.5 yr, n=6) and post-pubertal (3-7 yr, n=5) stallions. Germ cells were isolated using a two-enzyme digestion procedure and cryopreserved in minimal essential medium alpha containing 10% fetal bovine serum and 10% DMSO with or without addition of trehalose (50, 100, or 200mM) or polyethylene glycol (PEG, 2.5, 5, or 10%). Viability, cell population, and viable population were assessed after 1 and 3 months of cryopreservation. The viable UTF1-positive population of pre-pubertal stallion germ cells was also measured using immunocytochemistry after 1 and 3 months of cryopreservation. As expected, the viability, cell population, and viable cell population were significantly reduced after 1 and 3 months of cryopreservation. At the pre-pubertal stage, the addition of trehalose or PEG to 10% DMSO did not show any effect on the viability, cell population, viable cell population, or viable UTF1-positive germ cells at either 1 or 3 months after cryopreservation. However, at the post-pubertal stage, the viable population was significantly higher in germ cells that were cryopreserved with 5% or 10% PEG, than in the cells cryopreserved with 10% DMSO only. In conclusion, PEG at 5% or 10% added to 10% DMSO serves as an optimal cryoprotectant agent for the cryopreservation of germ cells from post-pubertal stallions. PMID:27546795

  19. Dazl is a target RNA suppressed by mammalian NANOS2 in sexually differentiating male germ cells

    PubMed Central

    Kato, Yuzuru; Katsuki, Takeo; Kokubo, Hiroki; Masuda, Aki; Saga, Yumiko

    2016-01-01

    Evolutionally conserved Nanos RNA-binding proteins play crucial roles in germ cell development. While a mammalian Nanos family protein, NANOS2, is required for sexual differentiation of male (XY) germ cells in mice, the underlying mechanisms and the identities of its target RNAs in vivo remain elusive. Using comprehensive microarray analysis and a bacterial artificial chromosome transgenic system, here we identify Dazl, a germ cell-specific gene encoding an RNA-binding protein implicated in translation, as a crucial target of NANOS2. Importantly, removal of the Dazl 3′-untranslated region in XY germ cells stabilizes the Dazl mRNA, resulting in elevated meiotic gene expression, abnormal resumption of the cell cycle and impaired processing-body formation, reminiscent of Nanos2-knockout phenotypes. Furthermore, our data suggest that NANOS2 acts as an antagonist of the DAZL protein. We propose a dual system of NANOS2-mediated suppression of Dazl expression as a pivotal molecular mechanism promoting sexual differentiation of XY germ cells. PMID:27072294

  20. Dazl is a target RNA suppressed by mammalian NANOS2 in sexually differentiating male germ cells.

    PubMed

    Kato, Yuzuru; Katsuki, Takeo; Kokubo, Hiroki; Masuda, Aki; Saga, Yumiko

    2016-01-01

    Evolutionally conserved Nanos RNA-binding proteins play crucial roles in germ cell development. While a mammalian Nanos family protein, NANOS2, is required for sexual differentiation of male (XY) germ cells in mice, the underlying mechanisms and the identities of its target RNAs in vivo remain elusive. Using comprehensive microarray analysis and a bacterial artificial chromosome transgenic system, here we identify Dazl, a germ cell-specific gene encoding an RNA-binding protein implicated in translation, as a crucial target of NANOS2. Importantly, removal of the Dazl 3'-untranslated region in XY germ cells stabilizes the Dazl mRNA, resulting in elevated meiotic gene expression, abnormal resumption of the cell cycle and impaired processing-body formation, reminiscent of Nanos2-knockout phenotypes. Furthermore, our data suggest that NANOS2 acts as an antagonist of the DAZL protein. We propose a dual system of NANOS2-mediated suppression of Dazl expression as a pivotal molecular mechanism promoting sexual differentiation of XY germ cells. PMID:27072294

  1. Gender differences in the induction of chromosomal aberrations and gene mutations in rodent germ cells

    SciTech Connect

    Adler, Ilse-Dore; Carere, Angelo; Eichenlaub-Ritter, Ursula

    2007-05-15

    Germ cell mutagenicity testing provides experimental data to quantify genetic risk for exposed human populations. The majority of tests are performed with exposure of males, and female data are relatively rare. The reason for this paucity lies in the differences between male and female germ cell biology. Male germ cells are produced throughout reproductive life and all developmental stages can be ascertained by appropriate breeding schemes. In contrast, the female germ cell pool is limited, meiosis begins during embryogenesis and oocytes are arrested over long periods of time until maturation processes start for small numbers of oocytes during the oestrus cycle in mature females. The literature data are reviewed to point out possible gender differences of germ cells to exogenous agents such as chemicals or ionizing radiation. From the limited information, it can be concluded that male germ cells are more sensitive than female germ cells to the induction of chromosomal aberrations and gene mutations. However, exceptions are described which shed doubt on the extrapolation of experimental data from male rodents to the genetic risk of the human population. Furthermore, the female genome may be more sensitive to mutation induction during peri-conceptional stages compared to the male genome of the zygote. With few exceptions, germ cell experiments have been carried out under high acute exposure to optimize the effects and to compensate for the limited sample size in animal experiments. Human exposure to environmental agents, on the other hand, is usually chronic and involves low doses. Under these conditions, gender differences may become apparent that have not been studied so far. Additionally, data are reviewed that suggest a false impression of safety when responses are negative under high acute exposure of male rodents while a mutational response is induced by low chronic exposure. The classical (morphological) germ cell mutation tests are not performed anymore

  2. Ectopic expression of Cvh (Chicken Vasa homologue) mediates the reprogramming of chicken embryonic stem cells to a germ cell fate.

    PubMed

    Lavial, Fabrice; Acloque, Hervé; Bachelard, Elodie; Nieto, M Angela; Samarut, Jacques; Pain, Bertrand

    2009-06-01

    When they are derived from blastodermal cells of the pre-primitive streak in vitro, the pluripotency of Chicken Embryonic Stem Cells (cESC) can be controlled by the cPouV and Nanog genes. These cESC can differentiate into derivatives of the three germ layers both in vitro and in vivo, but they only weakly colonize the gonads of host embryos. By contrast, non-cultured blastodermal cells and long-term cultured chicken primordial germ cells maintain full germline competence. This restriction in the germline potential of the cESC may result from either early germline determination in the donor embryos or it may occur as a result of in vitro culture. We are interested in understanding the genetic determinants of germline programming. The RNA binding protein Cvh (Chicken Vasa Homologue) is considered as one such determinant, although its role in germ cell physiology is still unclear. Here we show that the exogenous expression of Cvh, combined with appropriate culture conditions, induces cESC reprogramming towards a germ cell fate. Indeed, these cells express the Dazl, Tudor and Sycp3 germline markers, and they display improved germline colonization and adopt a germ cell fate when injected into recipient embryos. Thus, our results demonstrate that Vasa can drive ES cell differentiation towards the germ cell lineage, both in vitro and in vivo.

  3. Chemokine-Dependent pH Elevation at the Cell Front Sustains Polarity in Directionally Migrating Zebrafish Germ Cells.

    PubMed

    Tarbashevich, Katsiaryna; Reichman-Fried, Michal; Grimaldi, Cecilia; Raz, Erez

    2015-04-20

    Directional cell migration requires cell polarization with respect to the distribution of the guidance cue. Cell polarization often includes asymmetric distribution of response components as well as elements of the motility machinery. Importantly, the function and regulation of most of these molecules are known to be pH dependent. Intracellular pH gradients were shown to occur in certain cells migrating in vitro, but the functional relevance of such gradients for cell migration and for the response to directional cues, particularly in the intact organism, is currently unknown. In this study, we find that primordial germ cells migrating in the context of the developing embryo respond to the graded distribution of the chemokine Cxcl12 by establishing elevated intracellular pH at the cell front. We provide insight into the mechanisms by which a polar pH distribution contributes to efficient cell migration. Specifically, we show that Carbonic Anhydrase 15b, an enzyme controlling the pH in many cell types, including metastatic cancer cells, is expressed in migrating germ cells and is crucial for establishing and maintaining an asymmetric pH distribution within them. Reducing the level of the protein and thereby erasing the pH elevation at the cell front resulted in abnormal cell migration and impaired arrival at the target. The basis for the disrupted migration is found in the stringent requirement for pH conditions in the cell for regulating contractility, for the polarization of Rac1 activity, and hence for the formation of actin-rich structures at the leading edge of the migrating cells.

  4. DDX4 (VASA) is conserved in germ cell development in marsupials and monotremes.

    PubMed

    Hickford, Danielle E; Frankenberg, Stephen; Pask, Andrew J; Shaw, Geoff; Renfree, Marilyn B

    2011-10-01

    DDX4 (VASA) is an RNA helicase expressed in the germ cells of all animals. To gain greater insight into the role of this gene in mammalian germ cell development, we characterized DDX4 in both a marsupial (the tammar wallaby) and a monotreme (the platypus). DDX4 is highly conserved between eutherian, marsupial, and monotreme mammals. DDX4 protein is absent from tammar fetal germ cells but is present from Day 1 postpartum in both sexes. The distribution of DDX4 protein during oogenesis and spermatogenesis in the tammar is similar to eutherians. Female tammar germ cells contain DDX4 protein throughout all stages of postnatal oogenesis. In males, DDX4 is in gonocytes, and during spermatogenesis it is present in spermatocytes and round spermatids. A similar distribution of DDX4 occurs in the platypus during spermatogenesis. There are several DDX4 isoforms in the tammar, resulting from both pre- and posttranslational modifications. DDX4 in marsupials and monotremes has multiple splice variants and polyadenylation motifs. Using in silico analyses of genomic databases, we found that these previously unreported splice variants also occur in eutherians. In addition, several elements implicated in the control of Ddx4 expression in the mouse, including RGG (arginine-glycine-glycine) and dimethylation of arginine motifs and CpG islands within the Ddx4 promoter, are also highly conserved. Collectively these data suggest that DDX4 is essential for the regulation of germ cell proliferation and differentiation across all three extant mammalian groups-eutherians, marsupials, and monotremes.

  5. Retroperitoneal metastatic germ cell tumor presenting as a psoas abscess: a diagnostic pitfall.

    PubMed

    Dieker, Carrie A; De Las Casas, Luis E; Davis, Brian R

    2013-07-01

    Most testicular neoplasms are germ cell tumors, the vast majority of which represent seminomas. Most seminomas present localized to the testis, whereas nonseminomatous germ cell tumors more often present with lymph node metastases. Psoas abscesses generally arise from a contiguous intra-abdominal or pelvic infectious process, an adjacent focus of osteomyelitis or septic emboli from distant infectious foci. In this study, the case of a 24-year-old man who presented with a right psoas mass presumptively diagnosed as an abscess secondary to fever and leukocytosis is presented. The patient had a history of right testicular seminoma, and normal serum levels of alpha-fetoprotein and human chorionic gonadotropin. Surgical exploration and biopsy demonstrated seminoma metastasis. This case represents an extremely unusual clinical presentation of metastatic germ cell tumor presenting as a psoas abscess. This unique case represents an unusual presentation of a recurrent germ cell tumor mimicking a psoas abscess. Awareness of possible metastatic testicular germ cell neoplasm as a psoas abscess could prevent diagnosis delay before retroperitoneal tumor debulking. PMID:23360792

  6. Is the Blood-Brain Barrier Relevant in Metastatic Germ Cell Tumor?

    SciTech Connect

    Azar, Jose M. Schneider, Bryan P.; Einhorn, Lawrence H.

    2007-09-01

    Purpose: Germ cell tumors are uniquely chemosensitive and curable, even with advanced metastatic disease. Central nervous system recurrence can terminate a complete remission in other chemosensitive tumors, such as small cell lung cancer, because of the blood-brain barrier (BBB). We propose to document that the BBB is also relevant in germ cell tumors despite their dramatic chemosensitivity. Methods and Materials: We present five cases illustrating the concept of the BBB in patients with metastatic testicular cancer treated with chemotherapy. Results: In our large series of patients with metastatic testicular cancer treated with chemotherapy, we identified 5 unique patients. These patients were rendered free of disease only to experience relapse in the brain alone. This included 1 patient who initially had good-risk metastatic disease by means of the International Germ Cell Collaborative Group staging system at the onset of chemotherapy. Conclusions: The BBB is relevant in patients with metastatic testicular cancer.

  7. DNA Methylation Profiling of Embryonic Stem Cell Differentiation into the Three Germ Layers

    PubMed Central

    Isagawa, Takayuki; Nagae, Genta; Shiraki, Nobuaki; Fujita, Takanori; Sato, Noriko; Ishikawa, Shumpei; Kume, Shoen; Aburatani, Hiroyuki

    2011-01-01

    Embryogenesis is tightly regulated by multiple levels of epigenetic regulation such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent developmental reprogramming occurs by de novo methylation and demethylation. Variance in DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analyzed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions are methylated across all three germ layers and in the three adult somatic tissues. This commonly methylated gene set is enriched in germ cell-associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns by global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Our combined findings indicate that differentiation of ES cells into the three germ layers is accompanied by an increased number of commonly methylated DNA regions and that these tissue-specific alterations in methylation occur for only a small number of genes. DNA methylation at the proximal promoter regions of commonly methylated genes thus appears to be an irreversible mark which functions to fix somatic lineage by repressing the transcription of germ cell-specific genes. PMID:22016810

  8. Quantification of human telomerase reverse transcriptase mRNA in testicular germ cell tumors by quantitative fluorescence real-time RT-PCR.

    PubMed

    Schrader, Mark; Burger, Angelika M; Müller, Markus; Krause, Hans; Straub, Bernd; Smith, Gilian L; Newlands, Eward S; Miller, Kurt

    2002-01-01

    Telomerase is a ribonucleoprotein enzyme which is endogenously expressed in germ, stem and tumor cells, but absent in benign somatic cells. The two major telomerase components are human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). It has been shown that hTERT is rate-limiting for telomerase activity and that it plays a central role in human carcinogenesis. Here, we investigated the potential of hTERT and hTR gene expression as diagnostic markers in testicular germ cell tumors (TGCT). hTERT mRNA and hTR expression were quantified in 55 testicular germ cell tumors comprising 36 primary and 19 germ cell tumors from retroperitonal sides by fluorescence real-time RT-PCR using the LightCycler technology. Porphobilinogen deaminase (PBGD) was used as housekeeping gene and to enable relative quantification. For comparison to TGCTs, 38 benign testicular biopsies from patients with fertility disorders were assayed. hTERT expression was detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content (N(hTERT) 38-127). In contrast, mature teratomas from primary and post-chemotherapy masses, which are characterized by well-differentiated tissue components showed a nearly complete downregulation of hTERT expression (N(hTERT) 2-4, p<0.001). hTR levels however, were high in all tumors and independently of the presence of germ cells also in the benign tissue control group. hTERT mRNA is expressed in all undifferentiated TGCTs but repressed in mature teratomas. This suggests an inverse correlation between the differentiation status of germ cell tumors and hTERT expression. Thus, detection of hTERT expression in tumors histopathologically classified as mature teratomas enables a molecular-diagnostic confirmation and might aid decision making for treatment of patients presenting with this tumor subtype.

  9. Pluripotent stem cells derived from mouse primordial germ cells by small molecule compounds.

    PubMed

    Kimura, Tohru; Kaga, Yoshiaki; Sekita, Yoichi; Fujikawa, Keita; Nakatani, Tsunetoshi; Odamoto, Mika; Funaki, Soichiro; Ikawa, Masahito; Abe, Kuniya; Nakano, Toru

    2015-01-01

    Primordial germ cells (PGCs) can give rise to pluripotent stem cells known as embryonic germ cells (EGCs) when cultured with basic fibroblast growth factor (bFGF), stem cell factor (SCF), and leukemia inhibitory factor. Somatic cells can give rise to induced pluripotent stem cells (iPSCs) by introduction of the reprogramming transcription factors Oct4, Sox2, and Klf4. The effects of Sox2 and Klf4 on somatic cell reprogramming can be reproduced using the small molecule compounds, transforming growth factor-β receptor (TGFβR) inhibitor and Kempaullone, respectively. Here we examined the effects of TGFβR inhibitor and Kempaullone on EGC derivation from PGCs. Treatment of PGCs with TGFβR inhibitor and/or Kempaullone generated pluripotent stem cells under standard embryonic stem cell (ESC) culture conditions without bFGF and SCF, which we termed induced EGCs (iEGCs). The derivation efficiency of iEGCs was dependent on the differentiation stage and sex. DNA methylation levels of imprinted genes in iEGCs were reduced, with the exception of the H19 gene. The promoters of genes involved in germline development were generally hypomethylated in PGCs, but three germline genes showed comparable DNA methylation levels among iEGs, ESCs, and iPSCs. These results show that PGCs can be reprogrammed into pluripotent state using small molecule compounds, and that DNA methylation of these germline genes is not maintained in iEGCs. PMID:25186651

  10. The insulin sensitiser metformin regulates chicken Sertoli and germ cell populations.

    PubMed

    Faure, M; Guibert, E; Alves, S; Pain, B; Ramé, C; Dupont, J; Brillard, J P; Froment, P

    2016-05-01

    Metformin, an insulin sensitiser from the biguanide family of molecules, is used for the treatment of insulin resistance in type 2 diabetes individuals. It increases peripheral glucose uptake and may reduce food intake. Based on the tight link between metabolism and fertility, we investigated the role of metformin on testicular function using in vitro culture of Sertoli cells and seminiferous tubules, complemented by in vivo data obtained following metformin administration to prepubertal chickens. In vitro, metformin treatment reduced Sertoli cell proliferation without inducing apoptosis and morphological changes. The metabolism of Sertoli cells was affected because lactate secretion by Sertoli cells increased approximately twofold and intracellular free ATP was negatively impacted. Two important pathways regulating proliferation and metabolism in Sertoli cells were assayed. Metformin exposure was not associated with an increased phosphorylation of AKT or ERK. There was a 90% reduction in the proportion of proliferating germ cells after a 96-h exposure of seminiferous tubule cultures to metformin. In vivo, 6-week-old chickens treated with metformin for 3 weeks exhibited reduced testicular weight and a 50% decrease in testosterone levels. The expression of a marker of undifferentiated germ cells was unchanged in contrast to the decrease in expression of 'protamine', a marker of differentiated germ cells. In conclusion, these results suggest that metformin affects the testicular energy content and the proliferative ability of Sertoli and germ cells. PMID:26917452

  11. Human primordial germ cell-derived progenitors give rise to neurons and glia in vivo

    SciTech Connect

    Teng, Yincheng; Chen, Bin; Tao, Minfang

    2009-12-18

    We derived a cell population from cultured human primordial germ cells from early human embryos. The derivates, termed embryoid body-derived (EBD) cells, displayed an extensive capacity for proliferation and expressed a panel of markers in all three germ layers. Interestingly, EBD cells were also positive for markers of neural stem/progenitor cells, such as nestin and glial fibrillary acidic protein. When these cells were transplanted into the brain cavities of fetal sheep and postnatal NOD-SCID mice or nerve-degenerated tibialis anterior muscles, they readily gave rise to neurons or glial cells. To our knowledge, our data are the first to demonstrate that EBD cells can undergo further neurogenesis under suitable environments in vivo. Hence, with the abilities of extensive expansion, self-renewal, and differentiation, EBD cells may provide a useful donor source for neural stem/progenitor cells to be used in cell-replacement therapies for diseases of the nervous system.

  12. New perspective on molecular markers as promising therapeutic targets in germ cell tumors

    PubMed Central

    Chieffi, Paolo

    2016-01-01

    Summary Testicular germ cell tumors (TGCTs) are the most frequent solid malignant tumors in men 20–40 years of age and the most frequent cause of death from solid tumors in this age group. TGCTs comprise two major histologic groups: seminomas and non-seminomas germ cell tumors (NSGCTs). NSGCTs can be further divided into embryonal carcinoma, Teratoma, yolk sac tumor, and choriocarcinoma. Seminomas and NSGCTs present significant differences in clinical features, therapy, and prognosis, and both show characteristics of the Primordial Germ Cells (PGCs). Many discovered biomarkers including HMGA1, GPR30, Aurora-B, estrogen receptor β, and others have given further advantages to discriminate between histological subgroups and could represent useful therapeutic targets. PMID:27195201

  13. Heterozygosity for a Bub1 mutation causes female-specific germ cell aneuploidy in mice

    SciTech Connect

    Leland, Shawn; Nagarajan, Prabakaran; Polyzos, Aris; Thomas, Sharon; Samaan, George; Donnell, Robert; Marchetti, Francesco; Venkatachalam, Sundaresan

    2009-06-24

    Aneuploidy, the most common chromosomal abnormality at birth and the main ascertained cause of pregnancy loss in humans, originates primarily from chromosome segregation errors during oogenesis. Here we report that heterozygosity for a mutation in the mitotic checkpoint kinase gene, Bub1, induces aneuploidy in female germ cells of mice, and that the effect increases with advancing maternal age. Analysis of Bub1 heterozygous oocytes showed that aneuploidy occurred primarily during the first meiotic division and involved premature sister chromatid separation. Furthermore, aneuploidy was inherited in zygotes and resulted in the loss of embryos after implantation. The incidence of aneuploidy in zygotes was sufficient to explain the reduced litter size in matings with Bub1 heterozygous females. No effects were seen in germ cells from heterozygous males. These findings show that Bub1 dysfunction is linked to inherited aneuploidy in female germ cells and may contribute to the maternal age-related increase in aneuploidy and pregnancy loss.

  14. Primordial germ cell biology at the beginning of the XXI century.

    PubMed

    De Felici, Massimo

    2009-01-01

    At the XIV Workshop on the Development and Function of the Reproductive Organs held at the Congress Centre of the University of Rome Tor Vergata, Monteporzio Catone, Rome, Italy, the introduction to the first session entitled Mammalian primordial germ cells dedicated to the memory of Anne McLaren, was the occasion for a concise review of the state of art of research on the biology of primordial germ cells (PGCs). This great, unforgettable scientist, who died in a car accident in July 2007, dedicated most of her studies to this field over the last 25 years. Topics briefly reviewed in this Meeting Report are: 1) how the germ line is determined; 2) what are the mechanisms underlying PGC migration; 3) to what extent PGC survival, proliferation and differentiation are cell autonomous or environmentally controlled processes and 4) how the potential for totipotency is retained in PGCs.

  15. Sufficient Numbers of Early Germ Cells Are Essential for Female Sex Development in Zebrafish

    PubMed Central

    Dai, Xiangyan; Jin, Xia; Chen, Xiaowen; He, Jiangyan; Yin, Zhan

    2015-01-01

    The sex determination for zebrafish is controlled by a combination of genetic and environmental factors. The determination of sex in zebrafish has been suggested to rely on a mechanism that is affected by germ cell-derived signals. To begin our current study, a simplified and efficient germ cell-specific promoter of the dead end (dnd) gene was identified. Utilizing the metrodinazole (MTZ)/ bacterial nitroreductase (NTR) system for inducible germ cell ablation, several stable Tg (dnd:NTR-EGFP-3'UTR) and Tg (dnd:NTR-EGFP+3'UTR) zebrafish lines were then generated with the identified promoter. A thorough comparison of the expression patterns and tissue distributions of endogenous dnd and ntr-egfp transcripts in vivo revealed that the identified 2032-bp zebrafish dnd promoter can recapitulate dnd expression faithfully in stable transgenic zebrafish. The correlation between the levels of the germ cell-derived signals and requirement for maintaining the female fate has been also explored with different durations of the MTZ treatments. Our results revealed the decreasing ratios of female presented in the treated transgenic group are fairly associated with the reducing levels of the early germ cell-derived signals. After the juvenile transgenic fish treated with 5 mM MTZ for 20 days, all MTZ-treated transgenic fish exclusively developed into males with subfertilities. Taken together, our results identified here a simplified and efficient dnd promoter, and provide clear evidence indicating that it was not the presence but the sufficiency of signals derived from germ cells that is essential for female sex development in zebrafish. Our model also provides a unique system for sex control in zebrafish studies. PMID:25679390

  16. The emerging role of matrix metalloproteases of the ADAM family in male germ cell apoptosis

    PubMed Central

    Urriola-Muñoz, Paulina; Lagos-Cabré, Raúl

    2011-01-01

    Constitutive germ cell apoptosis during mammalian spermatogenesis is a key process for controlling sperm output and to eliminate damaged or unwanted cells. An increase or decrease in the apoptosis rate has deleterious consequences and leads to low sperm production. Apoptosis in spermatogenesis has been widely studied, but the mechanism by which it is induced under physiological or pathological conditions has not been clarified. We have recently identified the metalloprotease ADAM17 (TACE) as a putative physiological inducer of germ cell apoptosis. The mechanisms involved in regulating the shedding of the ADAM17 extracellular domain are still far from being understood, although they are important in order to understand cell-cell communications. Here, we review the available data regarding apoptosis during mammalian spermatogenesis and the localization of ADAM proteins in the male reproductive tract. We propose an integrative working model where ADAM17, p38 MAPK, protein kinase C (PKC) and the tyrosine kinase c-Abl participate in the physiological signalling cascade inducing apoptosis in germ cells. In our model, we also propose a role for the Sertoli cell in regulating the Fas/FasL system in order to induce the extrinsic pathway of apoptosis in germ cells. This working model could be applied to further understand constitutive apoptosis in spermatogenesis and in pathological conditions (e.g., varicocele) or following environmental toxicants exposure (e.g., genotoxicity or xenoestrogens). PMID:22319668

  17. Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras

    PubMed Central

    Keighren, Margaret A.; Flockhart, Jean H.

    2016-01-01

    ABSTRACT The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1−/− null mouse embryos die but a previous study showed that some homozygous Gpi1−/− null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1−/−↔Gpi1c/c chimaera with functional Gpi1−/− null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1−/− null cells in adult Gpi1−/−↔Gpi1c/c chimaeras and determine if Gpi1−/− null germ cells are functional. Analysis of adult Gpi1−/−↔Gpi1c/c chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1−/− null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1−/− null oocytes in one female Gpi1−/−↔Gpi1c/c chimaera were functional and provided preliminary evidence that one male putative Gpi1−/−↔Gpi1c/c chimaera produced functional spermatozoa from homozygous Gpi1−/− null germ cells. Although the male chimaera was almost certainly Gpi1−/−↔Gpi1c/c, this part of the study is considered preliminary because only blood was typed for GPI. Gpi1−/− null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1−/− null germ cells, it successfully identified functional Gpi1−/− null oocytes and revealed that some Gpi1−/− null cells could survive in many adult tissues. PMID:27103217

  18. Propagation of human germ stem cells in long-term culture

    PubMed Central

    Akhondi, Mohammad Mehdi; Mohazzab, Arash; Jeddi-Tehrani, Mahmood; Sadeghi, Mohammad Reza; Eidi, Akram; Khodadadi, Abbas; Piravar, Zeinab

    2013-01-01

    Background: Spermatogonial stem cells (SSCs), a subset of undifferentiated type A spermatogonia, are the foundation of complex process of spermatogenesis and could be propagated in vitro culture conditions for long time for germ cell transplantation and fertility preservation. Objective: The aim of this study was in vitro propagation of human spermatogonial stem cells (SSCs) and improvement of presence of human Germ Stem Cells (hGSCs) were assessed by specific markers POU domain, class 5, transcription factor 1 (POU5F1), also known as Octamer-binding transcription factor 4 (Oct-4) and PLZF (Promyelocytic leukaemia zinc finger protein). Materials and Methods: Human testicular cells were isolated by enzymatic digestion (Collagenase IV and Trypsin). Germ cells were cultured in Stem-Pro 34 media supplemented by growth factors such as glial cell line-derived neurotrophic factor, basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor to support self-renewal divisions. Germline stem cell clusters were passaged and expanded every week. Immunofluorecent study was accomplished by Anti-Oct4 antibody through the culture. The spermatogonial stem cells genes expression, PLZF, was studied in testis tissue and germ stem cells entire the culture. Results: hGSCs clusters from a brain dead patient developed in testicular cell culture and then cultured and propagated up to 6 weeks. During the culture Oct4 were a specific marker for identification of hGSCs in testis tissue. Expression of PLZF was applied on RNA level in germ stem cells. Conclusion: hGSCs indicated by SSCs specific marker can be cultured and propagated for long-term in vitro conditions. This article extracted from Ph.D. Thesis. (Zeinab Piravar) PMID:24639790

  19. Effect of methotrexate on cell proliferation in developing hamster molar tooth germs in vitro.

    PubMed

    Wöltgens, J H; Lyaruu, D M; Bronckers, A L; van Duin, M A; Bervoets, T J

    1998-01-01

    Amongst the most frequently used drugs for the treatment of acute lymphoblastic leukaemia (ALL) belongs methotrexate (MTX), an inhibitor of pyrimidine (thymidine) synthesis. We examined effects of MTX on cell proliferation during tooth morphogenesis in organ culture by exposing hamster molar tooth germs to 10(-7) to 10(-3) M MTX for 24 h. In the presence of serum, only the highest concentration of MTX (10(-3) M) induced a small, nonsignificant decrease in cell mass without histological changes but, unexpectedly, increased uptake of [3H]thymidine. In serumless conditions increase in cell mass (dry weight) and incorporation of [3H]thymidine was lower than in serum-supplemented conditions. Exposure to MTX in serumless conditions reduced the increase in cell mass even further without histological changes and, again, strongly enhanced incorporation of [3H]thymidine to the same proportion as measured in the serum-supplemented cultures exposed to MTX. The data suggest that only exposure to high levels of MTX reduces proliferation activity, shown by reduction in cell mass. The enhanced [3H]thymidine uptake under MTX exposure was explained by blockage of the internal biosynthesis of thymidine, by which action more radiolabel was taken up from the medium. The data also suggest that serum contains (growth) factors that stimulate cell proliferation, thereby increasing cell mass and [3H]thymidine incorporation.

  20. HIPSTR and thousands of lncRNAs are heterogeneously expressed in human embryos, primordial germ cells and stable cell lines

    PubMed Central

    Yunusov, Dinar; Anderson, Leticia; DaSilva, Lucas Ferreira; Wysocka, Joanna; Ezashi, Toshihiko; Roberts, R. Michael; Verjovski-Almeida, Sergio

    2016-01-01

    Eukaryotic genomes are transcribed into numerous regulatory long non-coding RNAs (lncRNAs). Compared to mRNAs, lncRNAs display higher developmental stage-, tissue-, and cell-subtype-specificity of expression, and are generally less abundant in a population of cells. Despite the progress in single-cell-focused research, the origins of low population-level expression of lncRNAs in homogeneous populations of cells are poorly understood. Here, we identify HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel lncRNA gene in the developmentally regulated TFAP2A locus. HIPSTR has evolutionarily conserved expression patterns, its promoter is most active in undifferentiated cells, and depletion of HIPSTR in HEK293 and in pluripotent H1BP cells predominantly affects the genes involved in early organismal development and cell differentiation. Most importantly, we find that HIPSTR is specifically induced and heterogeneously expressed in the 8-cell-stage human embryos during the major wave of embryonic genome activation. We systematically explore the phenomenon of cell-to-cell variation of gene expression and link it to low population-level expression of lncRNAs, showing that, similar to HIPSTR, the expression of thousands of lncRNAs is more highly heterogeneous than the expression of mRNAs in the individual, otherwise indistinguishable cells of totipotent human embryos, primordial germ cells, and stable cell lines. PMID:27605307

  1. HIPSTR and thousands of lncRNAs are heterogeneously expressed in human embryos, primordial germ cells and stable cell lines

    NASA Astrophysics Data System (ADS)

    Yunusov, Dinar; Anderson, Leticia; Dasilva, Lucas Ferreira; Wysocka, Joanna; Ezashi, Toshihiko; Roberts, R. Michael; Verjovski-Almeida, Sergio

    2016-09-01

    Eukaryotic genomes are transcribed into numerous regulatory long non-coding RNAs (lncRNAs). Compared to mRNAs, lncRNAs display higher developmental stage-, tissue-, and cell-subtype-specificity of expression, and are generally less abundant in a population of cells. Despite the progress in single-cell-focused research, the origins of low population-level expression of lncRNAs in homogeneous populations of cells are poorly understood. Here, we identify HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel lncRNA gene in the developmentally regulated TFAP2A locus. HIPSTR has evolutionarily conserved expression patterns, its promoter is most active in undifferentiated cells, and depletion of HIPSTR in HEK293 and in pluripotent H1BP cells predominantly affects the genes involved in early organismal development and cell differentiation. Most importantly, we find that HIPSTR is specifically induced and heterogeneously expressed in the 8-cell-stage human embryos during the major wave of embryonic genome activation. We systematically explore the phenomenon of cell-to-cell variation of gene expression and link it to low population-level expression of lncRNAs, showing that, similar to HIPSTR, the expression of thousands of lncRNAs is more highly heterogeneous than the expression of mRNAs in the individual, otherwise indistinguishable cells of totipotent human embryos, primordial germ cells, and stable cell lines.

  2. HIPSTR and thousands of lncRNAs are heterogeneously expressed in human embryos, primordial germ cells and stable cell lines.

    PubMed

    Yunusov, Dinar; Anderson, Leticia; DaSilva, Lucas Ferreira; Wysocka, Joanna; Ezashi, Toshihiko; Roberts, R Michael; Verjovski-Almeida, Sergio

    2016-01-01

    Eukaryotic genomes are transcribed into numerous regulatory long non-coding RNAs (lncRNAs). Compared to mRNAs, lncRNAs display higher developmental stage-, tissue-, and cell-subtype-specificity of expression, and are generally less abundant in a population of cells. Despite the progress in single-cell-focused research, the origins of low population-level expression of lncRNAs in homogeneous populations of cells are poorly understood. Here, we identify HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel lncRNA gene in the developmentally regulated TFAP2A locus. HIPSTR has evolutionarily conserved expression patterns, its promoter is most active in undifferentiated cells, and depletion of HIPSTR in HEK293 and in pluripotent H1BP cells predominantly affects the genes involved in early organismal development and cell differentiation. Most importantly, we find that HIPSTR is specifically induced and heterogeneously expressed in the 8-cell-stage human embryos during the major wave of embryonic genome activation. We systematically explore the phenomenon of cell-to-cell variation of gene expression and link it to low population-level expression of lncRNAs, showing that, similar to HIPSTR, the expression of thousands of lncRNAs is more highly heterogeneous than the expression of mRNAs in the individual, otherwise indistinguishable cells of totipotent human embryos, primordial germ cells, and stable cell lines. PMID:27605307

  3. Germ cell cluster organization and oogenesis in the tardigrade Dactylobiotus parthenogeneticus Bertolani, 1982 (Eutardigrada, Murrayidae).

    PubMed

    Poprawa, Izabela; Hyra, Marta; Rost-Roszkowska, Magdalena Maria

    2015-07-01

    Germ cell cluster organization and the process of oogenesis in Dactylobiotus parthenogeneticus have been described using transmission electron microscopy and light microscopy. The reproductive system of D. parthenogeneticus is composed of a single, sac-like, meroistic ovary and a single oviduct that opens into the cloaca. Two zones can be distinguished in the ovary: a small germarium that is filled with oogonia and a vitellarium that is filled with germ cell clusters. The germ cell cluster, which has the form of a modified rosette, consists of eight cells that are interconnected by stable cytoplasmic bridges. The cell that has the highest number of stable cytoplasmic bridges (four bridges) finally develops into the oocyte, while the remaining cells become trophocytes. Vitellogenesis of a mixed type occurs in D. parthenogeneticus. One part of the yolk material is produced inside the oocyte (autosynthesis), while the second part is synthesized in the trophocytes and transported to the oocyte through the cytoplasmic bridges. The eggs are covered with two envelopes: a thin vitelline envelope and a three-layered chorion. The surface of the chorion forms small conical processes, the shape of which is characteristic for the species that was examined. In our paper, we present the first report on the rosette type of germ cell clusters in Parachela.

  4. In vitro generation and characterization of chicken long-term germ cells from different embryonic origins.

    PubMed

    Raucci, Franca; Fuet, Aurelie; Pain, Bertrand

    2015-09-15

    Primordial germ cells (PGCs) are the precursors of differentiated germ cells. Located in the epiblast of a stage X (EG&K) embryo, the PGCs translocate anteriorly to the germinal crescent and migrate, within 48 to 56 hours of development, through the blood vascular system to the germinal ridges where they become the gonadal germ cells (GGCs). We aim to generate, compare, and determine the basic characters of the in vitro long-term cultured PGCs derived from (1) the chicken blastodermal cells (at stages IX-XII); (2) the chicken blood of a 2-day old embryo (stages 14-17 Hamburger Hamilton [HH]); and (3) the long-term cultured gonocytes taken from male gonads of a 5- to 6-day-old embryo (stages 29-30 HH). In presence of fibroblast growth factor, chicken blastodermal cells are able to long-term proliferate and generate small, round, alkaline phosphatase-positive cell clusters. Molecular characterization shows that these selected and amplified clusters show a PGC-like cell profile, as they express cPOUV (a pluripotent-associated marker), NR6A1/GCNF and DDX4/CVH (germ cell-specific genes). Both chicken PGCs and GGCs, obtained from embryonic blood and gonads, at 14 to 17 HH and 29 to 30 HH, respectively, generate long-term germ cell cultures and positively react in vitro to periodic acid-Schiff. Immunochemical analyses reveal that these cell lines are specifically recognized by anti-SSEA-1, anti-EMA-1, anti-CVH, anti-β1-integrin, and anti-CEACAM antibodies. The presence of surrounding cells may suggest a stronger dependency toward the niche process for the GGCs. The reactivity of chicken embryonic germ cells obtained from the two different sources to the specific markers used in this study was not altered through the culture. In conclusion, the morphologic analysis specific for chicken PGCs and GGCs will further contribute to quick and reliable characterization of long-term cultured in vitro chicken germ cells.

  5. Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells.

    PubMed

    Gazave, Eve; Béhague, Julien; Laplane, Lucie; Guillou, Aurélien; Préau, Laetitia; Demilly, Adrien; Balavoine, Guillaume; Vervoort, Michel

    2013-10-01

    Like most bilaterian animals, the annelid Platynereis dumerilii generates the majority of its body axis in an anterior to posterior temporal progression with new segments added sequentially. This process relies on a posterior subterminal proliferative body region, known as the "segment addition zone" (SAZ). We explored some of the molecular and cellular aspects of posterior elongation in Platynereis, in particular to test the hypothesis that the SAZ contains a specific set of stem cells dedicated to posterior elongation. We cloned and characterized the developmental expression patterns of orthologs of 17 genes known to be involved in the formation, behavior, or maintenance of stem cells in other metazoan models. These genes encode RNA-binding proteins (e.g., tudor, musashi, pumilio) or transcription factors (e.g., myc, id, runx) widely conserved in eumetazoans. Most of these genes are expressed both in the migrating primordial germ cells and in overlapping ring-like patterns in the SAZ, similar to some previously analyzed genes (piwi, vasa). The SAZ patterns are coincident with the expression of proliferation markers cyclin B and PCNA. EdU pulse and chase experiments suggest that new segments are produced through many rounds of divisions from small populations of teloblast-like posterior stem cells. The shared molecular signature between primordial germ cells and posterior stem cells in Platynereis thus corresponds to an ancestral "stemness" program. PMID:23891818

  6. The influence of scaffold elasticity on germ layer specification of human embryonic stem cells.

    PubMed

    Zoldan, Janet; Karagiannis, Emmanouil D; Lee, Christopher Y; Anderson, Daniel G; Langer, Robert; Levenberg, Shulamit

    2011-12-01

    Mechanical forces are critical to embryogenesis, specifically, in the lineage-specification gastrulation phase, whereupon the embryo is transformed from a simple spherical ball of cells to a multi-layered organism, containing properly organized endoderm, mesoderm, and ectoderm germ layers. Several reports have proposed that such directed and coordinated movements of large cell collectives are driven by cellular responses to cell deformations and cell-generated forces. To better understand these environmental-induced cell changes, we have modeled the germ layer formation process by culturing human embryonic stem cells (hESCs) on three dimensional (3D) scaffolds with stiffness engineered to model that found in specific germ layers. We show that differentiation to each germ layer was promoted by a different stiffness threshold of the scaffolds, reminiscent of the forces exerted during the gastrulation process. The overall results suggest that three dimensional (3D) scaffolds can recapitulate the mechanical stimuli required for directing hESC differentiation and that these stimuli can play a significant role in determining hESC fate. PMID:21963156

  7. Germ-cell nondisjunction in testes biopsies of men with idiopathic infertility.

    PubMed Central

    Huang, W J; Lamb, D J; Kim, E D; de Lara, J; Lin, W W; Lipshultz, L I; Bischoff, F Z

    1999-01-01

    Intracytoplasmic sperm injection (ICSI) has been used in combination with testicular sperm extraction to achieve pregnancies in couples with severe male-factor infertility, yet many of the underlying genetic mechanisms remain largely unknown. To investigate nondisjunction in mitotic and meiotic germ cells, we performed three-color FISH to detect numeric chromosome aberrations in testicular tissue samples from infertile men confirmed to have impaired spermatogenesis of unknown cause. FISH was employed to determine the rate of sex-chromosome aneuploidy in germ cells. Nuclei were distinguished as haploid or diploid, respectively. The overall incidence of sex-chromosome aneuploidy in germ cells was found to be significantly higher (P<.00001) in all three abnormal histopathologic patterns (range 39.0%-43.5%) as compared with normal controls (29.1%). The relative ratio of normal to aneuploid nuclei in the diploid cells of patients with impaired spermatogenesis was approximately 1.0, a >300% decrease when compared with the 4.42 ratio detected in patients with normal spermatogenesis. These results provide direct evidence of an increased incidence of sex-chromosome aneuploidy observed in germ cells of men with severely impaired spermatogenesis who might be candidates for ICSI with sperm obtained directly from the testis. The incidence of aneuploidy was significantly greater among the diploid nuclei, which suggests that chromosome instability is a result of altered genetic control during mitotic cell division and proliferation during spermatogenesis. PMID:10330350

  8. Benzo(a)pyrene Induced p53 Mediated Male Germ Cell Apoptosis: Synergistic Protective Effects of Curcumin and Resveratrol

    PubMed Central

    Banerjee, Bhaswati; Chakraborty, Supriya; Ghosh, Debidas; Raha, Sanghamitra; Sen, Parimal C.; Jana, Kuladip

    2016-01-01

    Benzo(a)pyrene (B(a)P) is an environmental toxicant that induces male germ cell apoptosis. Curcumin and resveratrol are phytochemicals with cytoprotective and anti-oxidative properties. At the same time resveratrol is also a natural Aryl hydrocarbon Receptor (AhR) antagonist. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted the synergistic protective effect of curcumin and resveratrol against B(a)P induced p53 mediated germ cell apoptosis. Curcumin-resveratrol significantly prevented B(a)P induced decrease in sperm cell count and motility, as well as increased serum testosterone level. Curcumin-resveratrol co-treatment actively protected B(a)P induced testicular germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3, 8 and 9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, and Apaf1. B(a)P induced testicular reactive oxygen species (ROS) generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 (Cytochrome P4501A1) expression. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ

  9. Benzo(a)pyrene Induced p53 Mediated Male Germ Cell Apoptosis: Synergistic Protective Effects of Curcumin and Resveratrol.

    PubMed

    Banerjee, Bhaswati; Chakraborty, Supriya; Ghosh, Debidas; Raha, Sanghamitra; Sen, Parimal C; Jana, Kuladip

    2016-01-01

    Benzo(a)pyrene (B(a)P) is an environmental toxicant that induces male germ cell apoptosis. Curcumin and resveratrol are phytochemicals with cytoprotective and anti-oxidative properties. At the same time resveratrol is also a natural Aryl hydrocarbon Receptor (AhR) antagonist. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted the synergistic protective effect of curcumin and resveratrol against B(a)P induced p53 mediated germ cell apoptosis. Curcumin-resveratrol significantly prevented B(a)P induced decrease in sperm cell count and motility, as well as increased serum testosterone level. Curcumin-resveratrol co-treatment actively protected B(a)P induced testicular germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3, 8 and 9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, and Apaf1. B(a)P induced testicular reactive oxygen species (ROS) generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 (Cytochrome P4501A1) expression. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ

  10. Differentiation of Human Umbilical Cord Matrix-Derived Mesenchymal Stem Cells into Germ-Like Cells

    PubMed Central

    Latifpour, Mostafa; Shakiba, Yadollah; Amidi, Fardin; Mazaheri, Zohreh; Sobhani, Aligholi

    2014-01-01

    Background Mesenchymal Stem Cells (MSCs) are multipotent cells that can be collected from different sources. Under specific conditions, MSCs can be differentiated to tissue specific cells in vitro. Human Umbilical Cord mesenchymal Stem Cells (hUCMSCs) can easily be harvested and cultured in in vitro conditions. Production of germ cells from mesenchymal stem cells is a very interesting and promising area in the field of reproductive medicine. In the present study, the possible trans-differentiation of hUCMSCs into Primordial like Germ Cell (PGC) was performed in vitro under specific condition. Methods Human umbilical cord mesenchymal stem cells were cultured and expanded in DMEM medium containing 10% FBS. The cultured cells were studied for differentiation ability to adipocytes and osteocytes. Furthermore, MSCs related markers were identified by flow cytometry method. For PGC differentiation, hUCMS cells were cultured in differentiation medium containing Bone Morphogenetic Protein 4 (BMP4) and it was followed by retinoic acid (RA). Real time PCR and immunocytochemistry analysis were performed to evaluate the expression of PGC specific genes and proteins, respectively. Results Our results showed that hUCMSCs cultured in the presence of BMP4 and RA are able to transdifferentiate in to PGC like cells in vitro. Real time PCR and immunocytochemistry results showed that differentiated cells expressed PGC specific markers after 14 days of culture. Conclusion Based on these results, it was concluded that hUCMSC may be considered as a promising alternative cell source in reproductive medicine. More studies including laboratory and also animal models are needed to evaluate the functionality of differentiated PGCs before introducing them to clinical applications. PMID:25414784

  11. Complete Meiosis from Embryonic Stem Cell-Derived Germ Cells In Vitro.

    PubMed

    Zhou, Quan; Wang, Mei; Yuan, Yan; Wang, Xuepeng; Fu, Rui; Wan, Haifeng; Xie, Mingming; Liu, Mingxi; Guo, Xuejiang; Zheng, Ying; Feng, Guihai; Shi, Qinghua; Zhao, Xiao-Yang; Sha, Jiahao; Zhou, Qi

    2016-03-01

    In vitro generation of functional gametes is a promising approach for treating infertility, although faithful replication of meiosis has proven to be a substantial obstacle to deriving haploid gamete cells in culture. Here we report complete in vitro meiosis from embryonic stem cell (ESC)-derived primordial germ cells (PGCLCs). Co-culture of PGCLCs with neonatal testicular somatic cells and sequential exposure to morphogens and sex hormones reproduced key hallmarks of meiosis, including erasure of genetic imprinting, chromosomal synapsis and recombination, and correct nuclear DNA and chromosomal content in the resulting haploid cells. Intracytoplasmic injection of the resulting spermatid-like cells into oocytes produced viable and fertile offspring, showing that this robust stepwise approach can functionally recapitulate male gametogenesis in vitro. These findings provide a platform for investigating meiotic mechanisms and the potential generation of human haploid spermatids in vitro.

  12. Cytoreductive surgery in disseminated non-seminomatous germ cell tumours of testis.

    PubMed

    Kulkarni, R P; Reynolds, K W; Newlands, E S; Dawson, P M; Makey, A R; Theodorou, N A; Bradley, J; Begent, R H; Rustin, G J; Bagshawe, K D

    1991-02-01

    Between 1977 and 1988, 67 patients underwent surgical removal of residual metastatic deposits following an aggressive chemotherapy regimen (cisplatin, vincristine, methotrexate and bleomycin alternating with etoposide, actinomycin D and cyclophosphamide) for disseminated germ cell tumours of the testis (stage IIB or above). Ninety-one surgical procedures were performed. There were 63 (69 per cent) retroperitoneal lymph node dissections, 16 (18 per cent) thoracotomies, three (3 per cent) hepatic resections, three (3 per cent) craniotomies, five (5 per cent) delayed orchidectomies and one anterolateral decompression of the vertebral column. Nine (13 per cent) patients required a repeat retroperitoneal node dissection and one patient needed a repeat thoracotomy to remove recurrent metastatic deposits during the period of follow-up. Multivisceral resections and vascular reconstruction procedures were required in 20 (30 per cent) patients undergoing retroperitoneal node dissection. Fifty-five (82 per cent) patients remain in complete remission with a mean follow-up period of 49.6 months (range 2-121 months). Nine (13 per cent) patients died with metastatic disease between 2 months to 4 years after operation. There were three deaths in the perioperative period (4 per cent). The histology of the resected metastases revealed undifferentiated active tumour in 20 (30 per cent) patients, differentiated mature teratoma in 29 (43 per cent) patients and fibrosis/necrosis in 18 (27 per cent) patients. Twelve (60 per cent) patients with undifferentiated elements and 15 patients (60 per cent) with raised preoperative tumour markers (poor prognostic categories) are in complete remission. Cytoreductive surgery in patients with metastatic germ cell tumours offers the best chance of remission following chemotherapy even in poor prognostic group categories.

  13. Origin of germ cells and formation of new primary follicles in adult human ovaries

    PubMed Central

    Bukovsky, Antonin; Caudle, Michael R; Svetlikova, Marta; Upadhyaya, Nirmala B

    2004-01-01

    Recent reports indicate that functional mouse oocytes and sperm can be derived in vitro from somatic cell lines. We hypothesize that in adult human ovaries, mesenchymal cells in the tunica albuginea (TA) are bipotent progenitors with a commitment for both primitive granulosa and germ cells. We investigated ovaries of twelve adult women (mean age 32.8 ± 4.1 SD, range 27–38 years) by single, double, and triple color immunohistochemistry. We show that cytokeratin (CK)+ mesenchymal cells in ovarian TA differentiate into surface epithelium (SE) cells by a mesenchymal-epithelial transition. Segments of SE directly associated with ovarian cortex are overgrown by TA, forming solid epithelial cords, which fragment into small (20 micron) epithelial nests descending into the lower ovarian cortex, before assembling with zona pellucida (ZP)+ oocytes. Germ cells can originate from SE cells which cover the TA. Small (10 micron) germ-like cells showing PS1 meiotically expressed oocyte carbohydrate protein are derived from SE cells via asymmetric division. They show nuclear MAPK immunoexpression, subsequently divide symmetrically, and enter adjacent cortical vessels. During vascular transport, the putative germ cells increase to oocyte size, and are picked-up by epithelial nests associated with the vessels. During follicle formation, extensions of granulosa cells enter the oocyte cytoplasm, forming a single paranuclear CK+ Balbiani body supplying all the mitochondria of the oocyte. In the ovarian medulla, occasional vessels show an accumulation of ZP+ oocytes (25–30 microns) or their remnants, suggesting that some oocytes degenerate. In contrast to males, adult human female gonads do not preserve germline type stem cells. This study expands our previous observations on the formation of germ cells in adult human ovaries. Differentiation of primitive granulosa and germ cells from the bipotent mesenchymal cell precursors of TA in adult human ovaries represents a most

  14. Ultrastructural Studies of Germ Cell Development and the Functions of Leydig Cells and Sertoli Cells associated with Spermatogenesis in Kareius bicoloratus (Teleostei, Pleuronectiformes, Pleuronectidae)

    PubMed Central

    Kang, Hee-Woong; Kim, Sung Hwan; Chung, Jae Seung

    2016-01-01

    The ultrastructures of germ cells and the functions of Leydig cells and Sertoli cells during spermatogenesis inmale Kareius bicoloratus (Pleuronectidae) were investigated by electron microscope observation. Each of the well-developed Leydig cells during active maturation division and before spermiation contained an ovoid vesicular nucleus, a number of smooth endoplasmic reticula, well-developed tubular or vesicular mitochondrial cristae, and several lipid droplets in the cytoplasm. It is assumed that Leydig cells are typical steroidogenic cells showing cytological characteristics associated with male steroidogenesis. No cyclic structural changes in the Leydig cells were observed through the year. However, although no clear evidence of steroidogenesis or of any transfer of nutrients from the Sertoli cells to spermatogenic cells was observed, cyclic structural changes in the Sertoli cells were observed over the year. During the period of undischarged germ cell degeneration after spermiation, the Sertoli cells evidenced a lysosomal system associated with phagocytic function in the seminiferous lobules. In this study, the Sertoli cells function in phagocytosis and the resorption of products originating from degenerating spermatids and spermatozoa after spermiation. The spermatozoon lacks an acrosome, as have been shown in all teleost fish spermatozoa. The flagellum or sperm tail of this species evidences the typical 9+2 array of microtubules. PMID:27294207

  15. GermlncRNA: a unique catalogue of long non-coding RNAs and associated regulations in male germ cell development.

    PubMed

    Luk, Alfred Chun-Shui; Gao, Huayan; Xiao, Sizhe; Liao, Jinyue; Wang, Daxi; Tu, Jiajie; Rennert, Owen M; Chan, Wai-Yee; Lee, Tin-Lap

    2015-01-01

    Spermatogenic failure is a major cause of male infertility, which affects millions of couples worldwide. Recent discovery of long non-coding RNAs (lncRNAs) as critical regulators in normal and disease development provides new clues for delineating the molecular regulation in male germ cell development. However, few functional lncRNAs have been characterized to date. A major limitation in studying lncRNA in male germ cell development is the absence of germ cell-specific lncRNA annotation. Current lncRNA annotations are assembled by transcriptome data from heterogeneous tissue sources; specific germ cell transcript information of various developmental stages is therefore under-represented, which may lead to biased prediction or fail to identity important germ cell-specific lncRNAs. GermlncRNA provides the first comprehensive web-based and open-access lncRNA catalogue for three key male germ cell stages, including type A spermatogonia, pachytene spermatocytes and round spermatids. This information has been developed by integrating male germ transcriptome resources derived from RNA-Seq, tiling microarray and GermSAGE. Characterizations on lncRNA-associated regulatory features, potential coding gene and microRNA targets are also provided. Search results from GermlncRNA can be exported to Galaxy for downstream analysis or downloaded locally. Taken together, GermlncRNA offers a new avenue to better understand the role of lncRNAs and associated targets during spermatogenesis. Database URL: http://germlncrna.cbiit.cuhk.edu.hk/ PMID:25982314

  16. GermlncRNA: a unique catalogue of long non-coding RNAs and associated regulations in male germ cell development

    PubMed Central

    Luk, Alfred Chun-Shui; Gao, Huayan; Xiao, Sizhe; Liao, Jinyue; Wang, Daxi; Tu, Jiajie; Rennert, Owen M.; Chan, Wai-Yee; Lee, Tin-Lap

    2015-01-01

    Spermatogenic failure is a major cause of male infertility, which affects millions of couples worldwide. Recent discovery of long non-coding RNAs (lncRNAs) as critical regulators in normal and disease development provides new clues for delineating the molecular regulation in male germ cell development. However, few functional lncRNAs have been characterized to date. A major limitation in studying lncRNA in male germ cell development is the absence of germ cell-specific lncRNA annotation. Current lncRNA annotations are assembled by transcriptome data from heterogeneous tissue sources; specific germ cell transcript information of various developmental stages is therefore under-represented, which may lead to biased prediction or fail to identity important germ cell-specific lncRNAs. GermlncRNA provides the first comprehensive web-based and open-access lncRNA catalogue for three key male germ cell stages, including type A spermatogonia, pachytene spermatocytes and round spermatids. This information has been developed by integrating male germ transcriptome resources derived from RNA-Seq, tiling microarray and GermSAGE. Characterizations on lncRNA-associated regulatory features, potential coding gene and microRNA targets are also provided. Search results from GermlncRNA can be exported to Galaxy for downstream analysis or downloaded locally. Taken together, GermlncRNA offers a new avenue to better understand the role of lncRNAs and associated targets during spermatogenesis. Database URL: http://germlncrna.cbiit.cuhk.edu.hk/ PMID:25982314

  17. [Study on pluripotency and cultivation of ES-like cells derived from male germ stem cells of bovine fetuses].

    PubMed

    Dong, Wu-Zi; Shen, Wen-Zheng; Hua, Jin-Lian; Dou, Zhong-Ying

    2007-07-01

    Male germ stem cells (mGSCs), which is in testis after sex differentiation, derive from primordial germ cells. In this study, bovine mGSCs were isolated from testis of 20 weeks fetuses. Number of CD9 positive cells of the cells through two-steps adhering plates velocity different was 95.8% by flow cytometer. The carina-type cells clones and the plane-type cells clones appeared in co-cultured system. One cells lines had been successively maintained for 4 passages, and the cells clusters showed AKP positive staining. The cells clusters showed nest-shape in third passage showed SSEA1 and Oct-4 positive staining. These cells can also spontaneously differentiate into c-kit positive staining germ cells, and the cells were directional induced to formaactin positive staining cardiac-like cells cluster and NF positive staining neuron-like cells. The conclusion showed that male germ stem cells from 20 weeks bovine fetuses could be in vitro formed like embryonic stem cells. PMID:17822057

  18. [Study on pluripotency and cultivation of ES-like cells derived from male germ stem cells of bovine fetuses].

    PubMed

    Dong, Wu-Zi; Shen, Wen-Zheng; Hua, Jin-Lian; Dou, Zhong-Ying

    2007-07-01

    Male germ stem cells (mGSCs), which is in testis after sex differentiation, derive from primordial germ cells. In this study, bovine mGSCs were isolated from testis of 20 weeks fetuses. Number of CD9 positive cells of the cells through two-steps adhering plates velocity different was 95.8% by flow cytometer. The carina-type cells clones and the plane-type cells clones appeared in co-cultured system. One cells lines had been successively maintained for 4 passages, and the cells clusters showed AKP positive staining. The cells clusters showed nest-shape in third passage showed SSEA1 and Oct-4 positive staining. These cells can also spontaneously differentiate into c-kit positive staining germ cells, and the cells were directional induced to formaactin positive staining cardiac-like cells cluster and NF positive staining neuron-like cells. The conclusion showed that male germ stem cells from 20 weeks bovine fetuses could be in vitro formed like embryonic stem cells.

  19. Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers

    PubMed Central

    Imai, Hiroyuki; Kano, Kiyoshi; Fujii, Wataru; Takasawa, Ken; Wakitani, Shoichi; Hiyama, Masato; Nishino, Koichiro; Kusakabe, Ken Takeshi; Kiso, Yasuo

    2015-01-01

    Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals. PMID:26091100

  20. The incidence and histological characteristics of intratubular germ cell neoplasia in postpubertal cryptorchid testis

    PubMed Central

    Ryang, Seung Hoon; Jung, Jae Hung; Eom, Minseob; Song, Jae Mann; Chung, Hyun Chul; Chae, Yunbyung; Lee, Chang Min

    2015-01-01

    Purpose It is well known that testicular germ cell tumors arise with increased frequency in patients with cryptorchidism. In addition, intratubular germ cell neoplasia (ITGCN) is a precursor lesion to testicular germ cell tumor. Approximately 50% of patients with ITGCN will develop an invasive of testicular germ cell tumors within 5 years. Therefore, we evaluated that the incidence of ITGCN in postpubertal cryptorchidism. Materials and Methods Between January 2002 and August 2012, orchiectomy specimens from 31 postpubertalpatients (aged 12 or over) with cryptorchid testis were reviewed. The specimens were evaluated for ITGCN using immunohistochemical stains of placental-like alkaline phosphatase and Oct 3/4 with routine hematoxylin-eosin stain. Additionally, the degree of spermatogenesis was assessed using the Johnsen score. Results Mean age was 34 years (range, 17 to 74 years) at surgery. All patients were diagnosed as unilateral cryptorchidism. One patient (3.2%) of 20-year-old had ITGCN in surgical specimen with all positive markers. Histological assessment of spermatogenesis showed that mean Johnsen score was 3.42 (range, 1 to 9). Majority of patients (27 of 31) presented impaired spermatogenesis with low Johnsen score lesser than 5. Conclusions Considering the risk of malignancy and low spermatogenesis, we should perform immunohistochemical stains and discuss preventative orchiectomy for the postpubertal cryptorchidism. PMID:26175870

  1. Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.

    PubMed

    Oulhen, Nathalie; Wessel, Gary M

    2016-10-01

    Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3'UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability.

  2. Boule Is Present in Fish and Bisexually Expressed in Adult and Embryonic Germ Cells of Medaka

    PubMed Central

    Xu, Hongyan; Li, Zhendong; Li, Mingyou; Wang, Li; Hong, Yunhan

    2009-01-01

    Background The DAZ family genes boule, daz and dazl encode RNA binding proteins essential for fertility of diverse animals including human. dazl has bisexual expression in both mitotic and meiotic germ cells, whereas daz has male premeiotic expression, and boule is largely a unisexual meiotic regulator. Although boule has been proposed as the ancestor for dazl/daz by gene duplication, it has been identified only in invertebrates and mammals. It has, however, remained unclear when and how the DAZ family has evolved in vertebrates. Methodology and Principal Findings This study was aimed at identifying and characterizing the DAZ family genes in fish as the basal vertebrate. We show that boule and dazl coexist in medaka and stickleback. Similar to the medaka dazl (Odazl), the medaka boule (Obol) is maternally supplied and segregates with primordial germ cells. Surprisingly, Obol is expressed in adult germ cells at pre-meiotic and meiotic stages of spermatogenesis and oogenesis. However, the maximal meiotic Obol expression in spermatocytes contrasts with the predominant pre-meiotic Odazl expression in spermatogonia, and the diffuse cytoplasmic Obol distribution in early oocytes contrasts with the Odazl concentration in the Balbinani's body. Conclusions The identification of fish boule and dazl genes provides direct evidence for the early gene duplication during vertebrate evolution. Our finding that Obol exhibits bisexual expression in both embryonic and adult germ cells considerably extends the diversity of boule expression patterns and offers a new insight into the evolutions of DAZ family members, expression patterns and functions in animal fertility. PMID:19564913

  3. Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.

    PubMed

    Oulhen, Nathalie; Wessel, Gary M

    2016-10-01

    Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3'UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability. PMID:27424271

  4. Utility of Dexrazoxane for the Attenuation of Epirubicin-Induced Genetic Alterations in Mouse Germ Cells

    PubMed Central

    Ahmad, Sheikh F.; Ansaria, Mushtaq A.; Nadeem, Ahmed; Al-Shabanah, Othman A.; Al-Harbi, Mohammed M.; Bakheet, Saleh A.

    2016-01-01

    Dexrazoxane has been approved to treat anthracycline-induced cardiomyopathy and extravasation. However, the effect of dexrazoxane on epirubicin-induced genetic alterations in germ cells has not yet been reported. Thus, the aim of this study was to determine whether dexrazoxane modulates epirubicin-induced genetic damage in the germ cells of male mice. Our results show that dexrazoxane was not genotoxic at the tested doses. Furthermore, it protected mouse germ cells against epirubicin-induced genetic alterations as detected by the reduction in disomic and diploid sperm, spermatogonial chromosomal aberrations, and abnormal sperm heads. The attenuating effect of dexrazoxane was greater at higher dose, indicating a dose-dependent effect. Moreover, sperm motility and count were ameliorated by dexrazoxane pretreatment. Epirubicin induced marked biochemical changes characteristic of oxidative DNA damage including elevated 8-hydroxy-2ʹ-deoxyguanosine levels and reduction in reduced glutathione. Pretreatment of mice with dexrazoxane before epirubicin challenge restored these altered endpoints. We conclude that dexrazoxane may efficiently mitigate the epirubicin insult in male germ cells, and prevent the enhanced risk of abnormal reproductive outcomes and associated health risks. Thus, pretreating patients with dexrazoxane prior to epirubicin may efficiently preserve not only sperm quality but also prevent the transmission of genetic damage to future generations. PMID:27690233

  5. Cell cycle gene-specific control of transcription has a critical role in proliferation of primordial germ cells

    PubMed Central

    Okamura, Daiji; Maeda, Ikuma; Taniguchi, Hirofumi; Tokitake, Yuko; Ikeda, Makiko; Ozato, Keiko; Mise, Nathan; Abe, Kuniya; Noce, Toshiaki; Izpisua Belmonte, Juan Carlos; Matsui, Yasuhisa

    2012-01-01

    Transcription elongation is stimulated by positive transcription elongation factor b (P-TEFb), for which activity is repressed in the 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. We show here a critical role of 7SK snRNP in growth control of primordial germ cells (PGCs). The expression of p15INK4b, a cyclin-dependent kinase inhibitor (CDKI) gene, in PGCs is selectively activated by P-TEFb and its recruiting molecule, Brd4, when the amount of active P-TEFb is increased due to reduction of the 7SK snRNP, and PGCs consequently undergo growth arrest. These results indicate that CDKI gene-specific control of transcription by 7SK snRNP plays a pivotal role in the maintenance of PGC proliferation. PMID:23154982

  6. Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

    SciTech Connect

    Ahn, Kwang Sung; Won, Ji Young; Park, Jin-Ki; Sorrell, Alice M.; Heo, Soon Young; Kang, Jee Hyun; Woo, Jae-Seok; Choi, Bong-Hwan; Chang, Won-Kyong; Shim, Hosup

    2010-10-01

    Research highlights: {yields} Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. {yields} hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. {yields} hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

  7. Expression of human hormone-sensitive lipase (HSL) in postmeiotic germ cells confers normal fertility to HSL-deficient mice.

    PubMed

    Wang, Shu Pei; Chung, Shari; Soni, Krishnakant; Bourdages, Hugo; Hermo, Louis; Trasler, Jacquetta; Mitchell, Grant A

    2004-12-01

    Hormone-sensitive lipase (HSL, Lipe, E.C.3.1.1.3) is a multifunctional fatty acyl esterase that is essential for male fertility and spermatogenesis and that also plays important roles in the function of adipocytes, pancreatic beta-cells, and adrenal cortical cells. Gene-targeted HSL-deficient (HSL-/-) male mice are infertile, have a 2-fold reduction in testicular mass, a 2-fold elevation of the ratio of esterified to free cholesterol in testis, and unique morphological abnormalities in round and elongating spermatids. Postmeiotic germ cells in the testis express a specific HSL isoform. We created transgenic mice expressing a normal human testicular HSL cDNA from the mouse protamine-1 promoter, which mediates expression specifically in postmeiotic germ cells. Testicular cholesteryl esterase activity was undetectable in HSL-/- mice, but in HSL-/- males expressing the testicular transgene, activity was 2-fold greater than normal. HSL transgene mRNA became detectable in testes between 19 and 25 days of age, coinciding with the first wave of postmeiotic transcription in round spermatids. In contrast to nontransgenic HSL-/- mice, HSL-/- males expressing the testicular transgene were normal with respect to fertility, testicular mass, testicular esterified/free cholesterol ratio, and testicular histology. Their cauda epididymides contained abundant, normal-appearing spermatozoa. We conclude that human testicular HSL is functional in mouse testis and that the mechanism of infertility in HSL-deficient males is cell autonomous and resides in postmeiotic germ cells, because HSL expression in these cells is in itself sufficient to restore normal fertility.

  8. MEETING REPORT ASSESSING HUMAN GERM-CELL MUTAGENESIS IN THE POST-GENOME ERA: A CELEBRATION OF THE LEGACY OF WILLIAM LAWSON (BILL) RUSSELL

    EPA Science Inventory

    Although numerous germ-cell mutagens have been identified in animal model systems, to date, no human germ-cell mutagens have been confirmed. Because the genomic integrity of our germ cells is essential for the continuation of the human species, a resolution of this enduring conu...

  9. DDX4 (VASA) is conserved in germ cell development in marsupials and monotremes.

    PubMed

    Hickford, Danielle E; Frankenberg, Stephen; Pask, Andrew J; Shaw, Geoff; Renfree, Marilyn B

    2011-10-01

    DDX4 (VASA) is an RNA helicase expressed in the germ cells of all animals. To gain greater insight into the role of this gene in mammalian germ cell development, we characterized DDX4 in both a marsupial (the tammar wallaby) and a monotreme (the platypus). DDX4 is highly conserved between eutherian, marsupial, and monotreme mammals. DDX4 protein is absent from tammar fetal germ cells but is present from Day 1 postpartum in both sexes. The distribution of DDX4 protein during oogenesis and spermatogenesis in the tammar is similar to eutherians. Female tammar germ cells contain DDX4 protein throughout all stages of postnatal oogenesis. In males, DDX4 is in gonocytes, and during spermatogenesis it is present in spermatocytes and round spermatids. A similar distribution of DDX4 occurs in the platypus during spermatogenesis. There are several DDX4 isoforms in the tammar, resulting from both pre- and posttranslational modifications. DDX4 in marsupials and monotremes has multiple splice variants and polyadenylation motifs. Using in silico analyses of genomic databases, we found that these previously unreported splice variants also occur in eutherians. In addition, several elements implicated in the control of Ddx4 expression in the mouse, including RGG (arginine-glycine-glycine) and dimethylation of arginine motifs and CpG islands within the Ddx4 promoter, are also highly conserved. Collectively these data suggest that DDX4 is essential for the regulation of germ cell proliferation and differentiation across all three extant mammalian groups-eutherians, marsupials, and monotremes. PMID:21653890

  10. DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells

    PubMed Central

    Spike, Caroline A.; Bader, Jason; Reinke, Valerie; Strome, Susan

    2008-01-01

    P granules are germ-cell-specific cytoplasmic structures containing RNA and protein, and required for proper germ cell development in C. elegans. PGL-1 and GLH-1 were previously identified as critical components of P granules. We have identified a new P-granule-associated protein, DEPS-1, the loss of which disrupts P-granule structure and function. DEPS-1 is required for the proper localization of PGL-1 to P granules, the accumulation of glh-1 mRNA and protein, and germ cell proliferation and fertility at elevated temperatures. In addition, DEPS-1 is required for RNA interference (RNAi) of germline-expressed genes, possibly because DEPS-1 promotes the accumulation of RDE-4, a dsRNA-binding protein required for RNAi. A genome wide analysis of gene expression in deps-1 mutant germ lines identified additional targets of DEPS-1 regulation, many of which are also regulated by the RNAi factor RDE-3. Our studies suggest that DEPS-1 is a key component of the P-granule assembly pathway and that its roles include promoting accumulation of some mRNAs, such as glh-1 and rde-4, and reducing accumulation of other mRNAs, perhaps by collaborating with RDE-3 to generate endogenous short interfering RNAs (endo-siRNAs). PMID:18234720

  11. Expression, sorting, and segregation of Golgi proteins during germ cell differentiation in the testis

    PubMed Central

    Au, Catherine E.; Hermo, Louis; Byrne, Elliot; Smirle, Jeffrey; Fazel, Ali; Simon, Paul H. G.; Kearney, Robert E.; Cameron, Pamela H.; Smith, Charles E.; Vali, Hojatollah; Fernandez-Rodriguez, Julia; Ma, Kewei; Nilsson, Tommy; Bergeron, John J. M.

    2015-01-01

    The molecular basis of changes in structure, cellular location, and function of the Golgi apparatus during male germ cell differentiation is unknown. To deduce cognate Golgi proteins, we isolated germ cell Golgi fractions, and 1318 proteins were characterized, with 20 localized in situ. The most abundant protein, GL54D of unknown function, is characterized as a germ cell–specific Golgi-localized type II integral membrane glycoprotein. TM9SF3, also of unknown function, was revealed to be a universal Golgi marker for both somatic and germ cells. During acrosome formation, several Golgi proteins (GBF1, GPP34, GRASP55) localize to both the acrosome and Golgi, while GL54D, TM9SF3, and the Golgi trafficking protein TMED7/p27 are segregated from the acrosome. After acrosome formation, GL54D, TM9SF3, TMED4/p25, and TMED7/p27 continue to mark Golgi identity as it migrates away from the acrosome, while the others (GBF1, GPP34, GRASP55) remain in the acrosome and are progressively lost in later steps of differentiation. Cytoplasmic HSP70.2 and the endoplasmic reticulum luminal protein-folding enzyme PDILT are also Golgi recruited but only during acrosome formation. This resource identifies abundant Golgi proteins that are expressed differentially during mitosis, meiosis, and postacrosome Golgi migration, including the last step of differentiation. PMID:25808494

  12. Increasing Notch signaling antagonizes PRC2-mediated silencing to promote reprograming of germ cells into neurons

    PubMed Central

    Seelk, Stefanie; Adrian-Kalchhauser, Irene; Hargitai, Balázs; Hajduskova, Martina; Gutnik, Silvia; Tursun, Baris; Ciosk, Rafal

    2016-01-01

    Cell-fate reprograming is at the heart of development, yet very little is known about the molecular mechanisms promoting or inhibiting reprograming in intact organisms. In the C. elegans germline, reprograming germ cells into somatic cells requires chromatin perturbation. Here, we describe that such reprograming is facilitated by GLP-1/Notch signaling pathway. This is surprising, since this pathway is best known for maintaining undifferentiated germline stem cells/progenitors. Through a combination of genetics, tissue-specific transcriptome analysis, and functional studies of candidate genes, we uncovered a possible explanation for this unexpected role of GLP-1/Notch. We propose that GLP-1/Notch promotes reprograming by activating specific genes, silenced by the Polycomb repressive complex 2 (PRC2), and identify the conserved histone demethylase UTX-1 as a crucial GLP-1/Notch target facilitating reprograming. These findings have wide implications, ranging from development to diseases associated with abnormal Notch signaling. DOI: http://dx.doi.org/10.7554/eLife.15477.001 PMID:27602485

  13. Wheat Germ Agglutinin Induces NADPH-Oxidase Activity in Human Neutrophils by Interaction with Mobilizable Receptors

    PubMed Central

    Karlsson, Anna

    1999-01-01

    Wheat germ agglutinin (WGA), a lectin with specificity for N-acetylglucosamine and sialic acid, was investigated with respect to its ability to activate the NADPH-oxidase of in vivo-exudated neutrophils (obtained from a skin chamber), and the activity was compared to that of peripheral blood neutrophils. The exudate cells responded to WGA, by both releasing reactive oxygen species into the extracellular milieu and producing oxygen metabolites intracellularly. The peripheral blood cells were unresponsive. To mimic the in vivo-exuded neutrophils with regards to receptor exposure, peripheral blood neutrophils were induced to mobilize their granules and vesicles to varying degrees (in vitro priming), prior to challenge with WGA. The oxidative response to WGA increased with increasing levels of granule mobilization, and the receptor(s) could be shown to reside in the secretory vesicles and/or the gelatinase granules in resting neutrophils. Several WGA-binding glycoproteins were detected in subcellular fractions containing these organelles. The extra- and intracellular NADPH-oxidase responses showed differences in sialic acid dependency, indicating that these two responses are mediated by different receptor structures. PMID:10377127

  14. Dispersed crude oil amplifies germ cell apoptosis in Caenorhabditis elegans, followed a CEP-1-dependent pathway.

    PubMed

    Polli, Joseph Ryan; Zhang, Yanqiong; Pan, Xiaoping

    2014-03-01

    The Deepwater Horizon oil spill is among the most severe environmental disasters in US history. The extent of crude oil released and the subsequent dispersant used for cleanup was unprecedented. The dispersed crude oil represents a unique form of environmental contaminant that warrants investigations of its environmental and human health impacts. Lines of evidence have demonstrated that dispersed oil affects reproduction in various organisms, in a more potent manner than oil- and dispersant-only exposures. However, the action mechanism of dispersed oil remains largely unknown. In this study, we utilized the model organism Caenorhabditis elegans to investigate impacts of dispersed oil exposure on sex cell apoptosis and related gene expressions. Worms were exposed to different diluted levels of crude oil-dispersant (oil-dis) mixtures (20:1, v/v; at 500×, 2,000×, and 5,000× dilutions). The dispersed crude oil significantly increases the number of apoptotic germ cells in treated worms when compared with control at all exposure levels (p < 0.05). Genes involved in the apoptosis pathway were dysregulated, which include ced-13, ced-3, ced-4, ced-9, cep-1, dpl-1, efl-1, efl-2, egl-1, egl-38, lin-35, pax-2, and sir-2.1. Many aberrant expressed genes encoding for core components in apoptosis machinery (cep-1/p53, ced-13/BH3, ced-9/Bcl-2, ced-4/Apaf-1, and ced-3/caspase) displayed consistent expression patterns across all exposure levels. Significantly ced-3/caspase was upregulated at all dispersed oil-treated groups, consistent with the observed apoptosis phenotype. Given cep-1/p53 was activated at all dispersed oil treatments and the germ cell apoptosis was suppressed in the CEP-1 loss of function mutant, the increased apoptosis is likely CEP-1 dependent. In addition, the anti-apoptotic ced-9/Bcl-2 was activated in response to the increase in cell death. This study provides a mechanism understanding of dispersed crude oil-induced reproductive toxicity. PMID:24496467

  15. STELLA Facilitates Differentiation of Germ Cell and Endodermal Lineages of Human Embryonic Stem Cells

    PubMed Central

    Wongtrakoongate, Patompon; Jones, Mark; Gokhale, Paul J.; Andrews, Peter W.

    2013-01-01

    Stella is a developmentally regulated gene highly expressed in mouse embryonic stem (ES) cells and in primordial germ cells (PGCs). In human, the gene encoding the STELLA homologue lies on chromosome 12p, which is frequently amplified in long-term cultured human ES cells. However, the role played by STELLA in human ES cells has not been reported. In the present study, we show that during retinoic acid (RA)-induced differentiation of human ES cells, expression of STELLA follows that of VASA, a marker of germline differentiation. By contrast, human embryonal carcinoma cells express STELLA at a higher level compared with both karyotypically normal and abnormal human ES cell lines. We found that over-expression of STELLA does not interfere with maintenance of the stem cell state of human ES cells, but following retinoic acid induction it leads to up-regulation of germline- and endodermal-associated genes, whereas neural markers PAX6 and NEUROD1 are down-regulated. Further, STELLA over-expression facilitates the differentiation of human ES cells into BE12-positive cells, in which the expression of germline- and endodermal-associated genes is enriched, and suppresses differentiation of the neural lineage. Taken together, this finding suggests a role for STELLA in facilitating germline and endodermal differentiation of human ES cells. PMID:23457636

  16. Modifications of wheat germ cell-free system for functional proteomics of plant membrane proteins.

    PubMed

    Nozawa, Akira; Tozawa, Yuzuru

    2014-01-01

    Functional proteomics of plant membrane proteins is an important approach to understand the comprehensive architecture of each metabolic pathway in plants. One bottleneck in the characterization of membrane proteins is the difficulty in producing sufficient quantities of functional protein for analysis. Here, we describe three methods for membrane protein production utilizing a wheat germ cell-free protein expression system. Owing to the open nature of cell-free synthesis reaction, protein synthesis can be modified with components necessary to produce functional protein. In this way we have developed modifications to a wheat germ cell-free system for the production of functional membrane proteins. Supplementation of liposomes or detergents allows the synthesis of functional integral membrane proteins. Furthermore, supplementation of myristic acid enables synthesis of N-myristylated peripheral membrane proteins. These modified cell-free synthesis methods facilitate the preparation and subsequent functional analyses of a wide variety of membrane proteins. PMID:24136528

  17. Distinct development patterns of c-mos protooncogene expression in female and male mouse germ cells

    SciTech Connect

    Mutter, G.L.; Wolgemuth, D.J.

    1987-08-01

    The protooncogene c-mos is expressed in murine reproductive tissues, producing transcripts of 1.7 and 1.4 kilobases in testis and ovary, respectively. In situ hybridization analysis of c-mos expression in histological sections of mouse ovaries revealed that oocytes are the predominant if not exclusive source of c-mos transcripts. /sup 35/S- or /sup 32/P-labelled RNA probes were transcribed. c-mos transcripts accumulate in growing oocytes, increasing 40- to 90-fold during oocyte and follicular development. c-mos transcripts were also detected in male germ cells and are most abundant after the cells have entered the haploid stage of spermatogenesis. This developmentally regulated pattern of c-mos expression in oocytes and spermatogenic cells suggest that the c-mos gene product may have a function in normal germ-cell differentiation or early embryogenesis.

  18. A misexpression screen reveals effects of bag-of-marbles and TGF beta class signaling on the Drosophila male germ-line stem cell lineage.

    PubMed Central

    Schulz, Cordula; Kiger, Amy A; Tazuke, Salli I; Yamashita, Yukiko M; Pantalena-Filho, Luiz C; Jones, D Leanne; Wood, Cricket G; Fuller, Margaret T

    2004-01-01

    Male gametes are produced throughout reproductive life by a classic stem cell mechanism. However, little is known about the molecular mechanisms for lineage production that maintain male germ-line stem cell (GSC) populations, regulate mitotic amplification divisions, and ensure germ cell differentiation. Here we utilize the Drosophila system to identify genes that cause defects in the male GSC lineage when forcibly expressed. We conducted a gain-of-function screen using a collection of 2050 EP lines and found 55 EP lines that caused defects at early stages of spermatogenesis upon forced expression either in germ cells or in surrounding somatic support cells. Most strikingly, our analysis of forced expression indicated that repression of bag-of-marbles (bam) expression in male GSC is important for male GSC survival, while activity of the TGF beta signal transduction pathway may play a permissive role in maintenance of GSCs in Drosophila testes. In addition, forced activation of the TGF beta signal transduction pathway in germ cells inhibits the transition from the spermatogonial mitotic amplification program to spermatocyte differentiation. PMID:15238523

  19. Expression of low density lipoprotein receptor-related protein 4 (Lrp4) gene in the mouse germ cells.

    PubMed

    Yamaguchi, Yasuka L; Tanaka, Satomi S; Kasa, Miyuki; Yasuda, Kunio; Tam, Patrick P L; Matsui, Yasuhisa

    2006-08-01

    The low density lipoprotein receptor-related protein 4 gene (Lrp4) was identified by subtractive screening of cDNAs of the migratory primordial germ cells (PGCs) of E8.5-9.5 embryo and E3.5 blastocysts. Lrp4 is expressed in PGCs in the hindgut and the dorsal mesentery of E9.5 embryos, and in germ cells in the genital ridges of male and female E10.5-13.5 embryos. Lrp4 is also expressed in spermatogonia of the neonatal and adult testes and in the immature oocytes and follicular cells of the adult ovary. The absence of Lrp4 expression in the blastocyst, embryonic stem cells and embryonic germ cells suggests the Lrp4 is a molecular marker that distinguishes the germ cells from embryo-derived pluripotent stem cells. PMID:16434236

  20. A replication-dependent passive mechanism modulates DNA demethylation in mouse primordial germ cells.

    PubMed

    Ohno, Rika; Nakayama, Megumi; Naruse, Chie; Okashita, Naoki; Takano, Osamu; Tachibana, Makoto; Asano, Masahide; Saitou, Mitinori; Seki, Yoshiyuki

    2013-07-01

    Germline cells reprogramme extensive epigenetic modifications to ensure the cellular totipotency of subsequent generations and to prevent the accumulation of epimutations. Notably, primordial germ cells (PGCs) erase genome-wide DNA methylation and H3K9 dimethylation marks in a stepwise manner during migration and gonadal periods. In this study, we profiled DNA and histone methylation on transposable elements during PGC development, and examined the role of DNA replication in DNA demethylation in gonadal PGCs. CpGs in short interspersed nuclear elements (SINEs) B1 and B2 were substantially demethylated in migrating PGCs, whereas CpGs in long interspersed nuclear elements (LINEs), such as LINE-1, were resistant to early demethylation. By contrast, CpGs in both LINE-1 and SINEs were rapidly demethylated in gonadal PGCs. Four major modifiers of DNA and histone methylation, Dnmt3a, Dnmt3b, Glp and Uhrf1, were actively repressed at distinct stages of PGC development. DNMT1 was localised at replication foci in nascent PGCs, whereas the efficiency of recruitment of DNMT1 into replication foci was severely impaired in gonadal PGCs. Hairpin bisulphite sequencing analysis showed that strand-specific hemi-methylated CpGs on LINE-1 were predominant in gonadal PGCs. Furthermore, DNA demethylation in SINEs and LINE-1 was impaired in Cbx3-deficient PGCs, indicating abnormalities in G1 to S phase progression. We propose that PGCs employ active and passive mechanisms for efficient and widespread erasure of genomic DNA methylation.

  1. Childhood Central Nervous System Germ Cell Tumors Treatment

    MedlinePlus

    ... cancer will come back, is called adjuvant therapy . High-dose chemotherapy with stem cell rescue High-dose chemotherapy with stem cell rescue is a way of giving high doses of chemotherapy and replacing blood -forming cells ...

  2. In vitro differentiation of human tooth germ stem cells into endothelial- and epithelial-like cells.

    PubMed

    Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

    2015-01-01

    Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell-lineage markers including CD31, VEGFR2, VE-Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)-17, CK-19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK-18, and CK-19 genes were detected in differentiated endothelial and epithelial-like cells. Endothelial-like cells derived from hTGSCs were cultured on Matrigel, tube-like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue.

  3. A non-surgical approach for male germ cell mediated gene transmission through transgenesis.

    PubMed

    Usmani, Abul; Ganguli, Nirmalya; Sarkar, Hironmoy; Dhup, Suveera; Batta, Suryaprakash R; Vimal, Manoj; Ganguli, Nilanjana; Basu, Sayon; Nagarajan, P; Majumdar, Subeer S

    2013-01-01

    Microinjection of foreign DNA in male pronucleus by in-vitro embryo manipulation is difficult but remains the method of choice for generating transgenic animals. Other procedures, including retroviral and embryonic stem cell mediated transgenesis are equally complicated and have limitations. Although our previously reported technique of testicular transgenesis circumvented several limitations, it involved many steps, including surgery and hemicastration, which carried risk of infection and impotency. We improved this technique further, into a two step non-surgical electroporation procedure, for making transgenic mice. In this approach, transgene was delivered inside both testes by injection and modified parameters of electroporation were used for in-vivo gene integration in germ cells. Using variety of constructs, germ cell integration of the gene and its transmission in progeny was confirmed by PCR, slot blot and immunohistochemical analysis. This improved technique is efficient, requires substantially less time and can be easily adopted by various biomedical researchers.

  4. CREM: a master-switch governing male germ cells differentiation and apoptosis.

    PubMed

    Sassone-Corsi, P

    1998-08-01

    Several endocrine and neuronal functions are governed by the cAMP-dependent signalling pathway. In eukaryotes, transcriptional regulation upon stimulation of the adenylyl cyclase signalling pathway is mediated by a family of cAMP-responsive nuclear factors. The CREM gene plays a key physiological and developmental role within the hypothalamic-pituitary-gonadal axis. CREM is highly expressed in postmeiotic cells upon a striking developmental switch regulated by the pituitary hormone FSH. CREM-mutant mice generated by homologous recombination reveal that spermatogenesis stops at the first step of spermiogenesis. Late spermatids are completely absent while there is a significant increase in apoptotic germ cells. A series of post-meiotic germ cell-specific genes are not expressed. Mutant male mice completely lack spermatozoa. This phenotype is reminiscent of cases of human infertility.

  5. Early Depletion of Primordial Germ Cells in Zebrafish Promotes Testis Formation

    PubMed Central

    Tzung, Keh-Weei; Goto, Rie; Saju, Jolly M.; Sreenivasan, Rajini; Saito, Taiju; Arai, Katsutoshi; Yamaha, Etsuro; Hossain, Mohammad Sorowar; Calvert, Meredith E.K.; Orbán, László

    2014-01-01

    Summary As complete absence of germ cells leads to sterile males in zebrafish, we explored the relationship between primordial germ cell (PGC) number and sexual development. Our results revealed dimorphic proliferation of PGCs in the early zebrafish larvae, marking the beginning of sexual differentiation. We applied morpholino-based gene knockdown and cell transplantation strategies to demonstrate that a threshold number of PGCs is required for the stability of ovarian fate. Using histology and transcriptomic analyses, we determined that zebrafish gonads are in a meiotic ovarian stage at 14 days postfertilization and identified signaling pathways supporting meiotic oocyte differentiation and eventual female fate. The development of PGC-depleted gonads appears to be restrained and delayed, suggesting that PGC number may directly regulate the variability and length of gonadal transformation and testicular differentiation in zebrafish. We propose that gonadal transformation may function as a developmental buffering mechanism to ensure the reproductive outcome. PMID:25434820

  6. Giant adrenal germ cell tumour in a 59-year-old woman

    PubMed Central

    Chen, Lei; Fang, Lu; Liu, Zhiqi; Yu, Dexin; Wang, Daming; Wang, Yi; Xie, Dongdong; Min, Jie; Ding, Demao; Zhang, Tao; Zou, Ci; Zhang, Zhiqiang

    2016-01-01

    Adrenal germ cell tumour is very rare. We report a case of a 59-year-old woman who presented with right flank discomfort. The laboratory examinations were normal and the chest computed tomography (CT) showed right pleural effusion. The abdominal CT scan revealed a large mass on the right adrenal gland. The patient underwent an adrenalectomy. Histopathologic examination and immunohistochemical findings were consistent with mixed germ cell tumour. Three months later following the operation, the patient was admitted to our hospital again with chest tightness and shortness of breath. The chest CT showed right pleural effusion recurrence and enlargement of mediastinal lymph nodes and right hilar lymph nodes. The patient had right supraclavicular lymphadenectasis on physical examination. Fine needle aspiration cytology from the supraclavicular lymph nodes showed groups of malignant tumour cells. The patient died within 6 months postoperatively. In this case, the lymph node pathway played an important role in the metastatic procedure.

  7. Male germ cell-specific expression of a novel Patched-domain containing gene Ptchd3

    SciTech Connect

    Fan Jun; Akabane, Hiroto; Zheng Xuehai; Zhou Xuan; Zhang Li; Liu Qiang; Zhang Yonglian; Yang Jing; Zhu Guozhang

    2007-11-23

    The Hedgehog (Hh) signaling pathway plays an important role in various biological processes, including pattern formation, cell fate determination, proliferation, and differentiation. Hh function is mediated through its membrane receptor Patched. Herein, we have characterized a novel Patched-domain containing gene Ptchd3 in mouse. Messenger RNA of Ptchd3 was exclusively detected in the testis, and existed in two isoforms Ptchd3a and Ptchd3b. The expression of these two mRNA isoforms was shown to be developmentally regulated in testes, and specifically found in male germ cells. Further analysis revealed that the Ptchd3 protein was located on the midpiece of mouse, rat and human sperm. Collectively, these results indicate that Ptchd3 is a novel male germ cell-specific gene and may be involved in the Hh signaling to regulate sperm development and/or sperm function.

  8. Vasa genes: Emerging roles in the germ line and in multipotent cells

    PubMed Central

    Gustafson, Eric A.; Wessel, Gary M.

    2011-01-01

    Sexually reproducing metazoans establish a cell lineage during development that is ultimately dedicated to gamete production. Work in a variety of animals suggests that a group of conserved molecular determinants function in this germ line maintenance and function. The most universal of these genes are vasa and vasa-like DEAD box RNA helicase genes. However, recent evidence indicates that vasa genes also function in other cell types, distinct from the germ line. Here we evaluate our current understanding of vasa function and its regulation during development, addressing vasa’s emerging role in multipotent cells. We also explore the evolutionary diversification of the amino-terminal domain of this gene and how this impacts the association of vasa with nuage-like perinuclear structures. PMID:20586054

  9. Transgenerational epigenetic effects of the Apobec1 cytidine deaminase deficiency on testicular germ cell tumor susceptibility and embryonic viability.

    PubMed

    Nelson, Vicki R; Heaney, Jason D; Tesar, Paul J; Davidson, Nicholas O; Nadeau, Joseph H

    2012-10-01

    Environmental agents and genetic variants can induce heritable epigenetic changes that affect phenotypic variation and disease risk in many species. These transgenerational effects challenge conventional understanding about the modes and mechanisms of inheritance, but their molecular basis is poorly understood. The Deadend1 (Dnd1) gene enhances susceptibility to testicular germ cell tumors (TGCTs) in mice, in part by interacting epigenetically with other TGCT modifier genes in previous generations. Sequence homology to A1cf, the RNA-binding subunit of the ApoB editing complex, raises the possibility that the function of Dnd1 is related to Apobec1 activity as a cytidine deaminase. We conducted a series of experiments with a genetically engineered deficiency of Apobec1 on the TGCT-susceptible 129/Sv inbred background to determine whether dosage of Apobec1 modifies susceptibility, either alone or in combination with Dnd1, and either in a conventional or a transgenerational manner. In the paternal germ-lineage, Apobec1 deficiency significantly increased susceptibility among heterozygous but not wild-type male offspring, without subsequent transgenerational effects, showing that increased TGCT risk resulting from partial loss of Apobec1 function is inherited in a conventional manner. By contrast, partial deficiency in the maternal germ-lineage led to suppression of TGCTs in both partially and fully deficient males and significantly reduced TGCT risk in a transgenerational manner among wild-type offspring. These heritable epigenetic changes persisted for multiple generations and were fully reversed after consecutive crosses through the alternative germ-lineage. These results suggest that Apobec1 plays a central role in controlling TGCT susceptibility in both a conventional and a transgenerational manner.

  10. Molecular Characteristics of Malignant Ovarian Germ Cell Tumors and Comparison With Testicular Counterparts: Implications for Pathogenesis

    PubMed Central

    Kraggerud, Sigrid Marie; Hoei-Hansen, Christina E.; Alagaratnam, Sharmini; Skotheim, Rolf I.; Abeler, Vera M.

    2013-01-01

    This review focuses on the molecular characteristics and development of rare malignant ovarian germ cell tumors (mOGCTs). We provide an overview of the genomic aberrations assessed by ploidy, cytogenetic banding, and comparative genomic hybridization. We summarize and discuss the transcriptome profiles of mRNA and microRNA (miRNA), and biomarkers (DNA methylation, gene mutation, individual protein expression) for each mOGCT histological subtype. Parallels between the origin of mOGCT and their male counterpart testicular GCT (TGCT) are discussed from the perspective of germ cell development, endocrinological influences, and pathogenesis, as is the GCT origin in patients with disorders of sex development. Integrated molecular profiles of the 3 main histological subtypes, dysgerminoma (DG), yolk sac tumor (YST), and immature teratoma (IT), are presented. DGs show genomic aberrations comparable to TGCT. In contrast, the genome profiles of YST and IT are different both from each other and from DG/TGCT. Differences between DG and YST are underlined by their miRNA/mRNA expression patterns, suggesting preferential involvement of the WNT/β-catenin and TGF-β/bone morphogenetic protein signaling pathways among YSTs. Characteristic protein expression patterns are observed in DG, YST and IT. We propose that mOGCT develop through different developmental pathways, including one that is likely shared with TGCT and involves insufficient sexual differentiation of the germ cell niche. The molecular features of the mOGCTs underline their similarity to pluripotent precursor cells (primordial germ cells, PGCs) and other stem cells. This similarity combined with the process of ovary development, explain why mOGCTs present so early in life, and with greater histological complexity, than most somatic solid tumors. PMID:23575763

  11. Molecular characteristics of malignant ovarian germ cell tumors and comparison with testicular counterparts: implications for pathogenesis.

    PubMed

    Kraggerud, Sigrid Marie; Hoei-Hansen, Christina E; Alagaratnam, Sharmini; Skotheim, Rolf I; Abeler, Vera M; Rajpert-De Meyts, Ewa; Lothe, Ragnhild A

    2013-06-01

    This review focuses on the molecular characteristics and development of rare malignant ovarian germ cell tumors (mOGCTs). We provide an overview of the genomic aberrations assessed by ploidy, cytogenetic banding, and comparative genomic hybridization. We summarize and discuss the transcriptome profiles of mRNA and microRNA (miRNA), and biomarkers (DNA methylation, gene mutation, individual protein expression) for each mOGCT histological subtype. Parallels between the origin of mOGCT and their male counterpart testicular GCT (TGCT) are discussed from the perspective of germ cell development, endocrinological influences, and pathogenesis, as is the GCT origin in patients with disorders of sex development. Integrated molecular profiles of the 3 main histological subtypes, dysgerminoma (DG), yolk sac tumor (YST), and immature teratoma (IT), are presented. DGs show genomic aberrations comparable to TGCT. In contrast, the genome profiles of YST and IT are different both from each other and from DG/TGCT. Differences between DG and YST are underlined by their miRNA/mRNA expression patterns, suggesting preferential involvement of the WNT/β-catenin and TGF-β/bone morphogenetic protein signaling pathways among YSTs. Characteristic protein expression patterns are observed in DG, YST and IT. We propose that mOGCT develop through different developmental pathways, including one that is likely shared with TGCT and involves insufficient sexual differentiation of the germ cell niche. The molecular features of the mOGCTs underline their similarity to pluripotent precursor cells (primordial germ cells, PGCs) and other stem cells. This similarity combined with the process of ovary development, explain why mOGCTs present so early in life, and with greater histological complexity, than most somatic solid tumors. PMID:23575763

  12. Sertoli-germ cell adherens junction dynamics in the testis are regulated by RhoB GTPase via the ROCK/LIMK signaling pathway.

    PubMed

    Lui, Wing-yee; Lee, Will M; Cheng, C Yan

    2003-06-01

    During spermatogenesis, cell-cell actin-based adherens junctions (AJs), such as ectoplasmic specializations (ESs), between Sertoli and germ cells undergo extensive restructuring in the seminiferous epithelium to facilitate germ cell movement across the epithelium. Although the mechanism(s) that regulates AJ dynamics in the testis is virtually unknown, Rho GTPases have been implicated in the regulation of these events in other epithelia. Studies have shown that the in vitro assembly of the Sertoli-germ cell AJs but not of the Sertoli cell tight junctions (TJs) is associated with a transient but significant induction of RhoB. Immunohistochemistry has shown that the localization of RhoB in the seminiferous epithelium is stage specific, being lowest in stages VII-VIII prior to spermiation, and displays cell-specific association during the epithelial cycle. Throughout the cycle, RhoB was localized near the site of basal and apical ESs but was restricted to the periphery of the nuclei in elongating (but not elongated) spermatids, spermatocytes, and Sertoli cells. However, RhoB was not detected near the site of apical ESs at stages VII-VIII. Furthermore, disruption of AJs in Sertoli-germ cell cocultures either by hypotonic treatment or by treatment with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) also induced RhoB expression. When adult rats were treated with AF-2364 to perturb Sertoli-germ cell AJs in vivo, a approximately 4-fold induction in RhoB in the testis, but not in kidney and brain, was detected within 1 h, at least approximately 1-4 days before germ cell loss from the epithelium could be detected by histological analysis. The signaling pathway(s) by which AF-2364 perturbed the Sertoli-germ cell AJs apparently began with an initial activation of integrin, which in turn activated RhoB, ROCK1, (Rho-associated protein kinase 1, also called ROKbeta), LIMK1 (LIM kinase 1, also called lin-11 isl-1 mec3 kinase 1), and cofilin but not p140mDia and profilin

  13. Glucose 6-phosphate dehydrogenase deficiency enhances germ cell apoptosis and causes defective embryogenesis in Caenorhabditis elegans

    PubMed Central

    Yang, H-C; Chen, T-L; Wu, Y-H; Cheng, K-P; Lin, Y-H; Cheng, M-L; Ho, H-Y; Lo, S J; Chiu, D T-Y

    2013-01-01

    Glucose 6-phosphate dehydrogenase (G6PD) deficiency, known as favism, is classically manifested by hemolytic anemia in human. More recently, it has been shown that mild G6PD deficiency moderately affects cardiac function, whereas severe G6PD deficiency leads to embryonic lethality in mice. How G6PD deficiency affects organisms has not been fully elucidated due to the lack of a suitable animal model. In this study, G6PD-deficient Caenorhabditis elegans was established by RNA interference (RNAi) knockdown to delineate the role of G6PD in animal physiology. Upon G6PD RNAi knockdown, G6PD activity was significantly hampered in C. elegans in parallel with increased oxidative stress and DNA oxidative damage. Phenotypically, G6PD-knockdown enhanced germ cell apoptosis (2-fold increase), reduced egg production (65% of mock), and hatching (10% of mock). To determine whether oxidative stress is associated with G6PD knockdown-induced reproduction defects, C. elegans was challenged with a short-term hydrogen peroxide (H2O2). The early phase egg production of both mock and G6PD-knockdown C. elegans were significantly affected by H2O2. However, H2O2-induced germ cell apoptosis was more dramatic in mock than that in G6PD-deficient C. elegans. To investigate the signaling pathways involved in defective oogenesis and embryogenesis caused by G6PD knockdown, mutants of p53 and mitogen-activated protein kinase (MAPK) pathways were examined. Despite the upregulation of CEP-1 (p53), cep-1 mutation did not affect egg production and hatching in G6PD-deficient C. elegans. Neither pmk-1 nor mek-1 mutation significantly affected egg production, whereas sek-1 mutation further decreased egg production in G6PD-deficient C. elegans. Intriguingly, loss of function of sek-1 or mek-1 dramatically rescued defective hatching (8.3- and 9.6-fold increase, respectively) induced by G6PD knockdown. Taken together, these findings show that G6PD knockdown reduces egg production and hatching in C. elegans

  14. MicroRNAs: From Female Fertility, Germ Cells, and Stem Cells to Cancer in Humans

    PubMed Central

    Virant-Klun, Irma; Ståhlberg, Anders; Kubista, Mikael; Skutella, Thomas

    2016-01-01

    MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an important regulatory role in gene expression. They are suggested to regulate a large proportion of protein encoding genes by mediating the translational suppression and posttranscriptional control of gene expression. Recent findings show that microRNAs are emerging as important regulators of cellular differentiation and dedifferentiation, and are deeply involved in developmental processes including human preimplantation development. They keep a balance between pluripotency and differentiation in the embryo and embryonic stem cells. Moreover, it became evident that dysregulation of microRNA expression may play a fundamental role in progression and dissemination of different cancers including ovarian cancer. The interest is still increased by the discovery of exosomes, that is, cell-derived vesicles, which can carry different proteins but also microRNAs between different cells and are involved in cell-to-cell communication. MicroRNAs, together with exosomes, have a great potential to be used for prognosis, therapy, and biomarkers of different diseases including infertility. The aim of this review paper is to summarize the existent knowledge on microRNAs related to female fertility and cancer: from primordial germ cells and ovarian function, germinal stem cells, oocytes, and embryos to embryonic stem cells. PMID:26664407

  15. Toward a More Precise and Informative Nomenclature Describing Fetal and Neonatal Male Germ Cells in Rodents1

    PubMed Central

    McCarrey, John R.

    2013-01-01

    ABSTRACT The germ cell lineages are among the best characterized of all cell lineages in mammals. This characterization includes precise nomenclature that distinguishes among numerous, often subtle, changes in function or morphology as development and differentiation of germ cells proceed to form the gametes. In male rodents, there are at least 41 distinct cell types that occur during progression through the male germ cell lineage that gives rise to spermatozoa. However, there is one period during male germ cell development—that which occurs immediately following the primordial germ cell stage and prior to the spermatogonial stage—for which the system of precise and informative cell type terminology is not adequate. Often, male germ cells during this period are referred to simply as “gonocytes.” However, this term is inadequate for multiple reasons, and it is suggested here that nomenclature originally proposed in the 1970s by Hilscher et al., which employs the terms M-, T1-, and T2-prospermatogonia, is preferable. In this Minireview, the history, proper utilization, and advantages of this terminology relative to that of the term gonocytes are described. PMID:23843236

  16. Cell proliferation in teeth reconstructed from dispersed cells of embryonic tooth germs in a three-dimensional scaffold.

    PubMed

    Iwatsuki, Shinji; Honda, Masaki J; Harada, Hidemitsu; Ueda, Minoru

    2006-08-01

    Tissue engineering can now reproduce tooth from postnatal tooth cells. However, crown formation is not accurately reconstituted, even when the complex structure of the enamel dentin is reproduced. Here, we showed that a tissue-engineered (TE) tooth, exhibiting morphogenesis according to regular crown-cusp pattern formation, was produced by embryonic tooth germ cells in a three-dimensional scaffold. Heterogeneous cells dissociated from embryonic day 14 (E14) mice tooth germs were seeded on a scaffold and implanted under a kidney capsule in adult mice. The developmental process of the implants was examined for up to 14 d. At 5 d, the cells had formed initial tooth germ, followed by enamel-covered dentin tissue formed symmetrically. To study the developmental process, we examined the growth pattern using 5-bromo-2'-deoxyuridine (BrdU)-labeling analysis. The initial cell-proliferation patterns of the TE teeth were similar to that at the cap and early bell stages in natural teeth. This was particularly true in the cervical loop, which showed a similar distribution pattern of BrdU-positive cells in TE- and natural teeth. These results suggested that even when embryonic tooth germs are dissociated, the single cells can reconstitute tooth, and that enamel organ morphogenesis proceeds as in natural teeth.

  17. Characterization of the Epigenetic Changes During Human Gonadal Primordial Germ Cells Reprogramming.

    PubMed

    Eguizabal, C; Herrera, L; De Oñate, L; Montserrat, N; Hajkova, P; Izpisua Belmonte, J C

    2016-09-01

    Epigenetic reprogramming is a central process during mammalian germline development. Genome-wide DNA demethylation in primordial germ cells (PGCs) is a prerequisite for the erasure of epigenetic memory, preventing the transmission of epimutations to the next generation. Apart from DNA demethylation, germline reprogramming has been shown to entail reprogramming of histone marks and chromatin remodelling. Contrary to other animal models, there is limited information about the epigenetic dynamics during early germ cell development in humans. Here, we provide further characterization of the epigenetic configuration of the early human gonadal PGCs. We show that early gonadal human PGCs are DNA hypomethylated and their chromatin is characterized by low H3K9me2 and high H3K27me3 marks. Similarly to previous observations in mice, human gonadal PGCs undergo dynamic chromatin changes concomitant with the erasure of genomic imprints. Interestingly, and contrary to mouse early germ cells, expression of BLIMP1/PRDM1 persists in through all gestational stages in human gonadal PGCs and is associated with nuclear lysine-specific demethylase-1. Our work provides important additional information regarding the chromatin changes associated with human PGCs development between 6 and 13 weeks of gestation in male and female gonads. Stem Cells 2016;34:2418-2428. PMID:27300161

  18. Testicular histology and germ cell cytology during spermatogenesis in the Mississippi map turtle, Graptemys pseudogeographica kohnii, from Northeast Arkansas

    PubMed Central

    Lancaster, Kelsey; Trauth, Stanley E; Gribbins, Kevin M

    2014-01-01

    The testicular histology and cytology of spermatogenesis in Graptemys pseudogeographica kohnii were examined using specimens collected between July 1996 and May 2004 from counties in northeastern Arkansas. A histological examination of the testes and germ cell cytology indicates a postnuptial testicular cycle of spermatogenesis and a major fall spermiation event. The majority of the germ cell populations in May and June specimens are represented by resting spermatogonia, type A spermatogonia, type B spermatogonia, pre-leptotene spermatocytes, and numerous Sertoli cell nuclei near the basement membrane. The start of proliferation is evident as spermatogonia in metaphase are present near the basal lamina and many of these germ cells have entered meiosis in June seminiferous tubules. Major spermatogenic events occur in the June and July specimens and result in an increased height of the seminiferous epithelium and increased diameter of the seminiferous tubules. The germ cell population during this time is represented by spermatogonia (type A, B, and resting), hypertrophic cells, large populations of early primary spermatocytes, and early round spermatids. By September, the major germ cell population has progressed past meiosis with abundant round and early elongating spermatids dominating the seminiferous epithelium. October seminiferous epithelia are marked by a decreas in height and mature spermatozoa fill the luminal space. Round and elongating spermatids constitute the largest portion of the germ cell population. Following the spermiation event, the testes enter a period of quiescence that lasts till the next spermatogenic cycle, which begins in the subsequent spring. Based on the cytological development of the seminiferous tubules revealed by our study, Graptemys pseudogeographica kohnii demonstrates a temporal germ cell development strategy similar to other temperate reptiles. A single major generation of germ cells progresses through spermatogenesis each year

  19. A genome-wide association study of testicular germ cell tumor.

    PubMed

    Rapley, Elizabeth A; Turnbull, Clare; Al Olama, Ali Amin; Dermitzakis, Emmanouil T; Linger, Rachel; Huddart, Robert A; Renwick, Anthony; Hughes, Deborah; Hines, Sarah; Seal, Sheila; Morrison, Jonathan; Nsengimana, Jeremie; Deloukas, Panagiotis; Rahman, Nazneen; Bishop, D Timothy; Easton, Douglas F; Stratton, Michael R

    2009-07-01

    We conducted a genome-wide association study for testicular germ cell tumor (TGCT), genotyping 307,666 SNPs in 730 cases and 1,435 controls from the UK and replicating associations in a further 571 cases and 1,806 controls. We found strong evidence for susceptibility loci on chromosome 5 (per allele OR = 1.37 (95% CI = 1.19-1.58), P = 3 x 10(-13)), chromosome 6 (OR = 1.50 (95% CI = 1.28-1.75), P = 10(-13)) and chromosome 12 (OR = 2.55 (95% CI = 2.05-3.19), P = 10(-31)). KITLG, encoding the ligand for the receptor tyrosine kinase KIT, which has previously been implicated in the pathogenesis of TGCT and the biology of germ cells, may explain the association on chromosome 12. PMID:19483681

  20. Saudi Oncology Society and Saudi Urology Association combined clinical management guidelines for testicular germ cell tumors.

    PubMed

    Alotaibi, Mohammed; Saadeddin, Ahmad; Bazarbashi, Shouki; Alkhateeb, Sultan; Alghamdi, Abdullah; Alghamdi, Khalid; Murshid, Esam; Abusamra, Ashraf; Rabah, Danny; Ahmad, Imran; Al-Mansour, Mubarak; Alsharm, Abdullah

    2016-01-01

    This is an update to the previously published Saudi guidelines for the evaluation, medical, and surgical management of patients diagnosed with testicular germ cell tumors. It is categorized according to the stage of the disease using the tumor-node-metastasis staging system 7(th) edition. The guidelines are presented with supporting evidence level, they are based on comprehensive literature review, several internationally recognized guidelines, and the collective expertise of the guidelines committee members (authors) who were selected by the Saudi Oncology Society and Saudi Urological Association. Considerations to the local availability of drugs, technology and expertise have been regarded. These guidelines should serve as a roadmap for the urologists, oncologists, general physicians, support groups, and health care policy makers in the management of patients diagnosed with testicular germ cell tumors. PMID:27141181

  1. Surgical Technique: Endoscopic Endonasal Transphenoidal Resection of a Large Suprasellar Mixed Germ Cell Tumor

    PubMed Central

    Chakravarthy, Vikram; Hanna, George; DeLos Reyes, Kennethy

    2016-01-01

    The endoscopic endonasal transphenoidal approach has proven to be a very versatile surgical approach for the resection of small midline skull base tumors. This is due to its minimally invasive nature, the potentially fewer neurological complications, and lower morbidity in comparison to traditional craniotomies. This surgical approach has been less commonly utilized for large midline tumors such as suprasellar germ cell tumors, due to numerous reasons including the surgeon’s comfort with the surgical approach, a higher chance of postoperative cerebrospinal fluid (CSF) leak, limited visualization due to arterial/venous bleeding, and limited working space. We present our surgical technique in the case of a large suprasellar and third ventricular mixed germ cell tumor that was resected via an endoscopic endonasal approach with favorable neurological outcome and no postoperative CSF leak. PMID:27014537

  2. Managing the risk of germ cell tumourigenesis in disorders of sex development patients.

    PubMed

    Cools, Martine; Looijenga, Leendert H J; Wolffenbuttel, Katja P; T'Sjoen, Guy

    2014-01-01

    The risk of germ cell cancer (GCC) is elevated in many disorders of sex development (DSD) patients, although not to the same extent. A number of risk factors have been identified recently, but their interplay and relative impact is currently not fully clear. This paper offers guidance on how theoretical knowledge on GCC risk can be translated to the clinical setting, taking into account individual patient characteristics. Guidelines for decision making in different patient groups, based on a literature review, epidemiological evidence, pathological and clinical research, and personal experience are offered. Until the advent of reliable screening tools for the detection of pre-invasive cancer lesions, managing germ cell tumour risk focuses on the question of if and when to perform biopsy or gonadectomy in most patients, and how to interpret the histological findings.

  3. Molecular targets, DNA breakage, DNA repair: Their roles in mutation induction in mammalian germ cells

    SciTech Connect

    Sega, G.A.

    1989-01-01

    Variability in genetic sensitivity among different germ-cell stages in the mammal to various mutagens could be the result of how much chemical reaches the different stages, what molecular targets may be affected in the different stages and whether or not repair of lesions occurs. Several chemicals have been found to bind very strongly to protamine in late-spermatid and early-spermatozoa stages in the mouse. The chemicals also produce their greatest genetic damage in these same germ-cell stages. While chemical binding to DNA has not been correlated with the level of induced genetic damage, DNA breakage in the sensitive stages has been shown to increase. This DNA breakage is believed to indirectly result from chemical binding to sulfhydryl groups in protamine which prevents normal chromatin condensation within the sperm nucleus. 22 refs., 5 figs.

  4. How do male germ cells handle DNA damage?

    SciTech Connect

    Olsen, Ann-Karin; Lindeman, Birgitte; Wiger, Richard; Duale, Nur; Brunborg, Gunnar . E-mail: gunnar.brunborg@fhi.no

    2005-09-01

    Male reproductive health has received considerable attention in recent years. In addition to declining sperm quality, fertility problems and increased incidence of testicular cancer, there is accumulating evidence that genetic damage, in the form of unrepaired DNA lesions or de novo mutations, may be transmitted via sperm to the offspring. Such genetic damage may arise from environmental exposure or via endogenously formed reactive species, in stem cells or during spermatogenesis. Damaged testicular cells not removed by apoptosis rely on DNA repair for their genomic integrity to be preserved. To identify factors with potentially harmful effects on testicular cells and to characterise associated risk, a thorough understanding of repair mechanisms in these cells is of particular importance. Based on results from our own and other laboratories, we discuss the current knowledge of different pathways of excision repair in rodent and human testicular cells. It has become evident that, in human spermatogenic cells, some repair functions are indeed non-functional.

  5. A rare cause in etiology of left atrial mass: metastatic testicular germ cell tumor

    PubMed Central

    Huseyin, Serhat; Okyay, Ahmet; Hacıbekiroğlu, İlhan; Tastekin, Ebru; Yılmaztepe, Mustafa; Taylan, Gökay; Canbaz, Suat; Çiçin, İrfan

    2016-01-01

    Although intracardiac metastasis of germ cell tumors is rare, it can be localized in the right or left heart by disseminating spread and give their cardiac symptoms depending on the location of metastatic mass. We present a 38-year-old male patient with a preliminary diagnosis of testicular tumor who was followed by the medical oncology clinic with cerebrovascular event and heart failure symptoms. PMID:27212979

  6. Evidence for endoreduplication: germ cell DNA levels prior to chromatin diminution in Mesocyclops edax.

    PubMed

    Rasch, E M; Wyngaard, G A

    2001-06-01

    We studied the functional significance of marked differences in the DNA content of somatic cells and germ line nuclei by static Feulgen-DNA cytophotometry for several species of microcrustaceans that exhibit chromatin diminution during very early stages of embryogenesis. Mature females and males showed many gonadal nuclei with elevated amounts of DNA that persist until dispersal of this "extra" DNA throughout the cytoplasm as fragments and coalescing droplets of chromatin during anaphase of the diminution division.

  7. Burden of testicular, paratesticular and extragonadal germ cell tumours in Europe.

    PubMed

    Trama, A; Mallone, S; Nicolai, N; Necchi, A; Schaapveld, M; Gietema, J; Znaor, A; Ardanaz, E; Berrino, F

    2012-01-01

    We provide updated estimates of survival, incidence, complete prevalence, and proportion cured for patients with testicular/paratesticular and extragonadal germ cell cancers in Europe, grouped according to the new list of cancer types developed by RARECARE. We collected data, archived in European cancer registries, with vital status information available to 31st December 2003. We analysed 26,000 cases of testicular, paratesticular and extragonadal germ cell cancers diagnosed 1995-2002, estimating that about 15,600 new testicular/paratesticular and 630 new extragonadal cancer cases occurred per year in EU27, with annual incidence rates of 31.5/1,000,000 and 1.27/1,000,000, respectively. Slightly more than 436,000 persons were alive at the beginning of 2008 with a diagnosis of testicular/paratesticular cancer, and about 17,000 with a diagnosis of extragonadal germ cell cancer. Five-year relative survival was 96% for testicular/paratesticular cancer and 71% for extragonadal germ cell cancer; the proportions cured were 95% and 69%, respectively. We found limited variation in survival between European regions except for non-seminomatous testicular cancer, for which five-year relative survival ranged from 86% in Eastern Europe to 96% in Northern Europe. Survival for all cancer types considered decreased with increasing age at diagnosis. Further investigation is required to establish the real reasons for the lower survival in Eastern Europe. Considering the high prevalence of these highly curable cancers, it is important to monitor patients long-term, so as to quantify treatment-related risks and develop treatments having limited impact on quality of life.

  8. Basigin null mutant male mice are sterile and exhibit impaired interactions between germ cells and Sertoli cells

    PubMed Central

    Bi, Jiajia; Li, Yanfen; Sun, Fengyun; Saalbach, Anja; Klein, Claudia; Miller, David J.; Hess, Rex; Nowak, Romana A.

    2013-01-01

    Basigin (BSG) is a multifunctional glycoprotein that plays an important role in male reproduction since male knockout (KO) mice are sterile. The Bsg KO testis lacks elongated spermatids and mature spermatozoa, a phenotype similar to that of alpha-mannosidase IIx (MX) KO mice. MX regulates formation of N-acetylglucosamine (GlcNAc) terminated N-glycans that participate in germ cell-Sertoli cell adhesion. Results showed that Bsg KO spermatocytes displayed normal homologous chromosome synapsis and progression through meiosis. However, only punctate expression of the round spermatid marker SP-10 in the acrosomal granule of germ cells of Bsg KO mice was detected indicating that spermatogenesis in Bsg KO mice was arrested at the early round spermatid stages. We observed a large increase in the number of germ cells undergoing apoptosis in Bsg KO testes. Using lectin blotting, we determined that GlcNAc terminated N-glycans are linked to BSG. GlcNAc terminated N-glycans were significantly reduced in Bsg KO testes. These observations indicate that BSG may act as a germ cell-Sertoli cell attachment molecule. Loss of BSG significantly reduced adhesion between GC-2 and SF7 cells. Moreover, wild type testes showed strong expression of N-cadherin (CDH2) while expression was greatly reduced in the testes of Bsg KO mice. In addition, the integrity of the blood-testis barrier (BTB) was compromised in Bsg KO testes. In conclusion, although some Bsg KO spermatogonia can undergo normal progression to the spermatocyte stage, BSG-mediated germ cell-Sertoli cell interactions appear to be necessary for integrity of the BTB and spermatocyte progression to mature spermatozoa. PMID:23727514

  9. Assessing Human Germ-Cell Mutagenesis in the Postgenome Era: A Celebration of the Legacy of William Lawson (Bill) Russell

    PubMed Central

    Wyrobek, Andrew J.; Mulvihill, John J.; Wassom, John S.; Malling, Heinrich V.; Shelby, Michael D.; Lewis, Susan E.; Witt, Kristine L.; Preston, R. Julian; Perreault, Sally D.; Allen, James W.; DeMarini, David M.; Woychik, Richard P.; Bishop, Jack B.

    2007-01-01

    Birth defects, de novo genetic diseases, and chromosomal abnormality syndromes occur in ~5% of all live births, and affected children suffer from a broad range of lifelong health consequences. Despite the social and medical impact of these defects, and the 8 decades of research in animal systems that have identified numerous germ-cell mutagens, no human germ-cell mutagen has been confirmed to date. There is now a growing consensus that the inability to detect human germ-cell mutagens is due to technological limitations in the detection of random mutations rather than biological differences between animal and human susceptibility. A multidisciplinary workshop responding to this challenge convened at The Jackson Laboratory in Bar Harbor, Maine. The purpose of the workshop was to assess the applicability of an emerging repertoire of genomic technologies to studies of human germ-cell mutagenesis. Workshop participants recommended large-scale human germ-cell mutation studies be conducted using samples from donors with high-dose exposures, such as cancer survivors. Within this high-risk cohort, parents and children could be evaluated for heritable changes in (a) DNA sequence and chromosomal structure, (b) repeat sequences and minisatellites, and (c) global gene expression profiles and pathways. Participants also advocated the establishment of a bio-bank of human tissue samples from donors with well-characterized exposure, including medical and reproductive histories. This mutational resource could support large-scale, multiple-endpoint studies. Additional studies could involve the examination of transgenerational effects associated with changes in imprinting and methylation patterns, nucleotide repeats, and mitochondrial DNA mutations. The further development of animal models and the integration of these with human studies are necessary to provide molecular insights into the mechanisms of germ-cell mutations and to identify prevention strategies. Furthermore, scientific

  10. Generation of progeny from embryonic stem cells by microinsemination of male germ cells from chimeric mice.

    PubMed

    Mizutani, Eiji; Ohta, Hiroshi; Kishigami, Satoshi; Van Thuan, Nguyen; Hikichi, Takafusa; Wakayama, Sayaka; Sato, Eimei; Wakayama, Teruhiko

    2005-09-01

    Mice chimeric for embryonic stem (ES) cells have not always successfully produced ES-derived offspring. Here we show that the male gametes from ES cells could be selected in male chimeric mice testes by labeling donor ES cells or host blastocytes with GFP. Male GFP-expressing ES-derived germ cells occurred as colonies in the chimeric testes, where the seminiferous tubules were separated into green and non-green regions. When mature spermatozoa from green tubules were used for microinsemination, GFP-expressing offspring were efficiently obtained. Using a reverse study, we also obtained ES-derived progeny from GFP-negative ES cells in GFP-labeled host chimeras. Furthermore, we showed this approach could be accelerated by using round spermatids from the testes of 20-day-old chimeric mice. Thus, this technique allowed us to generate the ES cell-derived progeny even from the low contributed chimeric mice, which cannot produce ES-origin offspring by natural mating.

  11. Complete in vitro generation of fertile oocytes from mouse primordial germ cells

    PubMed Central

    Morohaku, Kanako; Tanimoto, Ren; Sasaki, Keisuke; Kawahara-Miki, Ryouka; Kono, Tomohiro; Hayashi, Katsuhiko; Hirao, Yuji; Obata, Yayoi

    2016-01-01

    Reconstituting gametogenesis in vitro is a key goal for reproductive biology and regenerative medicine. Successful in vitro reconstitution of primordial germ cells and spermatogenesis has recently had a significant effect in the field. However, recapitulation of oogenesis in vitro remains unachieved. Here we demonstrate the first reconstitution, to our knowledge, of the entire process of mammalian oogenesis in vitro from primordial germ cells, using an estrogen-receptor antagonist that promotes normal follicle formation, which in turn is crucial for supporting oocyte growth. The fundamental events in oogenesis (i.e., meiosis, oocyte growth, and genomic imprinting) were reproduced in the culture system. The most rigorous evidence of the recapitulation of oogenesis was the birth of fertile offspring, with a maximum of seven pups obtained from a cultured gonad. Moreover, cryopreserved gonads yielded functional oocytes and offspring in this culture system. Thus, our in vitro system will enable both innovative approaches for a deeper understanding of oogenesis and a new avenue to create and preserve female germ cells. PMID:27457928

  12. Complete in vitro generation of fertile oocytes from mouse primordial germ cells.

    PubMed

    Morohaku, Kanako; Tanimoto, Ren; Sasaki, Keisuke; Kawahara-Miki, Ryouka; Kono, Tomohiro; Hayashi, Katsuhiko; Hirao, Yuji; Obata, Yayoi

    2016-08-01

    Reconstituting gametogenesis in vitro is a key goal for reproductive biology and regenerative medicine. Successful in vitro reconstitution of primordial germ cells and spermatogenesis has recently had a significant effect in the field. However, recapitulation of oogenesis in vitro remains unachieved. Here we demonstrate the first reconstitution, to our knowledge, of the entire process of mammalian oogenesis in vitro from primordial germ cells, using an estrogen-receptor antagonist that promotes normal follicle formation, which in turn is crucial for supporting oocyte growth. The fundamental events in oogenesis (i.e., meiosis, oocyte growth, and genomic imprinting) were reproduced in the culture system. The most rigorous evidence of the recapitulation of oogenesis was the birth of fertile offspring, with a maximum of seven pups obtained from a cultured gonad. Moreover, cryopreserved gonads yielded functional oocytes and offspring in this culture system. Thus, our in vitro system will enable both innovative approaches for a deeper understanding of oogenesis and a new avenue to create and preserve female germ cells. PMID:27457928

  13. Adnexal germ cell carcinoma with bone metastases in pregnant women: case report and review.

    PubMed

    Tenorio-Guadalupe, María Del Rosario; Arsentales-Montalva, Valeria; Yonz-Buendía, Yessabell Sonia; Fiestas-Saldarriaga, Fabián; Pimentel-Álvarez, Patricia

    2016-01-01

    Germ cell carcinoma during pregnancy is rare. However, its detection has increased due to the use of ultrasound fetal monitoring in the antenatal care program. In this article, we present the case of a Germ cell carcinoma during pregnancy is rare. However, its detection has increased due to the use of ultrasound fetal monitoring in the antenatal care program. In this article, we present the case of a pregnant 27-year-old diagnosed with an adnexal germ cell carcinoma at six weeks of gestation, whose initial approach was local resection (suboptimal cytoreduction). Four weeks after surgery, the patient presented with grade IV peripheral neuropathy in the lower limbs; magnetic resonance imaging scan indicated an infiltrative lesion at D5. The local medical board decided on chemotherapy starting on the 19th week of gestation. The rest of the pregnancy period was uneventful and the patient had a cesarean section at 34 weeks of gestation and a live newborn with no complications. Unfortunately, four days after caesarean section, the patient died of a septic shock with respiratory focus. PMID:27602713

  14. Extremely Low Frequency Magnetic Fields Induce Spermatogenic Germ Cell Apoptosis: Possible Mechanism

    PubMed Central

    Lee, Sang-Kon; Park, Sungman; Gimm, Yoon-Myoung; Kim, Yoon-Won

    2014-01-01

    The energy generated by an extremely low frequency electromagnetic field (ELF-EMF) is too weak to directly induce genotoxicity. However, it is reported that an extremely low frequency magnetic field (ELF-MF) is related to DNA strand breakage and apoptosis. The testes that conduct spermatogenesis through a dynamic cellular process involving meiosis and mitosis seem vulnerable to external stress such as heat, MF exposure, and chemical or physical agents. Nevertheless the results regarding adverse effects of ELF-EMF on human or animal reproductive functions are inconclusive. According to the guideline of the International Commission on Non-Ionizing Radiation Protection (ICNIRP; 2010) for limiting exposure to time-varying MF (1 Hz to 100 kHz), overall conclusion of epidemiologic studies has not consistently shown an association between human adverse reproductive outcomes and maternal or paternal exposure to low frequency fields. In animal studies there is no compelling evidence of causal relationship between prenatal development and ELF-MF exposure. However there is increasing evidence that EL-EMF exposure is involved with germ cell apoptosis in testes. Biophysical mechanism by which ELF-MF induces germ cell apoptosis has not been established. This review proposes the possible mechanism of germ cell apoptosis in testes induced by ELF-MF. PMID:25025060

  15. Adnexal germ cell carcinoma with bone metastases in pregnant women: case report and review.

    PubMed

    Tenorio-Guadalupe, María Del Rosario; Arsentales-Montalva, Valeria; Yonz-Buendía, Yessabell Sonia; Fiestas-Saldarriaga, Fabián; Pimentel-Álvarez, Patricia

    2016-08-24

    Germ cell carcinoma during pregnancy is rare. However, its detection has increased due to the use of ultrasound fetal monitoring in the antenatal care program. In this article, we present the case of a Germ cell carcinoma during pregnancy is rare. However, its detection has increased due to the use of ultrasound fetal monitoring in the antenatal care program. In this article, we present the case of a pregnant 27-year-old diagnosed with an adnexal germ cell carcinoma at six weeks of gestation, whose initial approach was local resection (suboptimal cytoreduction). Four weeks after surgery, the patient presented with grade IV peripheral neuropathy in the lower limbs; magnetic resonance imaging scan indicated an infiltrative lesion at D5. The local medical board decided on chemotherapy starting on the 19th week of gestation. The rest of the pregnancy period was uneventful and the patient had a cesarean section at 34 weeks of gestation and a live newborn with no complications. Unfortunately, four days after caesarean section, the patient died of a septic shock with respiratory focus.

  16. Retroperitoneal Leiomyosarcoma Associated with an Elevated β-HCG Serum Level Mimicking Extragonadal Germ Cell Tumor

    PubMed Central

    Gaertner, Hans-Juergen; Manseck, Andreas; Oehlschlaeger, Sven; Wirth, Manfred P.

    2000-01-01

    Patient. A 65-year-old man was admitted with a large primary retroperitoneal tumor and an increased β-human chorionic gonadotropin (β-HCG) serum level. A germ cell tumor was suspected; however, a computed tomography-guided biopsy failed to enable tumor classification. After two courses of chemotherapy, the β-HCG serum level had returned to the normal level and a diagnostic laparotomy with incisional biopsy was performed. The immunohistochemical examination of the specimen identified the tumor as a retroperitoneal pleomorphic leiomyosarcoma. Discussion. Tumor markers play only a marginal role in the work-up of patients with soft tissue sarcomas. In men with suspected retroperitoneal sarcomas, however, the determination of germ cell tumor markers occasionally enables a preoperative distinguishing of primary retroperitoneal germ cell tumors with considerable consequences for management. In this setting, a retroperitoneal tumor associated with a moderately elevated β-HCG is a diagnostic dilemma, and surgeons should be aware of the pitfall of a β-HCG-producing leiomyosarcoma in the differential diagnosis. PMID:18521299

  17. Exposure to Brefeldin A promotes initiation of meiosis in murine female germ cells.

    PubMed

    Zhang, Lian-Jun; Chen, Bo; Feng, Xin-Lei; Ma, Hua-Gang; Sun, Li-Lan; Feng, Yan-Min; Liang, Gui-Jin; Cheng, Shun-Feng; Li, Lan; Shen, Wei

    2015-01-01

    In mammals, ontogenesis starts from a fusion of spermatozoon and oocyte, which are produced by reductive nuclear division of a diploid germ cell in a specialised but complex biological process known as meiosis. However, little is known about the mechanism of meiotic initiation in germ cells, although many factors may be responsible for meiosis both in male and female gonads. In this study, 11.5 days post coitum (dpc) female fetal mouse genital ridges were cultured in vitro with exposure to Brefeldin A (BFA) for 6h, and the changes in meiosis were detected. Synaptonemal-complex analysis implied that BFA played a positive role in meiosis initiation and this hypothesis was confirmed by quantitative PCR of meiosis-specific genes: stimulated by retinoic acid gene 8 (Stra8) and deleted in a zoospermia-like (DAZL). At the same time, mRNA expression of retinoic acid synthetase (Raldh2) and retinoic acid (RA) receptors increased in female gonads with in vitro exposure to BFA. Transplanting genital ridges treated with BFA into the kidney capsule of immunodeficient mice demonstrated that the development capacity of female germ cells was normal, while formation of primordial follicles was seen to be a result of accelerated meiosis after exposure to BFA. In conclusion, the study indicated that BFA stimulated meiosis initiation partly by RA signalling and then promoted the development of follicles.

  18. A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

    NASA Astrophysics Data System (ADS)

    Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

    2000-05-01

    RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

  19. Granulosa cells and retinoic acid co-treatment enrich potential germ cells from manually selected Oct4-EGFP expressing human embryonic stem cells.

    PubMed

    Chen, Hsin-Fu; Jan, Pey-Shynan; Kuo, Hung-Chih; Wu, Fang-Chun; Lan, Chen-Wei; Huang, Mei-Chi; Chien, Chung-Liang; Ho, Hong-Nerng

    2014-09-01

    Differentiation of human embryonic stem (HES) cells to germ cells may become clinically useful in overcoming diseases related to germ-cell development. Niches were used to differentiate HES cell lines, NTU1 and H9 Oct4-enhanced green fluorescence protein (EGFP), including laminin, granulosa cell co-culture or conditioned medium, ovarian stromal cell co-culture or conditioned medium, retinoic acid, stem cell factor (SCF) and BMP4-BMP7-BMP8b treatment. Flow cytometry showed that granulosa cell co-culture (P < 0.001) or conditioned medium (P = 0.007) treatment for 14 days significantly increased the percentages of differentiated H9 Oct4-EGFP cells expressing early germ cell marker stage-specific embryonic antigen 1(SSEA1); sorted SSEA1[+] cells did not express higher levels of germ cell gene VASA and GDF9. Manually collected H9 Oct4-EGFP[+] cells expressed significantly higher levels of VASA (P = 0.005) and GDF9 (P = 0.001). H9 Oct4-EGFP[+] cells developed to ovarian follicle-like structures after culture for 28 days but with low efficiency. Unlike SCF and BMP4, retinoic acid co-treatment enhanced VASA, GDF9 and SCP3 expression. A protocol is recommended to enrich differentiated HES cells with germ-cell potential by culture with granulosa cells, conditioned medium or retinoic acid, manual selection of Oct4-EGFP[+] cells, and analysis of VASA, GDF9 expression, or both.

  20. Cytological study on Sertoli cells and their interactions with germ cells during annual reproductive cycle in turtle.

    PubMed

    Ahmed, Nisar; Yufei, Huang; Yang, Ping; Muhammad Yasir, Waqas; Zhang, Qian; Liu, Tengfei; Hong, Chen; Lisi, Hu; Xiaoya, Chu; Chen, Qiusheng

    2016-06-01

    Sertoli cells (SCs) play a central role in the development of germ cells within functional testes and exhibit varying morphology during spermatogenesis. This present study investigated the seasonal morphological changes in SCs in the reproductive cycle of Pelodiscus sinensis by light microscopy, transmission electron microscopy (TEM), and immunohistochemistry. During hibernation period with the quiescent of spermatogenesis, several autophagosomes were observed inside the SCs, the processes of which retracted. In early spermatogenesis, when the germ cells started to proliferate, the SCs contained numerous lipid droplets instead of autophagosomes. In late spermatogenesis, the SCs processes became very thin and contacted several round/elongated spermatids in pockets. At this time, abundant endoplasmic reticulum and numerous mitochondria were present in the SCs. The organization of the tight junctions and the adherens junctions between the SCs and germ cells also changed during the reproductive cycle. Moreover, SCs were involved in the formation of cytoplasmic bridges, phagophores, and exosome secretions during spermatogenesis. Tubulobulbar complexes (TBC) were also developed by SCs around the nucleus of the spermatid at the time of spermiation. Strong, positive expression of vimentin was noted on the SCs during late spermatogenesis compared with the hibernation stage and the early stage of spermatogenesis. These data provide clear cytological evidence about the seasonal changes in SCs, corresponding with their different roles in germ cells within the Chinese soft-shelled turtle Pelodiscus sinensis. PMID:27516863

  1. 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.

  2. Regulation of expression of mouse interferon-induced transmembrane protein like gene-3, Ifitm3 (mil-1, fragilis), in germ cells.

    PubMed

    Tanaka, Satomi S; Nagamatsu, Go; Tokitake, Yuko; Kasa, Miyuki; Tam, Patrick P L; Matsui, Yasuhisa

    2004-08-01

    Mouse interferon-induced transmembrane protein (IFITM) gene, Ifitm3 (previously known as mil-1 and fragilis), is expressed in primordial germ cells (PGCs), in their precursors, and in germ cells of the fetal gonads (Saitou et al. [2002] Nature 418:293-300; Tanaka and Matsui [2002] Mech Dev 119S:S261-S267). By examining the expression of green fluorescent protein transgene under the control of DNA sequences flanking exon 1, we have identified domains that direct Ifitm3 transcription in PGCs and their precursors in gastrula stage and 13.5 days post coitum embryos. Germ cell-specific expression is achieved by the activity of a consensus element unique to the Ifitm genes, which may act to suppress Ifitm3 expression in somatic tissues. The lack of any influence of the interferon-stimulable response elements on transgene expression in the germ-line suggests that interferon-mediated response is not critical for activating Ifitm3. PMID:15254899

  3. Glucose responsive insulin production from human embryonic germ (EG) cell derivatives

    SciTech Connect

    Clark, Gregory O.; Yochem, Robert L.; Axelman, Joyce; Sheets, Timothy P.; Kaczorowski, David J.; Shamblott, Michael J. . E-mail: mshambl1@jhmi.edu

    2007-05-11

    Type 1 diabetes mellitus subjects millions to a daily burden of disease management, life threatening hypoglycemia and long-term complications such as retinopathy, nephropathy, heart disease, and stroke. Cell transplantation therapies providing a glucose-regulated supply of insulin have been implemented clinically, but are limited by safety, efficacy and supply considerations. Stem cells promise a plentiful and flexible source of cells for transplantation therapies. Here, we show that cells derived from human embryonic germ (EG) cells express markers of definitive endoderm, pancreatic and {beta}-cell development, glucose sensing, and production of mature insulin. These cells integrate functions necessary for glucose responsive regulation of preproinsulin mRNA and expression of insulin C-peptide in vitro. Following transplantation into mice, cells become insulin and C-peptide immunoreactive and produce plasma C-peptide in response to glucose. These findings suggest that EG cell derivatives may eventually serve as a source of insulin producing cells for the treatment of diabetes.

  4. Re-irradiation of Recurrent Pineal Germ Cell Tumors with Radiosurgery: Report of Two Cases and Review of Literature.

    PubMed

    Wong, Kenneth; Opimo, Anthony B; Olch, Arthur J; All, Sean; Waxer, Jonathan F; Clark, Desirae; Cheng, Justine; Chlebik, Alisha; Erdreich-Epstein, Anat; Krieger, Mark D; Tamrazi, Benita; Dhall, Girish; Finlay, Jonathan L; Chang, Eric L

    2016-01-01

    Primary intracranial germ cell tumors are rare, representing less than 5% of all central nervous system tumors. Overall, the majority of germ cell tumors are germinomas and approximately one-third are non-germinomatous germ cell tumors (NGGCT), which include teratoma, embryonal carcinoma, yolk sac tumor (endodermal sinus tumor), choriocarcinoma, or mixed malignant germ cell tumor. Germ cell tumors may secrete detectable levels of proteins into the blood and/or cerebrospinal fluid, and these proteins can be used for diagnostic purposes or to monitor tumor recurrence. Germinomas have long been known to be highly curable with radiation therapy alone. However, many late effects of whole brain or craniospinal irradiation have been well documented. Strategies have been developed to reduce the dose and volume of radiation therapy, often in combination with chemotherapy. In contrast, patients with NGGCT have a poorer prognosis, with about 60% cured with multimodality chemoradiation. There are no standard approaches for relapsed germ cell tumors. Options may be limited by prior treatment. Radiation therapy has been utilized alone or in combination with chemotherapy or high-dose chemotherapy and transplant. We discuss two cases and review options for frameless radiosurgery or fractionated radiotherapy. PMID:27239400

  5. Reproductive Toxicity of Endosulfan: Implication From Germ Cell Apoptosis Modulated by Mitochondrial Dysfunction and Genotoxic Response Genes in Caenorhabditis elegans

    PubMed Central

    Du, Hua; Wang, Meimei; Wang, Lei; Dai, Hui; Wang, Min; Hong, Wei; Nie, Xinxin; Wu, Lijun; Xu, An

    2015-01-01

    Endosulfan as a new member of persistent organic pollutants has been shown to induce reproductive dysfunction in various animal models. However, the action mechanism of endosulfan-produced reproductive toxicity remains largely unknown. This study was focused on investigating the reproductive toxicity induced by α-endosulfan and clarifying the role of mitochondria and genotoxic response genes in germ cell apoptosis of Caenorhabditis elegans. Our data showed that endosulfan induced a dose-dependent decrease of life span, fecundity, and hatchability, whereas the germ cell apoptosis was dose-dependently increased. The mitochondria membrane potential was disrupted by endosulfan, leading to a significant increase of germ cell apoptosis in mev-1(kn-1) mutant. However, the apoptotic effects of endosulfan were blocked in mutants of cep-1(w40), egl-1(n487), and hus-1(op241), indicating conserved genotoxic response genes played an essential role in endosulfan-induced germ cell apoptosis. Furthermore, exposure to endosulfan induced the accumulation of HUS-1::GFP foci and the germ cell cycle arrest. These findings provided clear evidence that endosulfan caused significant adverse effects on the reproduction system of C. elegans and increased germ cell apoptosis, which was regulated by mitochondrial dysfunction and DNA damage response genes. This study may help to understand the signal transduction pathways involved in endosulfan-induced reproductive toxicity. PMID:25666835

  6. Re-irradiation of Recurrent Pineal Germ Cell Tumors with Radiosurgery: Report of Two Cases and Review of Literature

    PubMed Central

    Opimo, Anthony B; Olch, Arthur J; All, Sean; Waxer, Jonathan F; Clark, Desirae; Cheng, Justine; Chlebik, Alisha; Erdreich-Epstein, Anat; Krieger, Mark D; Tamrazi, Benita; Dhall, Girish; Finlay, Jonathan L; Chang, Eric L

    2016-01-01

    Primary intracranial germ cell tumors are rare, representing less than 5% of all central nervous system tumors. Overall, the majority of germ cell tumors are germinomas and approximately one-third are non-germinomatous germ cell tumors (NGGCT), which include teratoma, embryonal carcinoma, yolk sac tumor (endodermal sinus tumor), choriocarcinoma, or mixed malignant germ cell tumor. Germ cell tumors may secrete detectable levels of proteins into the blood and/or cerebrospinal fluid, and these proteins can be used for diagnostic purposes or to monitor tumor recurrence. Germinomas have long been known to be highly curable with radiation therapy alone. However, many late effects of whole brain or craniospinal irradiation have been well documented. Strategies have been developed to reduce the dose and volume of radiation therapy, often in combination with chemotherapy. In contrast, patients with NGGCT have a poorer prognosis, with about 60% cured with multimodality chemoradiation. There are no standard approaches for relapsed germ cell tumors. Options may be limited by prior treatment. Radiation therapy has been utilized alone or in combination with chemotherapy or high-dose chemotherapy and transplant. We discuss two cases and review options for frameless radiosurgery or fractionated radiotherapy. PMID:27239400

  7. Reproductive Toxicity of Endosulfan: Implication From Germ Cell Apoptosis Modulated by Mitochondrial Dysfunction and Genotoxic Response Genes in Caenorhabditis elegans.

    PubMed

    Du, Hua; Wang, Meimei; Wang, Lei; Dai, Hui; Wang, Min; Hong, Wei; Nie, Xinxin; Wu, Lijun; Xu, An

    2015-05-01

    Endosulfan as a new member of persistent organic pollutants has been shown to induce reproductive dysfunction in various animal models. However, the action mechanism of endosulfan-produced reproductive toxicity remains largely unknown. This study was focused on investigating the reproductive toxicity induced by α-endosulfan and clarifying the role of mitochondria and genotoxic response genes in germ cell apoptosis of Caenorhabditis elegans. Our data showed that endosulfan induced a dose-dependent decrease of life span, fecundity, and hatchability, whereas the germ cell apoptosis was dose-dependently increased. The mitochondria membrane potential was disrupted by endosulfan, leading to a significant increase of germ cell apoptosis in mev-1(kn-1) mutant. However, the apoptotic effects of endosulfan were blocked in mutants of cep-1(w40), egl-1(n487), and hus-1(op241), indicating conserved genotoxic response genes played an essential role in endosulfan-induced germ cell apoptosis. Furthermore, exposure to endosulfan induced the accumulation of HUS-1::GFP foci and the germ cell cycle arrest. These findings provided clear evidence that endosulfan caused significant adverse effects on the reproduction system of C. elegans and increased germ cell apoptosis, which was regulated by mitochondrial dysfunction and DNA damage response genes. This study may help to understand the signal transduction pathways involved in endosulfan-induced reproductive toxicity.

  8. Ovarian germ cell tumors with rhabdomyosarcomatous components and later development of growing teratoma syndrome: a case report

    PubMed Central

    2012-01-01

    Introduction Development of a sarcomatous component in a germ cell tumor is an uncommon phenomenon. Most cases reported have a grim prognosis. Growing teratoma syndrome is also an uncommon phenomenon and occurs in approximately 2% to 7% of non seminomatous germ cell tumors and should be treated surgically. Case presentation We report the case of a 12-year-old Asian girl with an ovarian mixed germ cell tumor containing a rhabdomyosarcomatous component. She was treated with a germ cell tumor chemotherapy regimen and rhabdomyosarcoma-specific chemotherapy. Towards the end of her treatment, she developed a retroperitoneal mass that was increasing in size. It was completely resected, revealing a mature teratoma, consistent with growing teratoma syndrome. She is still in complete remission approximately three years after presentation. Conclusion The presence of rhabdomyosarcoma in a germ cell tumor should be treated by a combined chemotherapy regimen (for germ cell tumor and rhabdomyosarcoma). In addition, development of a mass during or after therapy with normal serum markers should raise the possibility of growing teratoma syndrome that should be treated surgically. PMID:22248255

  9. PRMT5 Protects Genomic Integrity during Global DNA Demethylation in Primordial Germ Cells and Preimplantation Embryos

    PubMed Central

    Kim, Shinseog; Günesdogan, Ufuk; Zylicz, Jan J.; Hackett, Jamie A.; Cougot, Delphine; Bao, Siqin; Lee, Caroline; Dietmann, Sabine; Allen, George E.; Sengupta, Roopsha; Surani, M. Azim

    2014-01-01

    Summary Primordial germ cells (PGCs) and preimplantation embryos undergo epigenetic reprogramming, which includes comprehensive DNA demethylation. We found that PRMT5, an arginine methyltransferase, translocates from the cytoplasm to the nucleus during this process. Here we show that conditional loss of PRMT5 in early PGCs causes complete male and female sterility, preceded by the upregulation of LINE1 and IAP transposons as well as activation of a DNA damage response. Similarly, loss of maternal-zygotic PRMT5 also leads to IAP upregulation. PRMT5 is necessary for the repressive H2A/H4R3me2s chromatin modification on LINE1 and IAP transposons in PGCs, directly implicating this modification in transposon silencing during DNA hypomethylation. PRMT5 translocates back to the cytoplasm subsequently, to participate in the previously described PIWI-interacting RNA (piRNA) pathway that promotes transposon silencing via de novo DNA remethylation. Thus, PRMT5 is directly involved in genome defense during preimplantation development and in PGCs at the time of global DNA demethylation. PMID:25457166

  10. Translation of tobacco necrosis virus and its satellite in a cell-free wheat germ system.

    PubMed Central

    Salvato, M S; Fraenkel-Conrat, H

    1977-01-01

    Tobacco necrosis virus (TNV) and its satellite virus (STNV) each contain a single-stranded RNA genome, of about 1.4 X 10(6) and 0.4 X 10(6) daltons, respectively, which is very active in stimulating amino acid incorporation in a wheat germ cell-free system. With STNV RNA the predominant incorporation product of 22,000 daltons coelectrophoreses with viral coat protein and crossreacts with antibody to viral coat protein. A similar result is obtained with TNV RNA, the only major translation product being a 30,000-dalton protein which corresponds to the coat protein by gel sizing, serological tests, and tryptic peptide analysis. Other products appearing in smaller amounts are about 63,000, 43,000, and 26,000 daltons and smaller. The possible nature of these products is discussed, as well as the unusual feature of a large, presumably multigenic, viral RNA yielding the coat protein as the predominant translation product in a eukaryotic system. Much less STNV RNA than TNV RNA produces maximal translation. Cotranslation of both RNAs in vitro indicates that STNV RNA has a translational advantage over TNV RNA. The fact that these RNAs lack 5'-terminal capping and 3'-terminal poly(A) is discussed. PMID:267924

  11. Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis.

    PubMed

    Dores, C; Rancourt, D; Dobrinski, I

    2015-05-01

    To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density, or differential adhesion to substrates to separate germ cells from somatic cells. The objective of this study was to develop an approach that allowed germ cell enrichment while providing efficiency of handling large cell numbers. Here, we report the use of stirred suspension bioreactors (SSB) to exploit the adhesion properties of Sertoli cells to enrich cells obtained from pre-pubertal porcine testes for undifferentiated spermatogonia. We also compared the bioreactor approach with an established differential plating method and the combination of both: SSB followed by differential plating. After 66 h of culture, germ cell enrichment in SSBs provided 7.3 ± 1.0-fold (n = 9), differential plating 9.8 ± 2.4-fold (n = 6) and combination of both methods resulted in 9.1 ± 0.3-fold enrichment of germ cells from the initial germ cell population (n = 3). To document functionality of cells recovered from the bioreactor, we demonstrated that cells retained their functional ability to reassemble seminiferous tubules de novo after grafting to mouse hosts and to support spermatogenesis. These results demonstrate that the SSB allows enrichment of germ cells in a controlled and scalable environment providing an efficient method when handling large cell numbers while reducing variability owing to handling.

  12. Myosin-driven intercellular transportation of wheat germ agglutinin mediated by membrane nanotubes between human lung cancer cells.

    PubMed

    Wang, Zhi-Gang; Liu, Shu-Lin; Tian, Zhi-Quan; Zhang, Zhi-Ling; Tang, Hong-Wu; Pang, Dai-Wen

    2012-11-27

    Membrane nanotubes can facilitate direct intercellular communication between cells and provide a unique channel for intercellular transfer of cellular contents. However, the transport mechanisms of membrane nanotubes remain poorly understood between cancer cells. Also largely unknown is the transport pattern mediated by membrane nanotubes. In this work, wheat germ agglutinin (WGA), a widely used drug carrier and potential antineoplastic drug, was labeled with quantum dots (QDs-WGA) as a model for exploring the intercellular transportation via membrane nanotubes. We found that membrane nanotubes allowed effective transfer of QDs-WGA. Long-term single-particle tracking indicated that the movements of QDs-WGA exhibited a slow and directed motion pattern in nanotubes. Significantly, the transport of QDs-WGA was driven by myosin molecular motors in an active and unidirectional manner. These results contribute to a better understanding of cell-to-cell communication for cancer research.

  13. Dazl is a critical player for primordial germ cell formation in medaka

    PubMed Central

    Li, Mingyou; Zhu, Feng; Li, Zhendong; Hong, Ni; Hong, Yunhan

    2016-01-01

    The DAZ family genes boule, daz and dazl have conserved functions in primordial germ cell (PGC) migration, germ stem cell proliferation, differentiation and meiosis progression. It has remained unknown whether this family is required for PGC formation in developing embryos. Our recent study in the fish medaka (Oryzias latipes) has defined dnd as the critical PGC specifier and predicted the presence of additional factors essential for PGC formation. Here we report that dazl is a second key player for medaka PGC formation. Dazl knockdown did not prevent PGC formation even in the absence of normal somatic structures. It turned out that a high level of Dazl protein was maternally supplied and persisted until gastrulation, and hardly affected by two antisense morpholino oligos targeting the dazl RNA translation. Importantly, microinjection of a Dazl antibody remarkably reduced the number of PGCs and even completely abolished PGC formation without causing detectable somatic abnormality. Therefore, medaka PGC formation requires the Dazl protein as maternal germ plasm component, offering first evidence that dazl is a critical player in PGC formation in vivo. Our results demonstrate that antibody neutralization is a powerful tool to study the roles of maternal protein factors in PGC development in vivo. PMID:27328644

  14. Chicken stem cell factor enhances primordial germ cell proliferation cooperatively with fibroblast growth factor 2.

    PubMed

    Miyahara, Daichi; Oishi, Isao; Makino, Ryuichi; Kurumisawa, Nozomi; Nakaya, Ryuma; Ono, Tamao; Kagami, Hiroshi; Tagami, Takahiro

    2016-04-22

    An in vitro culture system of chicken primordial germ cells (PGCs) has been recently developed, but the growth factor involved in the proliferation of PGCs is largely unknown. In the present study, we investigated the growth effects of chicken stem cell factor (chSCF) on the in vitro proliferation of chicken PGCs. We established two feeder cell lines (buffalo rat liver cells; BRL cells) that stably express the putative secreted form of chSCF (chSCF1-BRL) and membrane bound form of chSCF (chSCF2-BRL). Cultured PGC lines were incubated on chSCF1 or chSCF2-BRL feeder cells with fibroblast growth factor 2 (FGF2), and growth effects of each chSCF isoform were investigated. The in vitro proliferation rate of the PGCs cultured on chSCF2-BRL at 20 days of culture was more than threefold higher than those cultured on chSCF1-BRL cells and more than fivefold higher than those cultured on normal BRL cells. Thus, use of chSCF2-BRL feeder layer was effective for in vitro proliferation of chicken PGCs. However, the acceleration of PGC proliferation on chSCF2-BRL was not observed without FGF2, suggesting that chSCF2 would act as a proliferation co-factor of FGF2. We transferred the PGCs cultured on chSCF2-BRL cells to recipient embryos, generated germline chimeric chickens and assessed the germline competency of cultured PGCs by progeny test. Donor-derived progenies were obtained, and the frequency of germline transmission was 3.39%. The results of this study demonstrate that chSCF2 induces hyperproliferation of chicken PGCs retaining germline competency in vitro in cooperation with FGF2. PMID:26727404

  15. Cystic trophoblastic tumor: a nonaggressive lesion in postchemotherapy resections of patients with testicular germ cell tumors.

    PubMed

    Ulbright, Thomas M; Henley, John D; Cummings, Oscar W; Foster, Richard S; Cheng, Liang

    2004-09-01

    Cystic trophoblastic tumor (CTT) is an uncommon lesion that is usually seen after chemotherapy in patients with testicular germ cell tumors. Its clinical significance has not been well studied. We identified 17 patients with CTT in retroperitoneal lymph node dissections (RPLNDs) after cisplatin-based chemotherapy for testicular germ cell tumors. None had other forms of persistent germ cell tumor except for teratoma, and no patient received additional chemotherapy after RPLND. At the time of RPLND, 7 patients were known to have had normal serum levels of beta-subunit of human chorionic gonadotropin (beta-hCG), whereas 5 had relatively mild elevations (1.6-165 mIU/mL, median, 8.0 mIU/mL). The CTTs consisted of circumscribed, small cysts, usually multifocal, lined by mostly mononucleated trophoblast cells with abundant eosinophilic cytoplasm, often with smudged nuclei and showing only infrequent mitotic figures. Although the epithelial lining was often stratified to several layers in thickness or formed intracystic papillary tufts, solid proliferations of trophoblast cells within the stroma were absent, as were clearly biphasic admixtures of mononucleated and multinucleated trophoblast cells. The cysts were either empty or contained fibrinoid material and were set in a hypocellular, fibrous stroma with adjacent teratoma. Stains for hCG highlighted rare cells. On follow-up of 15 patients, 11 were disease free (mean, 80 months). Three recurred with serum alpha-fetoprotein elevations at 25, 31, and 107 months, respectively, and one with beta-hCG elevation at 2 months. The latter patient, however, also had unresected mediastinal tumor postchemotherapy. We conclude that the finding of CTT in postchemotherapy resections does not warrant additional chemotherapy. Its clinical significance appears similar to that of residual teratoma.

  16. Effects of cinnamon (Cinnamomum zeylanicum) bark oil on testicular antioxidant values, apoptotic germ cell and sperm quality.

    PubMed

    Yüce, A; Türk, G; Çeribaşi, S; Sönmez, M; Çiftçi, M; Güvenç, M

    2013-08-01

    Cinnamon and its contents have multifactorial properties such as antioxidant, anti-inflammatory and antidiabetic. Male infertility is one of the major health problems in life. The aim of this study was to investigate the effects of long-term cinnamon bark oil (CBO) ingestion on testicular antioxidant values, apoptotic germ cell and sperm quality of adult rats. Twelve male healthy Wistar rats were divided into two groups, each group containing six rats. While olive oil was given to control group, 100 mg kg(-1)  CBO was administered to the other group by gavage daily for 10 weeks. Body and reproductive organ weights, sperm characteristics, testicular lipid peroxidation and antioxidant enzyme activities, and testicular apoptosis via terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method were examined. A significant decrease in malondialdehyde level and marked increases in reduced glutathione level, glutathione peroxidase and catalase activities were observed in rats treated with CBO compared with the control group. CBO consumption provided a significant increase in weights of testes and epididymides, epididymal sperm concentration, sperm motility and diameter of seminiferous tubules when compared with the control group. However, CBO consumption tended to decrease the abnormal sperm rate and apoptotic germ cell count, but it did not reach statistical significance. It is concluded that CBO has improvement effect on testicular oxidant-antioxidant balance and sperm quality, and its consumption may be useful for asthenozoospermic men.

  17. Chlamydia muridarum infection-induced destruction of male germ cells and sertoli cells is partially prevented by Chlamydia major outer membrane protein-specific immune CD4 cells.

    PubMed

    Sobinoff, Alexander P; Dando, Samantha J; Redgrove, Kate A; Sutherland, Jessie M; Stanger, Simone J; Armitage, Charles W; Timms, Peter; McLaughlin, Eileen A; Beagley, Kenneth W

    2015-01-01

    Chlamydia trachomatis infections are increasingly prevalent worldwide. Male chlamydial infections are associated with urethritis, epididymitis, and orchitis; however, the role of Chlamydia in prostatitis and male factor infertility remains controversial. Using a model of Chlamydia muridarum infection in male C57BL/6 mice, we investigated the effects of chlamydial infection on spermatogenesis and determined the potential of immune T cells to prevent infection-induced outcomes. Antigen-specific CD4 T cells significantly reduced the infectious burden in the penile urethra, epididymis, and vas deferens. Infection disrupted seminiferous tubules, causing loss of germ cells at 4 and 8 wk after infection, with the most severely affected tubules containing only Sertoli cells. Increased mitotic proliferation, DNA repair, and apoptosis in spermatogonial cells and damaged germ cells were evident in atrophic tubules. Activated caspase 3 (casp3) staining revealed increased (6-fold) numbers of Sertoli cells with abnormal morphology that were casp3 positive in tubules of infected mice, indicating increased levels of apoptosis. Sperm count and motility were both decreased in infected mice, and there was a significant decrease in morphologically normal spermatozoa. Assessment of the spermatogonial stem cell population revealed a decrease in promyelocytic leukemia zinc finger (PLZF)-positive cells in the seminiferous tubules. Interestingly, adoptive transfer of antigen-specific CD4 cells, particularly T-helper 2-like cells, prior to infection prevented these effects in spermatogenesis and Sertoli cells. These data suggest that chlamydial infection adversely affects spermatogenesis and male fertility, and that vaccination can potentially prevent the spread of infection and these adverse outcomes.

  18. Insulin-Like Activity of Concanavalin A and Wheat Germ Agglutinin—Direct Interactions with Insulin Receptors

    PubMed Central

    Cuatrecasas, Pedro; Tell, Guy P. E.

    1973-01-01

    Concanavalin A and wheat germ agglutinin are as effective as insulin in enhancing the rate of glucose transport and in inhibiting epinephrine-stimulated lipolysis in isolated adipocytes. These lectins, also like insulin, inhibit basal as well as epinephrine-stimulated adenylate cyclase activity of membranes obtained from homogenates of fat cells. Low concentrations of wheat germ agglutinin enhance the specific binding of insulin to receptors of fat cells and liver membranes. Higher concentrations of this plant lectin, as well as of concanavalin A, competitively displace the binding of insulin to receptors in these tissues. These effects are equally apparent in insulin-binding proteins solubilized from membranes, indicating that the plant lectins interact directly with insulin receptors. All of the effects observed with the plant lectins are reversed by simple sugars that bind specifically to these plant proteins. Agarose derivatives of the plant lectins effectively adsorb solubilized insulin-binding proteins, and these can be eluted with buffers containing specific simple sugars. The possible implications of these findings to certain biological properties (mitogenicity) of these lectins and to the mechanism of action of other growth-promoting substances are considered. PMID:4510292

  19. Specific repertoire of olfactory receptor genes in the male germ cells of several mammalian species

    SciTech Connect

    Vanderhaeghen, P.; Schurmans, S.; Vassart, G.; Parmentier, M.

    1997-02-01

    Olfactory receptors constitute the largest family among G protein-coupled receptors, with up to 1000 members expected. We have previously shown that genes belonging to this family were expressed in the male germ line from both dog and human. We have subsequently demonstrated the presence of one of the corresponding olfactory receptor proteins during dog spermatogenesis and in mature sperm cells. In this study, we investigated whether the unexpected pattern of expression of olfactory receptors in the male germ line was conserved in other mammalian species. Using reverse transcription-PCR with primers specific for the olfactory receptor gene family, about 20 olfactory receptor cDNA fragments were cloned from the testis of each mammalian species tested. As a whole, they displayed no sequence specificity compared to other olfactory receptors, but highly homologous, possibly orthologous, genes were amplified from different species. Finally, their pattern of expression, as determined by RNase protection assay, revealed that many but not all of these receptors were expressed predominantly in testis. The male germ line from each mammalian species tested is thus characterized by a specific repertoire of olfactory receptors, which display a pattern of expression suggestive of their potential implication in the control of sperm maturation, migration, or fertilization. 34 refs., 4 figs., 1 tab.

  20. Salvage therapy in patients with germ cell tumors.

    PubMed

    Einhorn, Lawrence H

    2015-01-01

    Testicular cancer is the most curable metastatic solid tumor. Initial chemotherapy is evidence based with risk stratification into three prognostic categories: good, intermediate, and advanced disease. Guidelines for disease management following progression after initial cisplatin combination chemotherapy are less clear. Options include salvage surgery for patients with anatomically confined relapse, standard-dose cisplatin combination chemotherapy, or high-dose chemotherapy with carboplatin plus etoposide with peripheral blood stem cell transplantation. Proper interpretation of a presumed relapse can be complicated. Growing masses on imaging studies might reflect a growing teratoma. Persistent elevations of serum human chorionic gonadotropin (hCG) or alpha fetoprotein (AFP) are only an indication for salvage therapy if there is a definitive rise in the tumor marker. Elevated and rising serum hCG as the only evidence of recurrence can be because of cross reactivity with luteinizing hormone or usage of marijuana rather than progressive cancer. Elevated liver function tests can cause rising serum AFP. PMID:25993183

  1. Prenatal exposure to chromium induces early reproductive senescence by increasing germ cell apoptosis and advancing germ cell cyst breakdown in the F1 offspring

    PubMed Central

    Sivakumar, Kirthiram K.; Stanley, Jone A.; Arosh, Joe A.; Pepling, Melissa E.; Burghardt, Robert C.; Banu, Sakhila K.

    2014-01-01

    Hexavalent chromium (CrVI), one of the more toxic heavy metals, is widely used in more than 50 industries such as chrome plating, welding, wood processing and tanneries. As one of the world’s leading producers of chromium compounds, the U.S. is facing growing challenges in protecting human health against multiple adverse effects of CrVI. CrVI is rapidly converted to CrIII intracellularly, and can induce apoptosis through different mechanisms. Our previous studies demonstrated postnatal exposure to CrVI results in a delay or arrest in follicle development and puberty. Pregnant rats were treated with 25 ppm potassium dichromate (CrVI) from gestational day (GD) 9.5 to 14.5 through drinking water, placentae were removed on GD 20, and total Cr was estimated in the placentae; ovaries were removed from the F1 offspring on postnatal day (PND)-1 and various analyses were performed. Our results show that gestational exposure to CrVI resulted in (i) increased Cr concentration in the placenta, (ii) increased germ cell apoptosis by up-regulating p53/p27–Bax–caspase-3 proteins and by increasing p53–SOD-2 co-localization; (iii) accelerated germ cell cyst (GCC) breakdown; (iv) advanced primordial follicle assembly and primary follicle transition and (v) down regulation of p-AKT, p-ERK and XIAP. As a result of the above events, CrVI induced early reproductive senescence and decrease in litter size in F1 female progeny. PMID:24530425

  2. Current state of the opportunities for derivation of germ-like cells from pluripotent stem cells: are you a man, or a mouse?

    PubMed Central

    Petkova, Rumena; Arabadjiev, Borislav; Chakarov, Stoyan; Pankov, Roumen

    2014-01-01

    The concept of pluripotency as a prerogative of cells of early mammal embryos and cultured embryonic stem cells (ESC) has been invalidated with the advent of induced pluripotent stem cells. Later, it became clear that the ability to generate all cell types of the adult organism is also a questionable aspect of pluripotency, as there are cell types, such as germ cells, which are difficult to produce from pluripotent stem cells. Recently it has been proposed that there are at least two different states of pluripotency; namely, the naïve, or ground state, and the primed state, which may differ radically in terms of timeline of existence, signalling mechanisms, cell properties, capacity for differentiation into different cell types, etc. Germ-like male and female rodent cells have been successfully produced in vitro from ESC and induced pluripotent stem cells. The attempts to derive primate primordial germ cells (PGC) and germ cells in vitro from pluripotent stem cells, however, still have a low success rate, especially with the female germline. The paper reviews the properties of rodent and primate ESC with regard to their capacity for differentiation in vitro to germ-like cells, outlining the possible caveats to derivation of PGC and germ cells from primate and human pluripotent cells. PMID:26019504

  3. FACS-sorted putative oogonial stem cells from the ovary are neither DDX4-positive nor germ cells.

    PubMed

    Zarate-Garcia, Larissa; Lane, Simon I R; Merriman, Julie A; Jones, Keith T

    2016-01-01

    Whether the adult mammalian ovary contains oogonial stem cells (OSCs) is controversial. They have been isolated by a live-cell sorting method using the germ cell marker DDX4, which has previously been assumed to be cytoplasmic, not surface-bound. Furthermore their stem cell and germ cell characteristics remain disputed. Here we show that although OSC-like cells can be isolated from the ovary using an antibody to DDX4, there is no good in silico modelling to support the existence of a surface-bound DDX4. Furthermore these cells when isolated were not expressing DDX4, and did not initially possess germline identity. Despite these unremarkable beginnings, they acquired some pre-meiotic markers in culture, including DDX4, but critically never expressed oocyte-specific markers, and furthermore were not immortal but died after a few months. Our results suggest that freshly isolated OSCs are not germ stem cells, and are not being isolated by their DDX4 expression. However it may be that culture induces some pre-meiotic markers. In summary the present study offers weight to the dogma that the adult ovary is populated by a fixed number of oocytes and that adult de novo production is a rare or insignificant event. PMID:27301892

  4. Condensation behavior of the human x chromosome in male germ cells and Sertoli cells examined by flourescence in situ hybridisation

    SciTech Connect

    Kofman-Alfaro, S.; Cervantes, A.; Speed, R.M.

    1994-09-01

    The chromatin condensation behavior of the human x chromosome has been studied by FISH analysis in germ cells and Sertoli cells of the adult testes. Comparisons are made with previous findings for the human Y chromosome and for chromosome 7. In meiotic prophase, the X chromosome can be seen to extend greatly at zygotene and to contract through pachytene into the sex vesicle. Such extension, which has also been noted for the human Y chromosome at this state of meiosis, could be a prerequisite for XY pairing crossing-over. In patients with {open_quotes}Sertoli-cell-only{close_quotes} syndrome, the sex chromosomes, by in situ hybridization analysis, appear extremely contracted compared with their normal extended state seen in adult Sertoli cells of fertile men. By contrast, the state of expansion of chromosome 7 in Sertoli cells appears identical for sterile and fertile testes. This could suggest an association between gene-controlled germ cell losses and failure of expansion of the sex chromosome axes. The variable patterns of extension and contraction for the X and Y chromosome axes in germ cells and Sertoli cells might provide underlying clues to pattern of expression noted for sex-linked genes in the human testis.

  5. Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells.

    PubMed

    Schusser, Benjamin; Collarini, Ellen J; Yi, Henry; Izquierdo, Shelley Mettler; Fesler, Jeffrey; Pedersen, Darlene; Klasing, Kirk C; Kaspers, Bernd; Harriman, William D; van de Lavoir, Marie-Cecile; Etches, Robert J; Leighton, Philip A

    2013-12-10

    Gene targeting by homologous recombination or by sequence-specific nucleases allows the precise modification of genomes and genes to elucidate their functions. Although gene targeting has been used extensively to modify the genomes of mammals, fish, and amphibians, a targeting technology has not been available for the avian genome. Many of the principles of humoral immunity were discovered in chickens, yet the lack of gene targeting technologies in birds has limited biomedical research using this species. Here we describe targeting the joining (J) gene segment of the chicken Ig heavy chain gene by homologous recombination in primordial germ cells to establish fully transgenic chickens carrying the knockout. In homozygous knockouts, Ig heavy chain production is eliminated, and no antibody response is elicited on immunization. Migration of B-lineage precursors into the bursa of Fabricius is unaffected, whereas development into mature B cells and migration from the bursa are blocked in the mutants. Other cell types in the immune system appear normal. Chickens lacking the peripheral B-cell population will provide a unique experimental model to study avian immune responses to infectious disease. More generally, gene targeting in avian primordial germ cells will foster advances in diverse fields of biomedical research such as virology, stem cells, and developmental biology, and provide unique approaches in biotechnology, particularly in the field of antibody discovery. PMID:24282302

  6. Bone Formation from Porcine Dental Germ Stem Cells on Surface Modified Polybutylene Succinate Scaffolds.

    PubMed

    Abay, Nergis; Gurel Pekozer, Gorke; Ramazanoglu, Mustafa; Kose, Gamze Torun

    2016-01-01

    Designing and providing a scaffold are very important for the cells in tissue engineering. Polybutylene succinate (PBS) has high potential as a scaffold for bone regeneration due to its capacity in cell proliferation and differentiation. Also, stem cells from 3rd molar tooth germs were favoured in this study due to their developmentally and replicatively immature nature. In this study, porcine dental germ stem cells (pDGSCs) seeded PBS scaffolds were used to investigate the effects of surface modification with fibronectin or laminin on these scaffolds to improve cell attachment, proliferation, and osteogenic differentiation for tissue engineering applications. The osteogenic potentials of pDGSCs on these modified and unmodified foams were examined to heal bone defects and the effects of fibronectin or laminin modified PBS scaffolds on pDGSC differentiation into bone were compared for the first time. For this study, MTS assay was used to assess the cytotoxic effects of modified and unmodified surfaces. For the characterization of pDGSCs, flow cytometry analysis was carried out. Besides, alkaline phosphatase (ALP) assay, von Kossa staining, real-time PCR, CM-Dil, and immunostaining were applied to analyze osteogenic potentials of pDGSCs. The results of these studies demonstrated that pDGSCs were differentiated into osteogenic cells on fibronectin modified PBS foams better than those on unmodified and laminin modified PBS foams. PMID:27413380

  7. Bone Formation from Porcine Dental Germ Stem Cells on Surface Modified Polybutylene Succinate Scaffolds

    PubMed Central

    2016-01-01

    Designing and providing a scaffold are very important for the cells in tissue engineering. Polybutylene succinate (PBS) has high potential as a scaffold for bone regeneration due to its capacity in cell proliferation and differentiation. Also, stem cells from 3rd molar tooth germs were favoured in this study due to their developmentally and replicatively immature nature. In this study, porcine dental germ stem cells (pDGSCs) seeded PBS scaffolds were used to investigate the effects of surface modification with fibronectin or laminin on these scaffolds to improve cell attachment, proliferation, and osteogenic differentiation for tissue engineering applications. The osteogenic potentials of pDGSCs on these modified and unmodified foams were examined to heal bone defects and the effects of fibronectin or laminin modified PBS scaffolds on pDGSC differentiation into bone were compared for the first time. For this study, MTS assay was used to assess the cytotoxic effects of modified and unmodified surfaces. For the characterization of pDGSCs, flow cytometry analysis was carried out. Besides, alkaline phosphatase (ALP) assay, von Kossa staining, real-time PCR, CM-Dil, and immunostaining were applied to analyze osteogenic potentials of pDGSCs. The results of these studies demonstrated that pDGSCs were differentiated into osteogenic cells on fibronectin modified PBS foams better than those on unmodified and laminin modified PBS foams. PMID:27413380

  8. Global deletion of Trp53 reverts ovarian tumor phenotype of the germ cell-deficient white spotting variant (Wv) mice.

    PubMed

    Cai, Kathy Qi; Wang, Ying; Smith, Elizabeth R; Smedberg, Jennifer L; Yang, Dong-Hua; Yang, Wan-Lin; Xu, Xiang-Xi

    2015-01-01

    White spotting variant (Wv) mice are spontaneous mutants attributed to a point mutation in the c-Kit gene, which reduces the tyrosine kinase activity to around 1% and affects the development of melanocytes, mast cells, and germ cells. Homozygous mutant mice are sterile but can live nearly a normal life span. The female Wv mice have a greatly reduced ovarian germ cell and follicle reserve at birth, and the remaining follicles are largely depleted soon after the females reach reproductive stage at around 7 weeks of age. Consequently, ovarian epithelial tumors develop in 100% of Wv females by 3 to 4 months of age. These tumors, called tubular adenomas, are benign but can become invasive in older Wv mice. We tested if additional genetic mutation(s) could convert the benign ovarian epithelial tumors to malignant tumors by crossing the Wv mutant into the Trp53 knockout background. Surprisingly, we found that global deletion of Trp53 suppressed the development of ovarian tubular adenomas in Wv mice. The ovaries of Wv/Wv; Trp53 (-/-) mice were covered by a single layer of surface epithelium and lacked excessive epithelial proliferation. Rather, the ovaries contained a small number of follicles. The presence of ovarian follicles and granulosa cells, as indicated by Pgc7 and inhibin-alpha expression, correlated with the absence of epithelial lesions. A reduction of Pten gene dosage, as in Wv/Wv; Pten (+/-) mice, produced a similar, though less dramatic, phenotype. We conclude that deletion of Trp53 prolongs the survival of ovarian follicles in Wv mice and consequently prevents the proliferation of ovarian epithelial cells and development of ovarian tubular adenomas. The results suggest that various cell types within the ovary communicate and mutually modulate, and an intact tissue environment is required to ensure homeostasis of ovarian surface epithelial cells. Especially, the current finding emphasizes the importance of ovarian follicles in suppressing the hyperplastic

  9. Large, Male Germ Cell-Specific Hypomethylated DNA Domains With Unique Genomic and Epigenomic Features on the Mouse X Chromosome

    PubMed Central

    Ikeda, Rieko; Shiura, Hirosuke; Numata, Koji; Sugimoto, Michihiko; Kondo, Masayo; Mise, Nathan; Suzuki, Masako; Greally, John M.; Abe, Kuniya

    2013-01-01

    To understand the epigenetic regulation required for germ cell-specific gene expression in the mouse, we analysed DNA methylation profiles of developing germ cells using a microarray-based assay adapted for a small number of cells. The analysis revealed differentially methylated sites between cell types tested. Here, we focused on a group of genomic sequences hypomethylated specifically in germline cells as candidate regions involved in the epigenetic regulation of germline gene expression. These hypomethylated sequences tend to be clustered, forming large (10 kb to ∼9 Mb) genomic domains, particularly on the X chromosome of male germ cells. Most of these regions, designated here as large hypomethylated domains (LoDs), correspond to segmentally duplicated regions that contain gene families showing germ cell- or testis-specific expression, including cancer testis antigen genes. We found an inverse correlation between DNA methylation level and expression of genes in these domains. Most LoDs appear to be enriched with H3 lysine 9 dimethylation, usually regarded as a repressive histone modification, although some LoD genes can be expressed in male germ cells. It thus appears that such a unique epigenomic state associated with the LoDs may constitute a basis for the specific expression of genes contained in these genomic domains. PMID:23861320

  10. Study on the regulatory mechanism of the lipid metabolism pathways during chicken male germ cell differentiation based on RNA-seq.

    PubMed

    Zuo, Qisheng; Li, Dong; Zhang, Lei; Elsayed, Ahmed Kamel; Lian, Chao; Shi, Qingqing; Zhang, Zhentao; Zhu, Rui; Wang, Yinjie; Jin, Kai; Zhang, Yani; Li, Bichun

    2015-01-01

    Here, we explore the regulatory mechanism of lipid metabolic signaling pathways and related genes during differentiation of male germ cells in chickens, with the hope that better understanding of these pathways may improve in vitro induction. Fluorescence-activated cell sorting was used to obtain highly purified cultures of embryonic stem cells (ESCs), primitive germ cells (PGCs), and spermatogonial stem cells (SSCs). The total RNA was then extracted from each type of cell. High-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. Gene Ontology (GO) analysis and the KEGG database were used to identify lipid metabolism pathways and related genes. Retinoic acid (RA), the end-product of the retinol metabolism pathway, induced in vitro differentiation of ESC into male germ cells. Quantitative real-time PCR (qRT-PCR) was used to detect changes in the expression of the genes involved in the retinol metabolic pathways. From the results of RNA-seq and the database analyses, we concluded that there are 328 genes in 27 lipid metabolic pathways continuously involved in lipid metabolism during the differentiation of ESC into SSC in vivo, including retinol metabolism. Alcohol dehydrogenase 5 (ADH5) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) are involved in RA synthesis in the cell. ADH5 was specifically expressed in PGC in our experiments and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) persistently increased throughout development. CYP26b1, a member of the cytochrome P450 superfamily, is involved in the degradation of RA. Expression of CYP26b1, in contrast, decreased throughout development. Exogenous RA in the culture medium induced differentiation of ESC to SSC-like cells. The expression patterns of ADH5, ALDH1A1, and CYP26b1 were consistent with RNA-seq results. We conclude that the retinol metabolism pathway plays an important role in the process of chicken male germ cell differentiation.

  11. Identical Allelic Losses in Mature Teratoma and Other Histologic Components of Malignant Mixed Germ Cell Tumors of the Testis

    PubMed Central

    Kernek, Kevin M.; Ulbright, Thomas M.; Zhang, Shaobo; Billings, Steven D.; Cummings, Oscar W.; Henley, John D.; Michael, Helen; Brunelli, Matteo; Martignoni, Guido; Foster, Richard S.; Eble, John N.; Cheng, Liang

    2003-01-01

    Teratomas of the testis in post-pubertal patients are histologically diverse tumors that often coexist with other types of germ cell tumors. Using laser capture microdissection and loss of heterozygosity analysis, we investigated the clonality of mature teratoma and its relationship to other components of malignant mixed germ cell tumors to gain potential insight into the histogenetic relationship of teratoma with other germ cell tumor components. All 16 patients had mature teratoma as one component of their mixed germ cell tumors. The other histological subtypes included immature teratoma, seminoma, embryonal carcinoma, yolk sac tumor, and choriocarcinoma. Laser-assisted microdissection was performed on the formalin-fixed, paraffin-embedded tissue. Polymerase chain reaction was used to amplify genomic DNA at specific loci on chromosome 1p36.2 (D1S508), 2q22–32 (D2S156), 9p21–22 (D9S162), 11p13 (D11S903), 12q22–23 (D12S1051), and 18q21 (D18S46). Fourteen of 16 (88%) cases showed allelic loss in one or more components of the mixed germ cell tumors. Fourteen of 16 mature teratomas showed allelic loss in at least one of six microsatellite polymorphic markers analyzed. The frequency of allelic loss in mature teratoma was 50% (7 of 14) with D1S508, 33% (5 of 15) with D2S156, 58% (7 of 12) with D9S162, 43% (6 of 14) with D11S903, 20% (3 of 15) with D12S1051, and 33% (5 of 15) with D18S46. Completely concordant allelic loss patterns between mature teratoma and all of the other germ cell tumor components were seen in 10 of 14 tumors in which mature teratoma showed loss of heterozygosity. Our data support the common clonal origin of mature teratoma with other components of malignant mixed germ cell tumors of the testis. PMID:14633619

  12. Treatment-related Cardiovascular Late-effects and Exercise Training Countermeasures in Testicular Germ Cell Cancer Survivorship

    PubMed Central

    Christensen, Jesper F; Bandak, Mikkel; Campbell, Anna; Jones, Lee W.; Højman, Pernille

    2016-01-01

    Background Treatment of testicular germ cell cancer constitutes a major success story in modern oncology. Today, the vast majority of patients are cured by a therapeutic strategy using one or more highly effective components including surgery (orchiectomy), radiotherapy and/or chemotherapy. However, the excellent cancer specific survival comes at considerable costs, as individuals with a history of germ cell cancer experience serious long-term complications, including markedly increased risk of cardiovascular morbidities and premature cardiovascular death. The factors responsible, as well as their mode of action, are not fully understood and there is a lack of knowledge concerning optimal evidence-based long-term follow-up strategies. Results Here, we present the growing body of evidence suggesting that germ cell cancer patients as a consequence of the different treatment components, are subjected to toxicities, which individually, and synergistically, can cause physiological impairments leading to sub-clinical or clinical cardiovascular disorders the ‘multiple-hit hypothesis’). Furthermore, we discuss the efficacy and utility of structured exercise training to ameliorate treatment-induced cardiovascular dysfunction to prevent premature onset of clinical cardiovascular disease in germ cell cancer survivors, with a view towards highlighting future directions of exercise-based survivorship research in the germ cell cancer setting. Conclusion Since exercise training may have the potential to ameliorate and/or reverse long-term cardiovascular disease sequelae in germ cell cancer survivors, a strong rationale exists for the promotion of exercise-oncology research in this setting, in order to provide exercise-recommendations for optimal germ cell cancer survivorship. PMID:25751759

  13. Notch pathway regulates female germ cell meiosis progression and early oogenesis events in fetal mouse.

    PubMed

    Feng, Yan-Min; Liang, Gui-Jin; Pan, Bo; Qin, Xun-Si; Zhang, Xi-Feng; Chen, Chun-Lei; Li, Lan; Cheng, Shun-Feng; De Felici, Massimo; Shen, Wei

    2014-01-01

    A critical process of early oogenesis is the entry of mitotic oogonia into meiosis, a cell cycle switch regulated by a complex gene regulatory network. Although Notch pathway is involved in numerous important aspects of oogenesis in invertebrate species, whether it plays roles in early oogenesis events in mammals is unknown. Therefore, the rationale of the present study was to investigate the roles of Notch signaling in crucial processes of early oogenesis, such as meiosis entry and early oocyte growth. Notch receptors and ligands were localized in mouse embryonic female gonads and 2 Notch inhibitors, namely DAPT and L-685,458, were used to attenuate its signaling in an in vitro culture system of ovarian tissues from 12.5 days post coitum (dpc) fetus. The results demonstrated that the expression of Stra8, a master gene for germ cell meiosis, and its stimulation by retinoic acid (RA) were reduced after suppression of Notch signaling, and the other meiotic genes, Dazl, Dmc1, and Rec8, were abolished or markedly decreased. Furthermore, RNAi of Notch1 also markedly inhibited the expression of Stra8 and SCP3 in cultured female germ cells. The increased methylation status of CpG islands within the Stra8 promoter of the oocytes was observed in the presence of DAPT, indicating that Notch signaling is probably necessary for maintaining the epigenetic state of this gene in a way suitable for RA stimulation. Furthermore, in the presence of Notch inhibitors, progression of oocytes through meiosis I was markedly delayed. At later culture periods, the rate of oocyte growth was decreased, which impaired subsequent primordial follicle assembly in cultured ovarian tissues. Taken together, these results suggested new roles of the Notch signaling pathway in female germ cell meiosis progression and early oogenesis events in mammals.

  14. Genotoxicity evaluation of buprofezin, petroleum oil and profenofos in somatic and germ cells of male mice.

    PubMed

    Fahmy, M A; Abdalla, E F

    1998-01-01

    The two pest control agents, buprofezin and petroleum oil (Super Royal), were tested to evaluate their potential mutagenicity, in comparison with the organophosphorus insecticide profenofos. Chromosomal aberration analysis was used in both somatic and germ cells of male mice. Single oral treatment at three different dose levels (1/16, 1/8 and 1/4 LD50) for each insecticide induced an increase in the percentage of chromosomal aberrations in bone-marrow cells 24 h post-treatment, indicating a dose-dependent relationship. The percentage of chromosomal aberrations reached 23 +/- 0.73, 10.5 +/- 0.64 and 15 +/- 1.4 after treatment with the highest tested dose of profenofos, buprofezin and Super Royal, respectively. Such percentages did not exceed the corresponding value of the positive control, mitomycin C (29.2 +/- 0.69). The percentage of chromosomal aberrations induced by the different doses of profenofos was still highly significant even after excluding gaps. The same trend of results was noticed only at the highest tested dose of buprofezin and Super Royal. With respect to germ cells, profenofos is also a potent inducer of chromosomal aberrations in 1ry spermatocytes, giving percentages of 14 +/- 1.3 and 19 +/- 1.6 at the two higher doses of 4.25 and 8.5 mg kg(-1) body wt., respectively. Buprofezin and Super Royal had no significant effect on mouse spermatocytes at the tested concentrations. The various types of induced aberrations were examined and recorded in both somatic and germ cells. In conclusion, the present investigation indicates that the two pest control agents buprofezin and Super Royal are relatively much safer compounds than the conventional organophosphorus insecticides. PMID:9804428

  15. Reversal of informational entropy and the acquisition of germ-like immortality by somatic cells.

    PubMed

    Kyriazis, Marios

    2014-01-01

    We live within an increasingly technological, information-laden environment for the first time in human evolution. This subjects us (and will continue to subject us in an accelerating fashion) to an unremitting exposure to 'meaningful information that requires action'. Directly dependent upon this new environment are novel evolutionary pressures, which can modify existing resource allocation mechanisms and may eventually favour the survival of somatic cells (particularly neurons) at the expense of germ line cells. In this theoretical paper I argue that persistent, structured information-sharing in both virtual and real domains, leads to increased biological complexity and functionality, which reflects upon human survival characteristics. Certain biological immortalisation mechanisms currently employed by germ cells may thus need to be downgraded in order to enable somatic cells to manage these new energy demands placed by our modern environment. Relevant concepts from a variety of disciplines such as the evolution of complex adaptive systems, information theory, digital hyper-connectivity, and cell immortalisation will be reviewed. Using logical, though sometimes speculative arguments, I will attempt to describe a new biology. A biology not driven by sex and reproduction but by information and somatic longevity. PMID:24852017

  16. Perchlorate Exposure Reduces Primordial Germ Cell Number in Female Threespine Stickleback.

    PubMed

    Petersen, Ann M; Earp, Nathanial C; Redmond, Mandy E; Postlethwait, John H; von Hippel, Frank A; Buck, C Loren; Cresko, William A

    2016-01-01

    Perchlorate is a common aquatic contaminant that has long been known to affect thyroid function in vertebrates, including humans. More recently perchlorate has been shown to affect primordial sexual differentiation in the aquatic model fishes zebrafish and threespine stickleback, but the mechanism has been unclear. Stickleback exposed to perchlorate from fertilization have increased androgen levels in the embryo and disrupted reproductive morphologies as adults, suggesting that perchlorate could disrupt the earliest stages of primordial sexual differentiation when primordial germ cells (PGCs) begin to form the gonad. Female stickleback have three to four times the number of PGCs as males during the first weeks of development. We hypothesized that perchlorate exposure affects primordial sexual differentiation by reducing the number of germ cells in the gonad during an important window of stickleback sex determination at 14-18 days post fertilization (dpf). We tested this hypothesis by quantifying the number of PGCs at 16 dpf in control and 100 mg/L perchlorate-treated male and female stickleback. Perchlorate exposure from the time of fertilization resulted in significantly reduced PGC number only in genotypic females, suggesting that the masculinizing effects of perchlorate observed in adult stickleback may result from early changes to the number of PGCs at a time critical for sex determination. To our knowledge, this is the first evidence of a connection between an endocrine disruptor and reduction in PGC number prior to the first meiosis during sex determination. These findings suggest that a mode of action of perchlorate on adult reproductive phenotypes in vertebrates, including humans, such as altered fecundity and sex reversal or intersex gonads, may stem from early changes to germ cell development. PMID:27383240

  17. Perchlorate Exposure Reduces Primordial Germ Cell Number in Female Threespine Stickleback

    PubMed Central

    Petersen, Ann M.; Earp, Nathanial C.; Redmond, Mandy E.; Postlethwait, John H.; von Hippel, Frank A.; Buck, C. Loren; Cresko, William A.

    2016-01-01

    Perchlorate is a common aquatic contaminant that has long been known to affect thyroid function in vertebrates, including humans. More recently perchlorate has been shown to affect primordial sexual differentiation in the aquatic model fishes zebrafish and threespine stickleback, but the mechanism has been unclear. Stickleback exposed to perchlorate from fertilization have increased androgen levels in the embryo and disrupted reproductive morphologies as adults, suggesting that perchlorate could disrupt the earliest stages of primordial sexual differentiation when primordial germ cells (PGCs) begin to form the gonad. Female stickleback have three to four times the number of PGCs as males during the first weeks of development. We hypothesized that perchlorate exposure affects primordial sexual differentiation by reducing the number of germ cells in the gonad during an important window of stickleback sex determination at 14–18 days post fertilization (dpf). We tested this hypothesis by quantifying the number of PGCs at 16 dpf in control and 100 mg/L perchlorate-treated male and female stickleback. Perchlorate exposure from the time of fertilization resulted in significantly reduced PGC number only in genotypic females, suggesting that the masculinizing effects of perchlorate observed in adult stickleback may result from early changes to the number of PGCs at a time critical for sex determination. To our knowledge, this is the first evidence of a connection between an endocrine disruptor and reduction in PGC number prior to the first meiosis during sex determination. These findings suggest that a mode of action of perchlorate on adult reproductive phenotypes in vertebrates, including humans, such as altered fecundity and sex reversal or intersex gonads, may stem from early changes to germ cell development. PMID:27383240

  18. Delayed Effects of Whole Brain Radiotherapy in Germ Cell Tumor Patients With Central Nervous System Metastases

    SciTech Connect

    Doyle, Danielle M. Einhorn, Lawrence H.

    2008-04-01

    Purpose: Central nervous system (CNS) metastases are uncommon in patients with germ cell tumors, with an incidence of 2-3%. CNS metastases have been managed with whole brain radiotherapy (WBRT) and concomitant cisplatin-based combination chemotherapy. Our previous study did not observe serious CNS toxicity (Int J Radiat Oncol Biol Phys 1991;22:17-22). We now report on 5 patients who developed delayed significant CNS toxicity. Patients and Methods: We observed 5 patients with delayed CNS toxicity. The initial diagnosis was between 1981 and 2003. All patients had poor-risk disease according to the International Germ Cell Consensus Collaborative Group criteria. Of the 5 patients, 3 had CNS metastases at diagnosis and 2 developed relapses with CNS metastases. These 5 patients underwent WBRT to 4,000-5,000 cGy in 18-28 fractions concurrently with cisplatin-based chemotherapy. Results: All 5 patients developed delayed symptoms consistent with progressive multifocal leukoencephalopathy. The symptoms included seizures, hemiparesis, cranial neuropathy, headaches, blindness, dementia, and ataxia. The median time from WBRT to CNS symptoms was 72 months (range, 9-228). Head imaging revealed multiple abnormalities consistent with gliosis and diffuse cerebral atrophy. Of the 5 patients, 3 had progressive and 2 stable symptoms. Treatment with surgery and/or steroids had modest benefit. The progressive multifocal leukoencephalopathy resulted in significant debility in all 5 patients, resulting in death (3 patients), loss of work, steroid-induced morbidity, and recurrent hospitalizations. Conclusion: Whole brain radiotherapy is not innocuous in young patients with germ cell tumors and can cause late CNS toxicity.

  19. Protective Effects of Thymoquinone against Methotrexate-Induced Germ Cell Apoptosis in Male Mice

    PubMed Central

    Sheikhbahaei, Fatemeh; Khazaei, Mozafar; Rabzia, Arezou; Mansouri, Kamran; Ghanbari, Ali

    2016-01-01

    Background Toxic effects of anti-cancer and other drugs on the normal tissues could be reduced by the herbal plants and their fractions. This study investigated the protective effect of thymoquinone (TQ) as a fraction of Nigella sativa on methotrexate (MTX)- induced germ cell apoptosis in male mice. Materials and Methods In this experimental study, thirty male Balb/c mice were divided randomly into 5 groups (n=6). A single dose of MTX (20 mg/kg) and different concentrations of TQ were administrated for 4 consecutive days. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed on paraffin embedded tissue sections to analysis the occurrence of apoptosis in the testis. Reverse transcription polymerase chain reaction (RT-PCR) of apoptosis-related genes was performed with RNA extracted from testes of the mice. Statistical analysis was done using one-way ANOVA. Results In the MTX group, there was a significant increase in morphologic sign of germ cell degeneration of tubules (48 ± 0.6%), apoptotic index (AI; 2.3 ± 0.6%), as well as mRNA expression of p53 (P=0.008), caspase 8 (P=0.002), caspase 3 (P=0.005), caspase 9 (P=0.000), bax (P=0.004) and the ratio of bax/bcl-2 (P=0.000), whereas there was an decrease in the expression of bcl-2 (P=0.003), as compared to control group. In MTX+TQ groups, the data showed that different concentrations of TQ could improve the harmful effects caused by the MTX. The best protective effects were achieved in MTX+TQ (10 mg/kg). Conclusion TQ protects testicular germ cell against MTX-induced apoptosis by affecting related genes regulation. PMID:26985343

  20. [Construction of goat germ cell specific reporting system pVASA-EGFP].

    PubMed

    Yan, Guangyao; Li, Peizhen; Ren, Caifang; Wang, Feng; Zhang, Yanli

    2015-09-01

    To monitor the trans-differentiation from adult stem cells to germ cells, we analyzed the vasa expression of goat testicular tissues in different ages and constructed the germ cell specific reporting vector pVASA-EGFP. The expression of vasa was verified by RT-PCR and immunofluorescence. The vector pVASA-EGFP was constructed by molecular technology, then transfected into goat bone mesenchymal stem cells (BMSCs) by Lipofectamine 2000. Moreover, we observed the expression of the vector through green fluorescent protein (GFP). Immunofluorescence results show that Vasa was expressed in all groups of goat testicular tissues, RT-PCR results show that the levels of vasa mRNA in 3-month group and 10-month group were significantly higher than that in 10-day group. Sequencing and restriction enzyme results show that the vector was successfully constructed. After transfection and RA treatment, GFP expression was observed, which proved the validity of our reporting system. All the results proved that vasa was expressed in different ages in goat testicular tissues, and the vector pVASA-EGFP is efficient in monitoring the trans-differentiation in vitro, which paves the way for further characterization and screening of the trans-differentiation of goat BMSCs.

  1. Differential Expression of Conserved Germ Line Markers and Delayed Segregation of Male and Female Primordial Germ Cells in a Hermaphrodite, the Leech Helobdella

    PubMed Central

    Cho, Sung-Jin; Vallès, Yvonne; Weisblat, David A.

    2014-01-01

    In sexually reproducing animals, primordial germ cells (PGCs) are often set aside early in embryogenesis, a strategy that minimizes the risk of genomic damage associated with replication and mitosis during the cell cycle. Here, we have used germ line markers (piwi, vasa, and nanos) and microinjected cell lineage tracers to show that PGC specification in the leech genus Helobdella follows a different scenario: in this hermaphrodite, the male and female PGCs segregate from somatic lineages only after more than 20 rounds of zygotic mitosis; the male and female PGCs share the same (mesodermal) cell lineage for 19 rounds of zygotic mitosis. Moreover, while all three markers are expressed in both male and female reproductive tissues of the adult, they are expressed differentially between the male and female PGCs of the developing embryo: piwi and vasa are expressed preferentially in female PGCs at a time when nanos is expressed preferentially in male PGCs. A priori, the delayed segregation of male and female PGCs from somatic tissues and from one another increases the probability of mutations affecting both male and female PGCs of a given individual. We speculate that this suite of features, combined with a capacity for self-fertilization, may contribute to the dramatically rearranged genome of Helobdella robusta relative to other animals. PMID:24217283

  2. [Intra-Abdominal Germ Cell Tumor in Persistent Mullerian Duct Syndrome].

    PubMed

    Mishima, Takao; Harada, Jiro; Kawa, Gen; Sakurai, Takanori; Okada, Takuya

    2016-08-01

    A 46-year-old man was admitted to hospital presenting with a lower abdominal mass. The patient's testes were not palpable in the scrotum, and the levels of lactic dehydrogenase, α-fetoprotein and human chorionic gonadotropin were all elevated. Enhanced computed tomography revealed that the lumen of the mass had penetrated the prostate. Pathological analysis of biopsy tissue indicated that the mass was a seminoma. Residual tumor resection was performed after chemotherapy. On histological examination, the lumen proved to be a Mullerian structure. Our diagnosis was an intra-abdominal germ cell tumor and persistent Mullerian duct syndrome. PMID:27624113

  3. Acute slices of mice testis seminiferous tubules unveil spontaneous and synchronous Ca2+ oscillations in germ cell clusters.

    PubMed

    Sánchez-Cárdenas, Claudia; Guerrero, Adán; Treviño, Claudia Lydia; Hernández-Cruz, Arturo; Darszon, Alberto

    2012-10-01

    Spermatogenic cell differentiation involves changes in the concentration of cytoplasmic Ca(2+) ([Ca(2+)]i); however, very few studies exist on [Ca(2+)]i dynamics in these cells. Other tissues display Ca(2+) oscillations involving multicellular functional arrangements. These phenomena have been studied in acute slice preparations that preserve tissue architecture and intercellular communications. Here we report the implementation of intracellular Ca(2+) imaging in a sliced seminiferous tubule (SST) preparation to visualize [Ca(2+)]i changes of living germ cells in situ within the SST preparation. Ca(2+) imaging revealed that a subpopulation of male germ cells display spontaneous [Ca(2+)]i fluctuations resulting from Ca(2+) entry possibly throughout Ca(V)3 channels. These [Ca(2+)]i fluctuation patterns are also present in single acutely dissociated germ cells, but they differ from those recorded from germ cells in the SST preparation. Often, spontaneous Ca(2+) fluctuations of spermatogenic cells in the SST occur synchronously, so that clusters of cells can display Ca(2+) oscillations for at least 10 min. Synchronous Ca(2+) oscillations could be mediated by intercellular communication via gap junctions, although intercellular bridges could also be involved. We also observed an increase in [Ca(2+)]i after testosterone application, suggesting the presence of functional Sertoli cells in the SST. In summary, we believe that the SST preparation is suitable to explore the physiology of spermatogenic cells in their natural environment, within the seminiferous tubules, in particular Ca(2+) signaling phenomena, functional cell-cell communication, and multicellular functional arrangements. PMID:22914313

  4. Hypersensitivity of Primordial Germ Cells to Compromised Replication-Associated DNA Repair Involves ATM-p53-p21 Signaling

    PubMed Central

    Zeng, Ruizhu; Southard, Teresa L.; Shima, Naoko; Schimenti, John C.

    2014-01-01

    Genome maintenance in germ cells is critical for fertility and the stable propagation of species. While mechanisms of meiotic DNA repair and chromosome behavior are well-characterized, the same is not true for primordial germ cells (PGCs), which arise and propagate during very early stages of mammalian development. Fanconi anemia (FA), a genomic instability syndrome that includes hypogonadism and testicular failure phenotypes, is caused by mutations in genes encoding a complex of proteins involved in repair of DNA lesions associated with DNA replication. The signaling mechanisms underlying hypogonadism and testicular failure in FA patients or mouse models are unknown. We conducted genetic studies to show that hypogonadism of Fancm mutant mice is a result of reduced proliferation, but not apoptosis, of PGCs, resulting in reduced germ cells in neonates of both sexes. Progressive loss of germ cells in adult males also occurs, overlaid with an elevated level of meiotic DNA damage. Genetic studies indicated that ATM-p53-p21 signaling is partially responsible for the germ cell deficiency. PMID:25010009

  5. Rare inactivating PDE11A variants associated with testicular germ cell tumors.

    PubMed

    Pathak, Anand; Stewart, Douglas R; Faucz, Fabio R; Xekouki, Paraskevi; Bass, Sara; Vogt, Aurelie; Zhang, Xijun; Boland, Joseph; Yeager, Meredith; Loud, Jennifer T; Nathanson, Katherine L; McGlynn, Katherine A; Stratakis, Constantine A; Greene, Mark H; Mirabello, Lisa

    2015-12-01

    Germline inactivating mutations of isoform 4 of phosphodiesterase (PDE) 11A (coded by the PDE11A gene) have been associated with familial adrenocortical tumors and familial testicular cancer. Testicular tissue is unique in expressing all four isoforms of PDE11A. In a prior candidate gene study of 94 familial testicular germ cell tumor (TGCT) subjects, we identified a significant association between the presence of functionally abnormal variants in PDE11A and familial TGCT risk. To validate this novel observation, we sequenced the PDE11A coding region in 259 additional TGCT patients (both familial and sporadic) and 363 controls. We identified 55 PDE11A variants: 20 missense, four splice-site, two nonsense, seven synonymous, and 22 intronic. Ten missense variants were novel; nine occurred in transcript variant 4 and one in transcript variant 3. Five rare mutations (p.F258Y, p.G291R, p.V820M, p.R545X, and p.K568R) were present only in cases and were significantly more common in cases vs controls (P=0.0037). The latter two novel variants were functionally characterized and shown to be functionally inactivating, resulting in reduced PDE activity and increased cAMP levels. In further analysis of this cohort, we focused on white participants only to minimize confounding due to population stratification. This study builds upon our prior reports implicating PDE11A variants in familial TGCT, provides the first independent validation of those findings, extends that work to sporadic testicular cancer, demonstrates that these variants are uncommonly but reproducibly associated with TGCT, and refines our understanding regarding which specific inactivating PDE11A variants are most likely to be associated with TGCT risk. PMID:26459559

  6. BOULE, a Deleted in Azoospermia Homolog, Is Recruited to Stress Granules in the Mouse Male Germ Cells

    PubMed Central

    Kim, Byunghyuk; Rhee, Kunsoo

    2016-01-01

    High temperature adversely affects normal development of male germ cells in mammals. Acute heat stress induces the formation of stress granules (SGs) in a set of male germ cells, and the SGs have been proposed to protect those cells from heat-induced apoptosis. DAZL, one of DAZ (Deleted in Azoospermia) family proteins, was shown to be an essential component of SGs, which is required for SG formation in the mouse testis. In the present study, we asked whether BOULE, the founding member of DAZ family proteins, is a component of the SGs. We show that BOULE is recruited to the SGs upon heat stress, and that these SGs are developmental stage-specific. These results suggest that DAZ family proteins may have conserved roles in the SGs of male germ cells. PMID:27632217

  7. Temporally controlled site-specific mutagenesis in the germ cell lineage of the mouse testis.

    PubMed

    Weber, Philipp; Schuler, Michael; Gérard, Christelle; Mark, Manuel; Metzger, Daniel; Chambon, Pierre

    2003-02-01

    We have obtained a PrP-Cre-ER(T) transgenic mouse line (28.8) that selectively expresses in testis the tamoxifen-inducible Cre-ER(T) recombinase under the control of a mouse Prion protein (PrP) promoter-containing genomic fragment. Cre-ER(T) is expressed in spermatogonia and spermatocytes, but not in Sertoli and Leydig cells. We also established reporter PrP-L-EGFP-L transgenic mice harboring a LoxP-flanked enhanced green fluorescent protein (EGFP) Cre reporter cassette under the control of the same PrP promoter-containing genomic fragment that exhibits prominent EGFP expression in brain and testis. Using the PrP-L-EGFP-L as well as other Cre-reporter mice, we demonstrate that tamoxifen administration efficiently and selectively induces Cre-mediated recombination in the germ cell lineage. The established PrP-Cre-ER(T) line should provide a valuable tool for studying functions of germ cell-expressed genes involved in spermatogenesis. PMID:12533419

  8. A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells.

    PubMed

    Hehnly, Heidi; Canton, David; Bucko, Paula; Langeberg, Lorene K; Ogier, Leah; Gelman, Irwin; Santana, L Fernando; Wordeman, Linda; Scott, John D

    2015-09-25

    Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin(-/-) mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division.

  9. A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells

    PubMed Central

    Hehnly, Heidi; Canton, David; Bucko, Paula; Langeberg, Lorene K; Ogier, Leah; Gelman, Irwin; Santana, L Fernando; Wordeman, Linda; Scott, John D

    2015-01-01

    Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin−/− mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division. DOI: http://dx.doi.org/10.7554/eLife.09384.001 PMID:26406118

  10. Discrete somatic niches coordinate proliferation and migration of primordial germ cells via Wnt signaling.

    PubMed

    Cantú, Andrea V; Altshuler-Keylin, Svetlana; Laird, Diana J

    2016-07-18

    Inheritance depends on the expansion of a small number of primordial germ cells (PGCs) in the early embryo. Proliferation of mammalian PGCs is concurrent with their movement through changing microenvironments; however, mechanisms coordinating these conflicting processes remain unclear. Here, we find that PGC proliferation varies by location rather than embryonic age. Ror2 and Wnt5a mutants with mislocalized PGCs corroborate the microenvironmental regulation of the cell cycle, except in the hindgut, where Wnt5a is highly expressed. Molecular and genetic evidence suggests that Wnt5a acts via Ror2 to suppress β-catenin-dependent Wnt signaling in PGCs and limit their proliferation in specific locations, which we validate by overactivating β-catenin in PGCs. Our results suggest that the balance between expansion and movement of migratory PGCs is fine-tuned in different niches by the opposing β-catenin-dependent and Ror2-mediated pathways through Wnt5a This could serve as a selective mechanism to favor early and efficient migrators with clonal dominance in the ensuing germ cell pool while penalizing stragglers. PMID:27402951

  11. A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells.

    PubMed

    Hehnly, Heidi; Canton, David; Bucko, Paula; Langeberg, Lorene K; Ogier, Leah; Gelman, Irwin; Santana, L Fernando; Wordeman, Linda; Scott, John D

    2015-01-01

    Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin(-/-) mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division. PMID:26406118

  12. Differential expression of boule and dazl in adult germ cells of the Asian seabass.

    PubMed

    Dwarakanath, Manali; Lim, Menghuat; Xu, Hongyan; Hong, Yunhan

    2014-10-10

    Fertility genes boule and dazl constitute the evolutionarily conserved DAZ (Deleted in AZoospermia) family of RNA binding proteins essential for germline development across animal phyla. Here we report the cloning and expression analysis of boule and dazl from the Asian seabass (Lates calcarifer), a marine fish that undergoes sequential male-to-female sex reversal. Molecular cloning and sequence comparison led to the identification of boule and dazl cDNAs. RT-PCR analysis showed that both boule and dazl RNAs were restricted to the gonads among adult organs examined. Chromogenic in situ hybridization revealed germ cell-specific expression for both boule and dazl in female and male adults. Importantly, distinct differences were found between boule and dazl in terms of temporospatial expression and subcellular distribution. The boule RNA was abundant in late gametogenic cells except sperm. Interestingly, dazl expression increases in early oocytes and concentrates in a perinuclear speckle that appears to develop ultimately into the Balbiani body in advanced oocytes. The dazl RNA was found to be abundant in spermatocytes but hardly detectable in sperm. These data demonstrate that boule and dazl are germ cell markers in the adult Asian seabass, and that bisexual germline-specific expression has been conserved for boule and dazl in fish. PMID:25084124

  13. Intra-Testicular Signals Regulate Germ Cell Progression and Production of Qualitatively Mature Spermatozoa in Vertebrates

    PubMed Central

    Meccariello, Rosaria; Chianese, Rosanna; Chioccarelli, Teresa; Ciaramella, Vincenza; Fasano, Silvia; Pierantoni, Riccardo; Cobellis, Gilda

    2014-01-01

    Spermatogenesis, a highly conserved process in vertebrates, is mainly under the hypothalamic–pituitary control, being regulated by the secretion of pituitary gonadotropins, follicle stimulating hormone, and luteinizing hormone, in response to stimulation exerted by gonadotropin releasing hormone from hypothalamic neurons. At testicular level, gonadotropins bind specific receptors located on the somatic cells regulating the production of steroids and factors necessary to ensure a correct spermatogenesis. Indeed, besides the endocrine route, a complex network of cell-to-cell communications regulates germ cell progression, and a combination of endocrine and intra-gonadal signals sustains the production of high quality mature spermatozoa. In this review, we focus on the recent advances in the area of the intra-gonadal signals supporting sperm development. PMID:24847312

  14. Targeted expression in zebrafish primordial germ cells by Cre/loxP and Gal4/UAS systems.

    PubMed

    Xiong, Feng; Wei, Zhi-Qiang; Zhu, Zuo-Yan; Sun, Yong-Hua

    2013-10-01

    In zebrafish and other vertebrates, primordial germ cells (PGCs) are a population of embryonic cells that give rise to sperm and eggs in adults. Any type of genetically manipulated lines have to be originated from the germ cells of the manipulated founders, thus it is of great importance to establish an effective technology for highly specific PGC-targeted gene manipulation in vertebrates. In the present study, we used the Cre/loxP recombinase system and Gal4/UAS transcription system for induction and regulation of mRFP (monomer red fluorescent protein) gene expression to achieve highly efficient PGC-targeted gene expression in zebrafish. First, we established two transgenic activator lines, Tg(kop:cre) and Tg(kop:KalTA4), to express the Cre recombinases and the Gal4 activator proteins in PGCs. Second, we generated two transgenic effector lines, Tg(kop:loxP-SV40-loxP-mRFP) and Tg(UAS:mRFP), which intrinsically showed transcriptional silence of mRFP. When Tg(kop:cre) females were crossed with Tg(kop:loxP-SV40-loxP-mRFP) males, the loxP flanked SV40 transcriptional stop sequence was 100 % removed from the germ cells of the transgenic hybrids. This led to massive production of PGC-specific mRFP transgenic line, Tg(kop:loxP-mRFP), from an mRFP silent transgenic line, Tg(kop:loxP-SV40-loxP-mRFP). When Tg(kop:KalTA4) females were crossed with Tg(UAS:mRFP) males, the hybrid embryos showed PGC specifically expressed mRFP from shield stage till 25 days post-fertilization (pf), indicating the high sensitivity, high efficiency, and long-lasting effect of the Gal4/UAS system. Real-time PCR analysis showed that the transcriptional amplification efficiency of the Gal4/UAS system in PGCs can be about 300 times higher than in 1-day-pf embryos. More importantly, when the UAS:mRFP-nos1 construct was directly injected into the Tg(kop:KalTA4) embryos, it was possible to specifically label the PGCs with high sensitivity, efficiency, and persistence. Therefore, we have established two

  15. Localization of early germ cells in a stony coral, Euphyllia ancora: potential implications for a germline stem cell system in coral gametogenesis

    NASA Astrophysics Data System (ADS)

    Shikina, Shinya; Chung, Yi-Jou; Wang, Hsiang-Ming; Chiu, Yi-Ling; Shao, Zih-Fang; Lee, Yan-Horn; Chang, Ching-Fong

    2015-06-01

    Most corals exhibit annual or multiple gametogenic cycles. Thus far, coral gametogenesis has been studied in many species and locations during the past three decades; however, currently, only a few papers exist that describe the origin of germ cells, such as germline stem cells (GSCs), which support the continuous production of gametes in every reproductive cycle. To address this issue, in this study, we focused on and identified piwi gene, which has been used as a marker of germline cells, including GSCs, in various metazoans, in a scleractinian coral, Euphyllia ancora. Reverse-transcription PCR and Western blotting analyses revealed that E. ancora piwi-like ( Eapiwi) is expressed in mesentery tissues where the sites of gametogenesis are located for both sexes. Immunohistochemistry with a specific antibody against Eapiwi revealed strong immunoreactivity in the spermatogonia in males and in the oogonia and early oocytes in females, demonstrating that Eapiwi could be used as an early germ cell marker in E. ancora. Subsequent immunohistochemical analyses regarding the spatial and temporal distribution patterns of early germ cells in mesentery tissues revealed that early germ cells were present throughout the year in the mesentery tissue we examined, regardless of the sexual reproductive cycle. In particular, small numbers of early germ cells were observed in specific sites of mesentery tissues with fully matured gonads in both sexes. These early germ cells were not released together with mature gametes during the spawning period and remained in the mesentery tissues. These results suggested that these early germ cells most likely serve as a reservoir of germline cells and that some of these cells would produce differentiated germ cells for the upcoming sexual reproduction period; hence, these cells would function as GSCs. Our data provide new information for understanding continuous gamete production in corals.

  16. The differentiation potential of gingival mesenchymal stem cells induced by apical tooth germ cell-conditioned medium

    PubMed Central

    Chen, Yan; Liu, Hongwei

    2016-01-01

    Gingival-derived mesenchymal stem cells (GMSCs) have recently been harvested; however, the use of GMSCs in periodontal tissue engineering requires further study. The present study established an indirect co-culture system between rat apical tooth germ-conditioned medium (APTG-CM) and GMSCs, in order to determine the effects on periodontal tissue differentiation in vitro and in vivo. Using the limiting dilution technique, single-colony derived human GMSCs and periodontal ligament stem cells (PDLSCs) were isolated and expanded to obtain homogeneous populations. PDLSCs were used as a positive control group. Cell cycle distribution, alkaline phosphatase (ALP) activity, mineralization behavior, expression of genes associated with a cementoblast phenotype (osteocalcin, bone sialoprotein, ALP, type I collagen, cementum-derived protein 23), and in vivo differentiation capacities of GMSCs/PDLSCs co-cultured with APTG-CM were evaluated. Flow cytometry indicated that GMSCs and PDLSCs were positive for STRO-1 and CD105, whereas CD45 expression was negative. The cell types were capable of forming colonies, and of osteogenic and adipogenic differentiation in response to appropriate stimuli. The induced GMSCs and PDLSCs exhibited numerous characteristics associated with cementoblast lineages, as indicated by increased proliferation and ALP activity, and upregulated expression of cementum-associated genes in vitro. In vivo, cementum/periodontal ligament-like structures were shown to form along the dentin surface and ceramic bovine bone in GMSCs and PDLSCs induced by APTG-CM group. Conversely, vertical fibers could not insert in the control group, which was not co-cultured with APTG-CM. In conclusion, GMSCs are likely to have a role in periodontal tissue regeneration. In addition, APTG-CM was able to provide a cementogenic microenvironment and promote differentiation of GMSCs along the cementoblastic lineage. PMID:27600358

  17. NODAL secreted by male germ cells regulates the proliferation and function of human Sertoli cells from obstructive azoospermia and nonobstructive azoospermia patients

    PubMed Central

    Tian, Ru-Hui; Yang, Shi; Zhu, Zi-Jue; Wang, Jun-Long; Liu, Yun; Yao, Chencheng; Ma, Meng; Guo, Ying; Yuan, Qingqing; Hai, Yanan; Huang, Yi-Ran; He, Zuping; Li, Zheng

    2015-01-01

    This study was designed to explore the regulatory effects of male germ cell secreting factor NODAL on Sertoli cell fate decisions from obstructive azoospermia (OA) and nonobstructive azoospermia (NOA) patients. Human Sertoli cells and male germ cells were isolated using two-step enzymatic digestion and SATPUT from testes of azoospermia patients. Expression of NODAL and its multiple receptors in human Sertoli cells and male germ cells were characterized by reverse transcription-polymerase chain reaction (RT-PCR) and immunochemistry. Human recombinant NODAL and its receptor inhibitor SB431542 were employed to probe their effect on the proliferation of Sertoli cells using the CCK-8 assay. Quantitative PCR and Western blots were utilized to assess the expression of Sertoli cell functional genes and proteins. NODAL was found to be expressed in male germ cells but not in Sertoli cells, whereas its receptors ALK4, ALK7, and ACTR-IIB were detected in Sertoli cells and germ cells, suggesting that NODAL plays a regulatory role in Sertoli cells and germ cells via a paracrine and autocrine pathway, respectively. Human recombinant NODAL could promote the proliferation of human Sertoli cells. The expression of cell cycle regulators, including CYCLIN A, CYCLIN D1 and CYCLIN E, was not remarkably affected by NODAL signaling. NODAL enhanced the expression of essential growth factors, including GDNF, SCF, and BMP4, whereas SB431542 decreased their levels. There was not homogeneity of genes changes by NODAL treatment in Sertoli cells from OA and Sertoli cell-only syndrome (SCO) patients. Collectively, this study demonstrates that NODAL produced by human male germ cells regulates proliferation and numerous gene expression of Sertoli cells. PMID:26289399

  18. Phytoestrogens regulate the proliferation and expression of stem cell factors in cell lines of malignant testicular germ cell tumors.

    PubMed

    Hasibeder, Astrid; Venkataramani, Vivek; Thelen, Paul; Radzun, Heinz-Joachim; Schweyer, Stefan

    2013-11-01

    Phytoestrogens have been shown to exert anti-proliferative effects on different cancer cells. In addition it could be demonstrated that inhibition of proliferation is associated with downregulation of the known stem cell factors NANOG, POU5F1 and SOX2 in tumor cells. We demonstrate the potential of Belamcanda chinensis extract (BCE) and tectorigenin as anticancer drugs in cell lines of malignant testicular germ cell tumor cells (TGCT) by inhibition of proliferation and regulating the expression of stem cell factors. The TGCT cell lines TCam-2 and NTera-2 were treated with BCE or tectorigenin and MTT assay was used to measure the proliferation of tumor cells. In addition, the expression of stem cell factors was analyzed by quantitative PCR and western blot analysis. Furthermore, global expression analysis was performed by microarray technique. BCE and tectorigenin inhibited proliferation and downregulated the stem cell factors NANOG and POU5F1 in TGCT cells. In addition, gene expression profiling revealed induction of genes important for the differentiation and inhibition of oncogenes. Utilizing connectivity map in an attempt to elucidate mechanism underlying BCE treatments we found highly positive association to histone deacetylase inhibitors (HDACi) amongst others. Causing no histone deacetylase inhibition, the effects of BCE on proliferation and stem cell factors may be based on histone-independent mechanisms such as direct hyperacetylation of transcription factors. Based on these findings, phytoestrogens may be useful as new agents in the treatment of TGCT.

  19. Effect of lactoferrin on odontogenic differentiation of stem cells derived from human 3rd molar tooth germ.

    PubMed

    Taşlı, Pakize Neslihan; Sahin, Fikrettin

    2014-11-01

    Stem cell technology has been a great hope for the treatment of many common tissue regeneration-related diseases. Therefore, the main challenge in hard tissue engineering is to make a successful combination of stem cells and efficient inductors such as biomaterials or growth factors, in the concept of stem cell conversion into odontogenic cell. Even though lactoferrin has been reported to promote bone growth in vivo, the molecular mechanism of teeth formation has not been elucidated yet. Different concentrations of lactoferrin were prepared for the analysis of cell toxicity and differentiation evaluations. The odontogenic differentiation of human tooth germ stem cells (hTGSCs) was assessed by gene expression analysis, determination of protein levels in odontogenic differentiation-related protein, measuring alkaline phosphatase (ALP) activity, mineralization, and calcium deposit levels. Lactoferrin-treated group showed the highest ALP activity as opposed to the other groups which were untreated. In addition, the gene expression levels as well as the protein levels of odontogenic factors were found to be high in compared to the control groups. In the current study, it is shown for the first time that there is a significant increase in odontogenic differentiation capacity in hTGSCs when lactoferrin is applied in vitro. The study offers a considerable promise for the development of pulp regeneration by using stem cell technology combined with lactoferrin in functional tooth tissue engineering.

  20. Genome wide DNA methylation profiles provide clues to the origin and pathogenesis of germ cell tumors.

    PubMed

    Rijlaarsdam, Martin A; Tax, David M J; Gillis, Ad J M; Dorssers, Lambert C J; Koestler, Devin C; de Ridder, Jeroen; Looijenga, Leendert H J

    2015-01-01

    The cell of origin of the five subtypes (I-V) of germ cell tumors (GCTs) are assumed to be germ cells from different maturation stages. This is (potentially) reflected in their methylation status as fetal maturing primordial germ cells are globally demethylated during migration from the yolk sac to the gonad. Imprinted regions are erased in the gonad and later become uniparentally imprinted according to fetal sex. Here, 91 GCTs (type I-IV) and four cell lines were profiled (Illumina's HumanMethylation450BeadChip). Data was pre-processed controlling for cross hybridization, SNPs, detection rate, probe-type bias and batch effects. The annotation was extended, covering snRNAs/microRNAs, repeat elements and imprinted regions. A Hidden Markov Model-based genome segmentation was devised to identify differentially methylated genomic regions. Methylation profiles allowed for separation of clusters of non-seminomas (type II), seminomas/dysgerminomas (type II), spermatocytic seminomas (type III) and teratomas/dermoid cysts (type I/IV). The seminomas, dysgerminomas and spermatocytic seminomas were globally hypomethylated, in line with previous reports and their demethylated precursor. Differential methylation and imprinting status between subtypes reflected their presumed cell of origin. Ovarian type I teratomas and dermoid cysts showed (partial) sex specific uniparental maternal imprinting. The spermatocytic seminomas showed uniparental paternal imprinting while testicular teratomas exhibited partial imprinting erasure. Somatic imprinting in type II GCTs might indicate a cell of origin after global demethylation but before imprinting erasure. This is earlier than previously described, but agrees with the totipotent/embryonic stem cell like potential of type II GCTs and their rare extra-gonadal localization. The results support the common origin of the type I teratomas and show strong similarity between ovarian type I teratomas and dermoid cysts. In conclusion, we identified

  1. Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes

    PubMed Central

    Lee, Kyung Hoon; Lee, Won Young; Kim, Jin Hoi; Park, Chan Kyu; Do, Jeong Tae; Kim, Jae Hwan; Choi, Young Suk; Kim, Nam Hyung; Song, Hyuk

    2016-01-01

    Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs) have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (<2 months) limited their application to further investigations. To develop a culture method that allows for in vitro maintenance of GDCs for long periods, we subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs) and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4), which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells. PMID:26880974

  2. Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes.

    PubMed

    Lee, Kyung Hoon; Lee, Won Young; Kim, Jin Hoi; Park, Chan Kyu; Do, Jeong Tae; Kim, Jae Hwan; Choi, Young Suk; Kim, Nam Hyung; Song, Hyuk

    2016-01-01

    Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs) have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (<2 months) limited their application to further investigations. To develop a culture method that allows for in vitro maintenance of GDCs for long periods, we subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs) and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4), which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells.

  3. Telomeric repeat silencing in germ cells is essential for early development in Drosophila.

    PubMed

    Morgunova, Valeriya; Akulenko, Natalia; Radion, Elizaveta; Olovnikov, Ivan; Abramov, Yuri; Olenina, Ludmila V; Shpiz, Sergey; Kopytova, Daria V; Georgieva, Sofia G; Kalmykova, Alla

    2015-10-15

    The germline-specific role of telomeres consists of chromosome end elongation and proper chromosome segregation during early developmental stages. Despite the crucial role of telomeres in germ cells, little is known about telomere biology in the germline. We analyzed telomere homeostasis in the Drosophila female germline and early embryos. A novel germline-specific function of deadenylase complex Ccr4-Not in the telomeric transcript surveillance mechanism is reported. Depletion of Ccr4-Not complex components causes strong derepression of the telomeric retroelement HeT-A in the germ cells, accompanied by elongation of the HeT-A poly(A) tail. Dysfunction of transcription factors Woc and Trf2, as well as RNA-binding protein Ars2, also results in the accumulation of excessively polyadenylated HeT-A transcripts in ovaries. Germline knockdowns of Ccr4-Not components, Woc, Trf2 and Ars2, lead to abnormal mitosis in early embryos, characterized by chromosome missegregation, centrosome dysfunction and spindle multipolarity. Moreover, the observed phenotype is accompanied by the accumulation of HeT-A transcripts around the centrosomes in early embryos, suggesting the putative relationship between overexpression of telomeric transcripts and mitotic defects. Our data demonstrate that Ccr4-Not, Woc, Trf2 and Ars2, components of different regulatory pathways, are required for telomere protection in the germline in order to guarantee normal development.

  4. Relative susceptibilities of male germ cells to genetic defects induced by cancer chemotherapies

    SciTech Connect

    Wyrobek, A J; Schmid, T E; Marchetti, F

    2004-06-15

    Some chemotherapy regimens include agents that are mutagenic or clastogenic in model systems. This raises concerns that cancer survivors, who were treated before or during their reproductive years, may be at increased risks for abnormal reproductive outcomes. However, the available data from offspring of cancer survivors are limited, representing diverse cancers, therapies, time-to-pregnancies, and reproductive outcomes. Rodent breeding data after paternal exposures to individual chemotherapeutic agents illustrate the complexity of factors that influence the risk for transmitted genetic damage including agent, dose, endpoint, and the germ-cell susceptibility profiles that vary across agents. Direct measurements of chromosomal abnormalities in sperm of mice and humans by sperm FISH have corroborated the differences in germ-cell susceptibilities. The available evidence suggests that the risk of producing chromosomally defective sperm is highest during the first few weeks after the end of chemotherapy, and decays with time. Thus, sperm samples provided immediately after the initiation of cancer therapies may contain treatment-induced genetic defects that will jeopardize the genetic health of offspring.

  5. Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells.

    PubMed

    Vieillard, Jennifer; Paschaki, Marie; Duteyrat, Jean-Luc; Augière, Céline; Cortier, Elisabeth; Lapart, Jean-André; Thomas, Joëlle; Durand, Bénédicte

    2016-09-26

    The ciliary transition zone (TZ) is a complex structure found at the cilia base. Defects in TZ assembly are associated with human ciliopathies. In most eukaryotes, three protein complexes (CEP290, NPHP, and MKS) cooperate to build the TZ. We show that in Drosophila melanogaster, mild TZ defects are observed in the absence of MKS components. In contrast, Cby and Azi1 cooperate to build the TZ by acting upstream of Cep290 and MKS components. Without Cby and Azi1, centrioles fail to form the TZ, precluding sensory cilia assembly, and no ciliary membrane cap associated with sperm ciliogenesis is made. This ciliary cap is critical to recruit the tubulin-depolymerizing kinesin Klp59D, required for regulation of axonemal growth. Our results show that Drosophila TZ assembly in sensory neurons and male germ cells involves cooperative actions of Cby and Dila. They further reveal that temporal control of membrane cap assembly by TZ components and microtubule elongation by kinesin-13 is required for axoneme formation in male germ cells. PMID:27646273

  6. A Case of Mixed Germ Cell Tumor in the Intramedullary Spinal-cord.

    PubMed

    Nitta, Masahiro; Hoshi, Akio; Higure, Taro; Shimizu, Yuki; Nakajima, Nobuyuki; Hanai, Kazuya; Kawamura, Yoshiaki; Terachi, Toshiro

    2016-01-01

    A 28-year-old man was hospitalized with advancing paraplegia. Under the diagnosis of Guillain-Barre syndrome, steroid pulse therapy was administered and plasmapheresis was performed. However, the paraplegia gradually progressed. Subsequently, a spinal cord tumor was revealed by magnetic resonance imaging (MRI). The pathological diagnosis, obtained by open biopsy, confirmed a mixed germ cell tumor in the spinal cord. Multiple lung and lymph nodes metastases were also detected upon computed tomography, along with increased serum alpha-fetoprotein (33.9 ng/mL) and human chorionic gonadotropin (182.5 mIU/mL) levels. Consequently, he received chemotherapy comprising three courses of BEP (bleomycin, etoposide, and cisplatin) as first-line therapy, followed by four courses of TGN (paclitaxel, gemcitabine, and nedaplatin) as second-line treatment. As a result, the spinal cord lesion area was significantly decreased and the alpha-fetoprotein and human chorionic gonadotropin levels were normalized. Four years after chemotherapy, MRI revealed pituitary gland and pineal organ recurrence of the germ cell tumor and additional TGN chemotherapy was performed. PMID:27628608

  7. [Current status of therapy of CNS metastases of germ cell tumors].

    PubMed

    Clemm, C; Gerl, A; Wendt, T G; Pöllinger, B; Winkler, P A; Wilmanns, W

    1993-05-01

    The incidence of CNS metastases in germ cell tumors is 2-5