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Sample records for murine sertoli cells

  1. The murine seminiferous epithelial cycle is pre-figured in the Sertoli cells of the embryonic testis.

    PubMed

    Timmons, Paula M; Rigby, Peter W J; Poirier, Françoise

    2002-02-01

    The seminiferous epithelial cycle and spermatogenic wave are conserved features of vertebrate spermatogenic organisation that reflect the need for the rigorous maintenance of sperm production. Although the cycle and the wave of the adult seminiferous epithelium have been well characterised, particularly in rodent species, their developmental origins are unknown. We show that the Sertoli cells of the pre-pubertal mouse, including those of the germ cell-deficient XXSxra mutant, exhibit coordinated, cyclical patterns of gene expression, presaging the situation in the adult testis, where Sertoli cell function is coupled to the spermatogenic cycle. In the case of the galectin 1 gene (Lgals1), localised differential expression in the Sertoli cells can be traced back to neonatal and embryonic stages, making this the earliest known molecular marker of functional heterogeneity in mammalian testis cords. In addition, the timing of germ cell apoptosis in normal pre-pubertal testes is linked to the temporal cycle of the Sertoli cells. These data show that the cycle and wave of the murine seminiferous epithelium originate at a much earlier stage in development than was previously known, and that their maintenance in the early postnatal cords depends exclusively on the somatic cell lineages. PMID:11830565

  2. Sertoli cells as biochambers

    NASA Technical Reports Server (NTRS)

    Cameron, Don F. (Inventor); Sanberg, Paul R. (Inventor); Saporta, Samuel (Inventor); Hushen, Joelle J. (Inventor)

    2004-01-01

    According to the present invention, there is provided a biological chamber system having a biochamber defined by outer walls of Sertoli cells. Also provided is a transplantation facilitator including a biochamber. A method of making biochambers by co-culturing facilitator cells and therapeutic cells and then aggregating the facilitator celes is also provided. Also provided is a method of transplanting cells by incorporating transplant cells into a biochamber and transplanting the biochamber containing the transplant cells.

  3. Constitutive WNT/Beta-Catenin Signaling in Murine Sertoli Cells Disrupts Their Differentiation and Ability to Support Spermatogenesis1

    PubMed Central

    Tanwar, Pradeep S.; Kaneko-Tarui, Tomoko; Zhang, LiHua; Rani, Poonam; Taketo, Makoto M.; Teixeira, Jose

    2009-01-01

    Sertoli and germ cell interactions are essential for spermatogenesis and, thus, male fertility. Sertoli cells provide a specialized microenvironment for spermatogonial stem cells to divide, allowing both self-renewal and spermatogenesis. In the present study, we used mice with a conditional activated allele of the beta-catenin gene (Ctnnb1tm1Mmt/+) in Sertoli cells expressing Cre recombinase driven by the anti-Müllerian hormone (AMH; also known as Müllerian-inhibiting substance) type II receptor promoter (Amhr2tm3(cre)Bhr/+) to show that constitutively activated beta-catenin leads to their continuous proliferation and compromised differentiation. Compared to controls, Sertoli cells in mature mutant mice continue to express high levels of both AMH and glial cell-derived neurotrophic factor (GDNF), which normally are expressed only in immature Sertoli cells. We also show evidence that LiCl treatment, which activates endogenous nuclear beta-catenin activity, regulates both AMH and GDNF expression at the transcriptional level. The epididymides were devoid of sperm in the Amhr2tm3(cre)Bhr/+;Ctnnb1tm1Mmt/+ mice at all ages examined. We show that the mutant mice are infertile because of defective differentiation of germ cells and increased apoptosis, both of which are characteristic of GDNF overexpression in Sertoli cells. Constitutive activation of beta-catenin in Amhr2-null mice showed the same histology, suggesting that the phenotype was the result of persistent overexpression of GDNF. These results show that dysregulated wingless-related MMTV integration site/beta-catenin signaling in Sertoli cells inhibits their postnatal differentiation, resulting in increased germ cell apoptosis and infertility. PMID:19794154

  4. Unexpected requirement for a binding partner of the syntaxin family in phagocytosis by murine testicular Sertoli cells.

    PubMed

    Dong, Y-S; Hou, W-G; Li, Y; Liu, D-B; Hao, G-Z; Zhang, H-F; Li, J-C; Zhao, J; Zhang, S; Liang, G-B; Li, W

    2016-05-01

    Testicular phagocytosis by Sertoli cells (SCs) plays an essential role in the efficient clearance of apoptotic spermatogenic cells under both physiological and pathological conditions. However, the molecular mechanism underlying this unique process is poorly understood. Herein, we report for the first time that α-taxilin protein (TXLNA), a binding partner of the syntaxin family that functions as a central player in the intracellular vesicle traffic, was dominantly expressed in SCs. Induction of apoptosis in murine meiotic spermatocytes and haploid spermatids by busulfan treatment stimulated a significant increase of TXLNA in SCs at day (d) 14 and d 24 after busulfan treatment, respectively. Consistently, TXLNA expression was steadily upregulated when SCs were co-cultured with apoptotic germ cells (GCs). Moreover, using siRNA treatment, we found that ablation of endogenous TXLNA significantly impaired the phagocytotic capacity of SCs and thereby resulted in defective spermiogenesis and reduced fertility during the late recovery after testicular heat stress. Mechanistically, upregulation of TXLNA expression by apoptotic GCs was associated with the stabilization of ATP-binding cassette transporter 1 (ABCA1), a transporter-mediated lipid efflux from SCs and influencing male fertility. TXLNA acted as an upstream suppressor of ABCA1 ubiquitination and thus promoted ABCA1 stability and accumulation following GC apoptosis. We further provide in vitro evidence that epidermal growth factor receptor (EGFR)-mediated phosphorylation regulated ABCA1 ubiquitination and was enhanced by TXLNA deficiency during testicular phagocytosis. Taken together, the TXLNA/ABCA1 cascade may serve as an important feedback mechanism to modulate the magnitude of subsequent phagocytotic process of SCs in response to testicular injury. PMID:26494466

  5. The kinase DYRKIA regulates pre-mRNA splicing in spermatogonia and proliferation of spermatogonia and Sertoli cells by phosphorylating a spliceosomal component, SAP155, in postnatal murine testes.

    PubMed

    Eto, Ko; Sonoda, Yoshiyuki; Abe, Shin-Ichi

    2011-09-01

    SAP155 is an essential component of the spliceosome and its phosphorylation is required for splicing catalysis, but little is known concerning its function and regulation during spermatogenesis in postnatal murine testes. We report that inhibition of dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) IA strongly suppressed the mitogen-stimulated SAP155 phosphorylation and constitutive splicing of I?B pre-mRNA as well as the proliferation of spermatogonial and Sertoli cells in cultures of the 6-day post partum testes and a spermatogonial cell line, but not in a Sertoli cell line. Our findings suggest that the active spliceosome, containing SAP155 phosphorylated by DYRKIA, performs pre-mRNA splicing in spermatogonia during testicular development. PMID:21553260

  6. Postnatal development of bovine Sertoli cells.

    PubMed

    Sinowatz, F; Amselgruber, W

    1986-01-01

    The fine structure of bovine Sertoli cells was studied from the 4th to the 40th week post natum in order to correlate the progressive acquisition of normal adult morphology with functional development. The considerable increase in tubular size during the first 20 weeks is due to the proliferation of both presumptive Sertoli and germ cells. Aside from this, the presumptive Sertoli cells are seen to expand radially and lengthen considerably. From then on however, the observed increase in tubular diameter during the later period of postnatal development is solely due to the great increase in the number of germ cells. Presumptive Sertoli cells undergo morphological differentiation to mature Sertoli cells during the first 28 weeks of proliferative development. The maturation process includes distinct changes in cell shape, nucleus and cellular organelles, as well as an increase in and differentiation of Sertoli cell surface specializations. At 24 weeks the development of inter-Sertoli cell junctions has reached a point of differentiation where, in our opinion, a functional blood-testis barrier can be expected. During the first 8 weeks an extensive development of rough endoplasmic reticulum and a well-developed Golgi apparatus can be observed, which suggests a high secretory activity in the presumptive Sertoli cells at this time. We speculate that these secretory activities may play a role in the formation of the basal lamina which is extremely well developed during early postnatal life. The subsequent reduction of the basal lamina correlates well with diminished secretory activity in the Sertoli cells. PMID:3766996

  7. Cytoarchitectonical dynamic of Sertoli cells in Melanorivulus punctatus (Cyprinodontiformes: Rivulidae).

    PubMed

    Cassel, Mônica; Neves da Silva, Débora Fabiane; Ferreira, Adelina

    2013-02-01

    The Sertoli cell contributes to spermatogenesis acting in the differentiation of germ cells and being the only somatic cells present in the germinal compartment. So that spermatogenesis is primarily dependent of Sertoli-Sertoli and Sertoli-germ cell interactions once Sertoli cells provide critical factors necessary for a successful differentiation of germ cells to sperm. In teleost fish the cytoplasmic extensions of Sertoli cells support the cysts that remain closed until spermiogenesis. The number of Sertoli cells determines the testicular size, the number of testicular germ cells and the production capacity of spermatozoa. Our objective was to describe the morphology and the cytoarchitectonical dynamic of Sertoli cells in Melanorivulus punctatus, which were collected in the municipality of Chapada dos Guimarães, Mato Grosso, Brazil. The gonads were extracted and prepared according to histological routine for light microscopy and transmission electron microscopy. Sertoli cells have cytoplasmic extensions which provide the conformation of cysts in the interior of the lobes. These cells possess a polymorphic nucleus with a well-defined nuclear envelope and a prominent and eccentric nucleoli. Each cyst is sustained for more than one Sertoli cell and the cysts seem to share the Sertoli cells with each other regardless the stage of development of germ cells within these cysts. This disposal promotes a reticulated arrangement of Sertoli cells. The Sertoli cells lining the ducts assume rectangular shape with rounded nucleus. Thus, the morphological characteristics of Sertoli cells observed did not differ from what has been described for other teleosts. Despite the similarity in the morphology of these cells, we observed that its disposal in the extension of the gonad seems to differ from what is described for fish. The arrangement by which the cytoplasmic extensions of Sertoli cells connect the ends of lobes prevents the proliferation of spermatogonia on the lobe side walls and are only observed in the end of the lobes, which ensures the testicular characteristic-lobular restricted in Atherinomorpha. PMID:23245812

  8. Sertoli Cell Differentiation in Pubertal Boars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Meishan boars experience puberty at a younger age than crossbred (BX) boars in association with earlier cessation of Sertoli cell proliferation and smaller post pubertal testicular size. The current study defined changes in expression, assessed by immunohistochemistry, of anti-Mullerian hormone (AMH...

  9. Apoptosis of Sertoli cells after conditional ablation of murine double minute 2 (Mdm2) gene is p53-dependent and results in male sterility.

    PubMed

    Fouchécourt, S; Livera, G; Messiaen, S; Fumel, B; Parent, A-S; Marine, J-C; Monget, P

    2016-03-01

    Beside its well-documented role in carcinogenesis, the function of p53 family has been more recently revealed in development and female reproduction, but it is still poorly documented in male reproduction. We specifically tested this possibility by ablating Mdm2, an E3 ligase that regulates p53 protein stability and transactivation function, specifically in Sertoli cells (SCs) using the AMH-Cre line and created the new SC-Mdm2(-/-) line. Heterozygous SC-Mdm2(-/+) adult males were fertile, but SC-Mdm2(-/-) males were infertile and exhibited: a shorter ano-genital distance, an extra duct along the vas deferens that presents a uterus-like morphology, degenerated testes with no organized seminiferous tubules and a complete loss of differentiated germ cells. In adults, testosterone levels as well as StAR, P450c17 (Cyp17a1) and P450scc (Cyp11a1) mRNA levels decreased significantly, and both plasma LH and FSH levels increased. A detailed investigation of testicular development indicated that the phenotype arose during fetal life, with SC-Mdm2(-/-) testes being much smaller at birth. Interestingly, Leydig cells remained present until adulthood and fetal germ cells abnormally initiated meiosis. Inactivation of Mdm2 in SCs triggered p53 activation and apoptosis as early as 15.5 days post conception with significant increase in apoptotic SCs. Importantly, testis development occurred normally in SC-Mdm2(-/-) lacking p53 mice (SC-Mdm2(-/-)p53(-/-)) and accordingly, these mice were fertile indicating that the aforementioned phenotypes are entirely p53-dependent. These data not only highlight the importance of keeping p53 in check for proper testicular development and male fertility but also certify the critical role of SCs in the maintenance of meiotic repression. PMID:26470726

  10. Defined pattern of Sertoli cell differentiation in pubertal porcine testes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Number of Sertoli cells is a primary determinant of mature testicular size and sperm production. In boars, formation of the blood/testis barrier, which occurs by 4 mo of age in commercial breeds, signals the end of Sertoli cell proliferation. Previous studies established that expression of p27Kip1, ...

  11. Sertoli-Leydig cell tumor

    MedlinePlus

    ... the female ovaries, usually in younger women. The cancer cells release a male sex hormone. As a result, the woman may develop symptoms such as: A deep voice Enlarged clitoris Facial hair Loss in breast size Stopping of menstrual periods Pain in the lower ...

  12. Characterization and Functionality of Proliferative Human Sertoli Cells

    PubMed Central

    Chui, Kitty; Trivedi, Alpa; Cheng, C. Yan; Cherbavaz, Diana B.; Dazin, Paul F.; Huynh, Ai Lam Thu; Mitchell, James B.; Rabinovich, Gabriel A.; Noble-Haeusslein, Linda J.; John, Constance M.

    2014-01-01

    It has long been thought that mammalian Sertoli cells are terminally differentiated and nondividing postpuberty. For most previous in vitro studies immature rodent testes have been the source of Sertoli cells and these have shown little proliferative ability when cultured. We have isolated and characterized Sertoli cells from human cadaveric testes from seven donors ranging from 12 to 36 years of age. The cells proliferated readily in vitro under the optimized conditions used with a doubling time of approximately 4 days. Nuclear 5-ethynyl-2′-deoxyuridine (EdU) incorporation confirmed that dividing cells represented the majority of the population. Classical Sertoli cell ultrastructural features, lipid droplet accumulation, and immunoexpression of GATA-4, Sox9, and the FSH receptor (FSHr) were observed by electron and fluorescence microscopy, respectively. Flow cytometry revealed the expression of GATA-4 and Sox9 by more than 99% of the cells, and abundant expression of a number of markers indicative of multipotent mesenchymal cells. Low detection of endogenous alkaline phosphatase activity after passaging showed that few peritubular myoid cells were present. GATA-4 and SOX9 expression were confirmed by reverse transcription polymerase chain reaction (RT-PCR), along with expression of stem cell factor (SCF), glial cell line-derived neurotrophic factor (GDNF), and bone morphogenic protein 4 (BMP4). Tight junctions were formed by Sertoli cells plated on transwell inserts coated with fibronectin as revealed by increased transepithelial electrical resistance (TER) and polarized secretion of the immunoregulatory protein, galectin-1. These primary Sertoli cell populations could be expanded dramatically in vitro and could be cryopreserved. The results show that functional human Sertoli cells can be propagated in vitro from testicular cells isolated from adult testis. The proliferative human Sertoli cells should have important applications in studying infertility, reproductive toxicology, testicular cancer, and spermatogenesis, and due to their unique biological properties potentially could be useful in cell therapy. PMID:21054948

  13. Sertoli cells- Immunological sentinels of spermatogenesis

    PubMed Central

    Kaur, Gurvinder; Thompson, Lea Ann; Dufour, Jannette M.

    2014-01-01

    Testicular germ cells, which appear after the establishment of central tolerance, express novel cell surface and intracellular proteins that can be recognized as ‘foreign antigens’ by the host’s immune system. However, normally these germ cells do not evoke an auto-reactive immune response. The focus of this manuscript is to review the evidence that the Blood-Testis-Barrier (BTB)/Sertoli cell (SC) barrier along with the SCs ability to modulate the immune response is vital for protecting auto-antigenic germ cells. In normal testis, the BTB/SC barrier protects the majority of the auto-antigenic germ cells by limiting access by the immune system and sequestering these ‘new antigens’. SCs also modulate testis immune cells (induce regulatory immune cells) by expressing several immunoregulatory factors, thereby creating a local tolerogenic environment optimal for survival of nonsequesetred auto-antigenic germ cells. Collectively, the fortress created by the BTB/SC barrier along with modulation of the immune response is pivotal for completion of spermatogenesis and species survival. PMID:24603046

  14. Testicular histopathology associated with disruption of the Sertoli cell cytoskeleton

    PubMed Central

    Johnson, Kamin J

    2014-01-01

    Testicular histological alterations following Sertoli cell cytoskeleton disruption are numerous. The Sertoli cell cytoskeleton is comprised of intermediate filaments, microtubules, microfilaments and their direct interacting proteins and performs essential functions including structural support of the seminiferous epithelium, apicobasal movement of elongate spermatids, and release of elongate spermatids from the seminiferous epithelium during spermiation. This review summarizes the histological changes occurring after disruption of the Sertoli cell cytoskeleton, including the signature lesion of seminiferous epithelium sloughing. By presenting examples of histological changes after exposure to toxins or toxicants directly affecting the Sertoli cell cytoskeleton or genetic manipulations of this cytoskeleton, the toxicologist observing similar histological changes associated with exposure to novel compounds can use this information to generate hypotheses about a potential mode of action. PMID:26413393

  15. A Rare Cause of Prepubertal Gynecomastia: Sertoli Cell Tumor

    PubMed Central

    Dursun, Fatma; Su Dur, Şeyma Meliha; Şahin, Ceyhan; Kırmızıbekmez, Heves; Karabulut, Murat Hakan; Yörük, Asım

    2015-01-01

    Prepubertal gynecomastia due to testis tumors is a very rare condition. Nearly 5% of the patients with testicular mass present with gynecomastia. Sertoli cell tumors are sporadic in 60% of the reported cases, while the remaining is a component of multiple neoplasia syndromes such as Peutz-Jeghers syndrome and Carney complex. We present a 4-year-old boy with gynecomastia due to Sertoli cell tumor with no evidence of Peutz-Jeghers syndrome or Carney complex. PMID:26366315

  16. Retinol uptake and esterification in the rate sertoli cell

    SciTech Connect

    Shingleton, J.L.

    1989-01-01

    The mechanism by which Sertoli cells accumulate retinol from retinol-binding protein (RBP) and the cellular metabolism of the accumulated retinol were investigated here using primary cultures of Sertoli cells isolated from 20 day-old rats. Cells incubated with ({sup 3}H)retinol-RBP accumulated ({sup 3}H)retinol in a time- and temperature dependent manner. The rate of ({sup 3}H) retinol accumulation declined when cellular ({sup 3}H) retinol concentrations reached approximately 0.53 pmol of retinol per {mu}g of cellular DNA, equivalent to the cellular content of cellular retinol-binding protein (CRBP). Excess unlabeled retinol-RBP competed with ({sup 3}H) retinol-RBP for ({sup 3}H) retinol delivery to the cells but free retinol did not. Furthermore, free ({sup 3}H) retinol associated with Sertoli cells in a non-saturable manner. The transport constant for specific retinol accumulation from RBP was 1.9 {mu}M, suggesting that any change in the normal circulating retinol-RBP level would directly affect the rate of retinol accumulation. Competition studies and studies using labeled RBP, cellular energy inhibitors, and lysosomal poisons indicated that the specific retinol accumulation by Sertoli cells occurs by interaction with a cell-surface receptor that internalizes retinol without concomitant internalization of RBP. Extraction and HPLC analysis of the radioactivity associated with Sertoli cells after incubation with ({sup 3}H) retinol-RBP yielded retinol and retinyl esters.

  17. Autophagy is required for ectoplasmic specialization assembly in sertoli cells.

    PubMed

    Liu, Chao; Wang, Hongna; Shang, Yongliang; Liu, Weixiao; Song, Zhenhua; Zhao, Haichao; Wang, Lina; Jia, Pengfei; Gao, Fengyi; Xu, Zhiliang; Yang, Lin; Gao, Fei; Li, Wei

    2016-05-01

    The ectoplasmic specialization (ES) is essential for Sertoli-germ cell communication to support all phases of germ cell development and maturity. Its formation and remodeling requires rapid reorganization of the cytoskeleton. However, the molecular mechanism underlying the regulation of ES assembly is still largely unknown. Here, we show that Sertoli cell-specific disruption of autophagy influenced male mouse fertility due to the resulting disorganized seminiferous tubules and spermatozoa with malformed heads. In autophagy-deficient mouse testes, cytoskeleton structures were disordered and ES assembly was disrupted. The disorganization of the cytoskeleton structures might be caused by the accumulation of a negative cytoskeleton organization regulator, PDLIM1, and these defects could be partially rescued by Pdlim1 knockdown in autophagy-deficient Sertoli cells. Altogether, our works reveal that the degradation of PDLIM1 by autophagy in Sertoli cells is important for the proper assembly of the ES, and these findings define a novel role for autophagy in Sertoli cell-germ cell communication. PMID:26986811

  18. Endocytic activity of Sertoli cells grown in bicameral culture chambers

    SciTech Connect

    Dai, R.X.; Djakiew, D.; Dym, M.

    1987-07-01

    Immature rat Sertoli cells were cultured for 7 to 14 days on Millipore filters impregnated with a reconstituted basement membrane extract in dual-environment (bicameral) culture chambers. Electron microscopy of the cultured cells revealed the presence of rod-shaped mitochondria, Golgi apparatus, rough endoplasmic reticulum, and Sertoli-Sertoli tight junctions, typical of these cells in vivo. The endocytic activity of both the apical and basal surfaces of the Sertoli cells was examined by either adding alpha 2-macroglobulin (alpha 2-M) conjugated to 20 nm gold particles to the apical chamber or by adding /sup 125/I labeled alpha 2-M to the basal chamber. During endocytosis from the apical surface of Sertoli cells, the alpha 2-M-gold particles were bound initially to coated pits and then internalized into coated vesicles within 5 minutes. After 10 minutes, the alpha 2-M-gold was found in multi-vesicular bodies (MVBs) and by 30 minutes it was present in the lysosomes. The proportion of alpha 2-M-gold found within endocytic cell organelles after 1 hour of uptake was used to estimate the approximate time that this ligand spent in each type of organelle. The alpha 2-M-gold was present in coated pits, coated vesicles, multivesicular bodies, and lysosomes for approximately 3, 11, 22, and 24 minutes, respectively. This indicates that the initial stages of endocytosis are rapid, whereas MVBs and lysosomes are relatively long-lived.

  19. Sertoli Cell-Only Syndrome: Behind the Genetic Scenes.

    PubMed

    Stouffs, Katrien; Gheldof, Alexander; Tournaye, Herman; Vandermaelen, Deborah; Bonduelle, Maryse; Lissens, Willy; Seneca, Sara

    2016-01-01

    Sertoli cell-only syndrome is defined by the complete absence of germ cells in testicular tissues and always results in male infertility. The aetiology often remains unknown. In this paper, we have investigated possible causes of Sertoli cell-only syndrome with a special focus on genetic causes. Our results show that, for a large part of the patients (>23% in an unselected group), the sex chromosomes are involved. The majority of patients had a Klinefelter syndrome, followed by patients with Yq microdeletions. Array comparative genomic hybridization in a selected group of "idiopathic patients" showed no known infertility related copy number variations. PMID:26925412

  20. Sertoli Cell-Only Syndrome: Behind the Genetic Scenes

    PubMed Central

    Stouffs, Katrien; Gheldof, Alexander; Tournaye, Herman; Vandermaelen, Deborah; Bonduelle, Maryse; Lissens, Willy; Seneca, Sara

    2016-01-01

    Sertoli cell-only syndrome is defined by the complete absence of germ cells in testicular tissues and always results in male infertility. The aetiology often remains unknown. In this paper, we have investigated possible causes of Sertoli cell-only syndrome with a special focus on genetic causes. Our results show that, for a large part of the patients (>23% in an unselected group), the sex chromosomes are involved. The majority of patients had a Klinefelter syndrome, followed by patients with Yq microdeletions. Array comparative genomic hybridization in a selected group of “idiopathic patients” showed no known infertility related copy number variations. PMID:26925412

  1. Reprogramming of Sertoli cells to fetal-like Leydig cells by Wt1 ablation

    PubMed Central

    Zhang, Lianjun; Chen, Min; Wen, Qing; Li, Yaqiong; Wang, Yaqing; Wang, Yanbo; Qin, Yan; Cui, Xiuhong; Yang, Lin; Huff, Vicki; Gao, Fei

    2015-01-01

    Sertoli and Leydig cells, the two major somatic cell types in the testis, have different morphologies and functions. Both are essential for gonad development and spermatogenesis. However, whether these cells are derived from the same progenitor cells and the mechanism regulating the differentiation between these two cell types during gonad development remains unclear. A previous study showed that overactivation of Ctnnb1 (cadherin-associated protein, beta 1) in Sertoli cells resulted in Sertoli cell tumors. Surprisingly, in the present study, we found that simultaneous deletion of Wilms’ Tumor Gene 1 (Wt1) and overactivation of Ctnnb1 in Sertoli cells led to Leydig cell-like tumor development. Lineage tracing experiments revealed that the Leydig-like tumor cells were derived from Sertoli cells. Further studies confirmed that Wt1 is required for the maintenance of the Sertoli cell lineage and that deletion of Wt1 resulted in the reprogramming of Sertoli cells to Leydig cells. Consistent with this interpretation, overexpression of Wt1 in Leydig cells led to the up-regulation of Sertoli cell-specific gene expression and the down-regulation of steroidogenic gene expression. These results demonstrate that the distinction between Sertoli cells and Leydig cells is regulated by Wt1, implying that these two cell types most likely originate from the same progenitor cells. This study thus provides a novel concept for somatic cell fate determination in testis development that may also represent an etiology of male infertility in human patients. PMID:25775596

  2. The roles and regulation of Sertoli cells in fate determinations of spermatogonial stem cells and spermatogenesis.

    PubMed

    Hai, Yanan; Hou, Jingmei; Liu, Yun; Liu, Yang; Yang, Hao; Li, Zheng; He, Zuping

    2014-05-01

    Spermatogenesis is a complex process by which spermatogonial stem cells (SSCs) self-renew and differentiate into spermatozoa under the elaborate coordination of testicular microenvironment, namely, niche. Sertoli cells, which locate around male germ cells, are the most critical component of the niche. Significant progress has recently been made by peers and us on uncovering the effects of Sertoli cells on regulating fate determinations of SSCs. Here we addressed the roles and regulation of Sertoli cells in normal and abnormal spermatogenesis. Specifically, we summarized the biological characteristics of Sertoli cells, and we emphasized the roles of Sertoli cells in mediating the self-renewal, differentiation, apoptosis, de-differentiation, and trans-differentiation of SSCs. The association between abnormal function of Sertoli cells and impaired spermatogenesis was discussed. Finally, we highlighted several issues to be addressed for further investigation on the effects and mechanisms of Sertoli cells in spermatogenesis. Since Sertoli cells are the key supportive cells for SSCs and they are very receptive to modification, a better understanding of the roles and regulation of Sertoli cells in SSC biology and spermatogenesis would make it feasible to identify novel targets for gene therapy of male infertility as well as seek more efficient and safer strategies for male contraception. PMID:24718316

  3. Sertoli Cells Maintain Leydig Cell Number and Peritubular Myoid Cell Activity in the Adult Mouse Testis

    PubMed Central

    Monteiro, Ana; Milne, Laura; Cruickshanks, Lyndsey; Jeffrey, Nathan; Guillou, Florian; Freeman, Tom C.; Mitchell, Rod T.; Smith, Lee B.

    2014-01-01

    The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health. PMID:25144714

  4. Lentiviral transduction of rat Sertoli cells as a means to modify gene expression

    PubMed Central

    Nicholls, Peter K.; Stanton, Peter G.; Rainczuk, Katarzyna E.; Qian, Hongwei; Gregorevic, Paul; Harrison, Craig A.

    2012-01-01

    Primary cell culture is an established and widely used technique to study Sertoli cell function in vitro. However, the relative difficulty of stably overexpressing or knocking down genes in Sertoli cell culture has limited progress in the field. In this technical report, we present a method to transduce 20 dpp rat Sertoli cell cultures with VSV-G pseudotyped lentiviral based vectors at a high rate (~80%), with stable reporter gene expression. Although high transgene expression is desirable, it was noted that at transduction rates > 60% inter-Sertoli cell tight junction integrity and, hence, Sertoli cell function, were transiently compromised. We envisage that this optimized procedure has the potential to stimulate Sertoli cell research, and motivate the use of Sertoli cells in various cell therapy applications. PMID:23248769

  5. Bilateral Sertoli cell adenoma in gonads, associated with serous cystadenoma.

    PubMed

    Fernandez-Vega, I; Santos-Juanes, J; García-Pravia, C

    2014-06-01

    Complete androgen insensitivity syndrome is an extremely infrequent disease. The patients exhibit female phenotype because of insensitivity to the androgen receptor and may develop tumors, especially in their undescended gonads. We report a case of bilateral Sertoli cell adenoma in gonads with unilateral serous cystadenoma, in an elderly phenotypic woman with primary amenorrhea. We also provide radiological and pathological studies. PMID:25119177

  6. Loss of Gata4 in Sertoli cells impairs the spermatogonial stem cell niche and causes germ cell exhaustion by attenuating chemokine signaling

    PubMed Central

    Chen, Su-Ren; Tang, Ji-Xin; Cheng, Jin-Mei; Li, Jian; Jin, Cheng; Li, Xiao-Yu; Deng, Shou-Long; Zhang, Yan; Wang, Xiu-Xia; Liu, Yi-Xun

    2015-01-01

    Sertoli cells, the primary somatic cell in the seminiferous epithelium, provide the spermatogonial stem cell (SSC) microenvironment (niche) through physical support and the expression of paracrine factors. However, the regulatory mechanisms within the SSC niche, which is primarily controlled by Sertoli cells, remain largely unknown. GATA4 is a Sertoli cell marker, involved in genital ridge initiation, sex determination and differentiation during the embryonic stage. Here, we showed that neonatal mice with a targeted disruption of Gata4 in Sertoli cells (Gata4flox/flox; Amh-Cre; hereafter termed Gata4 cKO) displayed a loss of the establishment and maintenance of the SSC pool and apoptosis of both gonocyte-derived differentiating spermatogonia and meiotic spermatocytes. Thus, progressive germ cell depletion and a Sertoli-cell-only syndrome were observed as early as the first wave of murine spermatogenesis. Transplantation of germ cells from postnatal day 5 (P5) Gata4 cKO mice into KitW/W-v recipient seminiferous tubules restored spermatogenesis. In addition, microarray analyses of P5 Gata4 cKO mouse testes showed alterations in chemokine signaling factors, including Cxcl12, Ccl3, Cxcr4 (CXCL12 receptor), Ccr1 (CCL3 receptor), Ccl9, Xcl1 and Ccrl2. Deletion of Gata4 in Sertoli cells markedly attenuated Sertoli cell chemotaxis, which guides SSCs or prospermatogonia to the stem cell niche. Finally, we showed that GATA4 transcriptionally regulated Cxcl12 and Ccl9, and the addition of CXCL12 and CCL9 to an in vitro testis tissue culture system increased the number of PLZF+ undifferentiated spermatogonia within Gata4 cKO testes. Together, these results reveal a novel role for GATA4 in controlling the SSC niche via the transcriptional regulation of chemokine signaling shortly after birth. PMID:26473289

  7. Rictor Regulates Spermatogenesis by Controlling Sertoli Cell Cytoskeletal Organization and Cell Polarity in the Mouse Testis.

    PubMed

    Dong, Heling; Chen, Zhenguo; Wang, Caixia; Xiong, Zhi; Zhao, Wanlu; Jia, Chunhong; Lin, Jun; Lin, Yan; Yuan, Weiping; Zhao, Allan Z; Bai, Xiaochun

    2015-11-01

    Maintenance of cell polarity is essential for Sertoli cell and blood-testis barrier (BTB) function and spermatogenesis; however, the signaling mechanisms that regulate the integrity of the cytoskeleton and polarity of Sertoli cells are not fully understood. Here, we demonstrate that rapamycin-insensitive component of target of rapamycin (TOR) (Rictor), a core component of mechanistic TOR complex 2 (mTORC2), was expressed in the seminiferous epithelium during testicular development, and was down-regulated in a cadmium chloride-induced BTB damage model. We then conditionally deleted the Rictor gene in Sertoli cells and mutant mice exhibited azoospermia and were sterile as early as 3 months old. Further study revealed that Rictor may regulate actin organization via both mTORC2-dependent and mTORC2-independent mechanisms, in which the small GTPase, ras-related C3 botulinum toxin substrate 1, and phosphorylation of the actin filament regulatory protein, Paxillin, are involved, respectively. Loss of Rictor in Sertoli cells perturbed actin dynamics and caused microtubule disarrangement, both of which accumulatively disrupted Sertoli cell polarity and BTB integrity, accompanied by testicular developmental defects, spermiogenic arrest and excessive germ cell loss in mutant mice. Together, these findings establish the importance of Rictor/mTORC2 signaling in Sertoli cell function and spermatogenesis through the maintenance of Sertoli cell cytoskeletal dynamics, BTB integrity, and cell polarity. PMID:26360620

  8. Changes of heavy metal, metallothionein and heat shock proteins in Sertoli cells induced by cadmium exposure.

    PubMed

    Kusakabe, Takahiko; Nakajima, Katsuyuki; Nakazato, Kyoumi; Suzuki, Keiji; Takada, Hisashi; Satoh, Takahiro; Oikawa, Masakazu; Arakawa, Kazuo; Nagamine, Takeaki

    2008-09-01

    In this study, we examined the levels of Cadmium (Cd), iron (Fe) and zinc (Zn), which were considered to be involved in Sertoli cell damage caused by Cd exposure. We also examined metallothionein (MT), heat shock protein 70 (Hsp70) and heme oxygenase-1 (HO-1) expressions in Sertoli cells induced by Cd exposure. Evaluation by the in-air micro-particle induced X-ray emission (PIXE) method revealed that Cd and Fe distribution was increased in the cytoplasm of Sertoli cells after Cd exposure. By contrast, Zn was decreased in the cytoplasm of Sertoli cells after Cd exposure. It was suggested that the target of Cd toxicity was the cytoplasm of Sertoli cells, Fe was considered to enhance damage to Sertoli cells caused by Cd exposure. The DNA fragmentation rate was determined by ELISA after Cd exposure to Sertoli cells. It remained essentially unchanged with 2.5 microM Cd exposure of Sertoli cells; however, MT, Hsp70 and HO-1 were significantly increased by Cd exposure. As a result, Cd-induced MT was protected Sertoli cells against apoptosis, and Cd-induced HO-1 was involved in protection against oxidative stress. Incidentally, MT, Hsp70 and HO-1 showed similar responses to Cd exposure. PMID:18556172

  9. Characterization of swine testicular cell line as immature porcine Sertoli cell line.

    PubMed

    Ma, Changping; Song, Huibin; Guan, Kaifeng; Zhou, Jiawei; Xia, Xuanyan; Li, Fenge

    2016-04-01

    Swine testicular (ST) cell line is isolated from swine fetal testes and has been widely used in biomedical research fields related to pig virus infection. However, the potential benefit and utilization of ST cells in boar reproductive studies has not been fully explored. As swine fetal testes mainly contain multiple types of cells such as Leydig cells, Sertoli cells, gonocytes, and peritubular myoid cells, it is necessary to clarify the cell type of ST cell line. In this study, we identified ST cell line was a collection of Sertoli cells by analyzing the unique morphological characteristic with satellite karyosomes and determining the protein expression of two markers (androgen-binding protein, ABP; Fas ligand, FASL) of Sertoli cells. Then ST cells were further confirmed to be immature Sertoli cells by examining the expression of three markers (anti-Mullerian hormone, AMH; keratin 18, KRT18; follicle-stimulating hormone receptor, FSHR). In conclusion, ST cells are a collection of immature Sertoli cells which can be good experimental materials for the researches involved in Sertoli cell functions and maturation, or even in boar reproductions. PMID:26744029

  10. Effects of simulated microgravity on mouse Sertoli cells in culture

    NASA Astrophysics Data System (ADS)

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

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

  11. Deletion of the tyrosine phosphatase Shp2 in Sertoli cells causes infertility in mice

    PubMed Central

    Hu, Xiaopeng; Tang, Zhenzhou; Li, Yang; Liu, Wensheng; Zhang, Shuang; Wang, Bingyan; Tian, Yingpu; Zhao, Yinan; Ran, Hao; Liu, Wenjie; Feng, Gen-Sheng; Shuai, Jianwei; Wang, Haibin; Lu, Zhongxian

    2015-01-01

    The male’s ability to reproduce is completely dependent on Sertoli cells. However, the mechanisms governing the functional integrity of Sertoli cells have remained largely unexplored. Here, we demonstrate that deletion of Shp2 in Sertoli cells results in infertility in mice. In Shp2 knockout mice (SCSKO), a normal population of Sertoli cells was observed, but the blood-testis barrier (BTB) was not formed. Shp2 ablation initiated the untimely and excessive differentiation of spermatogonial stem cells (SSCs) by disturbing the expression of paracrine factors. As a consequence, the process of spermatogenesis was disrupted, and the germ cells were depleted. Furthermore, Shp2 deletion impaired the cell junctions of the primary Sertoli cells and failed to support the clonal formation of SSCs co-cultured with SCSKO Sertoli cells. As expected, Shp2 restoration largely restores the cell junctions of the primary Sertoli cells and the clonal formation of SSCs. To identify the underlying mechanism, we further demonstrated that the absence of Shp2 suppressed Erk phosphorylation, and thus, the expression of follicle-stimulating hormone (FSH)- and testosterone-induced target genes. These results collectively suggest that Shp2 is a critical signaling protein that is required to maintain Sertoli cell function and could serve as a novel target for male infertility therapies. PMID:26265072

  12. In vitro effects of simulated microgravity on Sertoli cell function

    NASA Astrophysics Data System (ADS)

    Masini, M. A.; Prato, P.; Scarabelli, L.; Lanza, C.; Palmero, S.; Pointis, G.; Ricci, F.; Strollo, F.

    2011-02-01

    With the advent of space flights questions concerning the effects of microgravity (0×G) on human reproductive physiology have received great attention. The aim of this study was to evaluate the influence of 0×G on Sertoli cells. A Sertoli cell line from mouse testis (42GPA9) was analyzed for cytoskeletal and Sex Hormone Binding Globilin (SHBG) changes by immunohistochemistry, for antioxidant content by RT-PCR and for culture medium lactate concentrations by protein chemistry. Cells were cultured for 6, 24 and 48 h on a three-dimensional Random Positioning Machine (3D-RPM); static controls (1×G) were positioned on the supporting frame. At the end of each experiment, cultured cells were either fixed in paraformaldehyde or lysed and RNA-extracted or used for culture medium lactate measurements as needed. At 0×G, Sertoli cytoskeleton became disorganized, microtubules fragmented and SHBG undetectable already after 24 h, with alterations worsening by 48 h. It was evident that various antioxidant systems appreciably increased during the first 24 h but significantly decreased at 48 h. No changes occurred in the 1×G samples. Initially, 0×G seemed to disturb antioxidant protection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate production at 0×G slightly decreased after 24 h. Further experiments are needed in space to investigate upon steroidogenesis and germ cell differentiation within the testis, to rule out male infertility as a possible consequence, which could be a problem, as life expectancy increases.

  13. Activin A, a product of fetal Leydig cells, is a unique paracrine regulator of Sertoli cell proliferation and fetal testis cord expansion

    PubMed Central

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

    2010-01-01

    Formation of tubular structures relies upon complex interactions between adjacent epithelium and mesenchyme. In the embryonic testes, dramatic compartmentalization leads to the formation of testis cords (epithelium) and the surrounding interstitium (mesenchyme). Sertoli cells, the epithelial cell type within testis cords, produce signaling molecules to orchestrate testis cord formation. The interstitial fetal Leydig cells, however, are thought only to masculinize the embryo and are not known to be involved in testis cord morphogenesis. Contrary to this notion, we have identified activin A, a member of the TGF-β protein superfamily, as a product of the murine fetal Leydig cells that acts directly upon Sertoli cells to promote their proliferation during late embryogenesis. Genetic disruption of activin βA, the gene encoding activin A, specifically in fetal Leydig cells resulted in a failure of fetal testis cord elongation and expansion due to decreased Sertoli cell proliferation. Conditional inactivation of Smad4, the central component of TGF-β signaling, in Sertoli cells led to testis cord dysgenesis and proliferative defects similar to those of Leydig cell-specific activin βA knockout testes. These results indicate that activin A is the major TGF-β protein that acts directly on Sertoli cells. Testicular dysgenesis in activin βA and Smad4 conditional knockout embryos persists into adulthood, leading to low sperm production and abnormal testicular histology. Our findings challenge the paradigm that fetal testis development is solely under the control of Sertoli cells, by uncovering an active and essential role of fetal Leydig cells during testis cord morphogenesis. PMID:20498064

  14. [Experimental Evidence of Proliferation and Reproduction of Highly Differentiated Sertoli Cells].

    PubMed

    Zakhidova, S T; Marshak, T L

    2015-01-01

    The main results of studies regarding the biology of Sertoli cells under various experimental conditions are considered. Possible potential mechanisms underlying the transition of highly differentiated Sertoli cells to dedifferentiation, limited by proliferation and reproduction and not accompanied by significant phenotypic changes, are discussed. PMID:26415275

  15. Enhanced mitophagy in Sertoli cells of ethanol-treated rats: morphological evidence and clinical relevance.

    PubMed

    Eid, Nabil; Ito, Yuko; Otsuki, Yoshinori

    2012-02-01

    Although chronic ethanol consumption results in Sertoli cell vacuolization and augmented testicular germ cell apoptosis via death receptor and mitochondrial pathways, Sertoli cells are resistant to apoptosis. The aim of this study was to examine whether the activation of autophagy in the Sertoli cells of ethanol-treated rats (ETR) may have a role in their survival. Adult Wistar rats were fed either 5% ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 12 weeks. The TUNEL method demonstrated that Sertoli cells were always TUNEL-negative despite the presence of many apoptotic germ cells in ETR, supporting our previous studies. Electron microscopy revealed the presence of large numbers of autophagic vacuoles (AVs) in Sertoli cells of ETR compared to few AVs in control testes. Most of the AVs in Sertoli cells of ETR enveloped and sequestered damaged and abnormally shaped mitochondria, without cytoplasm, indicating mitochondrial autophagy (mitophagy). Immuno-electron microscopy showed the localization of LC3, a specific marker of early AVs (autophagosomes), around AVs sequestering mitochondria in Sertoli cells of ETR. Immunohistochemical staining of LC3 demonstrated a punctate pattern in Sertoli cells of ETR, confirming the formation of autophagosomes, while LC3 puncta were almost absent in control testes. Moreover, increased immunoreactivity of LAMP-2, a lysosomal membrane protein and marker of late AVs (autolysosomes), was mainly observed in Sertoli cells of ETR, with weaker expression in control testes. Via the deletion of pro-apoptotic damaged mitochondria, enhanced Sertoli cell mitophagy in ETR may be an anti-apoptotic mechanism that is essential for spermatogenesis. PMID:22076330

  16. MiRNA-133b promotes the proliferation of human Sertoli cells through targeting GLI3

    PubMed Central

    Yao, Chencheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Chen, Zheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

    2016-01-01

    Sertoli cells play critical roles in regulating spermatogenesis and they can be reprogrammed to the cells of other lineages, highlighting that they have significant applications in reproductive and regenerative medicine. The fate determinations of Sertoli cells are regulated precisely by epigenetic factors. However, the expression, roles, and targets of microRNA (miRNA) in human Sertoli cells remain unknown. Here we have for the first time revealed that 174 miRNAs were distinctly expressed in human Sertoli cells between Sertoli-cell-only syndrome (SCOS) patients and obstructive azoospermia (OA) patients with normal spermatogenesis using miRNA microarrays and real time PCR, suggesting that these miRNAs may be associated with the pathogenesis of SCOS. MiR-133b is upregulated in Sertoli cells of SCOS patients compared to OA patients. Proliferation assays with miRNA mimics and inhibitors showed that miR-133b enhanced the proliferation of human Sertoli cells. Moreover, we demonstrated that GLI3 was a direct target of miR-133b and the expression of Cyclin B1 and Cyclin D1 was enhanced by miR-133b mimics but decreased by its inhibitors. Gene silencing of GLI3 using RNA inference stimulated the growth of human Sertoli cells. Collectively, miR-133b promoted the proliferation of human Sertoli cells by targeting GLI3. This study thus sheds novel insights into epigenetic regulation of human Sertoli cells and the etiology of azoospermia and offers new targets for treating male infertility PMID:26755652

  17. Long-term culture and analysis of cashmere goat Sertoli cells.

    PubMed

    Su, Huimin; Luo, Fenhua; Bao, Jiajing; Wu, Sachula; Zhang, Xueming; Zhang, Yan; Duo, Shuguang; Wu, Yingji

    2014-12-01

    Sertoli cells have important functions in the testis for spermatogenesis. Thus, Sertoli cell culture systems have been established in many animals, such as rat, mouse, human, dog, cow, and pig, but a goat culture has not been reported. This study describes the isolation and culture of Sertoli cells from 3- to 4-month-old cashmere goat (Capra hircus) testes. These proliferative cells were expanded for 20 passages and repeatedly cryopreserved in vitro, in contrast to previous study in human, of which maintain steady growth for up to seven passages and only passages 1 to 5 could be refrozen. The microstructure and ultrastructure of the culture were typical of Sertoli cells, bearing irregular nuclei and a cytoplasm that was rich in smooth and rough endoplasmic reticulum, mitochondria, Golgi, lysosomes, lipid drops, and glycogenosomes. By immunofluorescence analysis, the all cells expressed SRY-related HMG box gene 9 (Sox9). Growth curves and 5-bromo-2'-deoxyuridine (BrdU) incorporation were used to analyze the proliferation of the cultured cells. With increasing passage times, the proliferation of the Sertoli cells declined, but the transcription of glial cell-derived neurotrophic factor (GDNF), stem cell factor (SCF), and β1-integrin was constant. By flow cytometry, the cells retained the ability to proliferate after 5 yr of cryopreservation. Thus, cashmere goat Sertoli cells have significant proliferative potential in vitro, expressing germ cell regulatory factors and have important applications in studying Sertoli cell-germ cell interactions, spermatogenesis, reproductive toxicology, and male infertility. PMID:25164184

  18. Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

    SciTech Connect

    Fan, Ping; He, Lan; Pu, Dan; Lv, Xiaohong; Zhou, Wenxu; Sun, Yining; Hu, Nan

    2011-01-21

    Research highlights: {yields} The proliferation of dramatic increased by co-cultured with Sertoli cells. {yields} VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. {yields} The MHC expression of ECs induced by INF-{gamma} and IL-6, IL-8 and sICAM induced by TNF-{alpha} decreased respectively after co-cultured with Sertoli cells. {yields} ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10{sup 3}, 1 x 10{sup 4} or 1 x 10{sup 5} cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-{gamma} and TNF-{alpha} were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10{sup 4} cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 x 10{sup 4} cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-{gamma}-induced MHC II antigen expression in co-cultured ECs compared with single culture group (P < 0.05). TNF-{alpha} induced the expression of IL-6, IL-8 and sICAM in ECs. When co-cultured with Sertoli cells, their expressions were significantly lower than in the EC single culture group (P < 0.05). ECs co-cultured with Sertoli cells also did not significantly increase the stimulation index of spleen lymphocytes compared to the single culture group (P < 0.05). Our results suggested that co-culturing with Sertoli cells can significantly promote the proliferation of ECs, accelerate post-transplant angiogenesis, while reduce EC immunogenicity and stimulus to lymphocytes.

  19. Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

    SciTech Connect

    Zhang, Fenxi; Hong, Yan; Liang, Wenmei; Ren, Tongming; Jing, Suhua; Lin, Juntang

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). Black-Right-Pointing-Pointer Presence of SCs dramatically increased proliferation and migration of UCMSCs. Black-Right-Pointing-Pointer Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of 'nurse' cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.

  20. Roles of miRNAs in microcystin-LR-induced Sertoli cell toxicity

    SciTech Connect

    Zhou, Yuan; Wang, Hui; Wang, Cong; Qiu, Xuefeng; Benson, Mikael; Yin, Xiaoqin; Xiang, Zou; Li, Dongmei; and others

    2015-08-15

    Microcystin (MC)-LR, a cyclic heptapeptide, is a potent reproductive system toxin. To understand the molecular mechanisms of MC-induced reproductive system cytotoxicity, we evaluated global changes of miRNA and mRNA expression in mouse Sertoli cells following MC-LR treatment. Our results revealed that the exposure to MC-LR resulted in an altered miRNA expression profile that might be responsible for the modulation of mRNA expression. Bio-functional analysis indicated that the altered genes were involved in specific cellular processes, including cell death and proliferation. Target gene analysis suggested that junction injury in Sertoli cells exposed to MC-LR might be mediated by miRNAs through the regulation of the Sertoli cell-Sertoli cell pathway. Collectively, these findings may enhance our understanding on the modes of action of MC-LR on mouse Sertoli cells as well as the molecular mechanisms underlying the toxicity of MC-LR on the male reproductive system. - Highlights: • miRNAs were altered in Sertoli cells exposed to MC-LR. • Alerted genes were involved in different cell functions including the cell morphology. • MC-LR adversely affected Sertoli cell junction formation through the regulating miRNAs.

  1. Expression of Dominant-Negative Thyroid Hormone Receptor Alpha1 in Leydig and Sertoli Cells Demonstrates No Additional Defect Compared with Expression in Sertoli Cells Only

    PubMed Central

    Fumel, Betty; Froment, Pascal; Holzenberger, Martin; Livera, Gabriel; Monget, Philippe; Fouchécourt, Sophie

    2015-01-01

    Background In the testis, thyroid hormone (T3) regulates the number of gametes produced through its action on Sertoli cell proliferation. However, the role of T3 in the regulation of steroidogenesis is still controversial. Methods The TRαAMI knock-in allele allows the generation of transgenic mice expressing a dominant-negative TRα1 (thyroid receptor α1) isoform restricted to specific target cells after Cre-loxP recombination. Here, we introduced this mutant allele in both Sertoli and Leydig cells using a novel aromatase-iCre (ARO-iCre) line that expresses Cre recombinase under control of the human Cyp19(IIa)/aromatase promoter. Findings We showed that loxP recombination induced by this ARO-iCre is restricted to male and female gonads, and is effective in Sertoli and Leydig cells, but not in germ cells. We compared this model with the previous introduction of TRαAMI specifically in Sertoli cells in order to investigate T3 regulation of steroidogenesis. We demonstrated that TRαAMI-ARO males exhibited increased testis weight, increased sperm reserve in adulthood correlated to an increased proliferative index at P3 in vivo, and a loss of T3-response in vitro. Nevertheless, TRαAMI-ARO males showed normal fertility. This phenotype is similar to TRαAMI-SC males. Importantly, plasma testosterone and luteinizing hormone levels, as well as mRNA levels of steroidogenesis enzymes StAR, Cyp11a1 and Cyp17a1 were not affected in TRαAMI-ARO. Conclusions/Significance We concluded that the presence of a mutant TRαAMI allele in both Leydig and Sertoli cells does not accentuate the phenotype in comparison with its presence in Sertoli cells only. This suggests that direct T3 regulation of steroidogenesis through TRα1 is moderate in Leydig cells, and that Sertoli cells are the main target of T3 action in the testis. PMID:25793522

  2. Interleukin 1alpha-induced disruption of the Sertoli cell cytoskeleton affects gap junctional communication.

    PubMed

    Chojnacka, Katarzyna; Bilinska, Barbara; Mruk, Dolores D

    2016-05-01

    Gap junctions (GJ) are transmembrane channels that connect the cytoplasms of adjacent cells, thereby facilitating the rapid exchange of ions, second messengers, and metabolites smaller than 1kDa. Connexin 43, the best-studied GJ protein, is a component of the Sertoli cell barrier/blood-testis barrier (BTB). To gain insight into the biology of the BTB, we investigated the effects of interleukin 1alpha (IL1A), a pro-inflammatory cytokine that disrupts BTB function, on gap junctional communication (GJC) in Sertoli cells. Compared with controls, the levels of connexin 43 and connexin 43 (Ser 368) increased ~30- and 20-fold, respectively, at 24h after IL1A treatment. To assess GJ function, we investigated fluorescence recovery in photobleached Sertoli cells after vehicle or IL1A treatment. Compared with the control, IL1A affected the ability of calcein to return to photobleached cells, indicating that GJC was compromised. To explain the effects of IL1A on GJ function, the involvement of the Sertoli cell cytoskeleton was investigated. Stress fibers aggregated at the periphery of Sertoli cells treated with IL1A. These results were substantiated by a biochemical assay that showed IL1A to disrupt the bundling of exogenous F-actin by Sertoli cells. In summary, IL1A regulates GJC in Sertoli cells, which is critical for BTB restructuring. PMID:26879129

  3. Expression of Genomic Functional Estrogen Receptor 1 in Mouse Sertoli Cells

    PubMed Central

    Lin, Jing; Zhu, Jia; Li, Xian; Li, Shengqiang; Lan, Zijian; Ko, Jay

    2014-01-01

    There is no consensus whether Sertoli cells express estrogen receptor 1 (Esr1). Reverse transcription-polymerase chain reaction, Western blot, and immunofluorescence demonstrated that mouse Sertoli cell lines, TM4, MSC-1, and 15P-1, and purified primary mouse Sertoli cells (PSCs) contained Esr1 messenger RNA and proteins. Incubation of Sertoli cells with 17?-estradiol (E2) or ESR1 agonist stimulated the expression of an estrogen responsive gene Greb1, which was prevented by ESR inhibitor or ESR1 antagonist. Overexpression of Esr1 in MSC-1 enhanced E2-induced Greb1 expression, while knockdown of Esr1 by small interfering RNA in TM4 attenuated the response. Furthermore, E2-induced Greb1 expression was abolished in the PSCs isolated from Amh-Cre/Esr1-floxed mice in which Esr1 in Sertoli cells were selectively deleted. Chromatin immunoprecipitation assays indicated that E2-induced Greb1 expression in Sertoli cells was mediated by binding of ESR1 to estrogen responsive elements. In summary, ligand-dependent nuclear ESR1 was present in mouse Sertoli cells and mediates a classical genomic action of estrogens. PMID:24615934

  4. Structural characterization of proteoglycans produced by testicular peritubular cells and Sertoli cells

    SciTech Connect

    Skinner, M.K.; Fritz, I.B.

    1985-09-25

    The structural characteristics of proteoglycans produced by seminiferous peritubular cells and by Sertoli cells are defined. Peritubular cells secrete two proteoglycans designated PC I and PC II. PC I is a high molecular mass protein containing chondroitin glycosaminoglycan (GAG) chains (maximum 70 kDa). PC II has a protein core of 45 kDa and also contains chondroitin GAG chains (maximum 70 kDa). Preliminary results imply that PC II may be a degraded or processed form of PC I. Sertoli cells secrete two different proteoglycans, designated SC I and SC II. SC I is a large protein containing both chondroitin (maximum 62 kDa) and heparin (maximum 15 kDa) GAG chains. Results obtained suggest that this novel proteoglycan contains both chondroitin and heparin GAG chains bound to the same core protein. SC II has a 50-kDa protein core and contains chondroitin (maximum 25 kDa) GAG chains. A proteoglycan obtained from extracts of Sertoli cells is described which contains heparin (maximum 48 kDa) GAG chains. In addition, Sertoli cells secrete a sulfoprotein, SC III, which is not a proteoglycan. The stimulation by follicle-stimulating hormone of the incorporation of (TVS)SO2) U) into moieties secreted by Sertoli cells is shown to represent an increased production or sulfation of SC III, and not an increased production or sulfation of proteoglycans. Results are discussed in relation to the possible functions of proteoglycans in the seminiferous tubule.

  5. Male Differentiation of Germ Cells Induced by Embryonic Age-Specific Sertoli Cells in Mice1

    PubMed Central

    Ohta, Kohei; Yamamoto, Miyuki; Lin, Yanling; Hogg, Nathanael; Akiyama, Haruhiko; Behringer, Richard R.; Yamazaki, Yukiko

    2012-01-01

    ABSTRACT Retinoic acid (RA) is a meiosis-inducing factor. Primordial germ cells (PGCs) in the developing ovary are exposed to RA, resulting in entry into meiosis. In contrast, PGCs in the developing testis enter mitotic arrest to differentiate into prospermatogonia. Sertoli cells express CYP26B1, an RA-metabolizing enzyme, providing a simple explanation for why XY PGCs do not initiate meios/is. However, regulation of entry into mitotic arrest is likely more complex. To investigate the mechanisms that regulate male germ cell differentiation, we cultured XX and XY germ cells at 11.5 and 12.5 days postcoitus (dpc) with an RA receptor inhibitor. Expression of Stra8, a meiosis initiation gene, was suppressed in all groups. However, expression of Dnmt3l, a male-specific gene, during embryogenesis was elevated but only in 12.5-dpc XY germ cells. This suggests that inhibiting RA signaling is not sufficient for male germ cell differentiation but that the male gonadal environment also contributes to this pathway. To define the influence of Sertoli cells on male germ cell differentiation, Sertoli cells at 12.5, 15.5, and 18.5 dpc were aggregated with 11.5 dpc PGCs, respectively. After culture, PGCs aggregated with 12.5 dpc Sertoli cells increased Nanos2 and Dnmt3l expression. Furthermore, these PGCs established male-specific methylation imprints of the H19 differentially methylated domains. In contrast, PGCs aggregated with Sertoli cells at late embryonic ages did not commit to the male pathway. These findings suggest that male germ cell differentiation is induced both by inhibition of RA signaling and by molecule(s) production by embryonic age-specific Sertoli cells. PMID:22262692

  6. Induction of midbrain dopaminergic neurons from primate embryonic stem cells by coculture with sertoli cells.

    PubMed

    Yue, Fengming; Cui, Li; Johkura, Kohei; Ogiwara, Naoko; Sasaki, Katsunori

    2006-07-01

    The aim of this study was to produce dopaminergic neurons from primate embryonic stem (ES) cells following coculture with mouse Sertoli cells. After 3 weeks of induction, immunostaining revealed that 90% +/- 9% of the colonies contained tyrosine hydroxylase-positive (TH(+)) neurons, and 60% +/- 7% of the tubulin beta III-positive (Tuj III(+)) neurons were TH(+). Reverse transcription-polymerase chain reaction analyses showed that Sertoli-induced neurons expressed midbrain dopaminergic neuron markers, including TH, dopamine transporter, aromatic amino acid decarboxylase (AADC), receptors such as TrkB and TrkC, and transcription factors NurrI and Lmx1b. Neurons that had been differentiated on Sertoli cells were positive for Pax2, En1, and AADC, midbrain-related markers, and negative for dopamine-beta-hydroxylase, a marker of noradrenergic neurons. These Sertoli cell-induced dopaminergic cells can release dopamine when depolarized by high K(+). Sertoli cell-conditioned medium contained glial cell line-derived neurotrophic factor (GDNF) and supported neuronal differentiation. After pretreatment with anti-GDNF antibody, the percentage of Tuj III(+) colonies was reduced to 14%. Thus, GDNF contributed significantly to inducing primate ES cells into dopaminergic neurons. When transplanted into a 6-hydroxydopamine-treated Parkinson's disease model, primate-derived dopaminergic neurons integrated into the mouse striatum. Two weeks after transplantation, surviving TH(+) cells were present. These TH(+) cells survived for 2 months. Therefore, the induction method of coculture ES cells with Sertoli cells provides an unlimited source of primate cells for the study of pathogenesis and transplantation in Parkinson's disease. PMID:16822882

  7. The Warburg Effect Revisited—Lesson from the Sertoli Cell

    PubMed Central

    Oliveira, Pedro F.; Martins, Ana D.; Moreira, Ana C.; Cheng, C. Yan; Alves, Marco G.

    2016-01-01

    Otto Warburg observed that cancerous cells prefer fermentative instead of oxidative metabolism of glucose, although the former is in theory less efficient. Since Warburg’s pioneering works, special attention has been given to this difference in cell metabolism. The Warburg effect has been implicated in cell transformation, immortalization, and proliferation during tumorigenesis. Cancer cells display enhanced glycolytic activity, which is correlated with high proliferation, and thus, glycolysis appears to be an excellent candidate to target cancer cells. Nevertheless, little attention has been given to noncancerous cells that exhibit a “Warburg-like” metabolism with slight, but perhaps crucial, alterations that may provide new directions to develop new and effective anticancer therapies. Within the testis, the somatic Sertoli cell (SC) presents several common metabolic features analogous to cancer cells, and a clear “Warburg-like” metabolism. Nevertheless, SCs actively proliferate only during a specific time period, ceasing to divide in most species after puberty, when they become terminally differentiated. The special metabolic features of SC, as well as progression from the immature but proliferative state, to the mature nonproliferative state, where a high glycolytic activity is maintained, make these cells unique and a good model to discuss new perspectives on the Warburg effect. Herein we provide new insight on how the somatic SC may be a source of new and exciting information concerning the Warburg effect and cell proliferation. PMID:25043918

  8. Cytokines produced by microwave-radiated Sertoli cells interfere with spermatogenesis in rat testis.

    PubMed

    Wu, H; Wang, D; Shu, Z; Zhou, H; Zuo, H; Wang, S; Li, Y; Xu, X; Li, N; Peng, R

    2012-05-01

    Microwave radiation resulted in degeneration, apoptosis or necrosis in germ cells at different stages. The molecular mechanisms by which microwaves induce spermatogenesis disorder have not been completely understood. Sertoli cells play crucial roles in mammalian spermatogenesis. Cytokines produced by Sertoli cells play pleiotropic roles in different conditions. At physiologically low concentration, TNFα, IL-1β and IL-6 behave as survival factors; while under pathological condition, these cytokines can induce apoptosis in testis. The effects of cytokines produced by microwave-radiated Sertoli cells on spermatogenesis are poorly understood. The purpose of this study was to determine the effect of cytokines produced by microwave-radiated Sertoli cells on the germ cells. We focused the effect of TNFα, IL-1β and IL-6 on the germ cells. The results showed that TNFα, IL-1β and IL-6 were increased in Sertoli cells after exposure to microwave radiation. These up-regulated cytokines can induce apoptosis and lipid peroxidation in the membrane of germ cells. In addition, germ cell apoptosis was associated with the up-regulation of Bax/Bcl-2 and caspase-3. These results suggest that cytokines produced by microwave-radiated Sertoli cells may disrupt spermatogenesis. Our data provided novel insight into the injury mechanism of germ cells induced by microwave radiation. PMID:22211786

  9. Late morfofunctional alterations of the Sertoli cell caused by doxorubicin administered to prepubertal rats

    PubMed Central

    2012-01-01

    Background Doxorubicin is a potent chemotherapeutic drug used against a variety of cancers. It acts through interaction with polymerases and topoisomerase II and free radical production. Doxorubicin activity is not specific to cancer cells and can also damage healthy cells, especially those undergoing rapid proliferation, such as spermatogonia. In previous studies our group showed that etoposide, another topoisomarese II poison, causes irreversible damage to Sertoli cells. Thus, the aim of this study was to address the effects of doxorubicin on Sertoli cell morphology and function and on the seminiferous epithelium cycle when administered to prepubertal rats. Methods Prepubertal rats received the dose of 5?mg/Kg of doxorubicin, which was fractioned in two doses: 3?mg/Kg at 15dpp and 2?mg/Kg at 22dpp. The testes were collected at 40, 64 and 127dpp, fixed in Bouins liquid and submitted to transferrin immunolabeling for Sertoli cell function analysis. Sertoli cell morphology and the frequency of the stages of the seminiferous epithelium cycle were analyzed in PAS?+?H-stained sections. Results The rats treated with doxorubicin showed reduction of transferrin labeling in the seminiferous epithelium at 40 and 64dpp, suggesting that Sertoli cell function is altered in these rats. All doxorubicin-treated rats showed sloughing and morphological alterations of Sertoli cells. The frequency of the stages of the seminiferous epithelium cycle was also affected in all doxorubicin-treated rats. Conclusions and discussion These data show that doxorubicin administration during prepuberty causes functional and morphological late damage to Sertoli cells; such damage is secondary to the germ cell primary injury and contributed to enhance the spermatogenic harm caused by this drug. However, additional studies are required to clarify if there is also a direct effect of doxorubicin on Sertoli cells producing a primary damage on these cells. PMID:22967030

  10. Nxf3 is Expressed in Sertoli Cells, but is Dispensable for Spermatogenesis

    PubMed Central

    Zhou, Jian; Pan, Jieyan; Eckardt, Sigrid; Leu, N. Adrian; McLaughlin, K. John; Wang, P. Jeremy

    2011-01-01

    SUMMARY In eukaryotes, mRNA is actively exported to the cytoplasm by a family of nuclear RNA export factors (NXF). Four Nxf genes have been identified in the mouse: Nxf1, Nxf2, Nxf3, and Nxf7. Inactivation of Nxf2, a germ cell-specific gene, causes defects in spermatogenesis. Here we report that Nxf3 is expressed exclusively in Sertoli cells of the postnatal testis, in a developmentally regulated manner. Expression of Nxf3 coincides with the cessation of Sertoli cell proliferation and the beginning of their differentiation. Continued expression of Nxf3 in mature Sertoli cells of the adult is spermatogenesis stage-independent. Nxf3 is not essential for spermatogenesis, however, suggesting functional redundancy among Nxf family members. With its unique expression pattern in the testis, the promoter of Nxf3 can be used to drive postnatal Sertoli cell-specific expression of other proteins such as Cre recombinase. PMID:21308854

  11. The Sertoli cell: one hundred fifty years of beauty and plasticity.

    PubMed

    França, L R; Hess, R A; Dufour, J M; Hofmann, M C; Griswold, M D

    2016-03-01

    It has been one and a half centuries since Enrico Sertoli published the seminal discovery of the testicular 'nurse cell', not only a key cell in the testis, but indeed one of the most amazing cells in the vertebrate body. In this review, we begin by examining the three phases of morphological research that have occurred in the study of Sertoli cells, because microscopic anatomy was essentially the only scientific discipline available for about the first 75 years after the discovery. Biochemistry and molecular biology then changed all of biological sciences, including our understanding of the functions of Sertoli cells. Immunology and stem cell biology were not even topics of science in 1865, but they have now become major issues in our appreciation of Sertoli cell's role in spermatogenesis. We end with the universal importance and plasticity of function by comparing Sertoli cells in fish, amphibians, and mammals. In these various classes of vertebrates, Sertoli cells have quite different modes of proliferation and epithelial maintenance, cystic vs. tubular formation, yet accomplish essentially the same function but in strikingly different ways. PMID:26846984

  12. Sertoli cells in the testis of caecilians, Ichthyophis tricolor and Uraeotyphlus cf. narayani (Amphibia: Gymnophiona): light and electron microscopic perspective.

    PubMed

    Smita, Mathew; Oommen, Oommen V; George, Jancy M; Akbarsha, M A

    2003-12-01

    The caecilians have evolved a unique pattern of cystic spermatogenesis in which cysts representing different stages in spermatogenesis coexist in a testis lobule. We examined unsettled issues relating to the organization of the caecilian testis lobules, including the occurrence of a fatty matrix, the possibility of both peripheral and central Sertoli cells, the origin of Sertoli cells from follicular cells, and the disengagement of older Sertoli cells to become loose central Sertoli cells. We subjected the testis of Ichthyophis tricolor (Ichthyophiidae) and Uraeotyphlus cf. narayani (Uraeotyphliidae) from the Western Ghats of Kerala, India, to light and transmission electron microscopic studies. Irrespective of the functional state of the testis, whether active or regressed, Sertoli cells constitute a permanent feature of the lobules. The tall Sertoli cells adherent to the basal lamina with basally located pleomorphic nuclei extend deeper into the lobule to meet at the core. There they provide for association of germ cells at different stages of differentiation, an aspect that has earlier been misconceived as the fatty matrix. Germ cells up to the 4-cell stage remain in the intercalating region of the Sertoli cells and they are located at the apices of the Sertoli cells from the 8-cell stage onwards. The developing germ cells are intimately associated with the Sertoli cell adherent to the basal lamina until spermiation. There are ameboid cells in the core of the lobules that appear to interact with the germ cells at the face opposite to their attachment with the Sertoli cells. Adherence of the Sertoli cells to the basal lamina is a permanent feature of the caecilian testicular lobules. The ameboid cells in the core are neither Sertoli cells nor their degeneration products. PMID:14584033

  13. Testicular cytology indicates differences in Sertoli cell counts between "good freezer" and "poor freezer" bulls.

    PubMed

    Rajak, Shailendra Kumar; Thippeswamy, Vijetha Bajjalli; Kumaresan, Arumugam; Layek, Siddhartha Shankar; Mohanty, Tushar Kumar; Gaurav, Mukesh Kumar; Chakravarty, Atish Kumar; Datta, Tirtha Kumar; Manimaran, Ayyasamy; Prasad, Shiv

    2016-01-01

    In artificial insemination, poor quality of semen unsuitable for cryopreservation and susceptibility of spermatozoa to cryodamage in crossbred bulls have been a matter of concern. Present study was designed to identify the testicular cytology indices that might be used to predict the semen quality and cryotolerance of spermatozoa in bulls. Based on the ejaculate rejection rate and sperm cryotolerance, bulls (Holstein Friesian X Tharparkar crossbred) were classified into either good (producing good quality semen with spermatozoa having good cryotolerance; n = 4) or poor (producing poor quality semen with spermatozoa having poor cryotolerance; n = 4). Testicular cytology was studied in all the 8 bulls using fine needle aspiration technique. Testicular cytology of good bulls and poor bulls differed significantly. The proportion of Sertoli cells was significantly higher in good bulls (25.3 ± 1.6) compared to poor bulls (11.0 ± 0.8). The Sertoli cell index was 46.1 ± 5.0 in good bulls while it was only 13.8 ± 1.3 in poor bulls. The cut off values, as determined using Receiver Operating Characteristics analysis, indicate that the bulls having testicular cytogram comprising of < 15.5% Sertoli cells, < 24.3 Sertoli cell index and > 4.0 spermatogenic cells to Sertoli cell ratio might be a poor bull in terms of semen quality and cryotolerance of spermatozoa. The proportion of Sertoli cells in the testicular cytology had positive (P < 0.05) relationship with semen quality and cryotolerance of spermatozoa. PMID:26891549

  14. Sertoli cells have a functional NALP3 inflammasome that can modulate autophagy and cytokine production

    PubMed Central

    Hayrabedyan, Soren; Todorova, Krassimira; Jabeen, Asma; Metodieva, Gergana; Toshkov, Stavri; Metodiev, Metodi V.; Mincheff, Milcho; Fernández, Nelson

    2016-01-01

    Sertoli cells, can function as non-professional tolerogenic antigen-presenting cells, and sustain the blood-testis barrier formed by their tight junctions. The NOD-like receptor family members and the NALP3 inflammasome play a key role in pro-inflammatory innate immunity signalling pathways. Limited data exist on NOD1 and NOD2 expression in human and mouse Sertoli cells. Currently, there is no data on inflammasome expression or function in Sertoli cells. We found that in primary pre-pubertal Sertoli cells and in adult Sertoli line, TLR4\\NOD1 and NOD2 crosstalk converged in NFκB activation and elicited a NALP3 activation, leading to de novo synthesis and inflammasome priming. This led to caspase-1 activation and IL-1β secretion. We demonstrated this process was controlled by mechanisms linked to autophagy. NOD1 promoted pro-IL-1β restriction and autophagosome maturation arrest, while NOD2 promoted caspase-1 activation, IL-1β secretion and autophagy maturation. NALP3 modulated NOD1 and pro-IL-1β expression, while NOD2 inversely promoted IL-1β. This study is proof of concept that Sertoli cells, upon specific stimulation, could participate in male infertility pathogenesis via inflammatory cytokine induction. PMID:26744177

  15. Depletion of the p43 Mitochondrial T3 Receptor Increases Sertoli Cell Proliferation in Mice

    PubMed Central

    Fumel, Betty; Roy, Stéphanie; Fouchécourt, Sophie; Livera, Gabriel; Parent, Anne-Simone; Casas, François; Guillou, Florian

    2013-01-01

    Among T3 receptors, TRα1 is ubiquitous and its deletion or a specific expression of a dominant-negative TRα1 isoform in Sertoli cell leads to an increase in testis weight and sperm production. The identification of a 43-kDa truncated form of the nuclear receptor TRα1 (p43) in the mitochondrial matrix led us to test the hypothesis that this mitochondrial transcription factor could regulate Sertoli cell proliferation. Here we report that p43 depletion in mice increases testis weight and sperm reserve. In addition, we found that p43 deletion increases Sertoli cell proliferation in postnatal testis at 3 days of development. Electron microscopy studies evidence an alteration of mitochondrial morphology observed specifically in Sertoli cells of p43−/− mice. Moreover, gene expression studies indicate that the lack of p43 in testis induced an alteration of the mitochondrial-nuclear cross-talk. In particular, the up-regulation of Cdk4 and c-myc pathway in p43−/− probably explain the extended proliferation recorded in Sertoli cells of these mice. Our finding suggests that T3 limits post-natal Sertoli cell proliferation mainly through its mitochondrial T3 receptor p43. PMID:24040148

  16. Primary rat Sertoli and interstitial cells exhibit a differential response to cadmium

    SciTech Connect

    Clough, S.R.; Welsh, M.J.; Payne, A.H.; Brown, C.D.; Brabec, M.J. )

    1990-01-01

    Two cell types central to the support of spermatogenesis, the Sertoli cell and the interstitial (Leydig) cell, were isolated from the same cohort of young male rats and challenged with cadmium chloride to compare their susceptibility to the metal. Both cell types were cultured under similar conditions, and similar biochemical endpoints were chosen to minimize experimental variability. These endpoints include the uptake of 109Cd, reduction of the vital tetrazolium dye MTT, incorporation of 3H-leucine, change in heat-stable cadmium binding capacity, and production of lactate. Using these parameters, it was observed that the Sertoli cell cultures were adversely affected in a dose-and time-dependent manner, while the interstitial cell cultures, treated with identical concentrations of CdCl2, were less affected. The 72-hr LC50's for Sertoli cells and interstitial cells were 4.1 and 19.6 microM CdCl2, respectively. Thus, different cell populations within the same tissue may differ markedly in susceptibility to a toxicant. These in vitro data suggest that the Sertoli cell, in relation to the interstitium, is particularly sensitive to cadmium. Because the Sertoli cell provides functional support for the seminiferous epithelium, the differential sensitivity of this cell type may, in part, explain cadmium-induced testicular dysfunction, particularly at doses that leave the vascular epithelium intact.

  17. Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus

    NASA Astrophysics Data System (ADS)

    Peng, Limin; Zheng, Yuan; You, Feng; Wu, Zhihao; Zou, Yuxia; Zhang, Peijun

    2015-11-01

    The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis. A new Sertoli cell line (POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages. Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P. olivaceus. Cells were optimally maintained at 25°C in DMEM/F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, and epidermal growth factor. The growth curve of POSC showed a typical "S" shape. Chromosome analysis revealed that the cell line possessed the normal P. olivaceus diploid karyotype of 2n=48t. POSC expressed dmrt1 but not vasa, which was detected using RT-PCR and sequencing. Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL. Therefore, POSC appeared to consist of testicular Sertoli cells. Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid, with the transfection efficiency reaching 10%. This research not only offers an ideal model for further gene expression and regulation studies on P. olivaceus, but also serves as valuable material in studying fish spermatogenesis, Sertoli cell-germ cell interactions, and the mechanism of growth and development of testis.

  18. The Sertoli cell of the water buffalo--an electron microscopic study.

    PubMed Central

    Azmi, T I; Bongso, T A; Harisah, M; Basrur, P K

    1990-01-01

    The ultrastructure of Sertoli cells in the seminiferous tubules of water buffaloes before and during sexual maturity was studied by transmission electron microscopy, with emphasis on the intranucleolar vesicular elements. Sertoli cells of animals under 12 months of age were distinguished from the germ cells by the presence of electron dense membrane bound bodies within their cytoplasm. These cells, referred to as basal indifferent supporting cells, were probably involved in the phagocytosis and elimination of degenerating spermatocytes, which failed to differentiate into spermatids and spermatozoa in animals under one year of age. In 12 month old animals, a few Sertoli cells exhibiting the vesicular elements appeared in the nucleolar region while in animals over 15 months of age Sertoli cells could be positively identified by the characteristic cytoplasm containing microtubules, elongated and electron dense mitochondria, extensive granular endoplasmic reticulum and the presence of spermatids in various stages of spermiogenesis. The vesicular elements in the nucleolar region of the Sertoli cells were most prominent at this stage. Ultrastructural features of the Sertoli cells revealed an abundance of ribosome-like particles surrounding the vesicles of varying size. Some of these vesicular elements contained amorphous material suggesting that they represent the products sequestered in the nuclear region for transport to the cytoplasm and that the process of spermiogenesis may be dependent on the ability of Sertoli cells to generate these products at sexual maturity. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9a. Fig. 9b. Fig. 10. Fig. 11. PMID:2306676

  19. Connexin-43: A possible mediator of heat stress effects on ram Sertoli cells

    PubMed Central

    Hassanpour, Hossain; Kadivar, Ali; Mirshokraei, Pejman; Nazari, Hassan; Afzali, Azita; Badisanaye, Maryam

    2015-01-01

    Sertoli cells are an essential group of cells in seminiferous epithelium which provide nutritional and structural supports for spermatogenic cells via cell junctions. In this study, the gene expression of connexin-43, the most abundantly distributed gap junction protein of cells, was investigated in ram Sertoli cells under mild and severe heat stresses with real-time quantitative PCR. Sertoli cells were isolated from testes of 10 lambs. After culture and 3 passages, they were treated with mild (39 ˚C) and severe (42 ˚C) heat stress for 6 hr. The results showed a significant reduction in the percentage of live cells under severe heat stress compared to the control group (32 ˚C), (p <0.05). Relative quantification analysis revealed significantly higher (3.80 fold increase) values of connexin-43 transcripts in severely heat stressed group than control group (p <0.05). It is concluded that challenging Sertoli cells with 42 ˚C heat could threaten their survival, and overexpression of connexin-43 may cause dysfunction of Sertoli cells due to heat stress. These findings can be useful to identify the molecular mechanisms involved in adverse effects of heat stress on male reproduction and enhance our understanding of its pathogenesis. PMID:26261707

  20. The interaction between Sertoli cells and luekemia inhibitory factor on the propagation and differentiation of spermatogonial stem cells in vitro

    PubMed Central

    Rastegar, Tayebeh; Habibi Roudkenar, Mehryar; Parvari, Soraya; Baazm, Maryam

    2015-01-01

    Background: Sertoli cells play a pivotal role in creating microenvironments essential for spermatogonial stem cells (SSCs) self-renewal and commitment to differentiation. Maintenance of SSCs and or induction of in vitro spermiogenesis may provide a therapeutic strategy to treat male infertility. Objective: This study investigated the role of luekemia inhibitory factor (LIF) on the propagation of SSCs and both functions of Sertoli cells on the proliferation and differentiation of these cells. Materials and Methods: SSCs were sorted from the testes of adult male mice by magnetic activated cell sorting and thymus cell antigen 1 antibody. On the other hand, isolated Sertoli cells were enriched using lectin coated plates. SSCs were cultured on Sertoli cells for 7 days in the absence or presence of LIF. The effects of these conditions were evaluated by microscopy and expression of meiotic and post meiotic transcripts by reverse transcriptase polymerase chain reaction. Results: Our data showed that SSCs co-cultured with Sertoli cells in the presence of LIF formed colonies on top of the Sertoli cells. These colonies had alkaline phosphatesase activity and expressed SSCs specific genes. SSCs were enjoyed limited development after the mere removal of LIF, and exhibiting expression of meiotic and postmeiotic transcript and loss of SSCs specific gene expression (p< 0.05). Conclusion: Our findings represent co-culture of SSCs with Sertoli cells provides conditions that may allow efficient proliferation and differentiation of SSCs for male infertility treatment. PMID:26730242

  1. Altered Lipid Homeostasis in Sertoli Cells Stressed by Mild Hyperthermia

    PubMed Central

    Vallés, Ana S.; Aveldaño, Marta I.; Furland, Natalia E.

    2014-01-01

    Spermatogenesis is known to be vulnerable to temperature. Exposures of rat testis to moderate hyperthermia result in loss of germ cells with survival of Sertoli cells (SC). Because SC provide structural and metabolic support to germ cells, our aim was to test the hypothesis that these exposures affect SC functions, thus contributing to germ cell damage. In vivo, regularly repeated exposures (one of 15 min per day, once a day during 5 days) of rat testes to 43°C led to accumulation of neutral lipids. This SC-specific lipid function took 1–2 weeks after the last of these exposures to be maximal. In cultured SC, similar daily exposures for 15 min to 43°C resulted in significant increase in triacylglycerol levels and accumulation of lipid droplets. After incubations with [3H]arachidonate, the labeling of cardiolipin decreased more than that of other lipid classes. Another specifically mitochondrial lipid metabolic function, fatty acid oxidation, also declined. These lipid changes suggested that temperature affects SC mitochondrial physiology, which was confirmed by significantly increased degrees of membrane depolarization and ROS production. This concurred with reduced expression of two SC-specific proteins, transferrin, and Wilms' Tumor 1 protein, markers of SC secretion and differentiation functions, respectively, and with an intense SC cytoskeletal perturbation, evident by loss of microtubule network (α-tubulin) and microfilament (f-actin) organization. Albeit temporary and potentially reversible, hyperthermia-induced SC structural and metabolic alterations may be long-lasting and/or extensive enough to respond for the decreased survival of the germ cells they normally foster. PMID:24690895

  2. The ultrastructure of the Sertoli cell of the vervet monkey, Chlorocebus aethiops.

    PubMed

    Lebelo, S L; van der Horst, G

    2010-12-01

    The ultrastructure of the Sertoli cell of the vervet monkey was studied using both scanning and transmission electron microscopic techniques. SEM micrographs revealed perforated sleeve-like processes which encased mature elongated spermatids which are ready for spermiation. TEM micrographs showed a large Sertoli cell nucleus characterized by many lobes (4-5) and consisting of a homogenous nucleoplasm and a distinctive nucleolus. The nucleus occupies a significant portion of the basal region of the cell. The distribution of chromatin clearly shows high activity of these cells. Lipid droplets and free ribosomes are also found scattered throughout the cytoplasm. Well-developed Golgi apparatus is found in the basal region of the cell. There is phagocytic activity in the Sertoli cells as revealed by the presence of numerous phagosomes. Numerous mitochondria with well-developed tubular cristae are found on the basal side of the nucleus, whereas few mitochondria are located on the apical side of the nucleus. Distinct desmosomes are located between cells. A well-developed smooth endoplasmic reticulum and granular endoplasmic reticulum are frequently found in the cytoplasm of the Sertoli cells. The results of this investigation showed that Sertoli cells of the vervet monkey are almost similar to those of humans and show many similarities with other mammalian species. PMID:20828773

  3. Uptake and metabolism of retinol in cultured sertoli cells: evidence for a kinetic model

    SciTech Connect

    Bishop, P.D.; Griswold, M.D.

    1987-11-17

    When cultured Sertoli cells derived from 20-day-old weanling rats were supplied (/sup 3/H) retinol bound to serum retinol binding protein-transthyretin complex, (/sup 3/H) retinol was rapidly incorporated and (/sup 3/H) retinyl esters were synthesized. Within 28 h after administration, 83% of the labeled retinoids were accounted for as retinyl esters (64% as retinyl palmitate). Sertoli cells derived from vitamin A deficient rats and supplied (/sup 3/H) retinol in culture under identical conditions likewise incorporated (/sup 3/H) retinol and synthesized retinyl esters. In contrast to normal Sertoli cells, vitamin A deficient Sertoli cells eventually metabolized virtually all of the cellular (/sup 3/H) retinol to retinyl esters. The primary metabolic fate of retinol administered to Sertoli cell cultures was the synthesis of retinyl esters under all conditions tested. However, administration of (/sup 3/H) retinol bound to serum retinol binding protein gave metabolic profiles having a higher proportion of retinyl esters and lower proportions of unresolved polar material than administration of (/sup 3/H) retinol alone. The kinetics of retinol uptake and intracellular retinyl ester synthesis in cultured Sertoli cells was complex. An initial, rapid phase of (/sup 3/H) retinol incorporation lasting 30 min was followed by a slower rate of incorporation and a concomitant decrease in the intracellular concentration of (/sup 3/H) retinol. During the time course the specific activity of (/sup 3/H) retinyl palmitate eventually exceeded that of intracellular (/sup 3/H) retinol. These observations suggest that two intracellular pools of retinol may exist in Sertoli cells.

  4. Reduced endogenous estrogen and hemicastration interact synergistically to increase porcine sertoli cell proliferation.

    PubMed

    Berger, Trish; Conley, Alan

    2014-05-01

    Both reduced endogenous estrogen and hemicastration stimulate proliferation of porcine Sertoli cells. The objective of these experiments was to compare the temporal patterns of response to each stimulus with the response to the combined stimuli as indications of shared or separate mechanisms. Within a replicate, one littermate was treated weekly with canola oil vehicle and remained intact; a second littermate was treated weekly with vehicle, and one testis was removed at Day 8; a third littermate was treated weekly with the aromatase inhibitor letrozole to reduce endogenous estrogens and remained intact; and the fourth littermate was treated weekly with letrozole, and one testis was removed at Day 8. Four replicates were evaluated at 2 wk of age, five replicates evaluated at 6.5 wk of age, and five replicates were evaluated at 11 wk of age, with treatment ceasing at 6 wk of age. Numbers of Sertoli cells were determined following GATA4 labeling using the optical dissector method. Levels of estradiol, estrogen conjugates, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and inhibin were determined by radioimmunoassay. Hemicastration appeared to have a rapid effect on Sertoli cell proliferation, but letrozole treatment had no apparent effect on Sertoli cell numbers at 2 wk of age. Both letrozole treatment and hemicastration had stimulated Sertoli cell proliferation by 6.5 wk of age, although the magnitude of the hemicastration response was much greater. Letrozole appeared to have minimal interaction with hemicastration at this age. Letrozole and hemicastration together increased Sertoli cell numbers at 11 wk of age compared with either treatment alone. Estradiol and estrogen conjugates were dramatically reduced by aromatase inhibition as anticipated; treatment-induced changes in inhibin, LH, or FSH were minimal. Differences in timing of response and positive interaction at 11 wk of age suggest that hemicastration and letrozole stimulate proliferation of Sertoli cells by two initially different pathways. PMID:24740600

  5. Receptors and signaling pathways involved in proliferation and differentiation of Sertoli cells

    PubMed Central

    Lucas, Thas FG; Nascimento, Aline R; Pisolato, Raisa; Pimenta, Maristela T; Lazari, Maria Fatima M; Porto, Catarina S

    2014-01-01

    The identification of the hormones and other factors regulating Sertoli cell survival, proliferation, and maturation in neonatal, peripubertal, and pubertal life remains one of the most critical questions in testicular biology. The regulation of Sertoli cell proliferation and differentiation is thought to be controlled by cellcell junctions and a set of circulating and local hormones and growth factors. In this review, we will focus on receptors and intracellular signaling pathways activated by androgen, follicle-stimulating hormone, thyroid hormone, activin, retinoids, insulin, insulin-like growth factor, relaxin, and estrogen, with special emphasis on estrogen receptors. Estrogen receptors activate intracellular signaling pathways that converge on cell cycle and transcription factors and play a role in the regulation of Sertoli cell proliferation and differentiation. PMID:25225624

  6. Roles of miRNAs in microcystin-LR-induced Sertoli cell toxicity.

    PubMed

    Zhou, Yuan; Wang, Hui; Wang, Cong; Qiu, Xuefeng; Benson, Mikael; Yin, Xiaoqin; Xiang, Zou; Li, Dongmei; Han, Xiaodong

    2015-08-15

    Microcystin (MC)-LR, a cyclic heptapeptide, is a potent reproductive system toxin. To understand the molecular mechanisms of MC-induced reproductive system cytotoxicity, we evaluated global changes of miRNA and mRNA expression in mouse Sertoli cells following MC-LR treatment. Our results revealed that the exposure to MC-LR resulted in an altered miRNA expression profile that might be responsible for the modulation of mRNA expression. Bio-functional analysis indicated that the altered genes were involved in specific cellular processes, including cell death and proliferation. Target gene analysis suggested that junction injury in Sertoli cells exposed to MC-LR might be mediated by miRNAs through the regulation of the Sertoli cell-Sertoli cell pathway. Collectively, these findings may enhance our understanding on the modes of action of MC-LR on mouse Sertoli cells as well as the molecular mechanisms underlying the toxicity of MC-LR on the male reproductive system. PMID:25986756

  7. Rat Sertoli cells acquire a. beta. -adrenergic response during primary culture

    SciTech Connect

    Kierszenbaum, A.L.; Spruill, W.A.; White, M.G.; Tres, L.L.; Perkins, J.P.

    1985-04-01

    Two-dimensional polyacrylamide gel electrophoresis and the radioligand (-)-(/sup 125/I)iodopindolol (/sup 125/I-Pin) have been used to study isoproterenol-dependent protein phosphorylation and ..beta..-adrenergic receptor availability, respectively, in cultured Sertoli cells and freshly isolated seminiferous tubular segments of sexually immature and mature rats. Sertoli cells prepared from sexually immature rats show progressive /sup 125/I-Pin binding in primary cultures that correlates with isoproterenol-induced cell shape changes, redistribution of immunoreactive vimentin, and phosphorylation of this intermediate filament protein. Seminiferous tubules do not show significant isoproterenol-dependent vimentin phosphorylation nor /sup 125/I-Pin binding. However, vimentin phosphorylation can be induced by follicle-stimulating hormone or a cyclic nucleotide analog. This study stresses the need for correlating pharmacological-induced responses observed in Sertoli cell primary cultures with those in the intact seminiferous tubule.

  8. Experimental Sertoli cell tumors in the mouse and their progression into a mixed germ cell-sex cord proliferation.

    PubMed Central

    Paquis-Flucklinger, V.; Rassoulzadegan, M.; Michiels, J. F.

    1994-01-01

    Males of transgenic families where the large T protein of polyoma virus is expressed in the seminiferous epithelium of the testis (Sertoli and germ cells) develop bilateral testicular tumors when they become old (15 to 18 months). The histological features of these tumors revealed a neoplastic proliferation of Sertoli cell origin. Occasional isolated germ cells arrested at premeiotic stages were seen in the tumor. They did not participate in tumoral proliferation and their malignant character could not at first be established. Tumor cells injected in athymic (nu/nu) mice generated secondary tumors. In this case, a proliferative component of non-Sertoli origin was clearly evident. Its ultrastructural characteristics and the expression of genes that are transcribed in vivo in male germ cells (c-kit, LDH-X, and Hox a-4) suggest the progression of an initial, apparently pure Sertoli cell tumor into a mixed proliferation. Images Figure 1 Figure 2 Figure 3 PMID:7510454

  9. Testosterone regulates the autophagic clearance of androgen binding protein in rat Sertoli cells

    PubMed Central

    Ma, Yi; Yang, Hao-Zheng; Xu, Long-Mei; Huang, Yi-Ran; Dai, Hui-Li; Kang, Xiao-Nan

    2015-01-01

    Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases. However, the ABP metabolism in Sertoli cells is largely unknown. We report that autophagy degrades ABP in rat Sertoli cells, and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. We report a novel finding with respect to the mechanisms by which ABP is cleared, and by which the process is regulated in Sertoli cells. PMID:25745956

  10. A substance secreted by rat Sertoli cells induces feminization of embryonic chick testes in vitro.

    PubMed

    Sánchez, A; Jiménez, R; Burgos, M; Díaz de la Guardia, R

    1994-06-01

    Male and female gonads from 7- to 9-day-old chick embryos were cultured for 6 days in Sertoli cell-conditioned medium or in serum-free medium to investigate the possible effect of substances secreted by rat Sertoli cells on chick gonad development. Histological analysis showed that whereas all female gonads proceed through normal ovarian development in both culture media, most of male gonads showed clear feminization only when cultured in Sertoli cell-conditioned medium; male gonads cultured in serum-free medium developed as normal testes. Because the only substance detected in our conditioned medium with the potential to cause these effects was sex-specific antigen (Sxs), our results provide further evidence that Sxs antigen may play a role in sexual differentiation in birds, and probably in mammals. PMID:7978357

  11. Sertoli cell tumor causing precocious puberty in a girl with Peutz-Jeghers syndrome.

    PubMed

    Zung, A; Shoham, Z; Open, M; Altman, Y; Dgani, R; Zadik, Z

    1998-09-01

    Distinctive ovarian and cervical tumors are associated with Peutz-Jeghers syndrome (PJS). The most common gynecological tumors in this syndrome are adenoma malignum of the uterine cervix and ovarian sex cord tumor, particularly sex cord tumor with annular tubules (SCTAT). Other kinds of ovarian tumors have been rarely reported in association of PJS, including Sertoli cell tumors. We report a case of a 4.5-year-old girl with PJS who presented with isosexual precocious puberty (IPP) due to ovarian lipid-rich Sertoli cell tumor. In addition to estrinizing effect of the tumor, the patient had decidual reaction secondary to tumor-derived progesterone secretion. The literature on gonadal tumors in PJS is reviewed, including one previous report of ovarian lipid-rich Sertoli cell tumor associated with this syndrome. PMID:9790799

  12. Specific deletion of AMP-activated protein kinase (?1AMPK) in mouse Sertoli cells modifies germ cell quality.

    PubMed

    Bertoldo, Michael J; Guibert, Edith; Faure, Melanie; Guillou, Florian; Ram, Christelle; Nadal-Desbarats, Lydie; Foretz, Marc; Viollet, Benoit; Dupont, Jolle; 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 ?1 gene (?1AMPK 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 ?1AMPK only in the Sertoli cells in mice (Sc-?1AMPK-KO mice). Specific deletion of the ?1AMPK 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 invivo 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-?1AMPK-KO Sertoli cells. These perturbations were associated with lower expression of mitochondrial markers (cytochrome c and PGC1-?). 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-?1AMPK-KO. In conclusion, these results suggest that dysregulation of the energy sensing machinery exclusively through disruption of ?1AMPK in Sertoli cells translates to a reduction in the quality of germ cells and fertility. PMID:26772142

  13. Specific deficiency of Plzf paralog, Zbtb20, in Sertoli cells does not affect spermatogenesis and fertility in mice

    PubMed Central

    Jiang, Xiaohua; Zhang, Huan; Yin, Shi; Zhang, Yuanwei; Yang, Weimei; Zheng, Wei; Wang, Liu; Wang, Zheng; Bukhari, Ihtisham; Cooke, Howard J.; Iqbal, Furhan; Shi, Qinghua

    2014-01-01

    Ztbt20 is a POK family transcription factor and primarily functions through its conserved C2H2 Krüppel type zinc finger and BTB/POZ domains. The present study was designed to define the function of the Zbtb20, in vivo, during mouse spermatogenesis. Immunohistochemical studies revealed that ZBTB20 protein was localized specifically in the nuclei of Sertoli cells in seminiferous tubules. To investigate its role during spermatogenesis, we crossed Amh-Cre transgenic mice with Zbtb20 floxp mice to generate conditionally knockout mice (cKO) in which Zbtb20 was specifically deleted in Sertoli cells. The cKO mice were fertile and did not show any detectable abnormalities in spermatogenesis. Taken together, though specific deletion of transcription factor Zbtb20 in Sertoli cells has no apparent influence on spermatogenesis, its specific localization in Sertoli cells makes Zbtb20 a useful marker for the identification of Sertoli cells in seminiferous tubules. PMID:25395169

  14. Characteristics of retinol accumulation from serum retinol-binding protein by cultured sertoli cells

    SciTech Connect

    Shingleton, J.L.; Skinner, M.K.; Ong, D.E. )

    1989-12-12

    The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoil cells accumulated ({sup 3}H)retinol in a time- and temperature-dependent manner. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/{mu}g of cellular DNA. Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with ({sup 3}H)retinol-RBP for ({sup 3}H)retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free ({sup 3}H)retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 {mu}M. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. Competition studies indicated, however, that protein recognition is important in the retinol uptake process. RBP, CRBP, and CRBP(II) competed with ({sup 3}H)retinol-RBP for ({sup 3}H)retinol accumulation, but free retinol, retinol-bovine serum albumin, and retinol-{beta}-lactoglobulin did not. These studies indicated that Sertoli cell uptake of retinol involved recognition of the retinol-RBP complex at the cell surface with subsequent internalization of retinol, but not RBP.

  15. 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

  16. Regulation of Sertoli-Germ Cell Adhesion and Sperm Release by FSH and Nonclassical Testosterone Signaling

    PubMed Central

    Shupe, John; Cheng, Jing; Puri, Pawan; Kostereva, Nataliya

    2011-01-01

    Testosterone and FSH act in synergy to produce the factors required to maximize the production of spermatozoa and male fertility. However, the molecular mechanisms by which these hormones support spermatogenesis are not well established. Recently, we identified a nonclassical mechanism of testosterone signaling in cultured rat Sertoli cells. We found that testosterone binding to the androgen receptor recruits and activates Src tyrosine kinase. Src then causes the activation of the epidermal growth factor receptor, which results in the phosphorylation and activation of the ERK MAPK and the cAMP response element-binding protein transcription factor. In this report, we find that FSH inhibits testosterone-mediated activation of ERK and the MAPK pathway in Sertoli cells via the protein kinase A-mediated inhibition of Raf kinase. In addition, FSH, as well as inhibitors of Src and ERK kinase activity, reduced germ cell attachment to Sertoli cells in culture. Using pathway-specific androgen receptor mutants we found that the nonclassical pathway is required for testosterone-mediated increases in germ cell attachment to Sertoli cells. Studies of seminiferous tubule explants determined that Src kinase, but not ERK kinase, activity is required for the release of sperm from seminiferous tubule explants. These findings suggest the nonclassical testosterone-signaling pathway acts via Src and ERK kinases to facilitate the adhesion of immature germ cells to Sertoli cells and through Src to permit the release of mature spermatozoa. In contrast, FSH acts to limit testosterone-mediated ERK kinase activity and germ cell attachment. PMID:21177760

  17. Sertoli cell junctional proteins as early targets for different classes of reproductive toxicants.

    PubMed

    Fiorini, Céline; Tilloy-Ellul, Anne; Chevalier, Stephan; Charuel, Claude; Pointis, Georges

    2004-05-01

    In the testis, Sertoli cells establish intercellular junctions that are essential for spermatogenesis. The SerW3 Sertoli cell line displays some features of native Sertoli cells. Western blot and immunofluorescence analyses showed that SerW3 Sertoli cells expressed typical components of tight (occludin and zonula occludens-1), anchoring (N-cadherin) and gap (connexin 43) junctions. Testicular toxicants (DDT, pentachlorophenol, dieldrin, dinitrobenzene, cadmium chloride, cisplatin, gossypol, bisphenol A and tert-octylphenol) affected intercellular junctions by either reducing the amount or inducing aberrant intracellular localization of these membranous proteins. Phosphodiesterase inhibitors (isobutyl methylxantine, rolipram, zaprinast, zardaverine) did not alter junctional-complex component levels but caused a rapid and reversible redistribution of these proteins to the cytoplasmic compartment. The present study showed that occludin, ZO-1, N-cadherin and specifically Cx43 could be early targets for testicular toxicants. The SerW3 cell line therefore appears as a useful in vitro model to evaluate molecules with potential anti-reproductive effects. PMID:15082077

  18. Basement membrane increases G-protein levels and follicle-stimulating hormone responsiveness of Sertoli cell adenylyl cyclase activity.

    PubMed

    Dym, M; Lamsam-Casalotti, S; Jia, M C; Kleinman, H K; Papadopoulos, V

    1991-02-01

    On a basement membrane substrate, Sertoli cells in culture have been shown to assume a phenotype similar to that of the in vivo differentiated cells. Sertoli cells from 10-day-old rats were cultured on plastic and on different extracellular matrix substrates [laminin, a reconstituted basement membrane (Matrigel), and a synthetic laminin peptide containing the arginine-glycine-aspartic acid (RGD) tripeptide sequence] to investigate the effects of the extracellular matrix on FSH responsiveness. Both laminin and Matrigel markedly enhanced the cAMP response to FSH and cholera toxin, indicating modifications at the level of guanine nucleotide-binding regulatory (G) proteins. Furthermore, Sertoli cell grown on either of these two substrates responded to physiological levels of FSH (25-50 ng/ml), whereas pharmacological levels of FSH (500 ng/ml) were required for cells grown on either plastic or on the RGD-containing laminin peptide. Immunoblotting of Sertoli cell plasma membranes with antibodies directed against the alpha-subunit of the stimulatory G-protein (Gs alpha) of adenylyl cyclase indicated that Sertoli cell culture on either laminin or Matrigel increased the amounts of Gs alpha. These results were further confirmed by immunoprecipitating the Gs alpha protein from the particulate fraction of [35S]methionine metabolically labeled Sertoli cells. However, Northern blot analysis using a cDNA probe for Gs alpha did not demonstrate changes in gene expression when Sertoli cells were grown on the various substrates. Immunofluorescent studies revealed that the Gs complex of adenylyl cyclase was preferentially located at the base of the Sertoli cells at the site of contact with the extracellular matrix. These data suggest that culture of epithelial Sertoli cells on basement membrane substrates enhances the Gs complex of adenylyl cyclase and the cAMP response to FSH, consistent with the more differentiated morphology and function of the cells. PMID:1846579

  19. Management of an invasive and metastatic Sertoli cell tumor with associated myelotoxicosis in a dog.

    PubMed

    Withers, Sita S; Lawson, Corinne M; Burton, Andrew G; Rebhun, Robert B; Steffey, Michele A

    2016-03-01

    We describe the surgical and post-operative management of a large, invasive, and metastatic functional Sertoli cell tumor in a 9-year-old cryptorchid male Labrador retriever dog. Despite residual disease after surgery, bone marrow recovery occurred without administration of bone marrow stimulants and serum estradiol accurately predicted tumor recurrence. PMID:26933269

  20. Effects of Gold Nanorods on Imprinted Genes Expression in TM-4 Sertoli Cells.

    PubMed

    Yuan, Beilei; Gu, Hao; Xu, Bo; Tang, Qiuqin; Wu, Wei; Ji, Xiaoli; Xia, Yankai; Hu, Lingqing; Chen, Daozhen; Wang, Xinru

    2016-01-01

    Gold nanorods (GNRs) are among the most commonly used nanomaterials. However, thus far, little is known about their harmful effects on male reproduction. Studies from our laboratory have demonstrated that GNRs could decrease glycine synthesis, membrane permeability, mitochondrial membrane potential and disrupt blood-testis barrier factors in TM-4 Sertoli cells. Imprinted genes play important roles in male reproduction and have been identified as susceptible loci to environmental insults by chemicals because they are functionally haploid. In this original study, we investigated the extent to which imprinted genes become deregulated in TM-4 Sertoli cells when treated with low dose of GNRs. The expression levels of 44 imprinted genes were analyzed by quantitative real-time PCR in TM-4 Sertoli cells after a low dose of (10 nM) GNRs treatment for 24 h. We found significantly diminished expression of Kcnq1, Ntm, Peg10, Slc22a2, Pwcr1, Gtl2, Nap1l5, Peg3 and Slc22a2, while Plagl1 was significantly overexpressed. Additionally, four (Kcnq1, Slc22a18, Pwcr1 and Peg3) of 10 abnormally expressed imprinted genes were found to be located on chromosome 7. However, no significant difference of imprinted miRNA genes was observed between the GNRs treated group and controls. Our study suggested that aberrant expression of imprinted genes might be an underlying mechanism for the GNRs-induced reproductive toxicity in TM-4 Sertoli cells. PMID:26938548

  1. Zearalenone impairs the male reproductive system functions via inducing structural and functional alterations of sertoli cells.

    PubMed

    Zheng, WangLong; Pan, ShunYe; Wang, Guangguang; Wang, Ya Jun; Liu, Qing; Gu, JianHong; Yuan, Yan; Liu, Xue Zhong; Liu, Zong Ping; Bian, Jian Chun

    2016-03-01

    The aim of this study was to investigate the effects of ZEA on the cytoskeletal structure, and factors specifically expressed by Sertoli cells. Primary Sertoli cells from rats aged 18-21 days were exposed to increasing ZEA concentrations (0, 5, 10, 20μgmL(-1)) for 24h. The results of immunofluorescence showed disruption of α-tubulin filaments and F-actin bundles, and damage to the nucleus of Sertoli cells on exposure to ZEA. In the control group, the protein level expression of androgen-binding protein (ABP), transferrin, vimentin, N-cadherin, and follicle-stimulating hormone receptor (FSHR) were decreased significantly (p<0.05, p<0.01). The mRNA levels of ABP, transferrin, vimentin, N-cadherin, and FSHR varied significantly in the experimental group (p<0.05). The results of enzyme-linked immunosorbent assay indicated a significant decrease in the levels of inhibin-β and transferrin in the cultural supernatants (p<0.05). Additionally, the ultrastructural analysis indicated the absence of mitochondria and Golgi apparatus, and presence of vacuoles in the cytoplasm. These findings showed that ZEA treatment can damage the cytoskeletal structure and affect specific secretory functions of Sertoli cells, which may be an underlying cause of ZEA-induced reproductive toxicity. PMID:26851377

  2. Effects of Gold Nanorods on Imprinted Genes Expression in TM-4 Sertoli Cells

    PubMed Central

    Yuan, Beilei; Gu, Hao; Xu, Bo; Tang, Qiuqin; Wu, Wei; Ji, Xiaoli; Xia, Yankai; Hu, Lingqing; Chen, Daozhen; Wang, Xinru

    2016-01-01

    Gold nanorods (GNRs) are among the most commonly used nanomaterials. However, thus far, little is known about their harmful effects on male reproduction. Studies from our laboratory have demonstrated that GNRs could decrease glycine synthesis, membrane permeability, mitochondrial membrane potential and disrupt blood-testis barrier factors in TM-4 Sertoli cells. Imprinted genes play important roles in male reproduction and have been identified as susceptible loci to environmental insults by chemicals because they are functionally haploid. In this original study, we investigated the extent to which imprinted genes become deregulated in TM-4 Sertoli cells when treated with low dose of GNRs. The expression levels of 44 imprinted genes were analyzed by quantitative real-time PCR in TM-4 Sertoli cells after a low dose of (10 nM) GNRs treatment for 24 h. We found significantly diminished expression of Kcnq1, Ntm, Peg10, Slc22a2, Pwcr1, Gtl2, Nap1l5, Peg3 and Slc22a2, while Plagl1 was significantly overexpressed. Additionally, four (Kcnq1, Slc22a18, Pwcr1 and Peg3) of 10 abnormally expressed imprinted genes were found to be located on chromosome 7. However, no significant difference of imprinted miRNA genes was observed between the GNRs treated group and controls. Our study suggested that aberrant expression of imprinted genes might be an underlying mechanism for the GNRs-induced reproductive toxicity in TM-4 Sertoli cells. PMID:26938548

  3. Dynamin 2 is required for actin assembly in phagocytosis in Sertoli cells

    SciTech Connect

    Otsuka, Atsushi; Abe, Tadashi; Watanabe, Masami; Yagisawa, Hitoshi; Takei, Kohji; Yamada, Hiroshi

    2009-01-16

    Dynamin 2 has been reported to be implicated in phagocytosis. However, the mode of action of dynamin is poorly understood. In this study, we examined whether dynamin 2 participates in actin assembly during phagocytosis in Sertoli cells. In the presence of dynasore, a dynamin inhibitor, phagocytosis was reduced by 60-70% in Sertoli cells and macrophages. Scanning electron microscopy revealed that Sertoli cells treated with dynasore were unable to form phagocytic cups. In addition, dysfunction of dynamin 2 reduced both actin polymerization and recruitment of actin and dynamin 2 to phosphatidylinositol (4,5) bisphosphate [PI(4,5)P{sub 2}]-containing liposomes. The formation of dynamin 2-positive ruffles of Sertoli cells was decreased by 60-70% by sequestering PI(4,5)P{sub 2} either by expression of PH domain of PLC{delta} or treatment with neomycin. These results strongly suggest that dynamin 2 is involved in actin dynamics and the formation of dynamin 2-positive ruffles during phagocytosis.

  4. MICRODISSECTION TESTICULAR SPERM EXTRACTION IN MEN WITH SERTOLI CELL ONLY TESTICULAR HISTOLOGY

    PubMed Central

    Berookhim, Boback M.; Palermo, Gianpiero D.; Zaninovic, Nikica; Rosenwaks, Zev; Schlegel, Peter N.

    2015-01-01

    Objective To study the outcomes of microdissection testicular sperm extraction (microTESE) among men with pure Sertoli cell only histology on diagnostic testicular biopsy. Design Retrospective cohort study. Setting Tertiary referral center. Patients 640 patients with pure Sertoli cell only histology on testicular biopsy who underwent microTESE by a single surgeon. Intervention MicroTESE. Main Outcome Measure Sperm retrieval rates. Results Overall, 44.5% of patients with Sertoli cell-only had sperm retrieved with microTESE. No difference was noted in sperm retrieval rates based on testis volume (? 15cc versus <15cc, 35.3% versus 46.1%, respectively). Patients with ? 15cc testicular volume and FSH 10-15 mU/mL had the worst prognosis, with a sperm retrieval rate of 6.7%. Conclusions Patients with previous testicular biopsy demonstrating Sertoli cell only histology can be counseled that they have a reasonable likelihood of sperm retrieval with the contemporary delivery of microTESE. Given this finding, the utility of testicular biopsy prior to microTESE is further questioned. PMID:25441063

  5. Ezrin is an Actin Binding Protein That Regulates Sertoli Cell and Spermatid Adhesion During Spermatogenesis

    PubMed Central

    Gungor-Ordueri, N. Ece; Tang, Elizabeth I.; Celik-Ozenci, Ciler

    2014-01-01

    During spermatogenesis, the transport of spermatids and the release of sperms at spermiation and the remodeling of the blood-testis barrier (BTB) in the seminiferous epithelium of rat testes require rapid reorganization of the actin-based cytoskeleton. However, the mechanism(s) and the regulatory molecule(s) remain unexplored. Herein we report findings that unfold the functional significance of ezrin in the organization of the testis-specific adherens junction at the spermatid-Sertoli cell interface called apical ectoplasmic specialization (ES) in the adluminal compartment and the Sertoli cell-cell interface known as basal ES at the BTB. Ezrin is expressed at the basal ES/BTB in all stages, except from late VIII to IX, of the epithelial cycle. Its knockdown by RNA interference (RNAi) in vitro perturbs the Sertoli cell tight junction-permeability barrier via a disruption of the actin microfilaments in Sertoli cells, which in turn impeded basal ES protein (eg, N-cadherin) distribution, perturbing the BTB function. These findings were confirmed by a knockdown study in vivo. However, the expression of ezrin at the apical ES is restricted to stage VIII of the cycle and limited only between step 19 spermatids and Sertoli cells. A knockdown of ezrin in vivo by RNAi was found to impede spermatid transport, causing defects in spermiation in which spermatids were embedded deep inside the epithelium, and associated with a loss of spermatid polarity. Also, ezrin was associated with residual bodies and phagosomes, and its knockdown by RNAi in the testis also impeded the transport of residual bodies/phagosomes from the apical to the basal compartment. In summary, ezrin is involved in regulating actin microfilament organization at the ES in rat testes. PMID:25051438

  6. Polycyclic aromatic hydrocarbon-induced cytotoxicity in cultured rat Sertoli cells involves differential apoptotic response.

    PubMed Central

    Raychoudhury, Samir S; Kubinski, Dana

    2003-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and persistent environmental contaminants. Some PAHs are carcinogens and may affect the male reproductive system. Therefore, we exposed cultured rat Sertoli cells to a variety of PAHs to determine possible direct toxic effects on the cells of the seminiferous epithelium. Sertoli cells were chosen because they support germ cell development and maintain spermatogenesis. Sertoli cells were isolated from 19-21-day-old male rats and cultured in medium containing 0.08% dimethylsulfoxide as vehicle or in the presence of a variety of PAHs. In the first set of experiments, cultured Sertoli cells were incubated in the presence of 10(-4) M, 10(-6 )M, 10(-8) M, 10(-12) M, and 10(-16) M fluoranthene (FL) for 24 hr. After 24 hr, FL at 10(4), 10(-6), and 10(-8) M killed significant numbers of Sertoli cells as revealed by cell viability determinations. Sertoli cells cultured in the presence of 10(-6) M and 10(-8) M FL showed morphologic changes. Cell protein levels were decreased and lactate production in the medium increased in a concentration-dependent manner. In addition, Sertoli cells exposed to 10(6) M and 10(-8) M FL exhibited altered F-actin and alpha-tubulin distributions compared with untreated controls. Because FL killed about 62% of cells at 10(-4) M (100 micro g/mL) and 48% of cells at 10(-6) M (1 micro g/mL), increased lactate production about 3-fold at both concentrations, and decreased cell protein by half at 10(-4) M (100 micro g/mL), we decided to use a range of concentrations between 10 and 100 micro g/mL for the second set of experiments using benz[a]anthracene (BaA), benzo[a]pyrene (BaP), or benzo[b]fluoranthene (BbF). After 24 hr, BaA (100 micro g/mL), BaP (50 and 100 micro g/mL), and BbF (100 micro g/mL) significantly increased lactate level in the medium in a concentration-dependent manner. In a third set of experiments, cells were treated in culture uniformly with only 10 micro g/mL FL, BaA, BaP, or BbF for 24 hr. The cytotoxic effects exerted by these PAHs tested resulted in different apoptotic responses as characterized by in situ fluorescence staining. Microscopic analysis of apoptotic cells demonstrated nuclei of reduced size and labeled 3 -OH DNA ends when Sertoli cells had been incubated for 24 hr with 10 micro g BaP or BbF, but not with vehicle, media, FL, or BaA. Thus, our results demonstrate that the toxic effects of BaA and BbF on Sertoli cells are exerted through apoptosis, whereas FL and BaA do not elicit the apoptotic response. PMID:12515676

  7. Bilateral retiform variant of sertoli leydig cell tumour of ovary: An uncommon tumor with review of literature

    PubMed Central

    Rathi, Monika; Budania, Satish Kumar; Khalid, Mohammad; Mittal, Ankur

    2015-01-01

    Sertoli-leydig cell tumors are the uncommon sex-cord stromal tumors of the ovary. We report a case of 42-year-old female with retiform variant of sertoli-leydig cell tumour. She presented with the complaint of mass in abdomen for 7 years. Ultrasound revealed bilateral ovarian mass suggestive of malignancy. Bilateral salpingo-oopherectomy with surgical staging was done. The tumor was diagnosed as stage I retiform variant of sertoli-leydig cell tumor on histopathology and immunohistochemistry. PMID:25861207

  8. The role of Pten/Akt signaling pathway involved in BPA-induced apoptosis of rat Sertoli cells.

    PubMed

    Wang, Chengmin; Fu, Wenjuan; Quan, Chao; Yan, Maosheng; Liu, Changjiang; Qi, Suqin; Yang, Kedi

    2015-07-01

    Bisphenol-A (BPA), one of endocrine-disrupting chemicals, is a male reproductive toxicant. Previous studies have revealed the direct cytotoxicity of BPA in many cultured cells, such as mitotic aneuploidy in embryonic cells and somatic cells, and apoptosis in neurons and testicular Sertoli cells. To understand the action of BPA and assess its risk, the Pten/Akt pathway was investigated in cultured Sertoli cells to elucidate the mechanism of the reproductive effects of BPA. The results showed that over 50 μM BPA treatment could decrease the viability of Sertoli cells and cause more apoptosis. In addition, BPA could induce the increase in mRNA levels of Pten and Akt. The protein level of Pten was increased; however, the protein levels of phospho-Akt and procaspase-3 were decreased after BPA exposure. Taken together, observed results suggested that the Pten/Akt pathway might be involved in the apoptotic effects of BPA on Sertoli cells. PMID:24464975

  9. 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.

  10. Age-dependent changes in the in-vitro response of a pig Sertoli cell-enriched population to FSH.

    PubMed

    Monet-Kuntz, C; Fontaine, I

    1989-07-01

    The response of pig Sertoli cell-enriched cultures to FSH was investigated by measuring plasminogen activator (PA) secretion in culture, throughout the nonpubertal and prepubertal periods. Sertoli cell-enriched populations could be isolated from birth until a testicular weight of 56 g. FSH elicited a dose-dependent increase in PA secretion by pig Sertoli cell-enriched cultures. The ED50 was minimal for cells coming from testes weighing 10-22 g, and increased more than 2-fold for cells from heavier testes. This suggests that, at the end of the non-pubertal period, an increased FSH sensitivity is important for initiation of spermatogenesis in this species, and that during the prepubertal period Sertoli cells become less sensitive to FSH. The FSH-stimulated PA secretion increased about 10-fold from a testicular weight of 25 g onwards, i.e. when primary spermatocytes appear in seminiferous tubules. PMID:2503609

  11. Sertoli cells are the target of environmental toxicants in the testis – a mechanistic and therapeutic insight

    PubMed Central

    Gao, Ying; Mruk, Dolores D; Cheng, C Yan

    2016-01-01

    Introduction Sertoli cells support germ cell development in the testis via an elaborate network of cell junctions that confers structural, communicating, and signaling support. However, Sertoli cell junctions and cytoskeletons are the target of environmental toxicants. Because germ cells rely on Sertoli cells for the provision of structural/functional/nutritional support, exposure of males to toxicants leads to germ cell exfoliation due to Sertoli cell injuries. Interestingly, the molecular mechanism(s) by which toxicants induce cytoskeletal disruption that leads to germ cell exfoliation is unclear, until recent years, which are discussed herein. This information can possibly be used to therapeutically manage toxicant-induced infertility/subfertility in human males. Areas covered In this review, we provide a brief update on the use of Sertoli cell system developed for rodents and humans in vitro, which can be deployed in any research laboratory with minimal upfront setup costs. These systems can be used to collect reliable data applicable to studies in vivo. We also discuss the latest findings on the mechanisms by which toxicants induce Sertoli cell injury, in particular cytoskeletal disruption. We also identify candidate molecules that are likely targets of toxicants. Expert opinion We provide two hypothetical models delineating the mechanism by which toxicants induce germ cell exfoliation and blood–testis barrier disruption. We also discuss molecules that are the targets of toxicants as therapeutic candidates. PMID:25913180

  12. Environmentally induced epigenetic transgenerational inheritance of altered Sertoli cell transcriptome and epigenome: molecular etiology of male infertility.

    PubMed

    Guerrero-Bosagna, Carlos; Savenkova, Marina; Haque, Md Muksitul; Nilsson, Eric; Skinner, Michael K

    2013-01-01

    Environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of adult onset disease, including testis disease and male infertility. The current study was designed to determine the impact of an altered sperm epigenome on the subsequent development of an adult somatic cell (Sertoli cell) that influences the onset of a specific disease (male infertility). A gestating female rat (F0 generation) was exposed to the agriculture fungicide vinclozolin during gonadal sex determination and then the subsequent F3 generation progeny used for the isolation of Sertoli cells and assessment of testis disease. As previously observed, enhanced spermatogenic cell apoptosis was observed. The Sertoli cells provide the physical and nutritional support for the spermatogenic cells. Over 400 genes were differentially expressed in the F3 generation control versus vinclozolin lineage Sertoli cells. A number of specific cellular pathways were identified to be transgenerationally altered. One of the key metabolic processes affected was pyruvate/lactate production that is directly linked to spermatogenic cell viability. The Sertoli cell epigenome was also altered with over 100 promoter differential DNA methylation regions (DMR) modified. The genomic features and overlap with the sperm DMR were investigated. Observations demonstrate that the transgenerational sperm epigenetic alterations subsequently alters the development of a specific somatic cell (Sertoli cell) epigenome and transcriptome that correlates with adult onset disease (male infertility). The environmentally induced epigenetic transgenerational inheritance of testis disease appears to be a component of the molecular etiology of male infertility. PMID:23555832

  13. Sertoli-germ cell junctions in the testis: a review of recent data.

    PubMed

    Kopera, Ilona A; Bilinska, Barbara; Cheng, C Yan; Mruk, Dolores D

    2010-05-27

    Spermatogenesis is a process that involves an array of cellular and biochemical events, collectively culminating in the formation of haploid spermatids from diploid precursor cells known as spermatogonia. As germ cells differentiate from spermatogonia into elongated spermatids, they also progressively migrate across the entire length of the seminiferous epithelium until they reach the luminal edge in anticipation of spermiation at late stage VIII of spermatogenesis. At the same time, these germ cells must maintain stable attachment with Sertoli cells via testis-unique intermediate filament- (i.e. desmosome-like junctions) and actin- (i.e. ectoplasmic specializations, ESs) based cell junctions to prevent sloughing of immature germ cells from the seminiferous epithelium, which may result in infertility. In essence, both desmosome-like junctions and basal ESs are known to coexist between Sertoli cells at the level of the blood-testis barrier where they cofunction with the well-studied tight junction in maintaining the immunological barrier. However, the type of anchoring device that is present between Sertoli and germ cells depends on the developmental stage of the germ cell, i.e. desmosome-like junctions are present between Sertoli and germ cells up to, but not including, step 8 spermatids after which this junction type is replaced by the apical ES. While little is known about the biology of the desmosome-like junction in the testis, we have a relatively good understanding of the molecular architecture and the regulation of the ES. Here, we discuss recent findings relating to these two junction types in the testis, highlighting prospective areas that should be investigated in future studies. PMID:20403872

  14. A spontaneously occurring malignant ovarian Sertoli cell tumor in a young Sprague Dawley rat.

    PubMed

    Kinoshita, Yuichi; Yoshizawa, Katsuhiko; Emoto, Yuko; Yuki, Michiko; Yuri, Takashi; Shikata, Nobuaki; Elmore, Susan A; Tsubura, Airo

    2016-01-01

    Primary ovarian tumors are generally uncommon in rats used in toxicologic studies. A malignant Sertoli cell tumor was present in the ovary of a 19-week-old female Sprague Dawley rat. Macroscopically, the mass was white and firm, 10 × 13 × 17 mm in size, and located in the right ovary. Histopathologically, the mass was composed of nests of pleomorphic cells, which formed seminiferous-like tubules separated by a thin fibrovascular stroma. The tubules were lined by tumor cells, which had basally located nuclei and abundant eosinophilic and vacuolated cytoplasm. In some areas, the tumor cells were arranged in a retiform growth pattern, mimicking a rete testis/ovarii. Disseminated metastases to the surfaces of the mesentery, spleen and liver were also present. Immunohistochemically, many tumor cells were strongly positive for vimentin, estrogen receptor α and Ki 67. Some tumor cells were positive for pancytokeratin and inhibin α. These findings closely resemble those of an ovarian-derived human malignant Sertoli cell tumor. From our review of the literature, we believe this is the first report of a spontaneous malignant Sertoli cell tumor in the ovary of a young laboratory rat. This case might provide useful historical control information for rat toxicity studies. PMID:26989303

  15. A spontaneously occurring malignant ovarian Sertoli cell tumor in a young Sprague Dawley rat

    PubMed Central

    Kinoshita, Yuichi; Yoshizawa, Katsuhiko; Emoto, Yuko; Yuki, Michiko; Yuri, Takashi; Shikata, Nobuaki; Elmore, Susan A.; Tsubura, Airo

    2015-01-01

    Primary ovarian tumors are generally uncommon in rats used in toxicologic studies. A malignant Sertoli cell tumor was present in the ovary of a 19-week-old female Sprague Dawley rat. Macroscopically, the mass was white and firm, 10 × 13 × 17 mm in size, and located in the right ovary. Histopathologically, the mass was composed of nests of pleomorphic cells, which formed seminiferous-like tubules separated by a thin fibrovascular stroma. The tubules were lined by tumor cells, which had basally located nuclei and abundant eosinophilic and vacuolated cytoplasm. In some areas, the tumor cells were arranged in a retiform growth pattern, mimicking a rete testis/ovarii. Disseminated metastases to the surfaces of the mesentery, spleen and liver were also present. Immunohistochemically, many tumor cells were strongly positive for vimentin, estrogen receptor α and Ki 67. Some tumor cells were positive for pancytokeratin and inhibin α. These findings closely resemble those of an ovarian-derived human malignant Sertoli cell tumor. From our review of the literature, we believe this is the first report of a spontaneous malignant Sertoli cell tumor in the ovary of a young laboratory rat. This case might provide useful historical control information for rat toxicity studies. PMID:26989303

  16. Expression of fetal-type intermediate filaments by 17-day-old rat Sertoli cells cultured on reconstituted basement membrane.

    PubMed

    Guillou, F; Monet-Kuntz, C; Fontaine, I; Flechon, J E

    1990-05-01

    The expression of cytokeratin- and vimentin-type intermediate filaments was studied by means of immunohistochemistry in Sertoli cells cultured on two types of reconstituted basement membrane in two-compartment culture chambers. In situ, the Sertoli cells of 17-day-old rats contained only vimentin intermediate filaments. During culture, a gradual reorganization of intermediate filaments accompanied by an increased cytokeratin immunoreactivity was observed. After 6 days, Sertoli cells contained both cytokeratin and vimentin, and the same cytokeratin type as in fetal and newborn testis was revealed by electrophoresis and immunoblotting. The present study shows that the isolation and culture of Sertoli cells causes, even in an improved culture system, qualitative changes in the expression of intermediate filament proteins. PMID:1694107

  17. Mumps virus-induced innate immune responses in mouse Sertoli and Leydig cells

    PubMed Central

    Wu, Han; Shi, Lili; Wang, Qing; Cheng, Lijing; Zhao, Xiang; Chen, Qiaoyuan; Jiang, Qian; Feng, Min; Li, Qihan; Han, Daishu

    2016-01-01

    Mumps virus (MuV) infection frequently causes orchitis and impairs male fertility. However, the mechanisms underlying the innate immune responses to MuV infection in the testis have yet to be investigated. This study showed that MuV induced innate immune responses in mouse Sertoli and Leydig cells through TLR2 and retinoic acid-inducible gene I (RIG-I) signaling, which result in the production of proinflammatory cytokines and chemokines, including TNF-α, IL-6, MCP-1, CXCL10, and type 1 interferons (IFN-α and IFN-β). By contrast, MuV did not induce the cytokine production in male germ cells. In response to MuV infection, Sertoli cells produced higher levels of proinflammatory cytokines and chemokines but lower levels of type 1 IFNs than Leydig cells did. The MuV-induced cytokine production by Sertoli and Leydig cells was significantly reduced by the knockout of TLR2 or the knockdown of RIG-I signaling. The local injection of MuV into the testis triggered the testicular innate immune responses in vivo. Moreover, MuV infection suppressed testosterone synthesis by Leydig cells. This is the first study examining the innate immune responses to MuV infection in testicular cells. The results provide novel insights into the mechanisms underlying the MuV-induced innate immune responses in the testis. PMID:26776505

  18. Prenatal and lactation nicotine exposure affects Sertoli cell and gonadotropin levels in rats.

    PubMed

    Paccola, C C; Miraglia, S M

    2016-02-01

    Nicotine is largely consumed in the world as a component of cigarettes. It can cross the placenta and reach the milk of smoking mothers. This drug induces apoptosis, affects sex hormone secretion, and leads to male infertility. To investigate the exposure to nicotine during the whole intrauterine and lactation phases in Sertoli cells, pregnant rats received nicotine (2 mg/kg per day) through osmotic minipumps. Male offsprings (30, 60, and 90 days old) had blood collected for hormonal analysis (FSH and LH) and their testes submitted for histophatological study, analysis of the frequency of the stages of seminiferous epithelium cycle, immunolabeling of apoptotic epithelial cells (TUNEL and Fas/FasL), analysis of the function and structure of Sertoli cells (respectively using transferrin and vimentin immunolabeling), and analysis of Sertoli-germ cell junctional molecule (β-catenin immunolabeling). The exposure to nicotine increased the FSH and LH plasmatic levels in adult rats. Although nicotine had not changed the number of apoptotic cells, neither in Fas nor FasL expression, it provoked an intense sloughing of epithelial cells and also altered the frequency of some stages of the seminiferous epithelium cycle. Transferrin and β-catenin expressions were not changed, but vimentin was significantly reduced in the early stages of the seminiferous cycle of the nicotine-exposed adult rats. Thus, we concluded that nicotine exposure during all gestational and lactation periods affects the structure of Sertoli cells by events causing intense germ cell sloughing observed in the tubular lumen and can compromise the fertility of the offspring. PMID:26556892

  19. Direct reprogramming of Sertoli cells into multipotent neural stem cells by defined factors

    PubMed Central

    Sheng, Chao; Zheng, Qinyuan; Wu, Jianyu; Xu, Zhen; Wang, Libin; Li, Wei; Zhang, Haijiang; Zhao, Xiao-Yang; Liu, Lei; Wang, Ziwei; Guo, Changlong; Wu, Hua-Jun; Liu, Zhonghua; Wang, Liu; He, Shigang; Wang, Xiu-Jie; Chen, Zhiguo; Zhou, Qi

    2012-01-01

    Multipotent neural stem/progenitor cells hold great promise for cell therapy. The reprogramming of fibroblasts to induced pluripotent stem cells as well as mature neurons suggests a possibility to convert a terminally differentiated somatic cell into a multipotent state without first establishing pluripotency. Here, we demonstrate that Sertoli cells derived from mesoderm can be directly converted into a multipotent state that possesses neural stem/progenitor cell properties. The induced neural stem/progenitor cells (iNSCs) express multiple NSC-specific markers, exhibit a global gene-expression profile similar to normal NSCs, and are capable of self-renewal and differentiating into glia and electrophysiologically functional neurons. iNSC-derived neurons stain positive for tyrosine hydroxylase (TH), ?-aminobutyric acid, and choline acetyltransferase. In addition, iNSCs can survive and generate synapses following transplantation into the dentate gyrus. Generation of iNSCs may have important implications for disease modeling and regenerative medicine. PMID:22064700

  20. Role of Endoplasmic reticulum apoptotic pathway in testicular Sertoli cells injury induced by Carbon disulfide.

    PubMed

    Guo, Yinsheng; Ji, Jiajia; Wang, Wei; Dong, Yu; Zhang, Zhen; Zhou, Yijun; Chen, Guoyuan; Cheng, Jinquan

    2015-08-01

    The exposure of Carbon disulfide (CS2) is associated with germ cell injury and male infertility in animals and humans. However, the molecular mechanism is currently unknown. This study show here that CS2-induced Sertoli cells injury via Endoplasmic reticulum (ER) apoptotic pathway. SD male rats were exposed to doses of CS2 (0, 50, 250, 1250mgm(-3)) for 4weeks. After treatment, loose structures of seminiferous tubules and disordered cell arrangements were observed by light microscopy. Ultrastructural lesions, deformed chromatins and vacuoles formed from swollen ER were observed by electron microscopy. After primary culture of Sertoli cells, a dose-dependent increased apoptosis were found. The increased activity of Caspase 3, accumulation of intracellular Ca(2+), up-regulation of mRNA and protein expressions of ER apoptotic relative molecules (Calpain 2, Cleaved-Caspase 12, GRP78 and CHOP) were also found in this study. Altogether, our findings indicated that ER apoptotic pathway played an important role in CS2-induced Sertoli cell impairment. PMID:25816788

  1. Impact of low molecular weight phthalates in inducing reproductive malfunctions in male mice: Special emphasis on Sertoli cell functions.

    PubMed

    Kumar, Narender; Srivastava, Swati; Roy, Partha

    2015-05-01

    Phthalates are commonly used as plasticizers in a variety of products. Since they have been identified as endocrine-disrupting chemicals (EDCs), effect of phthalates on human health is a major concern. In this study, we evaluated individual as well as combined mixture effects of three low molecular weight phthalates on the reproductive system of male mice, specifically on the Sertoli cell structure and function. In order to analyze the blood testes barrier (BTB) dynamics, primary culture of Sertoli cells from 3-weeks old male mice was used for mimicking typical tight junction structures. Male mice were exposed to long-term (45 days) and combined mixture of three phthalates, diethyl phthalate (DEP), diphenyl phthalate (DPP), and dimethyl isophthalate (DMIP) between pre-pubertal to adult stage. Our data showed significant decrease (p < 0.05) in the rates of transcription of certain prominent Sertoli cell specific genes like transferrin, testin and occludin. Moreover, we also observed significant decreases in the expression of proteins like 3β-HSD, connexin-43 and occludin in testicular lysates of treated animals (p < 0.05). The transmission electron microscopic analysis revealed that the test compounds significantly altered the structural integrity of Sertoli cells. The significant changes of Sertoli cell tight junction structure by test compounds were associated with phosphorylation of ERK. Taken together, our study suggests that low molecular weight phthalates may affect male fertility by altering both structural and functional integrity of Sertoli cells in testes. PMID:25268316

  2. TiO2 nanoparticles-induced apoptosis of primary cultured Sertoli cells of mice.

    PubMed

    Hong, Fashui; Zhao, Xiaoyang; Chen, Ming; Zhou, Yingjun; Ze, Yuguan; Wang, Ling; Wang, Yajing; Ge, Yushuang; Zhang, Qi; Ye, Lingqun

    2016-01-01

    Titanium dioxide nanoparticles (TiO2 NPs), as largest production and use of nanomaterials, have been demonstrated to have a potential toxicity on reproductive system. However, the mechanism underlying male reproductive toxicity of TiO2 NPs remains limited. Thus, our study was designed to examine the cellular viability, apoptosis, oxidative stress, antioxidant capacity, and expression of apoptotic cytokines in primary cultured Sertoli cells isolated from mice under TiO2 NPs exposure. Results showed that TiO2 NPs exposure from 5 to 30 μg/mL resulted in reduction of cell viability, lactate dehydrogenase release, and induction of apoptosis or death on Sertoli cells. TiO2 NPs could migrate to Sertoli cells, which induced mitochondria-mediated or endoplasmic-reticulum-mediated apoptotic changes including elevation in reactive oxygen species (ROS) generation and reductions in superoxide dismutase, catalase, and glutathione peroxidase activities, decreases in mitochondrial membrane potential (ΔΨm), and releases of cytochrome c into the cytosol. In addition, upregulation of cytochrome c, Bax, caspase-3, glucose-regulated protein 78, and C/EBP homologous protein and caspase-12 protein expression, and downregulation of bcl-2 protein expression in primary cultured Sertoli cells induced by TiO2 NPs treatment. All of the results suggested that ROS generation may play a critical role in the initiation of TiO2 NPs-induced apoptosis by mediation of the disruption of ΔΨm, the cytochrome c release, and further the activation of caspase cascade and unfolded protein response signaling pathway. PMID:26238530

  3. Androgen-Dependent Sertoli Cell Tight Junction Remodeling Is Mediated by Multiple Tight Junction Components

    PubMed Central

    Chakraborty, Papia; William Buaas, F.; Sharma, Manju; Smith, Benjamin E.; Greenlee, Anne R.; Eacker, Stephen M.

    2014-01-01

    Sertoli cell tight junctions (SCTJs) of the seminiferous epithelium create a specialized microenvironment in the testis to aid differentiation of spermatocytes and spermatids from spermatogonial stem cells. SCTJs must be chronically broken and rebuilt with high fidelity to allow the transmigration of preleptotene spermatocytes from the basal to adluminal epithelial compartment. Impairment of androgen signaling in Sertoli cells perturbs SCTJ remodeling. Claudin (CLDN) 3, a tight junction component under androgen regulation, localizes to newly forming SCTJs and is absent in Sertoli cell androgen receptor knockout (SCARKO) mice. We show here that Cldn3-null mice do not phenocopy SCARKO mice: Cldn3−/− mice are fertile, show uninterrupted spermatogenesis, and exhibit fully functional SCTJs based on imaging and small molecule tracer analyses, suggesting that other androgen-regulated genes must contribute to the SCARKO phenotype. To further investigate the SCTJ phenotype observed in SCARKO mutants, we generated a new SCARKO model and extensively analyzed the expression of other tight junction components. In addition to Cldn3, we identified altered expression of several other SCTJ molecules, including down-regulation of Cldn13 and a noncanonical tight junction protein 2 isoform (Tjp2iso3). Chromatin immunoprecipitation was used to demonstrate direct androgen receptor binding to regions of these target genes. Furthermore, we demonstrated that CLDN13 is a constituent of SCTJs and that TJP2iso3 colocalizes with tricellulin, a constituent of tricellular junctions, underscoring the importance of androgen signaling in the regulation of both bicellular and tricellular Sertoli cell tight junctions. PMID:24825397

  4. Identification of the Functions of Liver X Receptor-β in Sertoli Cells Using a Targeted Expression-Rescue Model.

    PubMed

    Maqdasy, Salwan; El Hajjaji, Fatim-Zohra; Baptissart, Marine; Viennois, Emilie; Oumeddour, Abdelkader; Brugnon, Florence; Trousson, Amalia; Tauveron, Igor; Volle, David; Lobaccaro, Jean-Marc A; Baron, Silvère

    2015-12-01

    Liver X receptors (LXRs) are key regulators of lipid homeostasis and are involved in multiple testicular functions. The Lxrα(-/-);Lxrβ(-/-) mice have illuminated the roles of both isoforms in maintenance of the epithelium in the seminiferous tubules, spermatogenesis, and T production. The requirement for LXRβ in Sertoli cells have been emphasized by early abnormal cholesteryl ester accumulation in the Lxrβ(-/-) and Lxrα(-/-);Lxrβ(-/-) mice. Other phenotypes, such as germ cell loss and hypogonadism, occur later in life in the Lxrα(-/-);Lxrβ(-/-) mice. Thus, LXRβ expression in Sertoli cells seems to be essential for normal testicular physiology. To decipher the roles of LXRβ within the Sertoli cells, we generated Lxrα(-/-);Lxrβ(-/-):AMH-Lxrβ transgenic mice, which reexpress Lxrβ in Sertoli cells in the context of Lxrα(-/-);Lxrβ(-/-) mice. In addition to lipid homeostasis, LXRβ is necessary for maintaining the blood-testis barrier and the integrity of the germ cell epithelium. LXRβ is also implicated in the paracrine action of Sertoli cells on Leydig cells to modulate T synthesis. The Lxrα(-/-);Lxrβ(-/-) and Lxrα(-/-);Lxrβ(-/-):AMH-Lxrβ mice exhibit lipid accumulation in germ cells after the Abcg8 down-regulation, suggesting an intricate LXRβ-dependent cooperation between the Sertoli cells and germ cells to ensure spermiogenesis. Further analysis revealed also peritubular smooth muscle defects (abnormal lipid accumulation and disorganized smooth muscle actin) and spermatozoa stagnation in the seminiferous tubules. Together the present work elucidates specific roles of LXRβ in Sertoli cell physiology in vivo beyond lipid homeostasis. PMID:26402841

  5. Hybrid GPCR/cadherin (Celsr) proteins in rat testis are expressed with cell type specificity and exhibit differential Sertoli cell-germ cell adhesion activity.

    PubMed

    Beall, Stephanie A; Boekelheide, Kim; Johnson, Kamin J

    2005-01-01

    Spermatogenesis requires Sertoli cell-germ cell adhesion for germ cell survival and maturation. Cadherins are a diverse superfamily of adhesion proteins; structurally unique members of this superfamily (celsr cadherins) are hybrid molecules containing extracellular cadherin repeats connected to a G protein-coupled receptor transmembrane motif. Here we demonstrate postnatal testicular mRNA expression of the 3 celsr paralogs (celsr1, celsr2, and celsr3), protein localization of celsr2 and celsr3, and functional analysis of celsr2 adhesion activity in primary Sertoli cell-germ cell co-cultures. Evaluation of celsr mRNA levels during a postnatal time course indicated that celsr1 and celsr2 were Sertoli cell and/or early-stage germ cell products, whereas celsr3 was expressed in later-stage germ cells. Cell type-specific expression was verified using the Sertoli cell line 93RS2, where celsr1 and celsr2 mRNA, but not celsr3, were detected. Immunostaining of testicular cryosections resulted in celsr2 protein localization to a spokelike pattern in the basal seminiferous epithelium and punctate figures in the apical epithelium, consistent with both Sertoli cell and germ cell expression. Celsr3 localized to punctate structures in the adluminal epithelium from postnatal day 40, consistent with elongate spermatid expression. The subcellular localization of celsr2 was examined further to define its localization in Sertoli cells and germ cells. Celsr2 localized to the Golgi complex in Sertoli cells and germ cells. In addition, germ cell celsr2 localized to a rab7-positive structure, which may be an endocytic compartment. Neither celsr2 nor celsr3 immunostaining was present at classic cadherin-based adhesion junctions. Nonetheless, the addition of a recombinant celsr2 protein fragment consisting of extracellular cadherin domains 4 through 8 to Sertoli cell-germ cell co-cultures resulted in germ cell detachment from Sertoli cells. Collectively, these data indicate that celsr cadherins have a cell type-specific expression pattern, and celsr2 may mediate Sertoli cell-germ cell adhesion outside of classic cadherin-based adhesion junctions. PMID:15955893

  6. The death of sertoli cells and the capacity to phagocytize elongated spermatids during testicular regression due to short photoperiod in Syrian hamster (Mesocricetus auratus).

    PubMed

    Seco-Rovira, Vicente; Beltrán-Frutos, Esther; Ferrer, Concepción; Sáez, Francisco José; Madrid, Juan Francisco; Pastor, Luis Miguel

    2014-05-01

    In the Syrian hamster (Mesocricetus auratus), an animal that displays testicular regression due to short photoperiod, germ cells are removed by apoptosis during this process and the apoptotic remains are phagocytized by Sertoli cells. The aim of this work was to investigate morphologically whether the testicular regression process due to short photoperiod leads to the apoptosis of Sertoli cells, and whether, during testicular regression, the elongated spermatids are eliminated through phagocytosis by Sertoli cells. To this end, we studied testis sections during testicular regression in Syrian hamster subjected to short photoperiod by means of several morphological techniques using conventional light microscopy (hematoxylin and eosin [H&E], semi-thin section vimentin, immunohistochemistry, SBA lectin, and TUNEL staining), fluorescence microscopy, and transmission electron microscopy (TEM). H&E and semi-thin sections identified Sertoli cells with a degenerated morphology. Greater portion of Sertoli cells that were positive for TUNEL staining were observed especially during the mild regression (MR) and strong regression (SR) phases. In addition, TEM identified the characteristic apoptotic changes in the nucleus and cytoplasm of Sertoli cells. Moreover, during testicular regression and using light microscopy, some elongated spermatids were seen in basal position next to the Sertoli cell nucleus. This Sertoli phagocytic activity was higher in MR and SR phases. TEM confirmed this to be the result of the phagocytic activity of Sertoli cells. In conclusion, during testicular regression in Syrian hamster due to short photoperiod, when germ cells are known to be lost through apoptosis, there is morphological evidences that Sertoli cells are also lost through apoptosis, while some elongated spermatids are phagocytized and eliminated by the Sertoli cells. PMID:24719257

  7. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cytotoxicity accompanied by oxidative stress in rat Sertoli cells: Possible role of mitochondrial fractions of Sertoli cells

    SciTech Connect

    Aly, Hamdy A.A.; Khafagy, Rasha M.

    2011-05-01

    TCDD, as an endocrine disruptor, is known to impair testicular functions and fertility. To elucidate the mechanism(s) underlying the testicular effects of TCDD, the potential toxicity of TCDD on Sertoli cells was investigated. Furthermore, the study aims to delineate whether mitochondrial fractions of Sertoli cells are involved in mediating the testicular effects of TCDD. Adult rat Sertoli cells were incubated with (5, 10 or 15 nM) of TCDD for 6, 12 or 24 h. Cell viability, lactate and LDH leakage into media along with lipid peroxidation, ROS generation, SOD, CAT, GPx, GR, {gamma}-GT and {beta}-glucuronidase activities, GSH content and {Delta}{psi}{sub m} were measured. Superoxide anion production, COX and cardiolipin content were measured in mitochondrial fractions. Cell viability was significantly decreased while lactate and LDH leakage into media were increased. ROS generation along with lipid peroxidation was also increased. SOD, CAT, GPx, GR activities and GSH content were significantly decreased. {gamma}-GT and {beta}-glucuronidase activities were also decreased. Superoxide anion production was increased while COX activity and cardiolipin content were decreased in mitochondrial fractions. Moreover, the {Delta}{psi}{sub m} was significantly decreased as measured in Sertoli cells. In conclusion, TCDD impairs Sertoli cell functions and this effect is, at least in part, attributed to oxidative stress. We have also found that TCDD increases mitochondrial superoxide anion production and decreases {Delta}{psi}{sub m}, COX activity and mitochondrial cardiolipin content. Our findings suggest that mitochondria may play an important role in ROS production, leading to the TCDD-induced oxidative stress response and resulting toxicological consequences in rat Sertoli cells.

  8. Early effects of Sertoli cell-selective androgen receptor ablation on testicular gene expression.

    PubMed

    Willems, A; De Gendt, K; Allemeersch, J; Smith, L B; Welsh, M; Swinnen, J V; Verhoeven, G

    2010-06-01

    Evidence from several models of hormone depletion and/or replacement and from knockout animals points to a key role of androgens in the control of spermatogenesis. In testes of mice with a Sertoli cell-selective ablation of the androgen receptor (SCARKO), transcriptional profiling, using microarray technology, revealed that, already on postnatal day 10,692 genes are differentially expressed compared with testes of control mice. Further evaluation of a subset of these genes by quantitative RT-PCR suggested that differences in expression may already be evident on day 8 or earlier. As the androgen receptor in mouse Sertoli cells becomes immunologically detectable around day 5, we tried to identify the earliest responses to androgens by a new transcriptional profiling study on testes from 6-day-old SCARKO and control mice. No obvious and novel early androgen response genes, potentially acting as mediators of subsequent indirect androgen actions, could be identified. However, several genes differentially expressed on day 10 already displayed a response to androgen receptor ablation on day 6. Quantitative RT-PCR studies for 12 of these genes on 10 paired SCARKO and control testes from 4-, 6-, 8-, 10-, 20- and 50-day-old mice revealed significant differences in expression level from day 4 onwards for three genes (Eppin, PCI, Cldn11) and from day 6 onwards for one more gene (Rhox5). For at least two of these genes (Rhox5 and Eppin), there is evidence for direct regulation via the androgen receptor. For three additional genes (Gpd1, Tubb3 and Tpd52l1) significantly lower expression in the SCARKO was noted from day 8 onwards. For all the studied genes, an impressive increase in transcript levels was observed between day 4-50 and differential expression was maintained in adulthood. It is concluded that the SCARKO model indicates incipient androgen action in mouse Sertoli cells from day 4 onwards. PMID:19392831

  9. Ovarian Sertoli-Leydig Cell Tumour with Heterologus Elements Masquerading as Mucinous Tumour on Frozen Section: A Case Report

    PubMed Central

    Valiathan, Manna; Kumar, Sandeep; Kapoor, Sukriti

    2015-01-01

    Sertoli-Leydig cell tumour (SLCT) is an extremely rare ovarian neoplasm. This tumour is characterized by excessive proliferation of normal testicular structures sertoli and leydig cells. These cells are seen in varying proportions and exhibit varying degrees of differentiation. We report a case of primary ovarian SLCT with heterologus elements in a 17-year-old girl which was misdiagnosed on frozen section as mucinous cystic neoplasm. We discuss the clinicopathologic features of SLCT along with the unusual features seen in this case. PMID:26393134

  10. Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis.

    PubMed

    Gelly, J L; Richoux, J P; Grignon, G

    1994-05-01

    The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV-VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis. PMID:8020066

  11. Retinoic acid induces Sertoli cell paracrine signals for spermatogonia differentiation but cell autonomously drives spermatocyte meiosis

    PubMed Central

    Raverdeau, Mathilde; Gely-Pernot, Aurore; Féret, Betty; Dennefeld, Christine; Benoit, Gérard; Davidson, Irwin; Chambon, Pierre; Mark, Manuel; Ghyselinck, Norbert B.

    2012-01-01

    Direct evidence for a role of endogenous retinoic acid (RA), the active metabolite of vitamin A in the initial differentiation and meiotic entry of spermatogonia, and thus in the initiation of spermatogenesis is still lacking. RA is synthesized by dedicated enzymes, the retinaldehyde dehydrogenases (RALDH), and binds to and activates nuclear RA receptors (RARA, RARB, and RARG) either within the RA-synthesizing cells or in the neighboring cells. In the present study, we have used a combination of somatic genetic ablations and pharmacological approaches in vivo to show that during the first, prepubertal, spermatogenic cycle (i) RALDH-dependent synthesis of RA by Sertoli cells (SC), the supporting cells of the germ cell (GC) lineage, is indispensable to initiate differentiation of A aligned into A1 spermatogonia; (ii) RARA in SC mediates the effects of RA, possibly through activating Mafb expression, a gene whose Drosophila homolog is mandatory to GC differentiation; (iii) RA synthesized by premeiotic spermatocytes cell autonomously induces meiotic initiation through controlling the RAR-dependent expression of Stra8. Furthermore, we show that RA of SC origin is no longer necessary for the subsequent spermatogenic cycles but essential to spermiation. Altogether, our data establish that the effects of RA in vivo on spermatogonia differentiation are indirect, via SC, but direct on meiotic initiation in spermatocytes, supporting thereby the notion that, contrary to the situation in the female, RA is necessary to induce meiosis in the male. PMID:23012458

  12. Metabolomic profiles reveal key metabolic changes in heat stress-treated mouse Sertoli cells.

    PubMed

    Xu, Bo; Chen, Minjian; Ji, Xiaoli; Yao, Mengmeng; Mao, Zhilei; Zhou, Kun; Xia, Yankai; Han, Xiao; Tang, Wei

    2015-10-01

    Heat stress (HS) is a potential harmful factor for male reproduction. However, the effect of HS on Sertoli cells is largely unknown. In this study, the metabolic changes in Sertoli cell line were analyzed after HS treatment. Metabolomic analysis revealed that carnitine, 2-hydroxy palmitic acid, nicotinic acid, niacinamide, adenosine monophosphate, glutamine and creatine were the key changed metabolites. We found the expression levels of BTB factors (Connexin43, ZO-1, Vimentin, Claudin1, Claudin5) were disrupted in TM-4 cells after HS treatment, which were recovered by the addition of carnitine. RT-PCR indicated that the mRNA levels of inflammatory cytokines (IL-1α, IL-1β, IL-6) were increased after HS treatment, and their related miRNAs (miR-132, miR-431, miR-543) levels were decreased. Our metabolomic data provided a novel understanding of metabolic changes in male reproductive cells after HS treatment and revealed that HS-induced changes of BTB factors and inflammatory status might be caused by the decreased carnitine after HS treatment. PMID:26165742

  13. Sox8 is a critical regulator of adult Sertoli cell function and male fertility.

    PubMed

    O'Bryan, Moira K; Takada, Shuji; Kennedy, Claire L; Scott, Greg; Harada, Shun-ichi; Ray, Manas K; Dai, Qunsheng; Wilhelm, Dagmar; de Kretser, David M; Eddy, E Mitch; Koopman, Peter; Mishina, Yuji

    2008-04-15

    Sox8 encodes a high-mobility group transcription factor that is widely expressed during development. Sox8, -9 and -10 form group E of the Sox gene family which has been implicated in several human developmental disorders. In contrast to other SoxE genes, the role of Sox8 is unclear and Sox8 mouse mutants reportedly showed only idiopathic weight loss and reduced bone density. The careful analysis of our Sox8 null mice, however, revealed a progressive male infertility phenotype. Sox8 null males only sporadically produced litters of reduced size at young ages. We have shown that SOX8 protein is a product of adult Sertoli cells and its elimination results in an age-dependent deregulation of spermatogenesis, characterized by sloughing of spermatocytes and round spermatids, spermiation failure and a progressive disorganization of the spermatogenic cycle, which resulted in the inappropriate placement and juxtaposition of germ cell types within the epithelium. Those sperm that did enter the epididymides displayed abnormal motility. These data show that SOX8 is a critical regulator of adult Sertoli cell function and is required for both its cytoarchitectural and paracrine interactions with germ cells. PMID:18342849

  14. BMP4 promotes human Sertoli cell proliferation via Smad1/5 and ID2/3 pathway and its abnormality is associated with azoospermia.

    PubMed

    Hai, Yanan; Sun, Min; Niu, Minghui; Yuan, Qingqing; Guo, Ying; Li, Zheng; He, Zuping

    2015-04-01

    Sertoli cell plays critical roles in regulating testis development and spermatogenesis. Any change in the number or biological functions of Sertoli cells can affect the normal formation of spermatozoa. However, the roles and molecular mechanisms of factors in controlling the fate determinations of human Sertoli cells and underlying male infertility remain unknown. Here we have for the first time explored the function and signaling pathway of BMP4 in regulating adult human Sertoli cells and their association with non-obstructive azoospermia (NOA) patients. Immunocytochemistry and immunohistochemistry revealed that BMP4 and its multiple receptors were present in human Sertoli cells. Cell proliferation and BrdU incorporation assays showed that BMP4 promoted DNA synthesis and proliferation of Sertoli cells. In contrast, BMP4 antagonist noggin and BMP4 knockdown reduced the division of Sertoli cells. Moreover, BMP4 knockdown inhibited the synthesis of FGF2, SCF, zonula occludens 1, and claudin 11 but enhanced p27kip1 transcription. BMP4 activated Smad1/5 phosphorylation and upregulated ID2 and ID3 transcription, whereas noggin counteracted these increases. Significantly, tissue arrays disclosed that overexpression of BMP4 may be associated with Sertoli cell-only syndrome and maturation arrest in spermatogonia or spermatocytes. BMP4 was identified as the first autocrine factor that regulates the proliferation and protein synthesis of human Sertoli cells via Smad1/5 and ID2/3 and its abnormality is associated with human non-obstructive azoospermia patients. This study thus provides novel insights into molecular mechanism underlying adult human Sertoli cell growth and offers new targets for gene therapy of male infertility. PMID:25977194

  15. FGF2 stimulates SDF-1 expression through the Erm transcription factor in Sertoli cells.

    PubMed

    Yoon, Kyung-Ae; Chae, Young-Mi; Cho, Je-Yoel

    2009-07-01

    Ets-related molecule (Erm) is a member of the Ets transcription factor family. Erm is known to be an important factor for the self-renewal of Spermatogonial stem cells (SSCs) and the maintenance of spermatogenesis. We investigated the molecular mechanism of Erm regulation on SDF-1 in TM4 Sertoli cells. Erm and Sdf-1 levels were up-regulated after FGF2 treatment in TM4 cells, whereas these levels were significantly decreased by FGF2 in ST2 bone marrow stromal cells. Knockdown of Erm by siRNA in the presence of FGF2 decreased the Sdf-1 levels in TM4 cells. The expression levels of Erm were similar and Erm overexpression increased the Sdf-1 in both TM4 and ST2 cells. FGFR subtype analysis revealed that FGFR4 was expressed in TM4 cells but not in ST2 cells. A blocking experiment also confirmed that FGFR4 is partly responsible for the up-regulation of Erm and SDF-1 induced by FGF2 stimulation in TM4 cells. FGF2 and ERM increased the activity of Sdf-1 gene promoter region in a dose-dependent manner. EMSA revealed that ERM strongly binds to the -846 to -851 nucleotide region of the potential Ets binding site (EBS) in the Sdf-1 promoter. In addition, CXCR4, the SDF-1 receptor, was expressed in spermatogonia and Sertoli cells in the seminiferous tubules of the mouse testis. Our results indicate that ERM directly regulates Sdf-1 gene expression by interacting with its cis-acting element in response to FGF2 stimulation in TM4 cells. PMID:19301256

  16. Morphometry and morphology of nucleus of the Sertoli and interstitial cells of the tambaqui Colossoma macropomum (Cuvier, 1881) (Pisces: Characidae) during the reproductive cycle.

    PubMed

    Nakaghi, L S O; Mitsuiki, D; Santos, H S L; Pacheco, M R; Ganeco, L N

    2003-02-01

    This study allowed the characterization of the tambaqui Colossoma macropomum testes structural organization, emphasizing Sertoli and interstitial cells and analyzing morphometrically the Sertoli cell nucleus diameter and the interstitial tissue area during the reproductive cycle. Fragments of tambaqui testes were collected in the following reproductive cycle stages: immature, resting, maturation I and II, mature, and regression, and were histologically processed. The Sertoli cells were found at the periphery of the cysts of germinative lineage cells and the nuclei were shown to be smaller as these cells developed. The interstitial cells were better observed between the seminiferous lobules next to vessels in the interstitial tissue of maturing testes. PMID:12914420

  17. SERTOLI CELLS IN THE BOAR TESTIS: CHANGES DURING DEVELOPMENT AND COMPENSATORY HYPERTROPHY AFTER HEMICASTRATION AT DIFFERENT AGES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Changes in Sertoli cell numbers and testicular structure during normal development and during compensatory hypertrophy were assessed in crossbred Meishan x White Composite males. Boars were assigned at birth to unilateral castration at 1, 10, 56 or 112 days, or remained as intact controls through 22...

  18. Modulation of Dhh signaling and altered Sertoli cell function in mice lacking the GPR37-prosaposin receptor.

    PubMed

    La Sala, Gina; Marazziti, Daniela; Di Pietro, Chiara; Golini, Elisabetta; Matteoni, Rafaele; Tocchini-Valentini, Glauco P

    2015-05-01

    The mammalian G-protein-coupled receptor 37 (GPR37) is expressed in brain, in adult testis, and during the early phase of gonad differentiation. Somatic Sertoli cells (SCs) are located within the seminiferous tubules where they support the germinal epithelium. An adequate number of SCs is required for the complete prepubertal differentiation of germ cells and adult fertility. This study shows that Gpr37 and its ligand prosaposin are both postnatally expressed by SCs, whose proliferation and maturation are affected in Gpr37-null mutant mice during postnatal testicular development. Mutant pups show a delayed timing in sperm cell development, with a partial arrest of spermatocytes at the meiotic pachytene (e.g., 1.5-fold increase in Gpr37(-/-) P21 pups) and their increased apoptosis (e.g., 1.8-fold and 3.5-fold increase in Gpr37(-/-) P21 and adult mice, respectively). Mutant adults have reduced testis weight (wild type, 299 ± 5 mg; knockout, 258 ± 16 mg; P < 0.05) and epididymal sperm count and motility (e.g., 1.5-fold and 1.45-fold decrease in Gpr37(-/-) mice, respectively). Lack of Gpr37 results in the reduction in androgen receptor levels during prepubertal testis development, alongside the altered expression of SC maturation markers. It also affects the prepubertal testis expression of desert hedgehog (Dhh) mitogenic cascade components (Dhh, 1.3-fold increase in Gpr37(-/-) P10 and P21 pups; Gli2, 1.4-fold and 1.6-fold increase in Gpr37(-/-) P10 and P21 pups, respectively) including patched homolog 1 (1.3-fold increase in Gpr37(-/-) P10 and P21 pups), which is found localized in prepubertal SCs and is associated with Gpr37 in cultured primary SC samples. These results indicate that Gpr37 is a specific modulator of murine testis Dhh mitogenic signaling and SC proliferation and maturation. PMID:25609427

  19. Testosterone deficiency induced by progressive stages of diabetes mellitus impairs glucose metabolism and favors glycogenesis in mature rat Sertoli cells.

    PubMed

    Rato, Luís; Alves, Marco G; Duarte, Ana I; Santos, Maria S; Moreira, Paula I; Cavaco, José E; Oliveira, Pedro F

    2015-09-01

    The incidence of type 2 diabetes mellitus and its prodromal stage, pre-diabetes, is rapidly increasing among young men, leading to disturbances in testosterone synthesis. However, the impact of testosterone deficiency induced by these progressive stages of diabetes on the metabolic behavior of Sertoli cells remains unknown. We evaluated the effects of testosterone deficiency associated with pre-diabetes and type 2 diabetes on Sertoli cells metabolism, by measuring (1) the expression and/or activities of glycolysis and glycogen metabolism-related proteins and (2) the metabolite secretion/consumption in Sertoli cells obtained from rat models of different development stages of the disease, to unveil the mechanisms by which testosterone deregulation may affect spermatogenesis. Glucose and pyruvate uptake were decreased in cells exposed to the testosterone concentration found in pre-diabetic rats (600nM), whereas the decreased testosterone concentrations found in type 2 diabetic rats (7nM) reversed this profile. Lactate production was not altered, although the expression and/or activity of lactate dehydrogenase and monocarboxylate transporter 4 were affected by progressive testosterone-deficiency. Sertoli cells exposed to type 2 diabetic conditions exhibited intracellular glycogen accumulation. These results illustrate that gradually reduced levels of testosterone, induced by progressive stages of diabetes mellitus, favor a metabolic reprogramming toward glycogen synthesis. Our data highlights a pivotal role for testosterone in the regulation of spermatogenesis metabolic support by Sertoli cells, particularly in individuals suffering from metabolic diseases. Such alterations may be in the basis of male subfertility/infertility associated with the progression of diabetes mellitus. PMID:26148570

  20. Immunohistochemical expression of SOX9 protein in immature, mature, and neoplastic canine Sertoli cells.

    PubMed

    Banco, Barbara; Palmieri, Chiara; Sironi, Giuseppe; Fantinato, Eleonora; Veronesi, Maria C; Groppetti, Debora; Giudice, Chiara; Martignoni, Benedetta; Grieco, Valeria

    2016-05-01

    Sex-determining region Y box9 gene (SOX9) protein plays a pivotal role in male sexual development. It regulates the transcription of the anti-Müllerian hormone gene promoting development of testis cords, multiplication, and maturation of Sertoli cells (SCs) and maintenance of spermatogenesis in adult testis. The immunohistochemical expression of SOX9 in normal testes has been reported in humans, mice, and rats. The present study aimed to investigate the expression of SOX9 in canine SCs during testicular maturation and neoplastic transformation. Canine testicular samples derived from three fetuses, four newborns, four prepubertal puppies, five adult dogs, 31 Sertoli cell tumors (SCTs) (one metastasizing), and five Leydig cell tumors (LCTs) were selected from departmental archive and tested immunohistochemically with a polyclonal antibody against SOX9 (1:150). All SCs from fetal, neonatal, and adult testes had a strong and exclusively nuclear labeling for SOX9. In SCs from prepubertal testes, SOX9 staining was highly variable with one negative sample (one of four), two samples with exclusively nuclear staining (two of four), and one with both nuclear and cytoplasmic labeling (one of four). Leydig cells (LCs) and LCTs were always negative. All 31 SCTs were positive for SOX9. The expression of SOX9 was nuclear, nuclear and cytoplasmic, and exclusively cytoplasmic in 18 of 31, 11 of 31, and two of 31 SCTs, respectively. This first report on the immunohistochemical expression of SOX9 in canine testes reports that in normal SCs from fetal, neonatal, and adult testes SOX9 labeled the nucleus, as in humans and laboratory animals. The cytoplasmic labeling observed in one prepubertal pairs of testes and in 11 SCTs could reflect SC immaturity or dedifferentiation, paralleling results observed in rat testes. The expression of SOX9 in SCs and SCTs and its absence in LCs and LCTs suggests that SOX9 is a reliable diagnostic marker for both normal and neoplastic SCs. PMID:26777558

  1. Ligand-dependent contribution of RXRβ to cholesterol homeostasis in Sertoli cells

    PubMed Central

    Mascrez, Bénédicte; Ghyselinck, Norbert B; Watanabe, Mitsuhiro; Annicotte, Jean-Sébastien; Chambon, Pierre; Auwerx, Johan; Mark, Manuel

    2004-01-01

    We show that mice expressing retinoid X receptor β (RXRβ) impaired in its transcriptional activation function AF-2 (Rxrbaf20 mutation) do not display the spermatid release defects observed in RXRβ-null mutants, indicating that the role of RXRβ in spermatid release is ligand-independent. In contrast, like RXRβ-null mutants, Rxrbaf20 mice accumulate cholesteryl esters in Sertoli cells (SCs) due to reduced ABCA1 transporter-mediated cholesterol efflux. We provide genetic and molecular evidence that cholesterol homeostasis in SCs does not require PPARα and β, but depends upon the TIF2 coactivator and RXRβ/LXRβ heterodimers, in which RXRβ AF-2 is transcriptionally active. Our results also indicate that RXRβ may be activated by a ligand distinct from 9-cis retinoic acid. PMID:14993927

  2. Changes in the morphology and protein expression of germ cells and Sertoli cells in plateau pikas testes during non-breeding season

    PubMed Central

    Liu, Ming; Cao, Guangming; Zhang, Yanming; Qu, Jiapeng; Li, Wei; Wan, Xinrong; Li, Yu-xia; Zhang, Zhibin; Wang, Yan-ling; Gao, Fei

    2016-01-01

    Plateau pikas are seasonally breeding small herbivores that inhabit the meadow ecosystem of the Qinghai-Tibetan Plateau. Testis regression in plateau pikas begins in early June, and the male pikas are completely infertile, with a dramatically reduced testis size, in late July. In this study, a decreased germ cell number in the testes was first noted in early June. By late June, only Sertoli cells and a small number of spermatogonia remained. Interestingly, large gonocyte-like germ cells were observed in early July. In late July, the number of gonocyte-like cells per tubule increased significantly, and most of the Sertoli cell nuclei moved to and clustered in the center of the seminiferous tubules. The gonocyte-like germ cells and Sertoli cells began to express AP-2γ and anti-Mullerian hormone (AMH) proteins, which were detected in the germ cells and Sertoli cells of juvenile pikas but not in adult testes. Simultaneously, LC3 puncta dramatically increased in the seminiferous tubules of the pikas’ testes during the non-breeding season. Our study found that spermatogonia and Sertoli cells in non-breeding adult pikas morphologically resembled those in juvenile pikas and expressed specific markers, indicating that de-differentiation-like transitions may occur during this process. PMID:26939551

  3. Changes in the morphology and protein expression of germ cells and Sertoli cells in plateau pikas testes during non-breeding season.

    PubMed

    Liu, Ming; Cao, Guangming; Zhang, Yanming; Qu, Jiapeng; Li, Wei; Wan, Xinrong; Li, Yu-Xia; Zhang, Zhibin; Wang, Yan-Ling; Gao, Fei

    2016-01-01

    Plateau pikas are seasonally breeding small herbivores that inhabit the meadow ecosystem of the Qinghai-Tibetan Plateau. Testis regression in plateau pikas begins in early June, and the male pikas are completely infertile, with a dramatically reduced testis size, in late July. In this study, a decreased germ cell number in the testes was first noted in early June. By late June, only Sertoli cells and a small number of spermatogonia remained. Interestingly, large gonocyte-like germ cells were observed in early July. In late July, the number of gonocyte-like cells per tubule increased significantly, and most of the Sertoli cell nuclei moved to and clustered in the center of the seminiferous tubules. The gonocyte-like germ cells and Sertoli cells began to express AP-2γ and anti-Mullerian hormone (AMH) proteins, which were detected in the germ cells and Sertoli cells of juvenile pikas but not in adult testes. Simultaneously, LC3 puncta dramatically increased in the seminiferous tubules of the pikas' testes during the non-breeding season. Our study found that spermatogonia and Sertoli cells in non-breeding adult pikas morphologically resembled those in juvenile pikas and expressed specific markers, indicating that de-differentiation-like transitions may occur during this process. PMID:26939551

  4. Rapid differentiation of NT2 cells in Sertoli-NT2 cell tissue constructs grown in the rotating wall bioreactor.

    PubMed

    Saporta, Samuel; Willing, Alison E; Shamekh, Rania; Bickford, Paula; Paredes, Daniel; Cameron, Don F

    2004-12-15

    Cell replacement therapy is of great interest as a long-term treatment of neurodegenerative diseases such as Parkinson's disease (PD). We have previously shown that Sertoli cells (SC) provide neurotrophic support to transplants of dopaminergic fetal neurons and NT2N neurons, derived from the human clonal precursors cell line NTera2/D1 (NT2), which differentiate into dopaminergic NT2N neurons when exposed to retinoic acid. We have created SC-NT2 cell tissue constructs cultured in the high aspect ratio vessel (HARV) rotating wall bioreactor. Sertoli cells, NT2, and SC plus NT2 cells combined in starting ratios of 1:1, 1:2, 1:4 and 1:8 were cultured in the HARV in DMEM with 10% fetal bovine serum and 1% growth factor reduced Matrigel for 3 days, without retinoic acid. Conventional, non-HARV, cultures grown in the same culture medium were used as controls. The presence of tyrosine hydroxylase (TH) was assessed in all culture conditions. Sertoli-neuron-aggregated-cell (SNAC) tissue constructs grown at starting ratios of 1:1 to 1:4 contained a significant amount of TH after 3 days of culture in the HARV. No TH was detected in SC HARV cultures, or SC, NT2 or SC-NT2 conventional co-cultures. Quantitative stereology of immunolabled 1:4 SNAC revealed that approximately 9% of NT2 cells differentiate into TH-positive (TH+) NT2N neurons after 3 days of culture in the HARV, without retinoic acid. SNAC tissue constructs also released dopamine (DA) when stimulated with KCl, suggesting that TH-positive NT2N neurons in the SNAC adopted a functional dopaminergic phenotype. SNAC tissue constructs may be an important source of dopaminergic neurons for neuronal transplantation. PMID:15561470

  5. Glycan composition of follicle (Sertoli) cells of the amphibian Pleurodeles waltl. A lectin histochemical study

    PubMed Central

    SÁEZ, FRANCISCO JOSÉ; MADRID, JUAN FRANCISCO; ALONSO, EDURNE; HERNÁNDEZ, FRANCISCO

    2001-01-01

    The glycan composition of the N- and O-linked oligosaccharides of the follicle (Sertoli) cells of the urodele amphibian Pleurodeles waltl testis were identified by lectin histochemistry, performed alone or in combination with enzymatic and chemical deglycosylation methods. The follicle cells were shown to contain: (1) Fuc, Galβ(1,4)GlcNAc, GalNAc and Neu5Acα(2,3)Galβ(1,4)GlcNAc in both N- and O-linked oligosaccharides; (2) Man in N-linked glycans; and (3) Galβ(1,3)GalNAc in O-linked sugar chains. The follicle cells at the pre- and postmeiotic stages showed some differences in the UEA-I-positive Fuc characterisation, suggesting differences in the glycan composition. In addition, the sequence Neu5Acα(2,6)Gal/GalNAc was shown in the follicle cells only after spermiation, in the sperm-empty lobules of the developing glandular tissue. These results suggest that the follicle cells modify their glycoprotein content, probably for the performance of new roles, as the spermatogenetic cells develop. Thus the follicle cells surrounding male germ cells at different spermatogenetic stages would contain different glycoproteins involved in specific roles during male germ cell proliferation and maturation. PMID:11465860

  6. Metallothionein gene expression under different time in testicular Sertoli and spermatogenic cells of rats treated with cadmium.

    PubMed

    Ren, Xu Yi; Zhou, Yong; Zhang, Jian Peng; Feng, Wei Hua; Jiao, Bing Hua

    2003-01-01

    The rodent testes are generally more susceptible to cadmium (Cd)-induced toxicity than the liver. To clarify the molecular mechanism underlying tissue and cell differences in Cd sensitivity, we compared metallothionein (MT) gene expression, MT protein accumulation, and Cd retention under different times in freshly isolated testicular Sertoli and spermatogenic cells and liver of rats treated with Cd. Adult male Sprague-Dawley rats received a s.c. injection of 4.0 micromol Cd/kg and 1, 3, 6, or 24h later and untreated animals (0h) tissue were sampled and testicular Sertoli and spermatogenic cells isolated. MT1 and MT2 mRNA levels were determined by semi-quantitative RT-PCR analysis followed by densitometry scanning, and MT was estimated by the enzyme-linked immunosorbent assay (ELISA) method. Cadmium content was determined by atomic absorption spectrophotometry. Testicular lesions were not grossly or histologically observed in rats treated with 4.0 micromol Cd/kg. In the present study, we demonstrated that the rat testis indeed expressed MT1 and MT2, the major isoforms. We also found that untreated animals contained relatively high basal levels of both isoform mRNA, which were increased after Cd treatment in liver and peaked at 3h, followed by a decline, in contrast, the mRNA levels in Sertoli cells peaked at 6h. Interestingly, the induction of MT1 mRNA was lower than MT2 mRNA in Sertoli cells and liver of rats treated with Cd. However, the MT1 mRNA levels of spermatogenic cells decreased 0-3h after Cd treatment, followed by an increase; in contrast, MT2 mRNA levels increased 0-3h after Cd treatment, followed by a reduction, but induced extents of them are lower than those of Sertoli cells and liver. Cd exposure substantially increased hepatic MT, but did not increase MT translation in Sertoli and spermatogenic cells. These results indicate: (1) that Cd-induced MT mRNA expression is cell- and time-dependent; (2) that the inability to induce the metal-detoxicating MT protein in response to Cd, might account for higher susceptibility of testes to Cd toxicity and carcinogenesis relative to liver. PMID:12642155

  7. Electrochemical sensors, MTT and immunofluorescence assays for monitoring the proliferation effects of cissus populnea extracts on Sertoli cells

    PubMed Central

    2011-01-01

    Background We describe the development of an electrochemical sensor array for monitoring the proliferation effects of cissus populnea plant extracts on TM4 Sertoli cells. Methods The proliferation activities of the extracts on Sertoli cells were studied using a high-throughput electrochemical sensor array (DOX-96) and the analytical sensor characteristics were compared with conventional colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and fluorescence spectroscopy. Results This work shows that there is a definite positive trend in the proliferation effect of the extract of Cissus populnea on the TM4 Sertoli cells. All of the three techniques confirmed that the most effective concentration for the proliferation is 10 ppm. At this concentration, the proliferation effect was established around 120% for both DOX-96 and MTT techniques, whereas fluorescence assays showed a higher level (120-150%). DOX-96 showed a lower limit of detection (1.25 × 10(4) cells/ml); whereas the LOD recorded for both MTT and fluorescence techniques was 2.5 × 10(4) cells/ml. Visual examination of the cells by means of confocal fluorescence microscopy confirmed the proliferation of Sertoli cells as was determined using the MTT assay. This investigation provides a confident interpretation of the results and proved that the most effective concentration for the proliferation using Cissus populnea plant extract is 10 ppm. Conclusions Overall, the DOX results compared well with the conventional methods of checking proliferation of cells. The fascinating feature of the sensor array is the ability to provide continuous proliferation experiments with no additional reagents including 96 simultaneous electrochemical experiments. The use of the DOX-96 could reduce a typical bioassay time by 20-fold. Thus the DOX-96 can be used as both a research tool and for practical cell culture monitoring. PMID:21575213

  8. Up-Regulation of SOX9 in Sertoli Cells from Testiculopathic Patients Accounts for Increasing Anti-Mullerian Hormone Expression via Impaired Androgen Receptor Signaling

    PubMed Central

    Lan, Kuo-Chung; Chen, Yen-Ta; Chang, Chawnshang; Chang, Yung-Chiao; Lin, Hsin-Jung; Huang, Ko-En; Kang, Hong-Yo

    2013-01-01

    Background Testosterone provokes Sertoli cell maturation and represses AMH production. In adult patients with Sertoli-cells-only syndrome (SCOS) and androgen insensitivity syndrome (AIS), high level of AMH expression is detected in Sertoli cells due to defect of androgen/AR signaling. Objective We postulated that up-regulation of SOX9 due to impairment of androgen/AR signaling in Sertoli cells might explain why high level of anti-Mullerian hormone (AMH) expression occur in these testiculopathic patients. Methods Biological research of testicular specimens from men with azoospermia or mouse. The serum hormone levels were studied in 23 men with obstructive azoospermia, 33 men with SCOS azoospermia and 21 volunteers with normal seminograms during a period of 4 years. Immunohistochemical staining and reverse-transcription PCR were used to examine the relationships among AR, SOX9 and AMH expression in adult human and mouse testes. The ability of AR to repress the expression of SOX9 and AMH was evaluated in vitro in TM4 Sertoli cells and C3H10T1/2 cells. Results SCOS specimens showed up-regulation of SOX9 and AMH proteins but down-regulation of AR proteins in Sertoli cells. The mRNA levels of AR were significantly lower and the SOX9, AMH mRNA levels higher in all SCOS patients compared to controls (P< 0.05). The testosterone levels in the SCOS patients were within the normal range, but most were below the median of the controls. Furthermore, our in vitro cell line experiments demonstrated that androgen/AR signaling suppressed the gene and protein levels of AMH via repression of SOX9. Conclusions Our data show that the functional androgen/AR signaling to repress SOX9 and AMH expression is essential for Sertoli cell maturation. Impairment of androgen/AR signaling promotes SOX9-mediated AMH production, accounts for impairments of Sertoli cells in SCOS azoospermic patients. PMID:24098470

  9. Rhox8 Ablation in the Sertoli Cells Using a Tissue-Specific RNAi Approach Results in Impaired Male Fertility in Mice.

    PubMed

    Welborn, Joshua P; Davis, Matthew G; Ebers, Steven D; Stodden, Genna R; Hayashi, Kanako; Cheatwood, Joseph L; Rao, Manjeet K; MacLean, James A

    2015-07-01

    The reproductive homeobox X-linked, Rhox, genes encode transcription factors that are selectively expressed in reproductive tissues. While there are 33 Rhox genes in mice, only Rhox and Rhox8 are expressed in Sertoli cells, suggesting that they may regulate the expression of somatic-cell gene products crucial for germ cell development. We previously characterized Rhox5-null mice, which are subfertile, exhibiting excessive germ cell apoptosis and compromised sperm motility. To assess the role of Rhox8 in Sertoli cells, we used a tissue-specific RNAi approach to knockdown RHOX8 in vivo, in which the Rhox5 promoter was used to drive Rhox8-siRNA transgene expression in the postnatal Sertoli cells. Western and immunohistochemical analysis confirmed Sertoli-specific knockdown of RHOX8. However, other Sertoli markers, Gata1 and Rhox5, maintained normal expression patterns, suggesting that the knockdown was specific. Interestingly, male RHOX8-knockdown animals showed significantly reduced spermatogenic output, increased germ cell apoptosis, and compromised sperm motility, leading to impaired fertility. Importantly, our results revealed that while some RHOX5-dependent factors were also misregulated in Sertoli cells of RHOX8-knockdown animals, the majority were not, and novel putative RHOX8-regulated genes were identified. This suggests that while reduction in levels of RHOX5 and RHOX8 in Sertoli cells elicits similar phenotypes, these genes are not entirely redundant. Taken together, our study underscores the importance of Rhox genes in male fertility and suggests that Sertoli cell-specific expression of Rhox5 and Rhox8 is critical for complete male fertility. PMID:25972016

  10. 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

  11. Hemicastration causes and testosterone prevents enhanced uptake of (/sup 3/H)thymidine by Sertoli cells in testes of immature rats

    SciTech Connect

    Orth, J.M.; Higginbotham, C.A.; Salisbury, R.L.

    1984-02-01

    Rat pups were hemicastrated and uptake of (/sup 3/H)thymidine by Sertoli cells in the remaining testis was compared to that in testes of sham-operated pups at intervals of from 8 h to 21 days after surgery. Labeled thymidine was administered subcutaneously 2 h before sacrifice. Testes were processed for light microscope autoradiography and the percent of Sertoli cell nuclei that had incorporated (/sup 3/H)thymidine was determined by scoring nuclei in tissue sections as labeled or unlabeled. The percentage of cells labeled was increased in hemicastrates over intact controls by 8 h after surgery and testicular hypertrophy became apparent in hemicastrates by the following day. Labeling of Sertoli cells in hemicastrates remained elevated for 4 days and then returned to normal. When plasma levels of gonadotropins were measured in both groups 4 days after surgery, follicle-stimulating hormone (FSH) was found to be more than twice normal in hemicastrates while luteinizing hormone (LH) was unchanged. The effect of testosterone on the response of Sertoli cells to hemicastration was also examined. In hemicastrates, 2 days of androgen therapy depressed, and an additional 2 days abolished, the proliferative response of the Sertoli cells. Our findings suggest that increased proliferation of Sertoli cells within the remaining testis is involved in the enlargement of the testis that follows hemicastration. They also imply that prevention of compensatory hypertrophy by testosterone involves interference with this response of Sertoli cells in some way. Finally, our data implicate FSH in control of Sertoli cell proliferation in vivo in immature rats.

  12. Vitamin A deprivation selectively lowers uridine nucleotide pools in cultured sertoli cells.

    PubMed

    Carson, D D; Lennarz, W J

    1983-02-10

    The effects of retinoid addition of vitamin A-depleted (UV-irradiated) culture medium on uridine metabolism in cultured Sertoli cells have been studied. After vitamin A depletion, a consistent 2- to 4-fold enhancement of [3H]uridine incorporation into RNA was observed. Several lines of evidence indicate that this enhancement is the result of an increase in the specific activity of the uridine-labeled precursors of RNA. Although vitamin A depletion did not affect either uridine uptake or alter cellular RNA content, a 5-fold increase in the specific activity of UMP was found in vitamin A-depleted cells. This increase results because the cellular content of uracil nucleosides plus nucleotides is selectively lowered in vitamin A-depleted cells. The decreased content of uridine derivatives could be accounted for by a 45-57% decrease in the activity of glutamine-dependent carbamylphosphate synthetase in vitamin A-depleted cells. The effects of vitamin A deprivation on uridine incorporation, as well as carbamylphosphate synthetase activity, could be completely restored to or above control values by supplementing vitamin A-depleted cell culture medium with either retinol or retinoic acid. This effect of vitamin A depletion appears to be highly specific. Under the same conditions, no gross alteration in either the pattern or extent of synthesis of cellular or secreted proteins, glycoproteins, glycosaminoglycans, and lipids was observed. In addition, vitamin A depletion/repletion had no effect on the growth rate or morphology of the cells. PMID:6822526

  13. Testosterone activates mitogen-activated protein kinase and the cAMP response element binding protein transcription factor in Sertoli cells

    PubMed Central

    Fix, Charity; Jordan, Cynthia; Cano, Patricia; Walker, William H.

    2004-01-01

    The androgen testosterone is essential for the Sertoli cell to support the maturation of male germ cells and the production of spermatozoa (spermatogenesis). In the classical view of androgen action, binding of androgen to the intracellular androgen receptor (AR) produces a conformational change in AR such that the receptor–steroid complex has high affinity for specific DNA regulatory elements and is able to stimulate gene transcription. Here, we demonstrate that testosterone can act by means of an alternative, rapid, and sustainable mechanism in Sertoli cells that is independent of AR–DNA interactions. Specifically, the addition of physiological levels of testosterone to Sertoli cells stimulates the mitogen-activated protein kinase signaling pathway and causes phosphorylation of the cAMP response element binding protein transcription factor on serine 133, a modification known to be required for Sertoli cells to support spermatogenesis. Androgen-mediated activation of mitogen-activated protein kinase and cAMP response element binding protein occurs within 1 min, extends for at least 12 h and requires AR. Furthermore, androgen induces endogenous cAMP response element binding protein-mediated transcription in Sertoli cells. These newly identified mechanisms of androgen action in Sertoli cells suggest new targets for developing male contraceptive agents. PMID:15263086

  14. New insights on hormones and factors that modulate Sertoli cell metabolism.

    PubMed

    Rato, Luís; Meneses, Maria João; Silva, Branca M; Sousa, Mário; Alves, Marco G; Oliveira, Pedro F

    2016-05-01

    Sertoli cells (SCs) play a key role in spermatogenesis by providing the physical support for developing germ cells and ensuring them the appropriate nutrients, energy sources, hormones, and growth factors. The control of SCs metabolism has been in the spotlight for reproductive biologists, since it may be crucial to determine germ cells' fate. Indeed, the maintenance of spermatogenesis is highly dependent on the metabolic cooperation established between SCs and germ cells, though this event has been overlooked. It depends on the orchestration of various metabolic pathways and an intricate network of signals. Several factors and/or hormones modulate the metabolic activity of SCs, which are major targets for the hormonal signalling that regulates spermatogenesis. Any alteration in the regulation of these cells' metabolic behaviour may compromise the normal development of spermatogenesis and consequently, male fertility. In this context, SC metabolism arises as a key regulation point for spermatogenesis. Herein, we present an up-to-date overview on the impact of hormones and factors that modulate SC metabolism, with special focus on glycolytic metabolism, highlighting their relevance in determining male reproductive potential. PMID:26711246

  15. Research resource: the dynamic transcriptional profile of sertoli cells during the progression of spermatogenesis.

    PubMed

    Zimmermann, Céline; Stévant, Isabelle; Borel, Christelle; Conne, Béatrice; Pitetti, Jean-Luc; Calvel, Pierre; Kaessmann, Henrik; Jégou, Bernard; Chalmel, Frédéric; Nef, Serge

    2015-04-01

    Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1β), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility. PMID:25710594

  16. A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates

    PubMed Central

    Yefimova, Marina G.; Messaddeq, Nadia; Harnois, Thomas; Meunier, Annie-Claire; Clarhaut, Jonathan; Noblanc, Anaïs; Weickert, Jean-Luc; Cantereau, Anne; Philippe, Michel; Bourmeyster, Nicolas; Benzakour, Omar

    2013-01-01

    Phagocytosis and autophagy are typically dedicated to degradation of substrates of extrinsic and intrinsic origins respectively. Although overlaps between phagocytosis and autophagy were reported, the use of autophagy for ingested substrate degradation by nonprofessional phagocytes has not been described. Blood-separated tissues use their tissue-specific nonprofessional phagocytes for homeostatic phagocytosis. In the testis, Sertoli cells phagocytose spermatid residual bodies produced during germ cell differentiation. In the retina, pigmented epithelium phagocytoses shed photoreceptor tips produced during photoreceptor renewal. Spermatid residual bodies and shed photoreceptor tips are phosphatidylserine-exposing substrates. Activation of the tyrosine kinase receptor MERTK, which is implicated in phagocytosis of phosphatidylserine-exposing substrates, is a common feature of Sertoli and retinal pigmented epithelial cell phagocytosis. The major aim of our study was to investigate to what extent phagocytosis by Sertoli cells may be tissue specific. We analyzed in Sertoli cell cultures that were exposed to either spermatid residual bodies (legitimate substrates) or retina photoreceptor outer segments (illegitimate substrates) the course of the main phagocytosis stages. We show that whereas substrate binding and ingestion stages occur similarly for legitimate or illegitimate substrates, the degradation of illegitimate but not of legitimate substrates triggers autophagy as evidenced by the formation of double-membrane wrapping, MAP1LC3A-II/LC3-II clustering, SQSTM1/p62 degradation, and by marked changes in ATG5, ATG9 and BECN1/Beclin 1 protein expression profiles. The recruitment by nonprofessional phagocytes of autophagy for the degradation of ingested cell-derived substrates is a novel feature that may be of major importance for fundamentals of both apoptotic substrate clearance and tissue homeostasis. PMID:23439251

  17. Immunohistochemical evaluation of the expression of anti-Mllerian hormone in mature, immature and neoplastic canine Sertoli cells.

    PubMed

    Banco, B; Veronesi, M C; Giudice, C; Rota, A; Grieco, V

    2012-01-01

    In mammals, the earliest specific protein expressed by Sertoli cells (SCs) is the anti-Mllerian hormone (AMH), which induces the regression of Mllerian ducts and is produced by SCs until the functional maturation of the testes. The aim of this study was to evaluate the expression of AMH by canine SCs during testicular maturation and neoplastic transformation. Testes from two fetuses, 18 newborn puppies, five puppies aged 43-180 days and six adult dogs, and 24 canine Sertoli cell tumours (SCTs) were studied immunohistochemically for expression of AMH. Fifteen of the 24 SCTs were classified as typical, eight as lipid-rich and one was considered malignant based on evidence of lymph node metastasis. SCs from fetuses and neonatal puppies and puppies up to 45 days old expressed AMH, while SCs from older puppies and adults were negative. All SCTs expressed AMH, suggesting that AMH expression is a useful marker of immature and neoplastic canine SCs. PMID:21571300

  18. Altered testicular development as a consequence of increase number of sertoli cell in male lambs exposed prenatally to excess testosterone.

    PubMed

    Rojas-García, Pedro P; Recabarren, Mónica P; Sir-Petermann, Teresa; Rey, Rodolfo; Palma, Sergio; Carrasco, Albert; Perez-Marin, Carlos C; Padmanabhan, Vasantha; Recabarren, Sergio E

    2013-06-01

    The reprograming effects of prenatal testosterone (T) treatment on postnatal reproductive parameters have been studied extensively in females of several species but similar studies in males are limited. We recently found that prenatal T treatment increases Sertoli cell number and reduced spermatogenesis in adult rams. If such disruptions are manifested early in life and involve changes in testicular paracrine environment remain to be explored. This study addresses the impact of prenatal T excess on testicular parameters in infant males, including Sertoli cell number and expression of critical genes [FSH receptor (FSHR), androgen receptor (AR), transforming growth factor beta 1 (TGFB1), 3 (TGFB3), transforming growth factor beta type 1 receptor, (TGFBR1), and anti-Müllerian hormone (AMH)] modulating testicular function. At 4 week of age, male lambs born to dams treated with 30 mg of T propionate twice weekly from day 30 to 90, followed by 40 mg of T propionate from day 90 to 120 of pregnancy (T-males), had a higher number of Sertoli cells/testis (P = 0.035) than control males (C-males) born to dams treated with the vehicle. While no differences were observed in the expression of FSHR and TGFB3, testicular TGFBR1 expression was found to be lower in T-males (P = 0.03) compared to C-males. Expression level of AMH, TGFB1, and AR also tended to be lower in T-males. These findings provide evidence that impact of fetal exposure to T excess is evident early in postnatal life, mainly characterized by an increase in Sertoli cell number. This could explain the testicular dysfunction observed in adult rams. PMID:23076741

  19. Retinoblastoma protein (RB) interacts with E2F3 to control terminal differentiation of Sertoli cells

    PubMed Central

    Rotgers, E; Rivero-Müller, A; Nurmio, M; Parvinen, M; Guillou, F; Huhtaniemi, I; Kotaja, N; Bourguiba-Hachemi, S; Toppari, J

    2014-01-01

    The retinoblastoma protein (RB) is essential for normal cell cycle control. RB function depends, at least in part, on interactions with the E2F family of DNA-binding transcription factors (E2Fs). To study the role of RB in the adult testis, a Sertoli cell (SC)-specific Rb knockout mouse line (SC-RbKO) was generated using the Cre/loxP recombination system. SC-RbKO mice exhibited an age-dependent testicular atrophy, impaired fertility, severe SC dysfunction, and spermatogenic defects. Removal of Rb in SC induced aberrant SC cycling, dedifferentiation, and apoptosis. Here we show that E2F3 is the only E2F expressed in mouse SCs and that RB interacts with E2F3 during mouse testicular development. In the absence of RB, the other retinoblastoma family members p107 and p130 began interacting with E2F3 in the adult testes. In vivo silencing of E2F3 partially restored the SC maturation and survival as well as spermatogenesis in the SC-RbKO mice. These results point to RB as a key regulator of SC function in adult mice and that the RB/E2F3 pathway directs SC maturation, cell cycle quiescence, and RB protects SC from apoptosis. PMID:24901045

  20. An Integrative Omics Strategy to Assess the Germ Cell Secretome and to Decipher Sertoli-Germ Cell Crosstalk in the Mammalian Testis

    PubMed Central

    Lavigne, Régis; Hernio, Nolwen; Teixeira-Gomes, Ana-Paula; Dacheux, Jean-Louis; Pineau, Charles

    2014-01-01

    Mammalian spermatogenesis, which takes place in complex testicular structures called seminiferous tubules, is a highly specialized process controlled by the integration of juxtacrine, paracrine and endocrine information. Within the seminiferous tubules, the germ cells and Sertoli cells are surrounded by testicular fluid (TF), which probably contains most of the secreted proteins involved in crosstalk between these cells. It has already been established that germ cells can modulate somatic Sertoli cell function through the secretion of diffusible factors. We studied the germ cell secretome, which was previously considered inaccessible, by analyzing the TF collected by microsurgery in an “integrative omics” strategy combining proteomics, transcriptomics, genomics and interactomics data. This approach identified a set of proteins preferentially secreted by Sertoli cells or germ cells. An interaction network analysis revealed complex, interlaced cell-cell dialog between the secretome and membranome of seminiferous cells, mediated via the TF. We then focused on germ cell-secreted candidate proteins, and we identified several potential interacting partners located on the surface of Sertoli cells. Two interactions, APOH/CDC42 and APP/NGFR, were validated in situ, in a proximity ligation assay (PLA). Our results provide new insight into the crosstalk between germ cells and Sertoli cells occurring during spermatogenesis. Our findings also demonstrate that this “integrative omics” strategy is powerful enough for data mining and highlighting meaningful cell-cell communication events between different types of cells in a complex tissue, via a biological fluid. This integrative strategy could be applied more widely, to gain access to secretomes that have proved difficult to study whilst avoiding the limitations of in vitro culture. PMID:25111155

  1. Immunohistochemical expression of markers of immaturity in sertoli and seminal cells in canine testicular atrophy.

    PubMed

    Giudice, C; Banco, B; Veronesi, M C; Ferrari, A; Di Nardo, A; Grieco, V

    2014-01-01

    During maturation from fetal to adult testis, both Sertoli cells (SCs) and germ cells (GCs) switch from an immature to a mature immunophenotype. Immature canine SCs express cytokeratins (CKs), desmin (DES), vimentin (VIM), anti-Mllerian hormone (AMH) and inhibin (INH)-?, while mature SCs retain only expression of VIM. Immature GCs express placental alkaline phosphatase (PLAP), which is lost in spermatocytes. Re-expression of markers of immaturity has been observed in human atrophic testes and in human and canine testicular tumours. In human medicine, testicular atrophy is considered a risk factor for testicular cancer. In the present study 13 canine atrophic testes were examined immunohistochemically. VIM was expressed in the SCs of all cases, while CK, DES, INH-? and AMH were expressed in a variable percentage of SCs in two, five, five and eight cases, respectively. PLAP was expressed by single GCs in one case. Markers of immaturity are therefore expressed by SCs and GCs in canine atrophic testes. Similar results were reported previously in canine testicular neoplasia, suggesting that testicular atrophy may represent a risk factor for tumour development in the dog. PMID:24064049

  2. Estrogenic regulation of bicarbonate transporters from SLC4 family in rat Sertoli cells.

    PubMed

    Bernardino, Raquel L; Martins, Ana D; Jesus, Tito T; Sá, Rosália; Sousa, Mário; Alves, Marco G; Oliveira, Pedro F

    2015-10-01

    The formation of competent spermatozoa is a complex event that depends on the establishment of adequate environments throughout the male reproductive tract. Bicarbonate is essential not only to ionic homeostasis but also to pH maintenance along the male reproductive tract. Previous studies support an association of high 17β-estradiol (E2) levels with modulation of specific ion transporters expression. Herein we determined the effect of E2 on the expression/functionality of SLC4 family bicarbonate transporters in rat Sertoli cells (SCs). All studied transporters [anion exchanger 2 (AE2), Na(+)-driven Cl(-)/HCO3 (-) exchanger (NDCBE), electrogenic Na(+)/HCO3 (-) co-transporters (NBCe1), and electroneutral Na(+)/HCO3 (-) co-transporters (NBCn1)] were identified in SCs, being AE2 and NBCn1 the most abundant. In E2-treated cells (100 nM), increases in AE2 and NBCn1 protein levels were observed, as well as altered transcellular transport. E2-treated SCs presented a significant perturbation of ATP-induced short-circuit current. This alteration was concurrent with augmented AE2 and NBCn1 levels. Overall, we report a relation between increased E2 levels and the expression/function of AE2 and NBCn1 in rat SCs, providing new evidence on the mechanisms by which E2 can regulate SCs physiology and consequently spermatogenesis, with direct influence on male reproductive potential. PMID:26100313

  3. Copy Number Variants in Patients with Severe Oligozoospermia and Sertoli-Cell-Only Syndrome

    PubMed Central

    Tüttelmann, Frank; Simoni, Manuela; Kliesch, Sabine; Ledig, Susanne; Dworniczak, Bernd; Wieacker, Peter; Röpke, Albrecht

    2011-01-01

    A genetic origin is estimated in 30% of infertile men with the common phenotypes of oligo- or azoospermia, but the pathogenesis of spermatogenic failure remains frequently obscure. To determine the involvement of Copy Number Variants (CNVs) in the origin of male infertility, patients with idiopathic severe oligozoospermia (N = 89), Sertoli-cell-only syndrome (SCOS, N = 37)) and controls with normozoospermia (N = 100) were analysed by array-CGH using the 244A/400K array sets (Agilent Technologies). The mean number of CNVs and the amount of DNA gain/loss were comparable between all groups. Ten recurring CNVs were only found in patients with severe oligozoospermia, three only in SCOS and one CNV in both groups with spermatogenic failure but not in normozoospermic men. Sex-chromosomal, mostly private CNVs were significantly overrepresented in patients with SCOS. CNVs found several times in all groups were analysed in a case-control design and four additional candidate genes and two regions without known genes were associated with SCOS (P<1×10−3). In conclusion, by applying array-CGH to study male infertility for the first time, we provide a number of candidate genes possibly causing or being risk factors for the men's spermatogenic failure. The recurring, patient-specific and private, sex-chromosomal CNVs as well as those associated with SCOS are candidates for further, larger case-control and re-sequencing studies. PMID:21559371

  4. Effects of intraperitoneal injection of microencapsulated Sertoli cells on chronic and presymptomatic dystrophic mice.

    PubMed

    Chiappalupi, Sara; Luca, Giovanni; Mancuso, Francesca; Madaro, Luca; Fallarino, Francesca; Nicoletti, Carmine; Calvitti, Mario; Arato, Iva; Falabella, Giulia; Salvadori, Laura; Di Meo, Antonio; Bufalari, Antonello; Giovagnoli, Stefano; Calafiore, Riccardo; Donato, Rosario; Sorci, Guglielmo

    2015-12-01

    We report data about the effects of intraperitoneal (i.p.) injection of specific pathogen-free (SPF) porcine Sertoli cells (SeC) encapsulated into clinical grade alginate-based microcapsules (SeC-MC) on muscles of chronic and presymptomatic dystrophic, mdx mice. Mdx mouse is the best characterized animal model of Duchenne muscular dystrophy (DMD), an X-linked lethal myopathy due to mutation in the gene of dystrophin, which is crucial for myofiber integrity during muscle contraction. Our data show that three weeks after i.p. injection of SeC-MC significantly reduced adipose and fibrous tissue deposition, reduced macrophage infiltrate, and reduced numbers of damaged myofibers are found in muscles of 12-month-old mdx mice, which reproduce chronic DMD conditions. Compared with muscles of mock-treated mdx mice muscles of SeC-MC-treated mice show upregulation of the dystrophin paralogue, utrophin which is localized to the periphery of myofibers. Moreover, our data show that i.p. injection of SeC-MC into presymptomatic, 2-week-old mdx mice, although not fully preventing myofiber degeneration, results in protection against myofiber necrosis and muscle inflammation. Extensive discussion of these data can be found in Ref. [1]. PMID:26759818

  5. Combined Leydig cell and Sertoli cell dysfunction in 46,XX males lacking the sex determining region Y gene

    SciTech Connect

    Turner, B.; Vordermark, J.S.; Fechner, P.Y.

    1995-07-03

    We have evaluated 3 individuals with a rare form of 46,XX sex reversal. All of them had ambiguous external genitalia and mixed wolffian and muellerian structures, indicating both Leydig cell and Sertoli cell dysfunction, similar to that of patients with true hermaphroditism. However, gonadal tissue was not ovotesticular but testicular with varying degrees of dysgenesis. SRY sequences were absent in genomic DNA from peripheral leukocytes in all 3 subjects. Y centromere sequences were also absent, indicating that testis development did not occur because of a low level mosaicism of Y-bearing cells. The subjects in this report demonstrate that there is a continuum in the extent of the testis determination in SRY-negative 46,XX sex reversal, ranging from nearly normal to minimal testicular development. 20 refs.

  6. Morphometric evaluation of seminiferous tubule and proportionate numerical analysis of Sertoli and spermatogenic cells indicate differences between crossbred and purebred bulls

    PubMed Central

    Tripathi, Utkarsh K.; Chhillar, Shivani; Kumaresan, A.; Aslam, M. K. Muhammad; Rajak, S. K.; Nayak, Samiksha; Manimaran, A.; Mohanty, T. K.; Yadav, Savita

    2015-01-01

    Aim: The present study compared the testicular cytology and histology between crossbred (Holstein–Friesian [HF] × Tharparkar) and purebred (HF and Tharparkar) bulls to find out differences if any. Materials and Methods: Four peripubertal bulls from each breed were utilized for the study. Through percutaneous needle aspiration biopsy, Sertoli and spermatogenic cells were extracted, and morphometry was studied. For histological studies, testicular tissues obtained through unilateral castration were utilized. Sertoli cells specific GATA4 antibody was used to study the population of Sertoli cells in the seminiferous tubule through immunofluorescence. Results: The testicular weight, volume, and scrotal circumference differed significantly among the breeds. The diameter and area of the seminiferous tubule was high in HF, followed by Karan Fries (KF), and Tharparkar bulls. However, the degree of compactness, based on qualitative evaluation, was high in Tharparkar followed by KF and HF bulls. The intensity of Leydig cells was higher in Tharparkar bulls followed by KF and HF. The proportion of Sertoli cells was higher (p<0.05) in HF and Tharparkar bulls compared to KF bulls. Conclusion: It may be concluded that variations exist in testicular components of the breeds studied and the proportion of Sertoli cells in relation to spermatogenic cells was significantly lower in crossbred bulls compared to purebred bulls. PMID:27047150

  7. UPF2, a nonsense-mediated mRNA decay factor, is required for prepubertal Sertoli cell development and male fertility by ensuring fidelity of the transcriptome

    PubMed Central

    Bao, Jianqiang; Tang, Chong; Yuan, Shuiqiao; Porse, Bo T.; Yan, Wei

    2015-01-01

    Nonsense-mediated mRNA decay (NMD) represents a highly conserved RNA surveillance mechanism through which mRNA transcripts bearing premature termination codons (PTCs) are selectively degraded to maintain transcriptomic fidelity in the cell. Numerous in vitro studies have demonstrated the importance of the NMD pathway; however, evidence supporting its physiological necessity has only just started to emerge. Here, we report that ablation of Upf2, which encodes a core NMD factor, in murine embryonic Sertoli cells (SCs) leads to severe testicular atrophy and male sterility owing to rapid depletion of both SCs and germ cells during prepubertal testicular development. RNA-Seq and bioinformatic analyses revealed impaired transcriptomic homeostasis in SC-specific Upf2 knockout testes, characterized by an accumulation of PTC-containing transcripts and the transcriptome-wide dysregulation of genes encoding splicing factors and key proteins essential for SC fate control. Our data demonstrate an essential role of UPF2-mediated NMD in prepubertal SC development and male fertility. PMID:25503407

  8. Starvation is more efficient than the washing technique for purification of rat Sertoli cells.

    PubMed

    Ghasemzadeh-Hasankolaei, Mohammad; Eslaminejad, Mohamadreza Baghaban; Sedighi-Gilani, Mohammadali; Mokarizadeh, Aram

    2014-09-01

    Sertoli cells (SCs), one of the most important components of seminiferous tubules, are vital for normal spermatogenesis and male fertility. In recent years, numerous in vitro studies have shown the potential and actual activities of SCs. However, pure SCs are necessary for various in vitro studies. In this study, we have evaluated the efficiency of the starvation method for SC purification as compared with the washing method. Seminiferous tubule-derived cells (STDCs) of rats' testes underwent two different techniques for SC purification. In the first group (washing group), the medium was changed every 3-4 d, and cells were washed twice with phosphate-buffered saline that lacked CaC12 and MgSO4 (PBS(-)) before the addition of fresh medium. In the second group (starvation), the medium was changed every 7-8 d. Primary culture (P0), passage 1 (P1), and passage 2 (P2) cells were analyzed for the expression of SC-specific genes, vimentin, Wilm's tumor 1 (WT1), germ cell gene (vasa), Leydig cell marker, 17beta-hydroxysteroid dehydrogenase type 3 (Hsd17b3), and a marker of peritubular myoid cells, alpha smooth muscle actin (αSma), by reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR. Gene expression analysis showed that P0 cells expressed all tested genes except Hsd17b3. The starvation method caused significant downregulation of vasa and αSma expression in P0, P1, and P2 cells, whereas vimentin and WT1 were upregulated. In contrast, the washing method was less effective than the starvation method for the removal of germ and pretubular myoid cells (p < 0.001). Totally, the results have revealed that although washing is the only common technique for elimination of contaminant cells in SC cultures, starvation has a stronger effect and is a suitable, affordable technique for SC purification. We propose that starvation is an efficient, inexpensive method that can be used for purification of SCs in animal species. PMID:24789729

  9. Activation of innate immune system in response to lipopolysaccharide in chicken Sertoli cells.

    PubMed

    Michailidis, Georgios; Anastasiadou, Maria; Guibert, Edith; Froment, Pascal

    2014-09-01

    Sertoli cells (SCs) play an important physiological role in the testis, as they support, nourish, and protect the germ cells. As protection of the developing spermatozoa is an emerging aspect of reproductive physiology, this study examined the expression pattern of innate immune-related genes, including avian β-defensins (AvBDs), Toll-like receptors (TLRs), and cytokines, and investigated the time course of an inflammatory response in rooster SCs triggered by exposure to the bacterial endotoxin lipopolysaccharide (LPS). SCs were isolated from 6-week-old chicken, cultured in vitro, and stimulated with 1 μg/ml LPS at different time courses (0, 6, 12, 24, and 48  h). Data on expression analysis revealed that all ten members of the chicken TLR family, nine members of the AvBD family, as well as eight cytokine genes were expressed in SCs. Quantitative real-time PCR analysis revealed that LPS treatment resulted in significant induction of the expression levels of six TLRs, six AvBDs, and four cytokine genes, while two cytokine genes were downregulated and two other genes were unchanged. The increasing interleukin 1β (IL1β) production was confirmed in the conditioned medium. Furthermore, the phagocytosis of SCs was increased after LPS treatment. In conclusion, these findings provide evidence that SCs express innate immune-related genes and respond directly to bacterial ligands. These genes represent an important component of the immune system, which could be integrated into semen, and present a distinctive constituent of the protective repertoire of the testis against ascending infections. PMID:24920664

  10. Aquaporin-4 as a molecular partner of cystic fibrosis transmembrane conductance regulator in rat Sertoli cells.

    PubMed

    Jesus, Tito T; Bernardino, Raquel L; Martins, Ana D; Sá, Rosália; Sousa, Mário; Alves, Marco G; Oliveira, Pedro F

    2014-04-18

    Sertoli cells (SCs) form the blood-testis barrier (BTB) that controls the microenvironment where the germ cells develop. The cystic fibrosis transmembrane conductance regulator (CFTR) plays an essential role to male fertility and it was recently suggested that it may promote water transport. Interestingly, Aquaporin-4 (AQP4) is widely expressed in blood barriers, but was never identified in SCs. Herein we hypothesized that SCs express CFTR and AQP4 and that they can physically interact. Primary SCs cultures from 20-day-old rats were maintained and CFTR and AQP4 mRNA and protein expression was assessed by RT-PCR and Western blot, respectively. The possible physical interaction between CFTR and AQP4 was studied by co-immunoprecipitation. We were able to confirm the presence of CFTR at mRNA and protein level in cultured rat SCs. AQP4 mRNA analysis showed that cultured rat SCs express the transcript variant c of AQP4, which was followed by immunodetection of the correspondent protein. The co-immunoprecipitation experiments showed a direct interaction between AQP4 and CFTR in cultured rat SCs. Our results suggest that CFTR physically interacts with AQP4 in rat SCs evidencing a possible mechanism by which CFTR can control water transport through BTB. The full enlightenment of this particular relation between CFTR and AQP4 may point towards possible therapeutic targets to counteract male subfertility/infertility in men with Cystic Fibrosis and mutations in CFTR gene, which are known to impair spermatogenesis due to defective water transport. PMID:24657265

  11. Regulation of the phosphoinositide pathway in cultured Sertoli cells from immature rats: effects of follicle-stimulating hormone and fluoride

    SciTech Connect

    Quirk, S.M.; Reichert, L.E. Jr.

    1988-07-01

    Many hormones elicit effects on target cells by stimulating the enzyme phospholipase-C, which catalyzes the hydrolysis of phosphoinositides to the intracellular second messengers diacylglycerol and inositol phosphates. The present study examined the roles of FSH and guanine nucleotide-binding proteins (G-proteins) in regulating the hydrolysis of phosphoinositides in Sertoli cells. Sertoli cell cultures prepared from 16- to 18-day-old rats were incubated for 24 h with myo-(2-3H) inositol to label endogenous phospholipids. Treatment of cells from 0.5-20 min with preparations of ovine FSH ranging in potency from 1-60 times that of NIH FSH S1 did not affect accumulation of inositol phosphates. Levels of total (3H)inositol phosphates ((3H)inositol mono-, di-, and triphosphates (IP, IP2, and IP3)) in FSH-treated cultures was 75-120% the levels in control cultures over the various time intervals studied. Addition of testosterone and the combination of testosterone plus retinoic acid, agents that have been shown to potentiate effects of FSH in other systems, did not affect accumulation of inositol phosphates in response to FSH. In contrast to the lack of effect on accumulation of inositol phosphates, FSH stimulated 4- to 11-fold increases in estradiol secretion over 24 h of culture, indicating that Sertoli cells were viable and responsive to FSH. AIF4- has been shown to activate G-proteins involved in regulation of adenylate cyclase activity. In the present study, AIF4- induced 4- to 5-fold increases in IP, IP2, and IP3 in experiments wherein FSH had no effect. Pretreatment of Sertoli cells with pertussis toxin (100 and 1000 ng/ml) for 24 h inhibited fluoride-induced generation of IP, IP2, and IP3 by 24-51%. Similar treatment with cholera toxin had no effect on basal or fluoride-induced generation of IP2 or IP3, but increased fluoride-induced generation of IP by 20-34%.

  12. Zinc and low-dose of cadmium protect sertoli cells against toxic-dose of cadmium: The role of metallothionein

    PubMed Central

    Kheradmand, Fatemeh; Nourmohammadi, Issa; Ahmadi-Faghih, Mohamad Amin; Firoozrai, Mohsen; Modarressi, Mohammad Hossein

    2013-01-01

    Background: The impact of cadmium (Cd) on male infertility may be related to the interaction with metal-binding proteins known as metallothioneins (Mts). Trace elements like zinc (Zn) have protective effects on testicular damage induced by Cd. Objective: We determined the effect of Zn and low-dose Cd pre-treatment on the expression of Mt1 and Mt2 genes on testicular Sertoli cells. Materials and Methods: The cultured TM4 mouse sertoli cells were treated with 50 μM ZnSO4 (Zn pre-treated group; ZnPG), 2 μM CdCl2 (Cd pre-treated group; CdPG), or distilled water (DW pre-treated group; DWPG). After 18 hour, all of these groups were exposed to 100 μM CdCl2 for different periods of time (1, 2, 3, and 6 hours). There was also a control group for all three groups, which was treated only with distilled water (without Cd or Zn pre-treatment). Cellular viability, Zn and Cd concentrations and gene expression were assessed by MTT, atomic absorption spectrometry and real time PCR methods, respectively. Results: The expression of Mt1 and Mt2 genes in ZnPG, CdPG, and DWPG was greater than the control group (p=0.02 and p=0.01, respectively). Cd concentrations in CdPG and DWPG were greater than the control group (p=0.00). Expression of both genes in ZnPG and CdPG increased after 3 hours of treatment and Cd concentration decreased simultaneously, which was more obvious in ZnPG. Conclusion: Zn and short term low-dose Cd pre-treatment might reduce the adverse effects of Cd by increasing expression of Mts genes in Sertoli cells. The protective effect of Zn was stronger than Cd. PMID:24639783

  13. Non-classic androgen actions in Sertoli cell membrane in whole seminiferous tubules: effects of nandrolone decanoate and catechin.

    PubMed

    Cavalari, Fernanda Carvalho; de Castro, Alexandre Luz; Fracasso, Bianca de Moraes; Loss, Eloísa da Silveira

    2012-01-01

    Studies show a mechanism of action of testosterone, nandrolone and catechin as agonists of the membrane androgen receptor. The aim of this work is to investigate the non-classical effect of androgens and catechin in Sertoli cells from immature rats. The membrane potential of Sertoli cells in whole seminiferous tubules was recorded using a standard single microelectrode technique. It was performed a topical application of testosterone (1 μM), nandrolone (0.1, 0.5 and 1 μM) and the flavonoid catechin (0.1, 0.5 and 1 μM) alone and also after infusion with flutamide (1 μM), diazoxide (100 μM) or U73122 (1 μM). The immature testes were incubated for 5 min in KRb with (45)Ca(2+), with or without nandrolone (1 μM). The results were given as mean±SEM. The data were analyzed using ANOVA for repeated measures with Bonferroni post-test. Testosterone produces a depolarization in the membrane potential at 120 s after application. Catechin (1 μM) and nandrolone (1 μM) have shown a similar response to testosterone: depolarization at 120 s after the application. The same response of catechin and nandrolone was observed at different doses. The effects of testosterone, catechin and nandrolone were not affected after perfusion with flutamide. Perfusion with diazoxide and U73122 nullified the effect of nandrolone (1 μM) and catechin (1 μM). Nandrolone and testosterone increased (45)Ca(2+) uptake with or without flutamide within 5min. These results indicate that nandrolone and catechin act through a receptor on the plasmatic membrane, as well as testosterone, showing a non-classical pathway in Sertoli cells from immature rat testes. PMID:22093481

  14. Perfluorooctanesulfonate (PFOS) Perturbs Male Rat Sertoli Cell Blood-Testis Barrier Function by Affecting F-Actin Organization via p-FAK-Tyr407: An in Vitro Study

    PubMed Central

    Wan, Hin-Ting; Mruk, Dolores D.; Wong, Chris K. C.

    2014-01-01

    Environmental toxicants such as perfluorooctanesulfonate (PFOS) have been implicated in male reproductive dysfunction, including reduced sperm count and semen quality, in humans. However, the underlying mechanism(s) remains unknown. Herein PFOS at 10–20 μM (∼5–10 μg/mL) was found to be more potent than bisphenol A (100 μM) in perturbing the blood-testis barrier (BTB) function by disrupting the Sertoli cell tight junction-permeability barrier without detectable cytotoxicity. We also delineated the underlying molecular mechanism by which PFOS perturbed Sertoli cell BTB function using an in vitro model that mimics the BTB in vivo. First, PFOS perturbed F-actin organization in Sertoli cells, causing truncation of actin filaments at the BTB. Thus, the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory and adhesion proteins at the cell-cell interface necessary to maintain BTB integrity. Second, PFOS was found to perturb inter-Sertoli cell gap junction (GJ) communication based on a dye-transfer assay by down-regulating the expression of connexin-43, a GJ integral membrane protein. Third, phosphorylated focal adhesion kinase (FAK)-Tyr407 was found to protect the BTB from the destructive effects of PFOS as shown in a study via an overexpression of an FAK Y407E phosphomimetic mutant. Also, transfection of Sertoli cells with an FAK-specific microRNA, miR-135b, to knock down the expression of phosphorylated FAK-Tyr407 was found to worsen PFOS-mediated Sertoli cell tight junction disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating phosphorylated FAK-Tyr407 and connexin-43, which in turn perturbed F-actin organization and GJ-based intercellular communication, leading to mislocalization of actin-regulatory and adhesion proteins at the BTB. PMID:24169556

  15. Formation and structure of transplantable tissue constructs generated in simulated microgravity from Sertoli cells and neuron precursors.

    PubMed

    Cameron, Don F; Hushen, J J; Colina, L; Mallery, J; Willing, A; Sanberg, P R; Saporta, Samuel

    2004-01-01

    Cell transplantation therapy for Parkinson's disease (PD) has received much attention as a potential treatment protocol for this neurodegenerative condition. Although there have been promising successes with this approach, it remains problematic, especially regarding the inability to provide immediate trophic support to the newly grafted cells and the inability to prevent acute and/or long-term graft rejection by the host. To address these issues of cell graftability, we have created a novel tissue construct from isolated rat Sertoli cells (SC) and the NTerra-2 immortalized human neuron precursor cell line (NT2) utilizing NASA-developed simulated microgravity technology. The two cell types were cocultured at a 1:4 (SC/NT2) ratio in the High Aspect Rotating Vessel (HARV) biochamber for 3 days, after which a disc-shaped aggregate (1-4 mm diameter) was formed. Sertoli neuron aggregated cells (SNAC) were collected by gravity sedimentation and processed either for light and electron microscopy or for fluorescent immunocytochemistry. Intra-SNAC clusters of SC and NT2 cells were identified by anti-human mitochondrial protein (huMT--specific for NT2 cells) and cholera toxin subunit B (CTb--specific for SC). There was little evidence of cell death throughout the aggregate and the absence of central necrosis, as might be expected in such a large aggregate in vitro. Ultrastructurally, SC did not express junctional modifications with NT2 cells nor with adjacent SC as is typical of SC in vivo and, in some protocols, in vitro. NT2 cells, however, showed distinct intercellular junction-like densities with adjacent NT2 cells, often defining canaliculi-like channels between the microvillus borders of the cells. The results show that the use of simulated microgravity coculture provides a culture environment suitable for the formation of a unique and viable Sertoli-NT2 (i.e., SNAC) tissue construct displaying intra-aggregate cellular organization. The structural integration of SC with NT2 cells provides a novel transplantable tissue source, which can be tested to determine if SC will suppress rejection of the grafted NT2 cells and provide for their short- and long-term trophic support in situ in the treatment of experimental PD. PMID:15690977

  16. Activation of inositol phospholipid turnover and calcium signaling in rat Sertoli cells by P2-purinergic receptors: modulation of follicle-stimulating hormone responses.

    PubMed

    Filippini, A; Riccioli, A; De Cesaris, P; Paniccia, R; Teti, A; Stefanini, M; Conti, M; Ziparo, E

    1994-03-01

    To study the role of extracellular nucleotides in the regulation of Sertoli cells, the effects of ATP and its analogs on the Ca(2+)-phospholipid- and cAMP-dependent pathways were tested. Cultured Sertoli cells from immature animals were incubated with ATP or structurally related compounds, and phosphoinositide (PI) turnover or cAMP accumulation was measured. Among the several nucleotide phosphate analogs tested, adenosine 5'-O-(3-thiotriphosphate) was the agonist most potent in stimulating inositol phosphate accumulation. The effects of purine nucleotides on PI turnover were time and concentration dependent. Because nonhydrolizable ATP analogs also stimulated PI turnover, ATP metabolites or metabolic products are not responsible for the observed stimulation. The order of potency of the different ATP analogs [adenosine 5'-O-(3-thiotriphosphate) > ATP approximately equal to UTP > beta, gamma-methyleneadenosine 5'-triphosphate, 2-methylthio-ATP > adenosine] was consistent with the presence of P2U receptors (nucleotide receptors) on the surface of the Sertoli cell. Augmented PI turnover was accompanied by a transient increase in Ca2+ concentration, measured in single Sertoli cells loaded with the intracellular Ca2+ indicator fura-2. When used alone, ATP and its analogs did not have a direct effect on cAMP levels in the Sertoli cell. However, ATP or its analogs inhibited FSH-dependent cAMP accumulation by more than 70%. Purine nucleotides also efficiently blocked the effects of FSH distal to cAMP accumulation, because extracellular ATP completely reversed the changes in Sertoli cell shape induced by FSH. The nucleotide-dependent inhibition of cAMP accumulation was blocked by pertussis toxin to a different degree depending on the purine or pirimidine nucleotide used. This indicated that more than one mechanism contributes to the purine nucleotide-dependent inhibition of cAMP accumulation. These data provide evidence that purine nucleotide receptors coupled to multiple pathways are present on the Sertoli cell in culture, and that extracellular ATP has profound biological effects on the FSH responsiveness of the Sertoli cell. PMID:8119196

  17. Modulation of m-dinitrobenzene and m-nitrosonitrobenzene toxicity in rat Sertoli--germ cell cocultures

    SciTech Connect

    Cave, D.A.; Foster, P.M. )

    1990-01-01

    Previous work has shown that m-dinitrobenzene is a testicular toxicant in rats in vivo, and in vitro produces comparable morphological changes in rat testicular Sertoli-germ cell cocultures. m-Dinitrobenzene is metabolized both in vivo and in the in vitro system to m-nitroaniline m-nitroaniline and m-nitroacetanilide. These metabolites do not provoke testicular toxicity in vivo or in vitro. We have therefore proposed a pathway for the metabolism of m-dinitrobenzene to m-nitroaniline and m-nitroacetanilide, which involved the intermediate m-nitrosonitrobenzene (1-nitroso-3-nitrobenzene, NNB). When tested, m-nitrosonitrobenzene, at equimolar doses to m-dinitrobenzene, produced similar morphological changes in the culture system to those exhibited by m-dinitrobenzene. However, m-nitrosonitrobenzene produced a greater toxicity than did m-dinitrobenzene (as measured by germ cell detachment). When the intracellular thiol levels were reduced in the cocultures pretreated with diethyl maleate, the toxicity of both m-dinitrobenzene and m-nitrosonitrobenzene was enhanced. In contrast, pretreatment of cocultures with agents known to increase cellular thiol (cysteamine) or scavenge reactive intermediates (cysteamine or ascorbate) reduced the toxicity of m-dinitrobenzene and m-nitrosonitrobenzene. We propose that m-dinitrobenzene requires metabolic activation before it can exert its toxicity to Sertoli cells, and it appears that the toxic species is m-nitrosonitrobenzene or a further metabolite of m-nitrosonitrobenzene.

  18. The Sertoli cell as the orchestra conductor of spermatogenesis: spermatogenic cells dance to the tune of testosterone.

    PubMed

    Dimitriadis, Fotios; Tsiampali, Chara; Chaliasos, Nikolaos; Tsounapi, Panagiota; Takenaka, Atsushi; Sofikitis, Nikolaos

    2015-10-01

    Spermatogenesis is contingent upon hormones and growth factors acting through endocrine and paracrine pathways either in vivo or in vitro. Sertoli cells (SCs) furnish essential factors for the successful advancement of spermatogenesis and spermiogenesis. Moreover, receptors for follicle stimulating hormone (FSH) and testosterone, which are the main hormonal regulators of spermatogenesis, are identified on SCs. Testosterone, FSH and luteinizing hormone are known to determine the destiny of germ cells and in their absence germ cells undergo apoptosis. Bcl-2 family proteins determine one signaling pathway which seems to be crucial for the homeostasis of male gametes. In addition to paracrine signals, germ cell development also relies on signals generated by SCs via direct membrane contact. The regulatory peptide somatostatin has an important role in the regulation of the proliferation of the male germ cells. Activin A, follistatin and FSH control germ cell development. In vitro culture systems have provided initial evidence supporting the achievement of the completion of the first and second male meiotic division in vitro. This review article provides an overview of the literature regarding the hormonal pathways governing spermatogenesis and spermiogenesis. PMID:26732153

  19. [The regeneration process in the Sertoli cells and Leydig cells of the rats undergoing the combined treatment with drinking mineral water and a magnetic field under stressful conditions].

    PubMed

    Korolev, Iu N; Bobrovnitskiĭ, I P; Geniatulina, M S; Nikulina, L A

    2014-01-01

    The experiments carried out on outbred white male rats have demonstrated that twice repeated immobilization stress resulted in the destruction of intracellular organelles and interfered with the regeneration processes in the Sertoli cells and Leydig cells. The combination of the consumption of drinking mineral sulfate water (MW) with the application of a constant or alternating magnetic field (MF) caused stimulation of intracellular regenerative reactions that were especially pronounced in the Sertoli cells in the form of enhanced organoid and intra-organoid regeneration of mitochondria. This in turn increased biological potential of the cells and thereby provided a basis for the further development of biosynthetic processes. The results of the study expose to a certain degree the causes and mechanisms responsible for the higher effectiveness of the combined action of mineral water and magnetic fields compared with the monofactor treatment (mineral water alone). PMID:25087420

  20. The Oncogenic Roles of DICER1 RNase IIIb Domain Mutations in Ovarian Sertoli-Leydig Cell Tumors12

    PubMed Central

    Wang, Yemin; Chen, Jiamin; Yang, Winnie; Mo, Fan; Senz, Janine; Yap, Damian; Anglesio, Michael S.; Gilks, Blake; Morin, Gregg B.; Huntsman, David G.

    2015-01-01

    DICER1, an endoribonuclease required for microRNA (miRNA) biogenesis, is essential for embryogenesis and the development of many organs including ovaries. We have recently identified somatic hotspot mutations in RNase IIIb domain of DICER1 in half of ovarian Sertoli-Leydig cell tumors, a rare class of sex-cord stromal cell tumors in young women. These hotspot mutations lost IIIb cleavage activity of DICER1 in vitro and failed to produce 5p-derived miRNAs in mouse Dicer1-null ES cells. However, the oncogenic potential of these hotspot DICER1 mutations has not been studied. Here, we further revealed that the global expression of 5p-derived miRNAs was dramatically reduced in ovarian Sertoli-Leydig cell tumors carrying DICER1 hotspot mutations compared with those without DICER1 hotspot mutation. The miRNA production defect was associated with the deregulation of genes controlling cell proliferation and the cell fate. Using an immortalized human granulosa cell line, SVOG3e, we determined that the D1709N-DICER1 hotspot mutation failed to produce 5p-derived miRNAs, deregulated the expression of several genes that control gonadal differentiation and cell proliferation, and promoted cell growth. Re-expression of let-7 significantly inhibited the growth of D1709N-DICER1 SVOG3e cells, accompanied by the suppression of key regulators of cell cycle control and ovarian gonad differentiation. Taken together, our data revealed that DICER1 hotspot mutations cause systemic loss of 5p-miRNAs that can both drive pseudodifferentiation of testicular elements and cause oncogenic transformation in the ovary. PMID:26408257

  1. The Sertoli Cell Only Syndrome and Glaucoma in a Sex – Determining Region Y (SRY) Positive XX Infertile Male

    PubMed Central

    Jain, Manish; V, Veeramohan; Chaudhary, Isha; Halder, Ashutosh

    2013-01-01

    The XX male syndrome is a rare genetic disorder. The phenotype is variable; it ranges from a severe impairment of the external genitalia to a normal male phenotype with infertility. It generally results from an unequal crossing over between the short arms of the sex chromosomes (X and Y). We are reporting a case of a 38-year-old man who presented with infertility and the features of hypogonadism and glaucoma. The examinations revealed normal external male genitalia, soft small testes, gynaecomastia and glaucoma. The semen analysis showed azoospermia. The serum gonadotropins were high, with low Anti Mullerian Hormone (AMH) and Inhibin B levels. The chromosomal analysis demonstrated a 46, XX karyotype. Fluorescent In-Situ Hybridization (FISH) and Polymerase Chain Reaction (PCR) revealed the presence of a Sex-determining Region Y (SRY). Testicular Fine Needle Aspiration Cytology (FNAC) revealed the Sertoli Cell Only Syndrome (SCOS). The presence of only Sertoli Cells in the testes, with glaucoma in the XX male syndrome, to our knowledge, has not been reported in the literature. PMID:23998093

  2. Sub-lethal concentrations of CdCl2 disrupt cell migration and cytoskeletal proteins in cultured mouse TM4 Sertoli cells.

    PubMed

    Egbowon, Biola F; Harris, Wayne; Arnott, Gordon; Mills, Chris Lloyd; Hargreaves, Alan J

    2016-04-01

    The aims of this study were to examine the effects of CdCl2 on the viability, migration and cytoskeleton of cultured mouse TM4 Sertoli cells. Time- and concentration-dependent changes were exhibited by the cells but 1?M CdCl2 was sub-cytotoxic at all time-points. Exposure to 1 and 12?M CdCl2 for 4h resulted in disruption of the leading edge, as determined by chemical staining. Cell migration was inhibited by both 1 and 12?M CdCl2 in a scratch assay monitored by live cell imaging, although exposure to the higher concentration was associated with cell death. Western blotting and immunofluorescence staining indicated that CdCl2 caused a concentration dependent reduction in actin and tubulin levels. Exposure to Cd(2+) also resulted in significant changes in the levels and/or phosphorylation status of the microtubule and microfilament destabilising proteins cofilin and stathmin, suggesting disruption of cytoskeletal dynamics. Given that 1-12?M Cd(2+) is attainable in vivo, our findings are consistent with the possibility that Cd(2+) induced impairment of testicular development and reproductive health may involve a combination of reduced Sertoli cell migration and impaired Sertoli cell viability depending on the timing, level and duration of exposure. PMID:26724415

  3. Transition from Preinvasive Carcinoma In Situ to Seminoma Is Accompanied by a Reduction of Connexin 43 Expression in Sertoli Cells and Germ Cells1

    PubMed Central

    Brehm, Ralph; Rüttinger, Christina; Fischer, Petra; Gashaw, Isabella; Winterhager, Elke; Kliesch, Sabine; Bohle, Rainer M; Steger, Klaus; Bergmann, Martin

    2006-01-01

    Abstract Carcinoma in situ (CIS) represents the preinvasive stage of human germ cell tumors, but the mechanism leading to pubertal proliferation and invasive malignancy remains unknown. Among testicular gap junctional proteins, connexin 43 (Cx43) represents the predominant Cx, and, previously, an inverse correlation between synthesis of Cx43 protein and progression of tumor development was detected. In the present study, using cDNA microarray analysis, in situ hybridization, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) from tissue homogenates, RT-PCR from microdissected tubules with normal spermatogenesis and CIS, and seminoma cells from invasive seminoma, we asked whether reduction of Cx43 protein is accompanied by a change of Cx43 transcripts. We detected a significant downregulation of Cx43 at mRNA level in Sertoli and germ cells starting in seminiferous tubules infiltrated with CIS and resulting in a complete loss in seminoma cells. It was demonstrated that downregulation of Cx43 expression in neoplastic human testis takes place at the transcriptional level and starts in CIS. This reduction of Cx43 expression further suggests that early intratubular derangement in Cx43 gene expression and disruption of intercellular communication between Sertoli cells and/or Sertoli and preinvasive tumor cells may play a role in the progression phase of human seminoma development. PMID:16820096

  4. The Dynamic of the Apical Ectoplasmic Specialization between Spermatids and Sertoli Cells: The Case of the Small GTPase Rap1

    PubMed Central

    2014-01-01

    Despite advances in assisted reproductive technologies, infertility remains a consistent health problem worldwide. Spermiation is the process through which mature spermatids detach from the supporting Sertoli cells and are released into the tubule lumen. Spermiation failure leads to lack of mature spermatozoa and, if not occasional, could result into azoospermia, major cause of male infertility in human population. Spermatids are led through their differentiation into spermatozoa by the apical ectoplasmic specialization (aES), a testis-specific, actin-based anchoring junction restricted to the Sertoli-spermatid interface. The aES helps spermatid movement across the seminiferous epithelium, promotes spermatid positioning, and prevents the release of immature spermatozoa. To accomplish its functions, aES needs to undergo tightly and timely regulated restructuring. Even if components of aES are partly known, the mechanism/s through which aES is regulated remains still elusive. In this review, we propose a model by which the small GTPase Rap1 could regulate aES assembly/remodelling. The characterization of key players in the dynamic of aES, such as Rap1, could open new possibility to develop prognostic, diagnostic, and therapeutic approaches for male patients under treatment for infertility as well as it could lead to the identification of new target for male contraception. PMID:24719879

  5. Isolated Large Cell Calcifying Sertoli Cell Tumor in a Young Boy, not Associated with Peutz-Jeghers Syndrome or Carney Complex

    PubMed Central

    Lai, Jin-Ping; Lee, Chyi-Chia; Crocker, Melissa; Najmuddin, Mufaddal; Lange, Eileen; Merino, Maria; Stratakis, Constantine A

    2015-01-01

    Background Large cell calcifying sertoli cell tumor (LCCSCT) is an exceedingly rare lesion of the testicle. It is most often seen in patients with Carney complex (CNC) or Peutz-Jeghers syndrome (PJS). We now report the first pediatric patient with what appears to be bilateral LCCSCT and no other conditions or a genetic syndrome, such as PJS or CNC, have been associated with it. Methods A 10-year-old boy was found to have a right testicular mass during a routine pediatric examination; he underwent right orchiectomy. He was then evaluated clinically for PJS or CNC and underwent genetic testing. His tumor was studied by immunohistochemistry for the expression of calretinin, NY-ESO-1, inhibin, CD99, S100, PLAP, AE1/AE3, Bcl-2, p53, and Mib1. Results Patient did not have clinical features or genetic abnormalities of CNC and PJS. Microscopic features showed large, round or cubical intratubular and aggregated tumor cells with prominent nuclear atypia, large and prominent nucleoli and extensive calcification. In the Immunohistochemical studies, calretinin and inhibin alpha were up regulated in LCCSCT as compared to the adjacent benign Sertoli cells. Meanwhile, NY-ESO-1 and CD99 were down-regulated in LCCSCT. Focally and weakly positive S100 was found in the tumor tissue, but no S100 expression was present in the adjacent Sertoli cells. There was no expression of PLAP, P53, Bcl-2, Mib1 and AE1/AE3 in LCCSCT and adjacent Sertoli cells. Micro-calcifications were found in the other gonad by ultrasonography, suggesting LCCSCT. Conclusion LCCSCT is a rare testicular neoplasm, and may present in isolated rather than in more typical association with syndromes such as CNC and PJS. PMID:26587565

  6. Postnatal testis development, Sertoli cell proliferation and number of different spermatogonial types in C57BL/6J mice made transiently hypo- and hyperthyroidic during the neonatal period.

    PubMed

    Auharek, Sarah Alves; de França, Luiz Renato

    2010-05-01

    The role of thyroid hormones in testis structure and function has been fairly well studied in laboratory rodents. However, there are no comprehensive data in the literature for mice regarding the effects of transiently induced neonatal hypo- and hyperthyroidism on testis and spermatogonial cell development from birth to adulthood. Our goals were to evaluate the effects of propylthiouracil (PTU) and triidothyronine (T3) on Sertoli cell proliferation/differentiation and to correlate these events with the evolution of the spermatogenic process, tubular lumen formation, blood vessel volume density, and size and number of different spermatogonial types. Although Sertoli cell maturation was accelerated or delayed, respectively, in T3- and PTU-treated mice, the pace of the germ cell maturation was only slightly altered before puberty and the period of Sertoli cell proliferation was apparently not affected by the treatments. However, compared with controls, the total number of Sertoli cells per testis from 10 days of age to adulthood was significantly increased and decreased in PTU- and T3-treated mice, respectively. In comparison to all other spermatogonia, type A(2) was the largest cell in all ages and groups investigated. The PTU-treated mice had a significantly increased total number of undifferentiated spermatogonia as well as volume and percentage of vessels/capillaries, probably due to the higher number of Sertoli cells, particularly at 10 days of age. Taken together, our results suggest that neonatal hypothyroidism may be a valuable tool for studying spermatogonial biology as well as a means for providing more spermatogonial stem cells that could potentially be used for spermatogonial transplantation, thereby optimizing the efficiency of this technique when young mice are used as donors. PMID:20525087

  7. Sertoli cells in culture secrete paracrine factor(s) that inhibit peritubular myoid cell proliferation: identification of heparinoids as likely candidates

    SciTech Connect

    Tung, P.S.; Fritz, I.B. )

    1991-06-01

    Conditioned medium from Sertoli cells, prepared from testes of 20-day-old rats, contains component(s) that inhibit the incorporation of (3H)-thymidine into DNA of peritubular myoid cells (PMC) and inhibit the proliferation of PMC. These components are trypsin-resistant, heat-stable compounds having a molecular weight less than 30,000. The active inhibitory components in Sertoli cell conditioned medium are inactivated by treatment with heparinase, but not by treatment with hyaluronidase or chondroitin sulfate lyases. Addition of heparin or heparan sulfate results in inhibition of DNA synthesis by PMC in a dose-dependent manner, whereas other glycosaminoglycans (GAGs) examined (hyaluronic acid, keratan sulfate, and chondroitin sulfate) have no detectable effects. Heparin and heparan sulfate are unique among GAGs tested in inhibiting the characteristic multilayer growth pattern of PMC following the attainment of confluence in serum-rich medium. On the basis of these and other data presented, it is concluded that heparin and other heparin-like GAGs synthesized by Sertoli cells are implicated in the modulation of growth of PMC in vitro during co-culture. It is postulated that heparin may play a similar role in maintaining the quiescent peritubular myoid cell phenotype in vivo.

  8. Androgen receptor binding to an androgen-responsive element in the promoter of the Srsf4 gene inhibits its expression in mouse Sertoli cells.

    PubMed

    Guo, Huan; Li, Yuchi; Luo, Manling; Lin, Shouren; Chen, Jianbo; Ma, Qian; Gu, Yanli; Jiang, Zhimao; Gui, Yaoting

    2015-12-01

    The serine/arginine-rich splicing actor 4 (SRSF4) is essential for pre-mRNA splicing and can influence alternative-splice-site choice. Little is known about the specific function of this gene in the reproductive system, although a recent study identified a SRSF4 polymorphism significantly associated with a decreased risk of non-obstructive azoospermia in Chinese men. We previously found that the expression of Srsf4 was up-regulated in the testes of Sertoli-cell-selective androgen receptor knockout (S-Ar(-/y) ) mice compared to wild-type mice using digital gene expression analysis. In this study, we confirmed and extended the selective gene expression data: SRSF4 was mainly located in the nucleus of Sertoli cells, and Srsf4 expression in the Sertoli-cell-derived cell line TM4 is down-regulation by testosterone. Moreover, androgen receptor directly binds the androgen-responsive element of the Srsf4 promoter. Taken together, these results demonstrate that Srsf4 is a direct downstream target of the androgen receptor in mouse Sertoli cells. Mol. Reprod. Dev. 82: 976-985, 2015. 2015 Wiley Periodicals, Inc. PMID:26308373

  9. Hormone responsiveness of cultured Sertoli cells obtained from adult rats after their rapid isolation under less harsh conditions.

    PubMed

    Gautam, M; Bhattacharya, I; Devi, Y S; Arya, S P; Majumdar, S S

    2016-05-01

    During adulthood, testicular Sertoli cells (Sc) coordinate all stages of germ cell (Gc) development involved in sperm production. However, our understanding about the functions of adult Sc is limited because of the difficulties involved in the process of isolating these cells from the adult testis, mainly because of the presence of large number of advanced Gc which interfere with Sc isolation at this age. Most of our knowledge about Sc function are derived from studies which used pre-pubertal rat Sc (18 ± 2-day old) as it is easy to isolate and culture Sc at this age. To this end, we established a less time consuming and less harsh procedure of isolating Sc from adult (60 days of age) rat testis for facilitating research on Sc-mediated regulation of spermatogenesis during adulthood. The cells were isolated using collagenase digestion at higher temperature, reducing the exposure time of cells to the enzyme. Step-wise digestion with intermittent removal of small clusters of tissue helped in increasing the yield of Sc. Isolated Sc were cultured and treated with FSH and testosterone (T) to evaluate their hormone responsiveness in terms of lactate, E2 , cAMP production. Adult Sc were found to be active and produced high amounts of lactate in a FSH-independent manner. FSH-mediated augmentation of cAMP and E2 production by adult Sc was less as compared with that by pre-pubertal Sc obtained from 18-day-old rats. Androgen-binding ability of adult Sc was significantly higher than pre-pubertal Sc. Although T treatment remarkably augmented expression of Claudin 11, it failed to augment lactate production by adult Sc. This efficient and rapid procedure for isolation and culture of functionally viable adult rat Sertoli cells may pave the way for determining their role in regulation and maintenance of spermatogenesis. PMID:26991307

  10. Follicle-stimulating hormone amplifies insulin-like growth factor I-mediated activation of AKT/protein kinase B signaling in immature rat Sertoli cells.

    PubMed

    Khan, Shafiq A; Ndjountche, Lilianne; Pratchard, Lauren; Spicer, L J; Davis, John S

    2002-06-01

    FSH and IGF-I are both important determinants of testicular development and Sertoli cell function. The present studies were performed to determine the actions of FSH and IGF-I on PI3K/AKT protein kinase signaling in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were prepared from 10-d-old rats. After 7 d in culture, Sertoli cells were treated with IGF-I, FSH, or IGF-I plus FSH. In some experiments cultures were treated with 8-bromo-cAMP (40 microM), (Bu)(2)cAMP (40 microM), or forskolin (10 microM). After treatments, cell lysates were prepared, and the activation state of AKT and cAMP response element-binding protein (CREB) was determined by Western blot analysis using phosphorylation site-specific antibodies. IGF-I had little effect on CREB phosphorylation, but rapidly increased the phosphorylation of AKT in a concentration-dependent manner. Maximal stimulatory effects of IGF-I were observed at 10-20 ng/ml. Treatment with FSH (0.9 IU/ml) or forskolin for 20 min increased CREB phosphorylation, but had little effect on AKT phosphorylation. However, FSH caused a concentration-dependent increase in IGF-I-induced AKT phosphorylation. Longer incubations (1-4 h) with FSH alone resulted in the elevation of AKT phosphorylation concomitant with an increased secretion of IGF-I and decreased production of IGF-binding protein-3, implicating endogenous IGF-I in the action of FSH on AKT phosphorylation. IGF-I- and FSH-dependent AKT phosphorylation was inhibited by LY29400 (10 microM), a PI3K inhibitor, and by IGF-binding protein 3, but not by a PKA inhibitor (H89). The present study demonstrates that immature rat Sertoli cells possess multiple protein kinase signaling cascades that are regulated by FSH. Furthermore, FSH amplifies IGF-I-mediated PI3K/AKT signaling in Sertoli cells. The results provide evidence for intracellular signaling mechanisms that may be required for the proliferation and differentiation of Sertoli cells. PMID:12021190

  11. Ultrastructural modifications in the mitochondrion of mouse Sertoli cells after inhalation of lead, cadmium or lead-cadmium mixture.

    PubMed

    Bizarro, Patricia; Acevedo, Sandra; Nio-Cabrera, Geraldine; Mussali-Galante, Patricia; Pasos, Francisco; Avila-Costa, Maria Rosa; Fortoul, Teresa I

    2003-01-01

    CD-1 mice inhaled 0.01 M lead acetate, 0.006 M cadmium chloride or Pb-Cd mixture during 1h twice a week during 4 weeks. Testes were processed for transmission electron microscopic analysis. The percentage of damaged mitochondria was related to exposure time and the type of metal inhaled, noticing more damage when the mixture was administered. A dose-time relationship was found. Cadmium chloride caused the most severe mitochondrial alteration compared to lead acetate, whereas the mixture was more aggressive compared with each metal alone. Our results suggest that the changes in Sertoli cell could lead to a transformation process that may interfere with spermatogenesis. PMID:14555194

  12. Anti-Müllerian hormone and inhibin B levels reflect altered Sertoli cell function in men with metabolic syndrome.

    PubMed

    Robeva, R; Tomova, A; Kirilov, G; Kumanov, P

    2012-05-01

    The aim of the present study was to investigate the Sertoli cell markers inhibin B and anti-Müllerian hormone (AMH) in men with metabolic syndrome (MS). Twenty patients with MS according to the criteria of the International Diabetes Federation and 20 non obese age-matched men were investigated. The levels of testosterone, sex hormone binding globulin (SHBG), gonadotropins, inhibin B and AMH were measured in all of them. In obese patients with MS total testosterone (15.74 ± 6.95 versus 27.84 ± 12.80 nmol l(-1), P = 0.001), SHBG (21.71 ± 11.08 versus 38.80 ± 17.51 nmol l(-1), P = 0.001) and free testosterone (430.35 ± 237.40 versus 613.85 ± 303.65 pmol l(-1), P = 0.040) were significantly lower than in the controls. Interestingly, the inhibin B (103.64 ± 56.77 versus 149.88 ± 68.31 pg ml(-1), P = 0.025) and AMH levels (30.84 ± 13.14 versus 43.14 ± 9.66 pmol l(-1), P = 0.002) were also significantly lower in MS group in comparison to the other participants. The lowest levels of AMH were found in patients with MS and carbohydrate disturbances. The decreased concentrations of testosterone, inhibin B and AMH in patients with MS could reflect an impaired Leydig and Sertoli cell function. Further studies in men with obesity, insulin resistance and diabetes type 2 could reveal more information about the interrelations between the metabolic disturbances and reproductive function in men. PMID:21749433

  13. A Sertoli cell-specific connexin43 knockout leads to altered interstitial connexin expression and increased Leydig cell numbers.

    PubMed

    Noelke, Joanna; Wistuba, Joachim; Damm, Oliver S; Fietz, Daniela; Gerber, Jonathan; Gaehle, Marion; Brehm, Ralph

    2015-08-01

    The Sertoli cell (SC)-specific knockout (KO) of connexin43 (Cx43) results in spermatogenic arrest at the level of spermatogonia and/or SC-only syndrome. Histology of the interstitial compartment suggests Leydig cell (LC) hyperplasia. Our aim has been to investigate possible effects of the SC-specific KO of Cx43 (SCCx43KO) on interstitial LC. We therefore counted LC via the optical dissector method (per microliter of testicular tissue and per testis) and found LC to be significantly increased in SCCx43KO(-/-) compared with wild-type mice. Semiquantitative western blot together with Cx43 and 3β-hydroxysteroid dehydrogenase immunohistochemistry showed that Cx43 protein was significantly reduced and barely detectable in LC in adult SCCx43KO(-/-) mice. This reduction of Cx43 protein was accompanied by a reduction of Cx43 mRNA as analyzed by laser-assisted microdissection of interstitial cells and subsequent quantitative real-time polymerase chain reaction (PCR). Interestingly, Cx45, another recently detected connexin in LC, was also downregulated. Preliminary qualitative data of LC differentiation markers (Thb2, Hsd3b6) and a steroidogenic marker (Hsd17b3) obtained by reverse transcription plus PCR revealed no obvious differences. Thus, the loss of Cx43 in SC also provokes the downregulation of connexins in interstitial LC at the transcriptional and translational levels. Moreover, SCCx43KO leads to alterations in LC numbers. Despite these alterations, steroidogenesis seems not to be impaired. Further studies, including ultrastructural analysis of the tissue as well as quantitative examination of additional LC markers and testosterone, and functional in vitro experiments, should provide more information about LC differentiation and function in SCCx43KO(-/-) mice. PMID:25693895

  14. EB1 Regulates Tubulin and Actin Cytoskeletal Networks at the Sertoli Cell Blood-Testis Barrier in Male Rats: An In Vitro Study

    PubMed Central

    Tang, Elizabeth I.; Mok, Ka-Wai; Lee, Will M.

    2015-01-01

    During spermatogenesis, developing germ cells are transported across the seminiferous epithelium. Studies propose that because microtubules (MTs) serve as the tracks for transporting cell organelles, they may also serve a similar function in the transport of developing germ cells. Polarized MTs may provide the tracks along which polarized actin microfilaments, which act as vehicles to transport cargo, such as preleptotene spermatocytes through the blood-testis barrier (BTB) and spermatids across the epithelium. Yet the molecular mechanism(s) underlying these events remain unknown. Using an established in vitro Sertoli cell system to study BTB function, we demonstrated herein that a MT regulatory protein end-binding protein 1 (EB1) regulates the MT- and also the actin-based cytoskeleton of the Sertoli cell BTB in the rat. EB1 serves as a coordinator between the two cytoskeletons by regulating MT polymerization and actin filament bundling to modulate germ cell transport at the Sertoli cell BTB. A knockdown of EB1 by RNA interference was found to perturb the tight junction (TJ)-permeability barrier, as evidenced by mislocalization of junctional proteins critical for barrier function to facilitate spermatocyte transport, which was likely achieved by two coordinated events. First, EB1 knockdown resulted in changes in MT polymerization, thereby perturbing MT organization in Sertoli cells in which polarized MT no longer stretched properly across the cell cytosol to serve as the tracks. Second, EB1 knockdown perturbed actin organization via its effects on the branched actin polymerization-inducing protein called Arp3 (actin-related protein 3), perturbing microfilament bundling capability based on a biochemical assay, thereby causing microfilament truncation and misorganization, disrupting the function of the vehicle. This reduced actin microfilament bundling capability thus perturbed TJ-protein distribution and localization at the BTB, destabilizing the TJ barrier, leading to its remodeling to facilitate spermatocyte transport. In summary, EB1 provides a functional link between tubulin- and actin-based cytoskeletons to confer spermatocyte transport at the BTB. PMID:25456071

  15. Enhanced Cultivation Of Stimulated Murine B Cells

    NASA Technical Reports Server (NTRS)

    Sammons, David W.

    1994-01-01

    Method of in vitro cultivation of large numbers of stimulated murine B lymphocytes. Cells electrofused with other cells to produce hybridomas and monoclonal antibodies. Offers several advantages: polyclonally stimulated B-cell blasts cultivated for as long as 14 days, hybridomas created throughout culture period, yield of hybridomas increases during cultivation, and possible to expand polyclonally in vitro number of B cells specific for antigenic determinants first recognized in vivo.

  16. Identification of genetic networks involved in the cell injury accompanying endoplasmic reticulum stress induced by bisphenol A in testicular Sertoli cells

    SciTech Connect

    Tabuchi, Yoshiaki . E-mail: ytabu@cts.u-toyama.ac.jp; Takasaki, Ichiro; Kondo, Takashi

    2006-07-07

    To identify detailed mechanisms by which bisphenol A (BPA), an endocrine-disrupting chemical, induces cell injury in mouse testicular Sertoli TTE3 cells, we performed genome-wide microarray and computational gene network analyses. BPA (200 {mu}M) significantly decreased cell viability and simultaneously induced an increase in mRNA levels of HSPA5 and DDIT3, endoplasmic reticulum (ER) stress marker genes. Of the 22,690 probe sets analyzed, BPA down-regulated 661 probe sets and up-regulated 604 probe sets by >2.0-fold. Hierarchical cluster analysis demonstrated nine gene clusters. In decreased gene clusters, two significant genetic networks were associated with cell growth and proliferation and the cell cycle. In increased gene clusters, two significant genetic networks including many basic-region leucine zipper transcription factors were associated with cell death and DNA replication, recombination, and repair. The present results will provide additional novel insights into the detailed molecular mechanisms of cell injury accompanying ER stress induced by BPA in Sertoli cells.

  17. Implication of actin microfilaments in maintenance of intercellular bridges and the Sertoli cell barrier in the rat seminiferous epithelium

    SciTech Connect

    Weber, J.E.

    1987-01-01

    Within the seminiferous epithelium, germ cells are connected to one another by intercellular bridges. Additionally, young germ cells are separated from more advanced germ cells by the Sertoli cell barrier, the occluding junctions of which are associated with actin microfilaments. To examine how microfilaments influence these structures in the rat the actin-disrupting agent cytochalasin D (CD) was injected intratesticularly (i.t.). In preliminary studies the optical injection volume was found to be 50 {mu}l and, by using the dye trypan blue, the injected solution was shown to enter the lymphatic system and rapidly spread throughout the testis. A 50% clearance of {sup 3}H-insulin from the testis was achieved at 3 hr and 95% by 24 hr. Vehicles with varying solubility properties did not cause testicular damage. Intercellular bridges were found to be dynamic structures. As spermatogenesis progressed, the bridge diameter gradually increased. The formation and degradation of bridge partitioning complexes within pre-existing bridges of dividing cells were described.

  18. Cytokines, polarity proteins, and endosomal protein trafficking and signaling-the sertoli cell blood-testis barrier system in vitro as a study model.

    PubMed

    Xiao, Xiang; Wong, Elissa W P; Lie, Pearl P Y; Mruk, Dolores D; Wong, Chris K C; Cheng, C Yan

    2014-01-01

    Endosomal signaling is emerging as one of the most important cellular events that regulate signaling function in mammalian cells or an epithelium in response to changes in environment such as the presence of stimuli mediated by cytokines, toxicants, heat, ions during growth and development, and other cellular processes such as cytokinesis and spermatogenesis. Recent studies have shown that protein endocytosis-the initial step of endosomal signaling-involves the participation of polarity proteins, such as partitioning defective protein 6 (Par6), Cdc42 and 14-3-3 (also known as Par5), which in turn is regulated by cytokines (e.g., TGF-β2, TGF-β3) and testosterone at the Sertoli cell blood-testis barrier (BTB) in the mammalian testis. In this short method paper, we provide a detailed protocol of assessing protein endocytosis, the initial and also the most critical step of endosomal signaling at the Sertoli cell BTB. This biochemical endocytosis assay summarizes our experience for the last decade, which should likely be performed in conjunction with the dual-labeled immunofluorescence analysis to assess protein endocytosis. While we are using a Sertoli cell in vitro system that mimics the BTB in vivo, this approach should be applicable to virtually all mammalian cells. PMID:24359954

  19. Dehydroepiandrosterone Sulfate Stimulates Expression of Blood-Testis-Barrier Proteins Claudin-3 and -5 and Tight Junction Formation via a Gnα11-Coupled Receptor in Sertoli Cells

    PubMed Central

    Papadopoulos, Dimitrios; Dietze, Raimund; Shihan, Mazen; Kirch, Ulrike; Scheiner-Bobis, Georgios

    2016-01-01

    Dehydroepiandrosterone sulfate (DHEAS) is a circulating sulfated steroid considered to be a pro-androgen in mammalian physiology. Here we show that at a physiological concentration (1 μM), DHEAS induces the phosphorylation of the kinase Erk1/2 and of the transcription factors CREB and ATF-1 in the murine Sertoli cell line TM4. This signaling cascade stimulates the expression of the tight junction (TJ) proteins claudin-3 and claudin-5. As a consequence of the increased expression, tight junction connections between neighboring Sertoli cells are augmented, as demonstrated by measurements of transepithelial resistance. Phosphorylation of Erk1/2, CREB, or ATF-1 is not affected by the presence of the steroid sulfatase inhibitor STX64. Erk1/2 phosphorylation was not observed when dehydroepiandrosterone (DHEA) was used instead of DHEAS. Abrogation of androgen receptor (AR) expression by siRNA did not affect DHEAS-stimulated Erk1/2 phosphorylation, nor did it change DHEAS-induced stimulation of claudin-3 and claudin-5 expression. All of the above indicate that desulfation and conversion of DHEAS into a different steroid hormone is not required to trigger the DHEAS-induced signaling cascade. All activating effects of DHEAS, however, are abolished when the expression of the G-protein Gnα11 is suppressed by siRNA, including claudin-3 and -5 expression and TJ formation between neighboring Sertoli cells as indicated by reduced transepithelial resistance. Taken together, these results are consistent with the effects of DHEAS being mediated through a membrane-bound G-protein-coupled receptor interacting with Gnα11 in a signaling pathway that resembles the non-classical signaling pathways of steroid hormones. Considering the fact that DHEAS is produced in reproductive organs, these findings also suggest that DHEAS, by acting as an autonomous steroid hormone and influencing the formation and dynamics of the TJ at the blood-testis barrier, might play a crucial role for the regulation and maintenance of male fertility. PMID:26938869

  20. Regulation of the proliferation of cocultured gonocytes and Sertoli cells by retinoids, triiodothyronine, and intracellular signaling factors: differences between fetal and neonatal cells.

    PubMed

    Boulogne, Barbara; Habert, Ren; Levacher, Christine

    2003-06-01

    The regulation of early fetal germ cell growth has not been studied in cell culture, probably due to the poor survival of these cells. However, cell culture is the only system in which the control of cell growth can be studied independently of the influence of secreted testicular factors, which are diluted in the medium. We successfully cultured dispersed testicular cells from 16.5-day-old rat fetuses in defined medium and compared the growth of these cells with that of cells from 3-day-old neonates. In this system, fetal gonocytes displayed low levels of mitotic activity and their numbers remained stable. In contrast, neonatal gonocytes displayed high levels of mitotic activity and increased in number, these characteristics resembling those observed in vivo. We found that retinoic acid had deleterious effects on the number of gonocytes but did not affect Sertoli cell proliferation in fetal and neonatal cell cultures. Moreover, in fetal cell cultures, the decrease in the number of gonocytes resulted from a decrease in mitotic activity, probably due to a direct effect of retinoids on fetal gonocytes. Among the selective agonists for the retinoic acid receptor (RARalpha agonist, RARbeta agonist, and RARgamma agonist) and the retinoic X receptor (pan-RXR agonist) tested, only the RARalpha agonist reproduced the effects of retinoic acid at concentrations lower than its Kd value in both fetal and neonatal cell cultures. As both RARalpha and RXRalpha are present in fetal and neonatal gonocytes, we suggest that retinoic acid exerts its effects on gonocytes via a RARalpha-RXRalpha heterodimer, with RARalpha functioning as an active partner and RXRalpha as a passive partner. In this culture system, we show for the first time that triiodothyronine (T3) inhibits testicular fetal Sertoli cell and germ cell growth. We also tested intracellular signaling factors and found that a cAMP analog increased Sertoli cell proliferation and germ cell survival in both fetal and neonatal cells whereas phorbol esters (PMA) strongly inhibited the proliferation of fetal but not of neonatal gonocytes. None of the tested factors (T3, dbcAMP, and PMA) seemed to interact with the all-trans retinoic acid pathway. Thus, fetal gonocytes and neonatal gonocytes differ in intrinsic properties, and their growth is not regulated in the same manner. Despite their low level of mitotic activity, fetal gonocytes were more sensitive to various factors than neonatal gonocytes. PMID:12704731

  1. The co-occurrence of an ovarian Sertoli-Leydig cell tumor with a thyroid carcinoma is highly suggestive of a DICER1 syndrome.

    PubMed

    Durieux, Emeline; Descotes, Françoise; Mauduit, Claire; Decaussin, Myriam; Guyetant, Serge; Devouassoux-Shisheboran, Mojgan

    2016-05-01

    The DICER1 gene encodes an endoribonuclease involved in the production of mature microRNAs which regulates gene expression through several mechanisms. Carriers of germline DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 syndrome. Pleuropulmonary blastoma is the most frequent lesion seen in this syndrome. Thyroid abnormalities are also a common finding, essentially concerning multinodular goiter. However, differentiated thyroid carcinoma is infrequently seen in such pedigrees. In addition to germline DICER1 mutations, specific somatic mutations have been identified in the DICER1 RNase IIIb catalytic domain in several tumor types, including ovarian Sertoli-Leydig cell tumors. We report two cases of differentiated thyroid carcinoma associated with ovarian Sertoli-Leydig cell tumor and with a heterozygous DICER1 gene mutation, occurring in two unrelated young girls without pleuropulmonary blastoma. Both thyroid carcinomas showed an E1813 mutation in exon 25 while the ovarian tumors harboured a somatic mutation in E1705 in exon 24 and a D1709 mutation in exon 25. Our observations confirm that the occurrence of an ovarian Sertoli-Leydig cell tumor with a thyroid carcinoma is highly suggestive of a DICER1 syndrome. We contend that the possibility of a relationship between sporadic thyroid carcinoma in young patients and somatic DICER1 gene mutation needs further investigation. PMID:26983701

  2. Retiform Sertoli-Leydig cell tumor of ovary in a 9-year-old girl: case report and review of the literature.

    PubMed

    Lou, Weizhen; Cao, Dongyan; Yang, Jiaxin; Guo, Lina; Shen, Keng

    2011-12-01

    The management of the retiform variant of Sertoli-Leydig cell tumor remains a challenge for the gynecologist. Surgery is the preferred treatment, but it is still inconclusive whether complete staging or postoperative adjuvant therapy is necessary. A 9-year-old girl was admitted with a well-circumscribed, solid cystic mass in the lower abdomen, of size corresponding to a 20-week gravid uterus, without any androgenic manifestations. Per-operatively, the mass arose from left ovary, which had a smooth outer surface with intact capsule. A cut section was almost multiloculated with cysts ranging from 0.5 to 2.5 cm in diameter and filled with thin yellow or brown serous fluid. Left salpingo-oophorectomy, bilateral lymph node dissection, infracolic omentectomy and appendectomy were performed. The pathological diagnosis was retiform pattern of intermediate to poorly differentiated Sertoli-Leydig cell tumor. The clinical stage was IA. The patient was followed up 3-monthly, and was disease-free at 18-month follow-up after the initial treatment. After review of the literature, we conclude that the retiform variant is a special subtype of Sertoli-Leydig cell tumors. Because of their young age, the uncertain malignant potential and rare bilaterality, patients should be treated conservatively whenever possible. There is at present no good evidence that postoperative adjuvant therapy is effective in preventing recurrence. PMID:21347632

  3. Genes involved in nonpermissive temperature-induced cell differentiation in Sertoli TTE3 cells bearing temperature-sensitive simian virus 40 large T-antigen

    SciTech Connect

    Tabuchi, Yoshiaki . E-mail: ytabu@ms.toyama-mpu.ac.jp; Kondo, Takashi; Suzuki, Yoshihisa; Obinata, Masuo

    2005-04-15

    Sertoli TTE3 cells, derived from transgenic mice bearing temperature-sensitive simian virus 40 large T (tsSV40LT)-antigen, proliferated continuously at a permissive temperature (33 deg C) whereas inactivation of the large T-antigen by a nonpermissive temperature (39 deg C) led to differentiation as judged by elevation of transferrin. To clarify the detailed mechanisms of differentiation, we investigated the time course of changes in gene expression using cDNA microarrays. Of the 865 genes analyzed, 14 genes showed increased levels of expression. Real-time quantitative PCR revealed that the mRNA levels of p21{sup waf1}, milk fat globule membrane protein E8, heat-responsive protein 12, and selenoprotein P were markedly elevated. Moreover, the differentiated condition induced by the nonpermissive temperature significantly increased mRNA levels of these four genes in several cell lines from the transgenic mice bearing the oncogene. The present results regarding changes in gene expression will provide a basis for a further understanding of molecular mechanisms of differentiation in both Sertoli cells and cell lines transformed by tsSV40LT-antigen.

  4. Smad2/3 Upregulates the Expression of Vimentin and Affects Its Distribution in DBP-Exposed Sertoli Cells

    PubMed Central

    Zhang, Xi; Wang, Xiaogang; Liu, Taixiu; Mo, Min; Ao, Lin; Liu, Jinyi; Cao, Jia; Cui, Zhihong

    2015-01-01

    Sertoli cells (SCs) in the testes provide physical and nutritional support to germ cells. The vimentin cytoskeleton in SCs is disrupted by dibutyl phthalate (DBP), which leads to SCs dysfunction. In a previous study, we found that peroxisome proliferator-activated receptor alpha (PPARα) influenced the distribution of vimentin by affecting its phosphorylation in DBP-exposed SCs. In the present study, we investigated the role of Smad2/3 in regulating the expression of vimentin in DBP-exposed SCs. We hypothesized that Smad2/3 affects the distribution of vimentin by regulating its expression and that there is cross talk between Smad2/3 and PPARα. The real-time PCR and ChIP-qPCR results showed that SB431542 (an inhibitor of Smad2/3) could significantly attenuate the expression of vimentin induced by DBP in SCs. Phosphorylated and soluble vimentin were both downregulated by SB431542 pretreatment. WY14643 (an agonist of PPARα) pretreatment stimulated, while GW6471 (an antagonist of PPARα) inhibited, the activity of Smad2/3; SB431542 pretreatment also inhibited the activity of PPARα, but it did not rescue the DBP-induced collapse in vimentin. Our results suggest that, in addition to promoting the phosphorylation of vimentin, DBP also stimulates the expression of vimentin by activating Smad2/3 in SCs and thereby induces irregular vimentin distribution. PMID:26819576

  5. Dose-dependent effects of caffeine in human Sertoli cells metabolism and oxidative profile: relevance for male fertility.

    PubMed

    Dias, Tânia R; Alves, Marco G; Bernardino, Raquel L; Martins, Ana D; Moreira, Ana C; Silva, Joaquina; Barros, Alberto; Sousa, Mário; Silva, Branca M; Oliveira, Pedro F

    2015-02-01

    Caffeine is a widely consumed substance present in several beverages. There is an increasing consumption of energetic drinks, rich in caffeine, among young individuals in reproductive age. Caffeine has been described as a modulator of cellular metabolism. Hence, we hypothesized that it alters human Sertoli cells (hSCs) metabolism and oxidative profile, which are essential for spermatogenesis. For that purpose, hSCs were cultured with increasing doses of caffeine (5, 50, 500 μM). Caffeine at the lowest concentrations (5 and 50 μM) stimulated lactate production, but only hSCs exposed to 50 μM showed increased expression of glucose transporters (GLUTs). At the highest concentration (500 μM), caffeine stimulated LDH activity to sustain lactate production. Notably, the antioxidant capacity of hSCs decreased in a dose-dependent manner and SCs exposed to 500 μM caffeine presented a pro-oxidant potential, with a concurrent increase of protein oxidative damage. Hence, moderate consumption of caffeine appears to be safe to male reproductive health since it stimulates lactate production by SCs, which can promote germ cells survival. Nevertheless, caution should be taken by heavy consumers of energetic beverages and food supplemented with caffeine to avoid deleterious effects in hSCs functioning and thus, abnormal spermatogenesis. PMID:25486098

  6. Melatonin regulates the development and function of bovine Sertoli cells via its receptors MT1 and MT2.

    PubMed

    Yang, Wu-Cai; Tang, Ke-Qiong; Fu, Chang-Zhen; Riaz, Hasan; Zhang, Qiong; Zan, Lin-Sen

    2014-06-10

    Melatonin and its receptors are found in the testis of many species, where they mediate testicular functions. The present study aimed to investigate the expression of melatonin receptors (MT1 and MT2) in bovine Sertoli cells (SCs), using reverse transcription polymerase chain reaction (RT-PCR) and western blot. In addition, we assessed the mRNA levels of spermatogenesis-related genes (real-time PCR) and secretion of inhibin B after treatment with various concentrations (0, 80, 160, and 320 pg/mL) of melatonin at different time points (24, 48, or 72 h). We found that bovine SCs express MT1 and MT2 receptors, which were regulated by melatonin in time- and dose-dependent manners after treatment with melatonin. Exogenous melatonin up-regulated the expression of spermatogenesis-related genes, including Cyclin D1, Cyclin E, Pdgfa, Dhh, Occludin, and Claudin, and decreased the mRNA levels of P21 and Kit1 in a time or dose-dependent manner. Meanwhile, melatonin supplementation significantly affected Inhba, Inhbb and Inha mRNA expression. These findings were consistent with inhibin B levels detected in the culture medium. In conclusion, exogenous melatonin acts via its receptors and appears to play regulatory roles in the development and function of bovine SCs. PMID:24768045

  7. Toxicogenomic Screening of Replacements for Di(2-Ethylhexyl) Phthalate (DEHP) Using the Immortalized TM4 Sertoli Cell Line.

    PubMed

    Nardelli, Thomas C; Erythropel, Hanno C; Robaire, Bernard

    2015-01-01

    Phthalate plasticizers such as di(2-ethylhexyl) phthalate (DEHP) are being phased out of many consumer products because of their endocrine disrupting properties and their ubiquitous presence in the environment. The concerns raised from the use of phthalates have prompted consumers, government, and industry to find alternative plasticizers that are safe, biodegradable, and have the versatility for multiple commercial applications. We examined the toxicogenomic profile of mono(2-ethylhexyl) phthalate (MEHP, the active metabolite of DEHP), the commercial plasticizer diisononyl cyclohexane-1,2-dicarboxylate (DINCH), and three recently proposed plasticizers: 1,4-butanediol dibenzoate (BDB), dioctyl succinate (DOS), and dioctyl maleate (DOM), using the immortalized TM4 Sertoli cell line. Results of gene expression studies revealed that DOS and BDB clustered with control samples while MEHP, DINCH and DOM were distributed far away from the control-DOS-BDB cluster, as determined by principle component analysis. While no significant changes in gene expression were found after treatment with BDB and DOS, treatment with MEHP, DINCH and DOM resulted in many differentially expressed genes. MEHP upregulated genes downstream of PPAR and targeted pathways of cholesterol biosynthesis without modulating the expression of PPAR's themselves. DOM upregulated genes involved in glutathione stress response, DNA repair, and cholesterol biosynthesis. Treatment with DINCH resulted in altered expression of a large number of genes involved in major signal transduction pathways including ERK/MAPK and Rho signalling. These data suggest DOS and BDB may be safer alternatives to DEHP/MEHP than DOM or the commercial alternative DINCH. PMID:26445464

  8. Characterization of a new superfusion, two-compartment culture system for Sertoli cells: influence of extracellular matrix on the cell permeability and dynamics of transferrin secretion.

    PubMed

    Guillou, F

    1990-01-01

    A new superfusion two-compartment culture system was developed in the laboratory of the author and was used to investigate the influence of testicular extracellular matrix on barrier formation by Sertoli cells in culture, and the acute dynamic changes in the bidirectional secretion of transferrin. Only Sertoli cells growing on extracellular matrix formed a monolayer that was specifically impermeable to inulin, peroxidase, and FSH, but did not affect the passage of testosterone. Moreover, in these conditions, they were highly polarized morphologically. The bidirectional secretion (basal/apical ratio) of transferrin was affected by the duration of the stationary culture preceding the superfusion. After 2 days of culture, the amount of transferrin secreted during the subsequent 20 h superfusion was higher in the basal chamber than in the apical chamber. In contrast, after 5 days of culture, the amount of secreted transferrin was higher in the apical compartment. The author compared his data with those previously reported by other authors, using stationary two-compartment chambers. PMID:2324005

  9. Effect of transglutaminase substrates and polyamines on the cellular sequestration and processing of follicle-stimulating hormone by rat Sertoli cells

    SciTech Connect

    Dias, J.A.

    1986-08-01

    Transglutaminase (TGase) substrates monodansyl cadaverine (MDC, monodansyl-1,5 diaminopentane) and methylamine (MA) and polyamines (PA) were tested for their effects on the cellular processing of radioiodinated human follicle-stimulating hormone (/sup 125/I-hFSH). Specifically bound /sup 125/I-hFSH that could be released from cells during 10-min incubation period with acidified (pH 3.9) Hanks balanced-salt solution was considered membrane-bound unsequestered hormone. The rate at which cells sequestered /sup 125/I-hFSH into cellular compartments resistant to acid dissociation depended on the length of time in which cells were incubated with hormone. Cells incubated with /sup 125/I-hFSH for 15, 60, and 120 min had half-lives of sequestration of 26, 55 and 67 min respectively. One hundred-micromolar MDC inhibited degradation of /sup 125/I-hFSH as measured by the presence of radioactivity in the medium that was soluble in trichloroacetic acid. The rate of sequestration was never slower than that of controls, indicating that MDC did not decrease the ability of Sertoli cells to sequester /sup 125/I-hFSH. Despite these two observations, radioactivity associated with cells (acid-resistant radioactivity) was lower in cells treated with MDC than in controls. No effect of MDC on specific binding of 125I-hFSH was observed. Similar results were observed with MA, albeit at higher levels (0.0025-0.0425 M), consistent with their relative potency to inhibit TGase activity. Polyamines, spermine, and putrescine also decreased cell-associated radioactivity despite decreasing degradation of hFSH. TGase substrates (MDC, MA, PA) prevented entry of sequestered 125I-hFSH into the degradative pathways of Sertoli cells. These data suggest that transglutamination may influence the fate of sequestered FSH in Sertoli cells but not the rate at which sequestration occurs.

  10. [INVESTIGATIONS OF SUBMICROSCOPIC ARCHITECTONICS SERTOLI AND LEYDIG CELLS AFTER HYDROCHLORIDE SEROTONIN DESTRUCTIVE IMPACT AND THE POSSIBILITY OF CORRECTION BY STIMULANTS OF METABOLIC PROCESSES].

    PubMed

    Brechka, N; Nevzorov, V; Bondarenko, V; Malova, N; Selyukova, N

    2015-01-01

    The results of study of ultrastructural changes in the Sertoli cells and Leydig's cells organelles after destructive influence of the serotonin hydrochloride and under influence bioglobin-U have been presented. It was shown that serotonin hydrochloride causes mitochondrial dysfunction and activates intracellular catabolic processes on the intracellular level. Bioglobin-U increases the activity and reparative synthetic reactions, reduced the degree of mitochondrial dysfunction and catabolic processes and activate the Leydig cell metabolism, and significantly reduces the number of foci destruction membranes of the endoplasmic reticulum, mitochondrial, and membranes of nucleus on the background of serotonin hydrochloride. PMID:26552310

  11. Leptin modulates human Sertoli cells acetate production and glycolytic profile: a novel mechanism of obesity-induced male infertility?

    PubMed

    Martins, Ana D; Moreira, Ana C; Sá, Rosália; Monteiro, Mariana P; Sousa, Mário; Carvalho, Rui A; Silva, Branca M; Oliveira, Pedro F; Alves, Marco G

    2015-09-01

    Human feeding behavior and lifestyle are gradually being altered, favoring the development of metabolic diseases, particularly type 2 diabetes and obesity. Leptin is produced by the adipose tissue acting as a satiety signal. Its levels have been positively correlated with fat mass and hyperleptinemia has been proposed to negatively affect male reproductive function. Nevertheless, the molecular mechanisms by which this hormone affects male fertility remain unknown. Herein, we hypothesize that leptin acts on human Sertoli cells (hSCs), the "nurse cells" of spermatogenesis, altering their metabolism. To test our hypothesis, hSCs were cultured without or with leptin (5, 25 and 50ng/mL). Leptin receptor was identified by qPCR and Western blot. Protein levels of glucose transporters (GLUT1, GLUT2 and GLUT3), phosphofructokinase, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 (MCT4) were determined by Western Blot. LDH activity was assessed and metabolite production/consumption determined by proton nuclear magnetic resonance. Oxidative damage was evaluated by assessing lipid peroxidation, protein carbonilation and nitration. Our data shows that leptin receptor is expressed in hSCs. The concentration of leptin found in lean, healthy patients, upregulated GLUT2 protein levels and concentrations of leptin found in lean and obese patients increased LDH activity. Of note, all leptin concentrations decreased hSCs acetate production illustrating a novel mechanism for this hormone action. Moreover, our data shows that leptin does not induce or protect hSCs from oxidative damage. We report that this hormone modulates the nutritional support of spermatogenesis, illustrating a novel mechanism that may be linked to obesity-induced male infertility. PMID:26071642

  12. rpS6 regulates blood-testis barrier dynamics through Arp3-mediated actin microfilament organization in rat sertoli cells. An in vitro study.

    PubMed

    Mok, Ka-Wai; Chen, Haiqi; Lee, Will M; Cheng, C Yan

    2015-05-01

    In the seminiferous epithelium of rat testes, preleptotene spermatocytes residing in the basal compartment are transported across the blood-testis barrier (BTB) to enter the adluminal compartment at stage VIII of the epithelial cycle. This process involves redistribution of tight junction (TJ) proteins via reorganization of actin cytoskeleton in Sertoli cells that serves as attachment site for adhesion protein complexes. Ribosomal protein S6 (rpS6), a downstream molecule of mTORC1 (mammalian target of rapamycin complex 1), participates in this process via a yet-to-be defined mechanism. Here, we constructed an rpS6 quadruple phosphomimetic mutant by converting Ser residues at 235, 236, 240, and 244 to Glu via site-directed mutagenesis, making this mutant constitutively active. When this rpS6 mutant was overexpressed in Sertoli cells cultured in vitro with an established TJ barrier mimicking the BTB in vivo, it perturbed the TJ permeability by down-regulating and redistributing TJ proteins at the cell-cell interface. These changes are mediated by a reorganization of actin microfilaments, which was triggered by a redistribution of activated actin-related protein 3 (Arp3) as well as changes in Arp3-neuronal Wiskott-Aldrich Syndrome protein (N-WASP) interaction. This in turn induced reorganization of actin microfilaments, converting them from a "bundled" to an "unbundled/branched" configuration, concomitant with a reduced actin bundling activity, thereby destabilizing the TJ-barrier function. These changes were mediated by Akt (transforming oncogene of v-akt), because an Akt knockdown by RNA interference was able to mimic the phenotypes of rpS6 mutant overexpression at the Sertoli cell BTB. In summary, this study illustrates a mechanism by which mTORC1 signal complex regulates BTB function through rpS6 downstream by modulating actin organization via the Arp2/3 complex, which may be applicable to other tissue barriers. PMID:25714812

  13. Dehydroepiandrosterone and 7-oxo-dehydroepiandrosterone in male reproductive health: Implications of differential regulation of human Sertoli cells metabolic profile.

    PubMed

    Dias, Tânia R; Alves, Marco G; Almeida, Susana P; Silva, Joaquina; Barros, Alberto; Sousa, Mário; Silva, Branca M; Silvestre, Samuel M; Oliveira, Pedro F

    2015-11-01

    Dehydroepiandrosterone (DHEA) is a precursor of androgen synthesis whose action is partially exerted through its metabolites. 7-Oxo-dehydroepiandrosterone (7-oxo-DHEA) is a common DHEA metabolite, non-convertible to androgens, which constitutes a promising therapeutic strategy for multiple conditions. Sertoli cells (SCs) are responsible for the support of spermatogenesis, having unique metabolic characteristics strongly modulated by androgens. Consequently, disruptions in androgen synthesis compromise SCs function and hence male fertility. We aimed to evaluate the effects of DHEA and 7-oxo-DHEA in human SCs (hSCs) metabolism and oxidative profile. To do so, hSCs were exposed to increasing concentrations of DHEA and 7-oxo-DHEA (0.025, 1 and 50 μM) that revealed to be non-cytotoxic in these experimental conditions. We measured hSCs metabolites consumption/production by (1)H NMR, the protein expression levels of key players of the glycolytic pathway by Western blot as well as the levels of carbonyl groups, nitration and lipid peroxidation by Slot blot. The obtained data demonstrated that 7-oxo-DHEA is a more potent metabolic modulator than DHEA since it increased hSCs glycolytic flux. DHEA seem to redirect hSCs metabolism to the Krebs cycle, while 7-oxo-DHEA has some inhibitory effect in this path. The highest 7-oxo-DHEA concentrations (1 and 50 μM) also increased lactate production, which is of extreme relevance for the successful progression of spermatogenesis in vivo. None of these steroids altered the intracellular oxidative profile of hSCs, illustrating that, at the concentrations used they do not have pro- nor antioxidant actions in hSCs. Our study represents a further step in the establishment of safe doses of DHEA and 7-oxo-DHEA to hSCs, supporting its possible use in hormonal and non-hormonal therapies against male reproductive problems. PMID:26134425

  14. Toxicogenomic Screening of Replacements for Di(2-Ethylhexyl) Phthalate (DEHP) Using the Immortalized TM4 Sertoli Cell Line

    PubMed Central

    Nardelli, Thomas C.; Erythropel, Hanno C.; Robaire, Bernard

    2015-01-01

    Phthalate plasticizers such as di(2-ethylhexyl) phthalate (DEHP) are being phased out of many consumer products because of their endocrine disrupting properties and their ubiquitous presence in the environment. The concerns raised from the use of phthalates have prompted consumers, government, and industry to find alternative plasticizers that are safe, biodegradable, and have the versatility for multiple commercial applications. We examined the toxicogenomic profile of mono(2-ethylhexyl) phthalate (MEHP, the active metabolite of DEHP), the commercial plasticizer diisononyl cyclohexane-1,2-dicarboxylate (DINCH), and three recently proposed plasticizers: 1,4-butanediol dibenzoate (BDB), dioctyl succinate (DOS), and dioctyl maleate (DOM), using the immortalized TM4 Sertoli cell line. Results of gene expression studies revealed that DOS and BDB clustered with control samples while MEHP, DINCH and DOM were distributed far away from the control-DOS-BDB cluster, as determined by principle component analysis. While no significant changes in gene expression were found after treatment with BDB and DOS, treatment with MEHP, DINCH and DOM resulted in many differentially expressed genes. MEHP upregulated genes downstream of PPAR and targeted pathways of cholesterol biosynthesis without modulating the expression of PPAR’s themselves. DOM upregulated genes involved in glutathione stress response, DNA repair, and cholesterol biosynthesis. Treatment with DINCH resulted in altered expression of a large number of genes involved in major signal transduction pathways including ERK/MAPK and Rho signalling. These data suggest DOS and BDB may be safer alternatives to DEHP/MEHP than DOM or the commercial alternative DINCH. PMID:26445464

  15. Monoclonal antibodies reacting with murine teratocarcinoma cells.

    PubMed Central

    Goodfellow, P N; Levinson, J R; Williams, V E; McDevitt, H O

    1979-01-01

    Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a rat immunized with the C3H mouse teratocarcinoma C86-S1. After the fusion two clones were chosen for further analysis. The first clone, 3C4-10, produced an antibody recognizing an antigen with a distribution restricted to teratocarcinoma cell lines, an endoderm cell line, and a neuroblastoma. The second clone, 4A1-9, produced an antibody that reacted with all cultured murine cells tested and adult brain. Neither antibody reacted with preimplantation embryos. The 3C4-10 antibody recognized an antigen associated with proteins. The apparent molecular weight of the 3C4-10 antigen was greater than 100,000. PMID:284353

  16. Pituitary follicle-stimulating hormone (FSH) induces CREM gene expression in Sertoli cells: involvement in long-term desensitization of the FSH receptor.

    PubMed Central

    Monaco, L; Foulkes, N S; Sassone-Corsi, P

    1995-01-01

    Transcription factor CREM (cAMP-responsive element modulator) plays a pivotal role in the nuclear response to cAMP in neuroendocrine cells. We have previously shown that follicle-stimulating hormone (FSH) directs CREM expression in male germ cells. The physiological importance of FSH in Sertoli cell function prompted us to analyze its effect on CREM expression in these cells. We observed a dramatic and specific increase in the CREM isoform ICER (inducible cAMP early repressor) expression, with a peak 4 h after FSH treatment of primary Sertoli cells. Interestingly, induced levels of ICER protein persist for a considerably longer time. Induction of the repressor ICER accompanies early down-regulation of the FSH receptor transcript, which leads to long-term desensitization. Here we show that ICER represses FSH receptor expression by binding to a CRE-like sequence in the regulatory region of the gene. Our results confirm the crucial role played by CREM in hormonal control and suggest its role in the long-term desensitization phenomenon of peptide membrane receptors. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7479863

  17. Anamnestic chemiluminescence of murine spleen cells.

    PubMed

    Wong, L S; Kiel, J L

    1984-01-01

    The oxidative response of murine spleen cells to secondary exposure to antigen was determined by luminol (5-amino-2,3-dihydro-1,4-pthalazinedione) amplified chemiluminescence, CL. BALB/cj and CBA/J mice were immunized with saline or an antigen solution of saline, luminol, and bovine serum albumin. Spleen cells were obtained from mice two and four days after immunization, and the CL response to in vitro antigenic exposure was measured for 35 minutes. At two days post-immunization, there was no difference in the CL of control and antigen-primed cells. By day four, the antigen-primed CL response differed significantly in both magnitude and time course from the primary antigen-stimulated response of the controls. This early development of differential CL response to antigenic challenge suggests a role for oxidative metabolic activity in the expression of the anamnestic immune response. PMID:6745991

  18. [The ultrastructural manifestations of the regenerative processes in the Sertoli cells under the action of low-intensity electromagnetic radiation in the rats subjected to stress].

    PubMed

    Korolev, Yu N; Geniatulina, M S; Nikulina, L A; Mikhailik, L V

    2015-01-01

    The experiments on the outbred female rats using the electron microscopic technique have demonstrated that the application of ultrahigh frequency low-intensity electromagnetic radiation (LIEMR) with a flux density below 1 mCW/Cm2 and a frequency of approximately 1,000 MHz in the regime of primary prophylaxis and therapeutic-preventive action suppressed the development of the post-stress pathological ultrastructural changes and increased the activity of the regenerative processes in the Sertoli cells. It was shown that the developing adaptive and compensatory changes in the Sertoli cells most frequently involve the energy-producing structures (mitochondria) that undergo the enlargement of their average and total dimensions. Simultaneously, the amount of granular endoplasmic reticulum and the number of ribosomes increased while the intracellular links between the organelles strengthened and the reserve potential of the cells improved. It is concluded that the observed effects may be due to the action of both local and systemic regulation mechanisms. PMID:26285333

  19. Assessment of testicular function after acute and chronic irradiation: Further evidence for an influence of late spermatids on Sertoli cell function in the adult rat

    SciTech Connect

    Pineau, C.; Velez de la Calle, J.F.; Pinon-Lataillade, G.; Jegou, B.

    1989-06-01

    To study cell to cell communications within the testis of adult Sprague-Dawley rats, we used acute whole body neutron plus gamma-irradiation over 7-121 days postirradiation and chronic whole body gamma-irradiation over 14-84 days of irradiation and 7-86 days postirradiation. Neither irradiation protocol had an effect on the body weight of the animals. Neutron plus gamma-rays induced dramatic damages to spermatogonia, preleptotene spermatocytes, spermatozoa, and, to a lesser extent, pachytene spermatocytes. In contrast, gamma-rays induced a selective destruction of spermatogonia. Subsequently, in both experiments a maturation-depletion process led to a marked decrease in all germ cell types. A complete or near complete recovery of the different germ cell types and spermatozoa took place during the two postirradiation periods. Under both irradiation protocols Sertoli cells number was unchanged. Androgen-binding protein and FSH levels were normal in spite of the disappearance of most germ cells from spermatogonia to early spermatids. However, the decline of androgen-binding protein as well as the rise of FSH and their subsequent recovery were highly correlated to the number of late spermatids and spermatozoa. Moreover, it appeared that spermatocytes may also interfere with the production of inhibin (Exp B). With neither irradiation was Leydig cell function altered, except in Exp B in which elevated LH levels were temporarily observed. Correlation analysis suggested a relationship between preleptotene spermatocytes and Leydig cell function. In conclusion, this study establishes that chronic gamma-irradiation is particularly useful in the study of intratesticular paracrine regulation in vivo and provides further support to the concept that late spermatids play a major role in controlling some aspects of Sertoli cell function in the adult rat.

  20. Isolation of Murine Valve Endothelial Cells

    PubMed Central

    Miller, Lindsey J.; Lincoln, Joy

    2014-01-01

    Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms. PMID:25177896

  1. Intratubular Large Cell Hyalinizing Sertoli Cell Tumor of the Testes in a 4-Year-Old Male With Peutz-Jeghers Syndrome.

    PubMed

    Armijo, Beeling; Bocklage, Theresa; Heideman, Richard

    2015-04-01

    Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant disorder that typically displays familial inheritance. Gastrointestinal polyposis and cutaneous pigmentation is a classic presentation of this syndrome. The reported lifetime cumulative cancer risk in PJS patients is >76% when compared with the general public with females affected more often than males. The prepubertal testicular tumor registry found Sertoli cell tumors (SCTs) to compose approximately 1% of all pediatric solid tumors. Prepubertal testicular masses are relatively rare. Only a small number of SCT cases have been reported in the first decade of life. The concurrence of PJS and feminizing SCTs of the testes is an increasingly recognized cause of prepubertal gynecomastia. The testicular lesions observed in patients with PJS primarily represent multifocal intratubular large cell hyalinizing SCTs with a distinct morphology that differs from large cell calcifying SCTs and sex cord tumors with annular tubules. Here, we describe the diagnosis and treatment course of a 4-year-old male with a SCT of the testes and diagnosis of PJS. PMID:25171448

  2. Xenograft of microencapsulated Sertoli cells for the cell therapy of type 2 diabetes mellitus in spontaneously diabetic nonhuman primates: preliminary data.

    PubMed

    Luca, G; Cameron, D F; Arato, I; Mancuso, F; Linden, E H; Calvitti, M; Falabella, G; Szekeres, K; Bodo, M; Ricci, G; Hansen, B C; Calafiore, R

    2014-01-01

    Insulin resistance in type 2 diabetes mellitus (T2DM) may be due to a chronic inflammation of the visceral adipose tissue (VAT) leading to local and systemic increases in proinflammatory cytokines. Microencapsulated porcine Sertoli cells (MC-pSC), by provision of immunomodulatory and trophic factors, have been successfully used to reduce such inflammation in rodent animal models of type 1 diabetes with no complications or deleterious side effects. Herein, we have begun to investigate this novel and safe therapeutic approach in the spontaneously obese nonhuman primate with spontaneous, insulin-dependent T2DM. After MC-pSC intraperitoneal injection we have evaluated, throughout a 6-month follow-up period, daily ad libitum fed glucose levels, daily exogenous insulin supplementation, biweekly body weight measurements, periodic fasting blood glucose concentrations, glycated hemoglobin (HbA1c) levels, glucose tolerance tests (GTT), and fluorescence-activated cell sorting cytometry (FACS) assessment of peripheral blood mononuclear cells. Very preliminarily, we have observed a slight reduction in fasting (FPG) and mean nonfasting (NF) plasma glucose levels. We found minimal changes, only in 1 animal, in daily exogenous insulin requirements and HbA1c levels. Flow cytometric analysis was associated with decrease in CD8(+) cells only in 1 recipient with a reduction in mean regulatory T Cells (Treg), whereas interestingly, decrease of B lymphocytes was observed in both animals. These results may suggest that this novel MC-SC-based transplantation protocol might possibly impact the metabolic status of T2DM in higher mammals that are close to humans. PMID:25131093

  3. Redefining Myeloid Cell Subsets in Murine Spleen

    PubMed Central

    Hey, Ying-Ying; Tan, Jonathan K. H.; O’Neill, Helen C.

    2016-01-01

    Spleen is known to contain multiple dendritic and myeloid cell subsets, distinguishable on the basis of phenotype, function and anatomical location. As a result of recent intensive flow cytometric analyses, splenic dendritic cell (DC) subsets are now better characterized than other myeloid subsets. In order to identify and fully characterize a novel splenic subset termed “L-DC” in relation to other myeloid cells, it was necessary to investigate myeloid subsets in more detail. In terms of cell surface phenotype, L-DC were initially characterized as a CD11bhiCD11cloMHCII−Ly6C−Ly6G− subset in murine spleen. Their expression of CD43, lack of MHCII, and a low level of CD11c was shown to best differentiate L-DC by phenotype from conventional DC subsets. A complete analysis of all subsets in spleen led to the classification of CD11bhiCD11cloMHCII−Ly6CloLy6G− cells as monocytes expressing CX3CR1, CD43 and CD115. Siglec-F expression was used to identify a specific eosinophil population, distinguishable from both Ly6Clo and Ly6Chi monocytes, and other DC subsets. L-DC were characterized as a clear subset of CD11bhiCD11cloMHCII−Ly6C−Ly6G− cells, which are CD43+, Siglec-F− and CD115−. Changes in the prevalence of L-DC compared to other subsets in spleens of mutant mice confirmed the phenotypic distinction between L-DC, cDC and monocyte subsets. L-DC development in vivo was shown to occur independently of the BATF3 transcription factor that regulates cDC development, and also independently of the FLT3L and GM-CSF growth factors which drive cDC and monocyte development, so distinguishing L-DC from these commonly defined cell types. PMID:26793192

  4. Murine Mueller cells are progenitor cells for neuronal cells and fibrous tissue cells

    SciTech Connect

    Florian, Christian; Langmann, Thomas; Weber, Bernhard H.F.; Morsczeck, Christian

    2008-09-19

    Mammalian Mueller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Mueller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Mueller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Mueller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Mueller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture.

  5. Telomere sister chromatid exchange in telomerase deficient murine cells

    SciTech Connect

    Wang, Yisong; Giannone, Richard J; Liu, Yie

    2005-01-01

    We have recently demonstrated that several types of genomic rearrangements (i.e., telomere sister chromatid exchange (T-SCE), genomic-SCE, or end-to-end fusions) were more often detected in long-term cultured murine telomerase deficient embryonic stem (ES) cells than in freshly prepared murine splenocytes, even through they possessed similar frequencies of critically short telomeres. The high rate of genomic rearrangements in telomerase deficient ES cells, when compared to murine splenocytes, may reflect the cultured cells' gained ability to protect chromosome ends with eroded telomeres allowing them to escape 'end crisis'. However, the possibility that ES cells were more permissive to genomic rearrangements than other cell types or that differences in the microenvironment or genetic background of the animals might consequentially determine the rate of T-SCEs or other genomic rearrangements at critically short telomeres could not be ruled out.

  6. Unusual Sertoli Cell Tumor Associated With Sex Cord Tumor With Annular Tubules in Peutz-Jeghers Syndrome: Report of a Case and Review of the Literature on Ovarian Tumors in Peutz-Jeghers Syndrome.

    PubMed

    Ravishankar, Sanjita; Mangray, Shamlal; Kurkchubasche, Arlet; Yakirevich, Evgeny; Young, Robert H

    2016-05-01

    We report the case of an 11-year-old girl with Peutz-Jeghers syndrome and a unilateral ovarian tumor most consistent with Sertoli cell tumor associated with sex cord tumor with annular tubules. The ovary was replaced by a lobular, solid, yellow tumor. Microscopic examination showed 2 components that focally merged. The first was composed of uniform, cytologically bland cells arranged mostly in diffuse sheets and focally in tubules. The second showed typical sex cord tumor with annular tubules with extensive calcification. The predominant component of the tumor clearly fell in the sex cord category and most closely resembled Sertoli cell tumor. This case adds to the limited information on ovarian sex cord tumors, other than typical sex cord tumor with annular tubules, arising in association with Peutz-Jeghers syndrome, a topic reviewed herein. PMID:26621753

  7. The Wilms Tumor Gene, Wt1, Is Critical for Mouse Spermatogenesis via Regulation of Sertoli Cell Polarity and Is Associated with Non-Obstructive Azoospermia in Humans

    PubMed Central

    Wang, Ya Qing; Chen, Min; Zhang, Jun; Hao, Jian Xiu; Wang, Yan Bo; Sha, Ri Na; Huang, Yi; Liu, Xiao; Hu, Jing Chu; Sun, Guang Qing; Li, Hong Gang; Xiong, Cheng Liang; Xie, Jun; Jiang, Zhi Mao; Cai, Zhi Ming; Wang, Jun; Wang, Jian; Huff, Vicki; Gui, Yao Ting; Gao, Fei

    2013-01-01

    Azoospermia is one of the major reproductive disorders which cause male infertility in humans; however, the etiology of this disease is largely unknown. In the present study, six missense mutations of WT1 gene were detected in 529 human patients with non-obstructive azoospermia (NOA), indicating a strong association between WT1 mutation and NOA. The Wilms tumor gene, Wt1, is specifically expressed in Sertoli cells (SCs) which support spermatogenesis. To examine the functions of this gene in spermatogenesis, Wt1 was deleted in adult testis using Wt1flox and Cre-ERTM mice strains. We found that inactivation of Wt1 resulted in massive germ cell death and only SCs were present in most of the seminiferous tubules which was very similar to NOA in humans. In investigating the potential mechanism for this, histological studies revealed that the bloodtestis barrier (BTB) was disrupted in Wt1 deficient testes. In vitro studies demonstrated that Wt1 was essential for cell polarity maintenance in SCs. Further studies found that the expression of cell polarity associated genes (Par6b and E-cadherin) and Wnt signaling genes (Wnt4, Wnt11) were downregulated in Wt1 deficient SCs, and that the expression of Par6b and E-cadherin was regulated by Wnt4. Our findings suggest that Wt1 is important in spermatogenesis by regulating the polarity of SCs via Wnt signaling pathway and that WT1 mutation is one of the genetic causes of NOA in humans. PMID:23935527

  8. Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model

    SciTech Connect

    Carette, Diane; Perrard, Marie-Hélène; Prisant, Nadia; Gilleron, Jérome; Pointis, Georges; Segretain, Dominique; Durand, Philippe

    2013-04-01

    Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12 days. Exposure to low concentrations of chromium (10 μg/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood–testis barrier dynamic is postulated. - Highlights: ► Influence of Cr(VI) on the Sertoli cell barrier and on junctional proteins ► Use of cultured seminiferous tubules in bicameral chambers ► Low concentrations of Cr(VI) (10 μg/l) altered the trans-epithelial resistance. ► Cr(VI) did not alter claudin-11 and N-cadherin. ► Cr(VI) delocalized connexin 43 from the membrane to the cytoplasm of Sertoli cells.

  9. Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

    SciTech Connect

    Johnson, G.P.

    1987-01-01

    Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of ({sup 3}H)GDP binding to plasma membranes suggested a single high affinity site with a K{sub d} = 0.24 uM. Competition studies indicated that GTP{sub {gamma}}S was 7-fold more potent than GDP{sub {beta}}S. Bound GDP could be released by FSH in the presence of GTP{sub {gamma}}S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP{sub {beta}}S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP{sub {beta}}S competitively inhibited GTP{sub {gamma}}S-stimulated adenylate cyclase activity with a K{sub i} = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP{sub {gamma}}S-bound form persisted even if GDP{sub {beta}}S previously occupied all available binding sites. Two membrane proteins, M{sub r} = 43,000 and 48,000, were ADP{centered dot}ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP{sub {gamma}}S but not by GDP{sub {beta}}S. The M{sub r} = 43,000 and 48,000 proteins represented variant forms of G{sub S}. A single protein of M{sub r} = 40,000 (G{sub i}) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC{sub 50} = 0.1 uM. The adenosine analog, N{sup 6}{centered dot}phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin.

  10. Improving in vitro Sertoli cell/Gonocyte co-culture model for assessing male reproductive toxicity: lessons learned from comparisons of cytotoxicity versus genomic responses to phthalates

    PubMed Central

    Yu, Xiaozhong; Hong, SungWoo; Moreira, Estefania G; Faustman, Elaine M

    2009-01-01

    Gonocytes exist in the neonatal testis and represent a transient population of male germ-line stem cells. It has been shown that stem cell self-renewal and progeny production is probably controlled by the neighboring differentiated cells and extracellular matrix (ECM) in vivo known as niches. Recently, we developed an in vitro three-dimensional (3D) Sertoli cell/Gonocyte coculture (SGC) model with ECM overlay, which creates an in vivo-like niche and supports germ-line stem cell functioning within a 3D environment. In this study, we applied morphological and cytotoxicity evaluations, as well as microarray-based gene expression to examine the effects of different phthalate esters (PE) on this model. Known in vivo male developmentally toxic PEs (DTPE) and developmentally nontoxic PEs (DNTPE) were evaluated. We observed that DTPE induced significantly greater dose-dependent morphological changes, a decrease in cell viability and an increase in cytotoxicity compared to those treated with DNTPE. Moreover, gene expression was more greatly altered by DTPE than by DNTPE and non-supervised cluster analysis allowed the discrimination of DTPE from the DNTPE. Our systems-based GO-Quant analysis showed significant alterations in gene pathways involved in cell cycle, phosphate transport and apoptosis regulation with DTPE but not with DNTPE treatment. Disruptions of steroidogenesis related-gene expression such as Star, Cyp19a1, Hsd17b8, and Nr4a3 were observed in the DTPE group, but not in the DNTPE group. In summary, our observation on cell viability, cytotoxicity, and microarray-based gene expression analysis induced by PEs demonstrate that our in vitro 3D-SGC system mimicked in vivo responses for PEs and suggests that the 3D-SGC system might be useful in identifying developmental reproductive toxicants. PMID:19560483

  11. Improving in vitro Sertoli cell/gonocyte co-culture model for assessing male reproductive toxicity: Lessons learned from comparisons of cytotoxicity versus genomic responses to phthalates

    SciTech Connect

    Yu Xiaozhong; Hong, Sung Woo; Moreira, Estefania G.; Faustman, Elaine M.

    2009-09-15

    Gonocytes exist in the neonatal testis and represent a transient population of male germ-line stem cells. It has been shown that stem cell self-renewal and progeny production is probably controlled by the neighboring differentiated cells and extracellular matrix (ECM) in vivo known as niches. Recently, we developed an in vitro three-dimensional (3D) Sertoli cell/gonocyte co-culture (SGC) model with ECM overlay, which creates an in vivo-like niche and supports germ-line stem cell functioning within a 3D environment. In this study, we applied morphological and cytotoxicity evaluations, as well as microarray-based gene expression to examine the effects of different phthalate esters (PE) on this model. Known in vivo male developmentally toxic PEs (DTPE) and developmentally non-toxic PEs (DNTPE) were evaluated. We observed that DTPE induced significantly greater dose-dependent morphological changes, a decrease in cell viability and an increase in cytotoxicity compared to those treated with DNTPE. Moreover, the gene expression was more greatly altered by DTPE than by DNTPE and non-supervised cluster analysis allowed the discrimination of DTPE from the DNTPE. Our systems-based GO-Quant analysis showed significant alterations in the gene pathways involved in cell cycle, phosphate transport and apoptosis regulation with DTPE but not with DNTPE treatment. Disruptions of steroidogenesis related-gene expression such as Star, Cyp19a1, Hsd17b8, and Nr4a3 were observed in the DTPE group, but not in the DNTPE group. In summary, our observation on cell viability, cytotoxicity, and microarray-based gene expression analysis induced by PEs demonstrate that our in vitro 3D-SGC system mimicked in vivo responses for PEs and suggests that the 3D-SGC system might be useful in identifying developmental reproductive toxicants.

  12. Insulin-like growth factor (IGF-I) mRNA and IGF-I receptor in trout testis and in isolated spermatogenic and Sertoli cells.

    PubMed

    Le Gac, F; Loir, M; le Bail, P Y; Ollitrault, M

    1996-05-01

    Few data exist concerning the occurrence and potential role of an insulin-like growth factor (IGF) system in fish gonads. Using Northern and slot blot hybridization with a specific salmon IGF-I cDNA, we confirmed that IGF-I transcription occurs in trout testis. Testicular IGF-I mRNA abundance may be increased by long-term GH treatment in juvenile fish, while shorter treatment with growth hormone (GH) or a gonadotropin (GTH-II) in maturing males had no statistically significant effect. Radiolabelled recombinant human IGF-I binds with high affinity to crude trout testis preparation, to cultured isolated testicular cells, and to a membrane fraction of these cells (Ka = 0.2 to 0.7 x 10(10) M-1; Bmax = 10 to 20 fmol/10(7) cells, and 68 fmol/mg protein of membrane). The binding site was identified as type 1 IGF receptor by its binding specificity (IGF-I > IGF-II > insulin) and the molecular size of its alpha-subunit labelled with 125I-IGF-I (M(r)125-140 kDa). 125I-IGF-II also bound to the type 1 receptor whereas IGF-II/ mannose 6 phosphate receptors could not be detected. Separation of isolated testicular cells by Percoll gradient and centrifugal elutriation provided populations enriched in different types of intratubular cells. IGF-I mRNA (detected by reverse transcription + polymerase chain reaction [PCR]) and IGF-I receptors (measured by competitive binding) were observed to a greater extent in Sertoli cell-enriched populations and in spermatogonia with primary spermatocytes. Therefore, IGF-I is a potential paracrine/autocrine regulator inside the spermatogenic compartment and appears as a possible mediator of GH action at the gonadal level in fish. PMID:8722689

  13. Novel Role for p110β PI 3-Kinase in Male Fertility through Regulation of Androgen Receptor Activity in Sertoli Cells

    PubMed Central

    Guillermet-Guibert, Julie; Smith, Lee B.; Halet, Guillaume; Whitehead, Maria A.; Pearce, Wayne; Rebourcet, Diane; León, Kelly; Crépieux, Pascale; Nock, Gemma; Strömstedt, Maria; Enerback, Malin; Chelala, Claude; Graupera, Mariona; Carroll, John; Cosulich, Sabina; Saunders, Philippa T. K.; Huhtaniemi, Ilpo; Vanhaesebroeck, Bart

    2015-01-01

    The organismal roles of the ubiquitously expressed class I PI3K isoform p110β remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110β, we document that full inactivation of p110β leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110β kinase-dead mice that survive into adulthood (maximum ~26% on a mixed genetic background) have no apparent phenotypes, other than subfertility in females and complete infertility in males. Systemic inhibition of p110β results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110β was previously suggested to signal downstream of the c-kit tyrosine kinase receptor in germ cells to regulate their proliferation and survival. We now report that p110β also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110β inactivation dampening expression of the SC-specific Androgen Receptor (AR) target gene Rhox5, a homeobox gene critical for spermatogenesis. All extragonadal androgen-dependent functions remain unaffected by global p110β inactivation. In line with a crucial role for p110β in SCs, selective inactivation of p110β in these cells results in male infertility. Our study is the first documentation of the involvement of a signalling enzyme, PI3K, in the regulation of AR activity during spermatogenesis. This developmental pathway may become active in prostate cancer where p110β and AR have previously been reported to functionally interact. PMID:26132308

  14. Procedures for the isolation and culture of Sertoli cells from the testes of infant, juvenile, and adult rhesus monkeys (Macaca mulatta).

    PubMed

    Majumdar, S S; Winters, S J; Plant, T M

    1998-03-01

    The purpose of the present study was to establish culture conditions for the in vitro study of the rhesus monkey Sertoli cell (Sc) at three major stages of development, namely infancy, adulthood, and the intervening prepubertal period. Conditions for the culture of Sc from juveniles were first established using collagenase and pancreatin digestion of seminiferous tubules. The addition of 1% fetal bovine serum for the first 24 h of culture was necessary for attachment of Sc clusters. Confluency of Sc from juveniles was reached as early as 4 days of culture. Histochemical and ultrastructural observations confirmed that the cultures were enriched with Sc and that contamination by peritubular cells was minimal (2%). Although application of similar culture conditions was successful in establishing cultures of Sc from infants, significant modification of the procedure was required before Sc from adults could be cultured. Specifically, adult testicular tissue required two sequential collagenase digestions at elevated temperature. The yield of adult Sc, however, remained low. Cultures of juvenile Sc produced substantial quantities of 31-kDa inhibin, which was bioactive as reflected by its ability to suppress FSH secretion from rat pituitary cells in vitro. Although aromatase activity in juvenile Sc cultures was stimulated by FSH, inhibin synthesis, as reflected by immunoactive inhibin production and steady-state levels of alpha inhibin mRNA, was not increased by FSH. The establishment of conditions for the culture of infant, juvenile, and adult Sc from the rhesus monkey will provide a model for study of the postnatal ontogeny of Sc function in higher primates. PMID:9510950

  15. MicroRNA-1285 Regulates 17β-Estradiol-Inhibited Immature Boar Sertoli Cell Proliferation via Adenosine Monophosphate-Activated Protein Kinase Activation.

    PubMed

    Jiao, Zhang Jiao; Yi, Wang; Rong, Yang Wei; Kee, Jeong Dong; Zhong, Wang Xian

    2015-11-01

    This study investigated the capacity of 10 μM 17β-estradiol to inhibit immature boar Sertoli cell (SC) proliferation and the involvement of microRNA (miR)-1285 in this process. SC viability and cell cycle progression were investigated using a cell counting kit-8 and flow cytometry, respectively. Expression of AMP-activated protein kinase (AMPK), S phase kinase-associated protein 2 (Skp2), and miR-1285 was analyzed by real-time RT-PCR and Western blotting. 17β-Estradiol (10 μM) reduced SC viability and miR-1285 expression and promoted AMPK phosphorylation. A double-stranded synthetic miR-1285 mimic promoted SC viability, increased levels of ATP, and phosphorylated mammalian target of rapamycin (mTOR) and Skp2 mRNA and protein, whereas p53 and p27 expression decreased, and 17β-estradiol-mediated effects on SCs were significantly attenuated. A single-stranded synthetic miR-1285 inhibitor produced the opposite effects on these measures. Activation of AMPK inhibited SC viability, reduced levels of ATP, phosphorylated mTOR and Skp2 mRNA and protein, and increased p53 and p27 expression. An AMPK inhibitor (compound C) attenuated the effects of 17β-estradiol on SCs. This indicated that 17β-estradiol (10 μM) reduced SC proliferation by inhibiting miR-1285 and thus activating AMPK. Phosphorylated AMPK is involved in the regulation of 17β-estradiol-mediated inhibition of SC viability through increasing p53 and p27 expression and inhibiting mTOR and Skp2 expression. Our findings also implicated Skp2 as the downstream integration point of p53 and mTOR. These findings indicated that miR-1285 may represent a target for the manipulation of boar sperm production. PMID:26287402

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

    PubMed Central

    KANG, Hoin; PARK, Jong Im; ROH, Sangho

    2015-01-01

    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

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

    PubMed

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

    2016-02-01

    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

  18. Human Pontine Glioma Cells Can Induce Murine Tumors

    PubMed Central

    Caretti, Viola; Sewing, A. Charlotte P.; Lagerweij, Tonny; Schellen, Pepijn; Bugiani, Marianna; Jansen, Marc H. A.; van Vuurden, Dannis G.; Navis, Anna C.; Horsman, Ilona; Vandertop, W. Peter; Noske, David P.; Wesseling, Pieter; Kaspers, Gertjan J.L.; Nazarian, Javad; Vogel, Hannes; Hulleman, Esther; Monje, Michelle; Wurdinger, Thomas

    2014-01-01

    Diffuse intrinsic pontine glioma (DIPG), with a median survival of only nine months, is the leading cause of pediatric brain cancer mortality. Dearth of tumor tissue for research has limited progress in this disease until recently. New experimental models for DIPG research are now emerging. To develop preclinical models of DIPG, two different methods were adopted: cells obtained at autopsy 1) were directly xenografted orthotopically into the pons of immunodeficient mice without an intervening cell culture step or 2) were first cultured in vitro and, upon successful expansion, injected in vivo. Both strategies resulted in pontine tumors histopathologically similar to the original human DIPG tumors. However, following the direct transplantation method all tumors proved to be composed of murine and not of human cells. This is in contrast to the indirect method that included initial in vitro culture and resulted in xenografts comprised of human cells. Of note, direct injection of cells obtained post mortem from the pons and frontal lobe of human brains not affected by cancer did not give rise to neoplasms. The murine pontine tumors exhibited an immunophenotype similar to human DIPG, but were also positive for microglia/macrophage markers, such as CD45, CD68 and CD11b. Serial orthotopic injection of these murine cells results in lethal tumors in recipient mice. Direct injection of human DIPG cells in vivo can give rise to malignant murine tumors. This represents an important caveat for xenotransplantation models of DIPG. In contrast, an initial in vitro culture step can allow establishment of human orthotopic xenografts. The mechanism underlying this phenomenon observed with direct xenotransplantation remains an open question. PMID:24777482

  19. Human pontine glioma cells can induce murine tumors.

    PubMed

    Caretti, Viola; Sewing, A Charlotte P; Lagerweij, Tonny; Schellen, Pepijn; Bugiani, Marianna; Jansen, Marc H A; van Vuurden, Dannis G; Navis, Anna C; Horsman, Ilona; Vandertop, W Peter; Noske, David P; Wesseling, Pieter; Kaspers, Gertjan J L; Nazarian, Javad; Vogel, Hannes; Hulleman, Esther; Monje, Michelle; Wurdinger, Thomas

    2014-01-01

    Diffuse intrinsic pontine glioma (DIPG), with a median survival of only 9 months, is the leading cause of pediatric brain cancer mortality. Dearth of tumor tissue for research has limited progress in this disease until recently. New experimental models for DIPG research are now emerging. To develop preclinical models of DIPG, two different methods were adopted: cells obtained at autopsy (1) were directly xenografted orthotopically into the pons of immunodeficient mice without an intervening cell culture step or (2) were first cultured in vitro and, upon successful expansion, injected in vivo. Both strategies resulted in pontine tumors histopathologically similar to the original human DIPG tumors. However, following the direct transplantation method all tumors proved to be composed of murine and not of human cells. This is in contrast to the indirect method that included initial in vitro culture and resulted in xenografts comprising human cells. Of note, direct injection of cells obtained postmortem from the pons and frontal lobe of human brains not affected by cancer did not give rise to neoplasms. The murine pontine tumors exhibited an immunophenotype similar to human DIPG, but were also positive for microglia/macrophage markers, such as CD45, CD68 and CD11b. Serial orthotopic injection of these murine cells results in lethal tumors in recipient mice. Direct injection of human DIPG cells in vivo can give rise to malignant murine tumors. This represents an important caveat for xenotransplantation models of DIPG. In contrast, an initial in vitro culture step can allow establishment of human orthotopic xenografts. The mechanism underlying this phenomenon observed with direct xenotransplantation remains an open question. PMID:24777482

  20. 7,12-dimethylbenz[a]anthracene induces sertoli-leydig-cell tumors in the follicle-depleted ovaries of mice treated with 4-vinylcyclohexene diepoxide.

    PubMed

    Craig, Zelieann R; Davis, John R; Marion, Samuel L; Barton, Jennifer K; Hoyer, Patricia B

    2010-02-01

    Ovarian cancer is associated with high mortality due to its late onset of symptoms and lack of reliable screening methods for early detection. Furthermore, the incidence of ovarian cancer is higher in postmenopausal women. Mice rendered follicle-depleted through treatment with 4-vinylcyclohexene diepoxide (VCD) are a model of ovary-intact menopause. The present study was designed to induce ovarian neoplasia in this model by treating mice with 7,12-dimethylbenz[a]anthracene (DMBA). Female B6C3F1 mice (age, 28 d) received intraperitoneal sesame oil (vehicle; VCD- groups) as a control or VCD (160 mg/kg; VCD+ groups) daily for 20 d to cause ovarian failure. Four months after the onset of dosing, mice from each group received a single injection of DMBA (VCD-DMBA+ and VCD+DMBA+ groups, n = 15 per group) or vehicle control (VCD-DMBA-, n = 15; VCD+ DMBA-, n = 14) under the bursa of the right ovary. Ovaries were collected 3 or 5 mo after injection and processed for histologic evaluation. Immunohistochemistry was used to confirm classification of neoplasms. None of the animals in the VCD-DMBA- and VCD-DMBA+ groups (that is, mice still undergoing estrus) had tumors at either time point. At the 3-mo time point, 12.5% of the VCD+DMBA+ mice had ovarian tumors; at 5 mo, 57.1% of the VCD+DMBA+ and 14.3% of VCD+DMBA- ovaries had neoplasms. Neoplasms stained positively for inhibin alpha (granulosa cells) and negatively for keratin 7 (surface epithelium), thus confirming classification of the lesions as Sertoli-Leydig cell tumors. These findings provide evidence for an increased incidence of DMBA-induced ovarian neoplasms in the ovaries of follicle-depleted mice compared with that in age-matched cycling controls. PMID:20158943

  1. Dickkopf Homolog 3 (DKK3) Plays a Crucial Role Upstream of WNT/?-CATENIN Signaling for Sertoli Cell Mediated Regulation of Spermatogenesis

    PubMed Central

    Kunj, Neetu; Sarda, Kanchan; Pradhan, Bhola Shankar; Majumdar, Subeer S.

    2013-01-01

    Testicular Sertoli cells (Sc) are main somatic component of seminiferous tubules that govern the differentiation of germ cells (Gc) and provide them physical support. Sc are the target of follicle stimulating hormone (FSH) and testosterone (T) which are known to regulate spermatogenesis. FSH and T levels in human and sub-human male primates remain high during infancy (46 months post birth), similar to those during puberty. Subsequently, juvenile phase is marked with low levels of these hormones. In spite of prolonged hormonal exposure, spermatogenesis is not discerned during infancy unlike that during puberty. Situation during infancy is similar to certain idiopathic male infertility, where prolonged hormone supplementation fails to initiate spermatogenesis. In our quest to determine non hormonal causes of idiopathic infertility which may reside within the Sc, we investigated the association between spermatogenesis and Sc specific gene(s) expressed differentially during puberty and infancy. Although products of several genes may be necessary for quantitatively normal spermatogenesis, one needs to investigate their roles one by one. Differential display and real time PCR analysis revealed higher expression of a known tumor suppressor, Dickkopf homolog 3 (DKK3), by pubertal monkey Sc as compared to infant Sc. To evaluate role of DKK3 in spermatogenesis, we generated DKK3 knock down mice (DKDM) using shRNA construct targeted to DKK3. In testis of adult DKDM, expression of DKK3 mRNA and protein were significantly (p<0.05) low and was associated with elevated WNT-4/?-CATENIN activity. Elevated ?-CATENIN activity is known to restrict Sc maturation. Abundant expression of infant Sc marker, Mullerian inhibiting substance (MIS), in the testes of adult DKDM confirmed lack of Sc maturation in DKDM. Gc differentiation and fertility was severely compromised in DKDM. This is the first report of role of DKK3 in the testis and DKK3 mediated regulation of spermatogenesis via WNT-4/?-CATENIN modulation. PMID:23667645

  2. Cadmium-induced Activation of Stress Signaling Pathways, Disruption of Ubiquitin-dependent Protein Degradation and Apoptosis in Primary Rat Sertoli Cell-Gonocyte Cocultures

    PubMed Central

    Yu, Xiaozhong; Hong, Sungwoo; Faustman, Elaine M.

    2008-01-01

    Cadmium (Cd) is a ubiquitous environmental pollutant that has been associated with male reproductive toxicity in both humans and animal models. The underlying mechanism of this response, however, is still uncharacterized. To address this issue, we employed a recently developed and optimized three-dimensional primary Sertoli cell-gonocyte coculture system and examined the time- and dose-dependent effects of Cd on morphological alterations, cell viability, activation of stress signaling pathway proteins, and the disruption of the ubiquitin proteasome system (UPS). Our results demonstrated that Cd exposure lead to time- and dose-dependent morphological changes that are associated with the induction of apoptosis. In response to Cd, we also saw a disruption of the UPS as evaluated through the accumulation of high–molecular weight polyubiquitinated proteins (HMW-polyUb) as well as alterations in proteasome activity. Robust activation of cellular stress response, measured through the increased phosphorylation of stress-activated protein kinase/c-jun N-terminal kinase and p38, paralleled the accumulation of HMW-polyUb. In addition, p53, a key regulatory protein, was upregulated and underwent increased ubiquitination in response to Cd. To further characterize the role of the UPS in Cd cellular response, we compared the above changes with two classic proteasomal inhibitors, lactacystin, and MG132. The stress response and the accumulation of HWM-polyUb induced by Cd were consistent with the response seen with MG132 but not with lactacystin. In addition, Cd treatment resulted in a dose- and time-dependent effect on proteasome activity, but the overall Cd-induced proteasomal inhibition was unique as compared to MG132 and lactacystin. Taken together, our studies further characterize Cd-induced in vitro testicular toxicity and highlight the potential role of the UPS in this response. PMID:18463101

  3. Differential effects of c-Src and c-Yes on the endocytic vesicle-mediated trafficking events at the Sertoli cell blood-testis barrier: an in vitro study.

    PubMed

    Xiao, Xiang; Mruk, Dolores D; Wong, Elissa W P; Lee, Will M; Han, Daishu; Wong, Chris K C; Cheng, C Yan

    2014-10-01

    The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. However, it undergoes cyclic restructuring during the epithelial cycle of spermatogenesis in which the "old" BTB located above the preleptotene spermatocytes being transported across the immunological barrier is "disassembled," whereas the "new" BTB found behind these germ cells is rapidly "reassembled," i.e., mediated by endocytic vesicle-mediated protein trafficking events. Thus, the immunological barrier is maintained when preleptotene spermatocytes connected in clones via intercellular bridges are transported across the BTB. Yet the underlying mechanism(s) in particular the involving regulatory molecules that coordinate these events remains unknown. We hypothesized that c-Src and c-Yes might work in contrasting roles in endocytic vesicle-mediated trafficking, serving as molecular switches, to effectively disassemble and reassemble the old and the new BTB, respectively, to facilitate preleptotene spermatocyte transport across the BTB. Following siRNA-mediated specific knockdown of c-Src or c-Yes in Sertoli cells, we utilized biochemical assays to assess the changes in protein endocytosis, recycling, degradation and phagocytosis. c-Yes was found to promote endocytosed integral membrane BTB proteins to the pathway of transcytosis and recycling so that internalized proteins could be effectively used to assemble new BTB from the disassembling old BTB, whereas c-Src promotes endocytosed Sertoli cell BTB proteins to endosome-mediated protein degradation for the degeneration of the old BTB. By using fluorescence beads mimicking apoptotic germ cells, Sertoli cells were found to engulf beads via c-Src-mediated phagocytosis. A hypothetical model that serves as the framework for future investigation is thus proposed. PMID:25117412

  4. Murine Cell Glycolipids Customization by Modular Expression of Glycosyltransferases

    PubMed Central

    Cid, Emili; Yamamoto, Miyako; Buschbeck, Marcus; Yamamoto, Fumiichiro

    2013-01-01

    Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins. PMID:23798992

  5. Nanoelectroablation Therapy for Murine Basal Cell Carcinoma

    PubMed Central

    Nuccitelli, Richard; Tran, Kevin; Athos, Brian; Kreis, Mark; Nuccitelli, Pamela; Chang, Kris S.; Epstein, Ervin H.; Tang, Jean Y.

    2012-01-01

    When skin tumors are exposed to non-thermal, low energy, nanosecond pulsed electric fields (nsPEF), apoptosis is initiated both in vitro and in vivo. This nanoelectroablation therapy has already been proven effective in treating subdermal murine allograft tumors. We wanted to determine if this therapy would be equally as effective in the treatment of autochthonous BCC tumors in Ptch1+/−K14-Cre-ER p53 fl/fl mice. These tumors are similar to human BCCs in histology [2;20] and in response to drug therapy [19]. We have treated 27 BCCs across 8 mice with either 300 pulses of 300 ns duration or 2700 pulses of 100 ns duration, all at 30 kV/cm and 5–7 pulses per second. Every nsPEF-treated BCC began to shrink within a day after treatment and their initial mean volume of 36 ± 5 (SEM) mm3 shrunk by 76 ± 3% over the ensuing two weeks. After four weeks, they were 99.8% ablated if the size of the treatment electrode matched the tumor size. If the tumor was larger than the 4 mm wide electrode, multiple treatments were needed for complete ablation. Treated tumors were harvested for histological analysis at various times after treatment and exhibited apoptosis markers. Specifically, pyknosis of nuclei was evident as soon as 2 days after nsPEF treatment, and DNA fragmentation as detected via TUNEL staining was also evident post treatment. Nanoelectroablation is effective in triggering apoptosis and remission of radiation-induced BCCs with a single 6 minute-long treatment of 2700 pulses. PMID:22771794

  6. Murine and human mast cell progenitors.

    PubMed

    Schmetzer, Oliver; Valentin, Patricia; Church, Martin K; Maurer, Marcus; Siebenhaar, Frank

    2016-05-01

    The development of mature mast cells (MCs) from hematopoietic progenitor cells as well as the identification and characterization of committed progenitor cells are a current focus of mast cell research. Most published reports in this area are on the origin and differentiation of MCs in mice. Evidence for the human system, i.e. derived from primary human MCs, is widely lacking. Based on the published data, MCs develop either from a committed progenitor or from a common basophil/mast cell precursor. This review summarizes the current knowledge on MC development and MC differentiation. PMID:26164789

  7. Intramyocardial Cell Delivery: Observations in Murine Hearts

    PubMed Central

    Poggioli, Tommaso; Sarathchandra, Padmini; Rosenthal, Nadia; Santini, Maria P.

    2014-01-01

    Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells. Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe. Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load. PMID:24513973

  8. Nanoelectroablation therapy for murine basal cell carcinoma

    SciTech Connect

    Nuccitelli, Richard; Tran, Kevin; Athos, Brian; Kreis, Mark; Nuccitelli, Pamela; Chang, Kris S.; Epstein, Ervin H.; Tang, Jean Y.; Stanford University, Stanford, CA 94305

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Nanoelectroablation is a new, non-thermal therapy that triggers apoptosis in tumors. Black-Right-Pointing-Pointer Low energy, ultrashort, high voltage pulses ablate the tumor with little or no scar. Black-Right-Pointing-Pointer Nanoelectroablation eliminates 99.8% of the BCC but may leave a few remnants behind. Black-Right-Pointing-Pointer Pilot clinical trials on human BCCs are ongoing and leave no remnants in most cases. -- Abstract: When skin tumors are exposed to non-thermal, low energy, nanosecond pulsed electric fields (nsPEF), apoptosis is initiated both in vitro and in vivo. This nanoelectroablation therapy has already been proven effective in treating subdermal murine allograft tumors. We wanted to determine if this therapy would be equally effective in the treatment of autochthonous BCC tumors in Ptch1{sup +/-}K14-Cre-ER p53 fl/fl mice. These tumors are similar to human BCCs in histology and in response to drug therapy . We have treated 27 BCCs across 8 mice with either 300 pulses of 300 ns duration or 2700 pulses of 100 ns duration, all at 30 kV/cm and 5-7 pulses per second. Every nsPEF-treated BCC began to shrink within a day after treatment and their initial mean volume of 36 {+-} 5 (SEM) mm{sup 3} shrunk by 76 {+-} 3% over the ensuing two weeks. After four weeks, they were 99.8% ablated if the size of the treatment electrode matched the tumor size. If the tumor was larger than the 4 mm wide electrode, multiple treatments were needed for complete ablation. Treated tumors were harvested for histological analysis at various times after treatment and exhibited apoptosis markers. Specifically, pyknosis of nuclei was evident as soon as 2 days after nsPEF treatment, and DNA fragmentation as detected via TUNEL staining was also evident post treatment. Nanoelectroablation is effective in triggering apoptosis and remission of radiation-induced BCCs with a single 6 min-long treatment of 2700 pulses.

  9. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    SciTech Connect

    Grasso, P.; Reichert, L.E. Jr. )

    1990-08-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.

  10. Impaired NK cell education diminishes resistance to murine CMV infection

    PubMed Central

    Wei, Hairong; Nash, William T.; Makrigiannis, Andrew P.; Brown, Michael G.

    2014-01-01

    Ly49G2 (G2+) NK cells mediate murine (M)CMV resistance in MHC Dk-expressing mice. Bone marrow transplantation (BMT) studies revealed that G2+ NK cell-mediated MCMV resistance requires Dk in both hematopoietic and nonhematopoietic cells. As a Ly49G2 ligand, Dk in both cell lineages may contribute to lysis of virus-infected cells. Alternatively, cellular differences in self-MHC Dk may have affected NK-cell education, and consequently NK cell-mediated viral clearance. We investigated the Dk-licensing effect on BM-derived NK cells in BMT recipients by analyzing cytokines, cytotoxicity and MCMV resistance. In BMT recipients with lineage-restricted Dk, G2+ NK-cell reactivity and cytotoxicity was diminished in comparison to BMT recipients with self-MHC in all cells. Reduced G2+ NK-mediated MCMV resistance in BMT recipients with lineage-restricted self-MHC indicates that licensing of G2+ NK cells is related to NK cell reactivity and viral control. Titrating donor BM with self-MHC-bearing hematopoietic cells, as well as adoptive transfer of mature G2+ NK cells into BMT recipients with self-MHC in non-hematopoietic cells only, enhanced NK cell licensing and rescued MCMV resistance. This disparate self-MHC NK cell education model would suggest that inadequately licensed NK cells corresponded to inefficient viral sensing and clearance. PMID:25187217

  11. Mesenchymal Stem Cells Reduce Murine Atherosclerosis Development

    PubMed Central

    Frodermann, Vanessa; van Duijn, Janine; van Pel, Melissa; van Santbrink, Peter J.; Bot, Ilze; Kuiper, Johan; de Jager, Saskia C. A.

    2015-01-01

    Mesenchymal stem cells (MSCs) have regenerative properties, but recently they were also found to have immunomodulatory capacities. We therefore investigated whether MSCs could reduce atherosclerosis, which is determined by dyslipidaemia and chronic inflammation. We adoptively transferred MSCs into low-density lipoprotein-receptor knockout mice and put these on a Western-type diet to induce atherosclerosis. Initially after treatment, we found higher levels of circulating regulatory T cells. In the long-term, overall numbers of effector T cells were reduced by MSC treatment. Moreover, MSC-treated mice displayed a significant 33% reduction in circulating monocytes and a 77% reduction of serum CCL2 levels. Most strikingly, we found a previously unappreciated effect on lipid metabolism. Serum cholesterol was reduced by 33%, due to reduced very low-density lipoprotein levels, likely a result of reduced de novo hepatic lipogenesis as determined by a reduced expression of Stearoyl-CoA desaturase-1 and lipoprotein lipase. MSCs significantly affected lesion development, which was reduced by 33% in the aortic root. These lesions contained 56% less macrophages and showed a 61% reduction in T cell numbers. We show here for the first time that MSC treatment affects not only inflammatory responses but also significantly reduces dyslipidaemia in mice. This makes MSCs a potent candidate for atherosclerosis therapies. PMID:26490642

  12. Antagonistic Effects of a Mixture of Low-Dose Nonylphenol and Di-N-Butyl Phthalate (Monobutyl Phthalate) on the Sertoli Cells and Serum Reproductive Hormones in Prepubertal Male Rats In Vitro and In Vivo

    PubMed Central

    Xiang, Zou; Qian, Weiping; Han, Xiaodong; Li, Dongmei

    2014-01-01

    The estrogenic chemical nonylphenol (NP) and the antiandrogenic agent di-n-butyl phthalate (DBP) are regarded as widespread environmental endocrine disruptors (EDCs) which at high doses in some species of laboratory animals, such as mice and rats, have adverse effects on male reproduction and development. Given the ubiquitous coexistence of various classes of EDCs in the environment, their combined effects warrant clarification. In this study, we attempted to determine the mixture effects of NP and DBP on the testicular Sertoli cells and reproductive endocrine hormones in serum in male rats based on quantitative data analysis by a mathematical model. In the in vitro experiment, monobutyl phthalate (MBP), the active metabolite of DBP, was used instead of DBP. Sertoli cells were isolated from 9-day-old Sprague-Dawley rats followed by treatment with NP and MBP, singly or combined. Cell viability, apoptosis, necrosis, membrane integrity and inhibin-B concentration were tested. In the in vivo experiment, rats were gavaged on postnatal days 23–35 with a single or combined NP and DBP treatment. Serum reproductive hormone levels were recorded. Next, Bliss Independence model was employed to analyze the quantitative data obtained from the in vitro and in vivo investigation. Antagonism was identified as the mixture effects of NP and DBP (MBP). In this study, we demonstrate the potential of Bliss Independence model for the prediction of interactions between estrogenic and antiandrogenic agents. PMID:24676355

  13. Dye-mediated photosensitization of murine neuroblastoma cells

    SciTech Connect

    Sieber, F.; Sieber-Blum, M.

    1986-04-01

    The purpose of this study was to determine if photosensitization mediated by the fluorescent dye, merocyanine 540, could be used to preferentially kill murine neuroblastoma cells in simulated autologous remission marrow grafts. Simultaneous exposure of Neuro 2a or NB41A3 neuroblastoma cells to merocyanine 540 and white light reduced the concentration of in vitro-clonogenic tumor cells 50,000-fold. By contrast, the same treatment had little effect on the graft's ability to rescue lethally irradiated syngeneic hosts. Lethally irradiated C57BL/6J X A/J F1 mice transplanted with photosensitized mixtures of neuroblastoma cells and normal marrow cells (1:100 or 1:10) survived without developing neuroblastomas. It is conceivable that merocyanine 540-mediated photosensitization will prove useful for the extracorporeal purging of residual neuroblastoma cells from human autologous remission marrow grafts.

  14. Theiler's murine encephalomyelitis virus induces tumour necrosis factor-alpha in murine astrocyte cell cultures.

    PubMed Central

    Sierra, A; Rubio, N

    1993-01-01

    Cytokines have been postulated to exert an important modulatory and recruiting role in demyelination induced by Theiler's murine encephalomyelitis virus (TMEV) in SJL/J mice. Using a cytolytic bioassay and ELISA, we have detected and quantified a cytokine, tumour necrosis factor-alpha (TNF-alpha), in supernatants from astrocyte cultures infected in vitro with TMEV. TNF was detected only after TMEV-specific infection of astrocyte cultures (approximately 200-400 U/ml). In vitro TNF synthesis appeared in a dose- and time-dependent manner and was produced by both SJL/J (a strain susceptible to TMEV-induced demyelination) and BALB/c (a resistant strain) astrocytes. The precise nature of TNF activity was further assessed by fast protein liquid chromatography (FPLC) and antibody neutralization. These results indicate an active role for astrocytes as accessory immune cells in our experimental model for multiple sclerosis. PMID:8478023

  15. Generation of Eosinophils from Cryopreserved Murine Bone Marrow Cells

    PubMed Central

    Schollaert, Kaila L.; Stephens, Michael R.; Gray, Jerilyn K.; Fulkerson, Patricia C.

    2014-01-01

    Eosinophils are produced in the bone marrow from CD34+ eosinophil lineagecommitted progenitors, whose levels in the bone marrow are elevated in a variety of human diseases. These findings suggest that increased eosinophil lineagecommitted progenitor production is an important process in disease-associated eosinophilia. The pathways central to the biology of the eosinophil lineagecommitted progenitor remain largely unknown. Thus, developing new methods to investigate the regulators of eosinophil lineagecommitted progenitor differentiation is needed to identify potential therapeutic targets to specifically inhibit eosinophil production. We tested cytokine regimens to optimize liquid cultures for the study of eosinophil lineagecommitted progenitor and eosinophil precursor differentiation into mature eosinophils. Stem cell factor (but not fms-related tyrosine kinase 3 ligand) was required for optimal yield of eosinophils. Furthermore, we evaluated the effects of cell preservation and scale on the culture, successfully culturing functional eosinophils from fresh and frozen murine bone marrow cells and in a standard-sized and 96-well culture format. In summary, we have developed an adaptable culture system that yields functionally competent eosinophils from murine low-density bone marrow cells and whose cytokine regime includes expansion of progenitors with stem cell factor alone with subsequent differentiation with interleukin 5. PMID:25551463

  16. Notch Signaling Pathway Regulates Progesterone Secretion in Murine Luteal Cells.

    PubMed

    Wang, Jing; Liu, Shuangmei; Peng, Lichao; Dong, Qiming; Bao, Riqiang; Lv, Qiulan; Tang, Min; Hu, Chuan; Li, Gang; Liang, Shangdong; Zhang, Chunping

    2015-10-01

    Notch signaling is an evolutionarily conserved pathway, which involves in various cell life activities. Other studies and our report showed that the Notch signaling plays very important role in follicle development in mammalian ovaries. In luteal cells, Notch ligand, delta-like ligand 4, is involved in normal luteal vasculature. In this study, murine luteal cells were cultured in vitro and treated with Notch signaling inhibitors, L-658,458 and N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester (DAPT). We found that L-658,458 and DAPT treatment decrease basal and human chorionic gonadotropin (hCG)-stimulated progesterone secretion. On the contrary, overexpression of intracellular domain of Notch3 increased basal and hCG-stimulated progesterone secretion. Further studies demonstrated that Notch signaling regulated the expression of steroidogenic acute regulatory protein and CYP11A, 2 key enzymes for progesterone synthesis. In conclusion, Notch signaling plays important role in regulating progesterone secretion in murine luteal cells. PMID:25701842

  17. Isolation and Characterization of Murine Multipotent Lung Stem Cells

    PubMed Central

    Gadepalli, Venkat S.; Vaughan, Catherine; Rao, Raj R.

    2016-01-01

    Cancer stem cells possess the ability to self-renew and differentiate into specific cells found in tumor types, a characteristic feature of normal multipotent stem cells. These cells harbor within the bulk of tumors and if the tumor suppressor p53 is mutated in these cells, can be more likely to cause relapse and metastasis by giving rise to new tumors. This new paradigm of oncogenesis has been observed in various cancers, including lung cancer. Determining the interaction of critical cellular pathways in the ontogeny of lung tumors is expected to lead to identification of molecular targets for effective therapeutic strategies. To achieve this, it is important to characterize and dissect the differences between the cancer cells with aberrant stem cell like properties and normal multipotent stem cells that contribute to regeneration. This could be accomplished by using cell surface markers unique for certain cell types by employing techniques such as flow cytometry and magnetic bead isolation. This chapter summarizes the isolation process of the resident stem cell Sca1 (+ve), CD-45 (−ve), and CD-31 (−ve) populations for its potential use in assessing correlations between specific p53 gain of function phenotypes in different murine lung cancer models. PMID:23150447

  18. A murine-ES like state facilitates transgenesis and homologous recombination in human pluripotent stem cells

    PubMed Central

    Buecker, Christa; Chen, Hsu-Hsin; Polo, Jose; Daheron, Laurence; Bu, Lei; Barakat, Tahsin Stefan; Okwieka, Patricia; Porter, Andrew; Gribnau, Joost; Hochedlinger, Konrad; Geijsen, Niels

    2010-01-01

    Murine embryonic stem cells have been shown to exist in two functionally distinct pluripotent states, embryonic stem cells (ES cell)- and epiblast stem cells (EpiSCs), which are defined by the culture growth factor conditions. Human ES cells appear to exist in an epiblast-like state, which in comparison to their murine counterparts, is relatively difficult to propagate and manipulate. As a result, gene targeting is difficult and to-date only a handful of human knock-in or knock-out cell lines exist. We explored whether an alternative stem cell state exists for human stem cells as well, and demonstrate that manipulation of the growth factor milieu allows the derivation of a novel human stem cell type that displays morphological, molecular and functional properties of murine ES cells and facilitates gene targeting. As such, the murine ES-like state provides a powerful tool for the generation of recombinant human pluripotent stem cell lines. PMID:20569691

  19. Murine mammary stem/progenitor cell isolation: Different method matters?

    PubMed

    Gao, Hui; Dong, Qiaoxiang; Chen, Yuanhong; Zhang, Fuchuang; Wu, Anqi; Shi, Yuanshuo; Bandyopadhyay, Abhik; Daniel, Benjamin J; Huang, Changjiang; Sun, Lu-Zhe

    2016-01-01

    Murine mammary stem/progenitor cell isolation has been routinely used in many laboratories, yet direct comparison among different methods is lacking. In this study, we compared two frequently used digestion methods and three sets of frequently used surface markers for their efficiency in enriching mammary stem and progenitor cells in two commonly used mouse strains, C57BL/6J and FVB. Our findings revealed that the slow overnight digestion method using gentle collagenase/hyaluronidase could be easily adopted and yielded reliable and consistent results in different batches of animals. In contrast, the different fast digestion protocols, as described in published studies, yielded high percent of non-epithelial cells with very few basal epithelial cells liberated in our hands. The three sets of markers tested in our hands reveal rather equally efficiency in separating luminal and basal cells if same fluorochrome conjugations were used. However, the tendency of non-epithelial cell inclusion in the basal cell gate was highest in samples profiled by CD24/CD29 and lowest in samples profiled by CD49f/EpCAM, this is especially true in mammary cells isolated from C57BL/6J mice. This finding will have significant implication when sorted basal cells are used for subsequent gene expression analysis. PMID:26933638

  20. The dynamics of murine mammary stem/progenitor cells

    PubMed Central

    DONG, Qiaoxiang; SUN, Lu-Zhe

    2014-01-01

    The stem/progenitor cells in the murine mammary gland are a highly dynamic population of cells that are responsible for ductal elongation in puberty, homeostasis maintenance in adult, and lobulo-alveolar genesis during pregnancy. In recent years understanding the epithelial cell hierarchy within the mammary gland is becoming particularly important as these different stem/progenitor cells were perceived to be the cells of origin for various subtypes of breast cancer. Although significant advances have been made in enrichment and isolation of stem/progenitor cells by combinations of antibodies against cell surface proteins together with flow cytometry, and in identification of stem/progenitor cells with multi-lineage differentiation and self-renewal using mammary fat pad reconstitution assay and in vivo genetic labeling technique, a clear understanding of how these different stem/progenitors are orchestrated in the mammary gland is still lacking. Here we discuss the different in vivo and in vitro methods currently available for stem/progenitor identification, their associated caveats, and a possible new hierarchy model to reconcile various putative stem/progenitor cell populations identified by different research groups. PMID:25580105

  1. Permissive and restricted virus infection of murine embryonic stem cells

    PubMed Central

    Wash, Rachael; Calabressi, Sabrina; Franz, Stephanie; Griffiths, Samantha J.; Goulding, David; Tan, E-Pien; Wise, Helen; Digard, Paul; Haas, Jürgen; Efstathiou, Stacey

    2012-01-01

    Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA ‘off-target’ effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host–pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host–virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host–virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence. PMID:22815272

  2. Permissive and restricted virus infection of murine embryonic stem cells.

    PubMed

    Wash, Rachael; Calabressi, Sabrina; Franz, Stephanie; Griffiths, Samantha J; Goulding, David; Tan, E-Pien; Wise, Helen; Digard, Paul; Haas, Jürgen; Efstathiou, Stacey; Kellam, Paul

    2012-10-01

    Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA 'off-target' effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host-pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host-virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host-virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence. PMID:22815272

  3. Control of Murine Cytomegalovirus Infection by ?? T Cells

    PubMed Central

    Sell, Sabrina; Dietz, Monika; Schneider, Andrea; Holtappels, Rafaela; Mach, Michael; Winkler, Thomas H.

    2015-01-01

    Infections with cytomegalovirus (CMV) can cause severe disease in immunosuppressed patients and infected newborns. Innate as well as cellular and humoral adaptive immune effector functions contribute to the control of CMV in immunocompetent individuals. None of the innate or adaptive immune functions are essential for virus control, however. Expansion of ?? T cells has been observed during human CMV (HCMV) infection in the fetus and in transplant patients with HCMV reactivation but the protective function of ?? T cells under these conditions remains unclear. Here we show for murine CMV (MCMV) infections that mice that lack CD8 and CD4 ??-T cells as well as B lymphocytes can control a MCMV infection that is lethal in RAG-1-/- mice lacking any T- and B-cells. ?? T cells, isolated from infected mice can kill MCMV infected target cells in vitro and, importantly, provide long-term protection in infected RAG-1-/- mice after adoptive transfer. ?? T cells in MCMV infected hosts undergo a prominent and long-lasting phenotypic change most compatible with the view that the majority of the ?? T cell population persists in an effector/memory state even after resolution of the acute phase of the infection. A clonotypically focused V?1 and V?2 repertoire was observed at later stages of the infection in the organs where MCMV persists. These findings add ?? T cells as yet another protective component to the anti-CMV immune response. Our data provide clear evidence that ?? T cells can provide an effective control mechanism of acute CMV infections, particularly when conventional adaptive immune mechanisms are insufficient or absent, like in transplant patient or in the developing immune system in utero. The findings have implications in the stem cell transplant setting, as antigen recognition by ?? T cells is not MHC-restricted and dual reactivity against CMV and tumors has been described. PMID:25658831

  4. FLOW CYTOMETRIC COMPARISON OF THE EFFECTS OF TRIALKYTING ON THE MURINE ERYTHROLEUKEMIC CELL

    EPA Science Inventory

    Cellular effects of exposure to tributyltin (TBT), triethyltin (TET), or trimethyltin (TMT) were investigated by flow cytometry employing the murine erythroleukemic cell (MELC) as a model cellular system. Cell viability was investigated by the carboxyfluorescein diacetate (CFDA) ...

  5. Toxicity of Calcium Hydroxide Nanoparticles on Murine Fibroblast Cell Line

    PubMed Central

    Dianat, Omid; Azadnia, Sina; Mozayeni, Mohammad Ali

    2015-01-01

    Introduction: One of the major contributing factors, which may cause failure of endodontic treatment, is the presence of residual microorganisms in the root canal system. For years, most dentists have been using calcium hydroxide (CH) as the intracanal medicament between treatment sessions to eliminate remnant microorganisms. Reducing the size of CH particles into nanoparticles enhances the penetration of this medicament into dentinal tubules and increases their antimicrobial efficacy. This in vitro study aimed to compare the cytotoxicity of CH nanoparticles and conventional CH on fibroblast cell line using the Mosmann’s Tetrazolium Toxicity (MTT) assay. Methods and Materials: This study was conducted on L929 murine fibroblast cell line by cell culture and evaluation of the direct effect of materials on the cultured cells. Materials were evaluated in two groups of 10 samples each at 24, 48 and 72 h. At each time point, 10 samples along with 5 positive and 5 negative controls were evaluated. The samples were transferred into tubes and exposed to fibroblast cells. The viability of cells was then evaluated. The Two-way ANOVA was used for statistical analysis and the level of significance was set at 0.05. Results: Cytotoxicity of both materials decreased over time and for conventional CH was lower than that of nanoparticles. However, this difference was not statistically significant (P>0.05). Conclusion: The cytotoxicity of CH nanoparticles was similar to that of conventional CH. PMID:25598810

  6. Differential expression of CD22 (Lyb8) on murine B cells

    PubMed Central

    Erickson, Loren D.; Tygrett, Lorraine T.; Bhatia, Sudershan K.; Grabstein, Kenneth H.; Waldschmidt, Thomas J.

    2010-01-01

    Previous studies have established the distribution, biochemistry and functional attributes of human CD22, a B cell-restricted glycoprotein. Recently, molecular cloning of the murine CD22 equivalent revealed this molecule to be the same as the previously described Lyb8 alloantigen. Using the antl-Lyb8 mAb Cy34.1.2, the present report documents the expression patterns of CD22 within the murine B cell compartment. The results demonstrate that in the bone marrow, murine CD22 is absent on the surface of pro-B cells, pre-B cells and newly emerging lgM+ B cells. CD22 is present at a low density on immature IgMhi B cells and fully expressed on mature recirculatlng B cells. In the periphery, murine CD22 is expressed at mature levels on all B cell subsets Including follicular, marginal zone, B1 and switched B cells. Further studies showed CD22 to be retained on activated murine B cells for extended periods. Finally, In combination with CD23 and heat stable antigen, CD22 can be used to delineate the immature splenic B cells, and distinguish them from follicular and marginal zone cells. Together, the results demonstrate murine CD22 to be a useful pan marker for all mature B cell subsets. PMID:8757957

  7. The kin17 Protein in Murine Melanoma Cells

    PubMed Central

    Ramos, Anelise C.; Gaspar, Vanessa P.; Kelmer, Sabrina M. G.; Sellani, Tarciso A.; Batista, Ana G. U.; De Lima Neto, Quirino A.; Rodrigues, Elaine G.; Fernandez, Maria A.

    2015-01-01

    kin17 has been described as a protein involved in the processes of DNA replication initiation, DNA recombination, and DNA repair. kin17 has been studied as a potential molecular marker of breast cancer. This work reports the detection and localization of this protein in the murine melanoma cell line B16F10-Nex2 and in two derived subclones with different metastatic potential, B16-8HR and B16-10CR. Nuclear and chromatin-associated protein fractions were analyzed, and kin17 was detected in all fractions, with an elevated concentration observed in the chromatin-associated fraction of the clone with low metastatic potential, suggesting that the kin17 expression level could be a marker of melanoma. PMID:26610484

  8. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by proteoliposomes and cultured rat sertoli cells: Evidence for involvement of voltage-activated and voltage-independent calcium channels

    SciTech Connect

    Grasso, P.; Reichert, L.E. Jr. )

    1989-12-01

    We have previously reported incorporation into liposomes of Triton X-100-solubilized FSH receptor-G-protein complexes derived from purified bovine calf testis membranes. In the present study we have used this model system to show that FSH induces flux of 45Ca2+ into such proteoliposomes in a hormone-specific concentration-dependent manner. FSH, inactivated by boiling, had no stimulatory effect on 45Ca2+ flux, nor did isolated alpha- or beta-subunits of FSH. Addition of GTP (or its analogs 5'-guanylylimidodiphosphate and guanosine-5'-O-(3-thiotriphosphate)) or sodium fluoride (in the presence or absence of GTP or its analogs) failed to induce 45Ca2+ flux into proteoliposomes, suggesting that the uptake of 45Ca2+ was receptor, and not G-protein, related. Voltage-independent (ruthenium red and gadolinium chloride) and voltage-activated (methyoxyverapamil and nifedipine) calcium channel-blocking agents reduced FSH-stimulated 45Ca2+ flux into proteoliposomes to control levels. FSH also induced uptake of 45Ca2+ by cultured rat Sertoli cells. Ruthenium red and gadolinium chloride had no effect on basal levels of 45Ca2+ uptake or estradiol secretion by cultured rat Sertoli cells, nor did methoxyverapamil or nifedipine. All four calcium channel blockers, however, were able to reduce FSH-induced 45Ca2+ uptake to basal levels and FSH-stimulated conversion of androstenedione to estradiol by up to 50%, indicating an involvement of Ca2+ in FSH-stimulated steroidogenesis. Our results suggest that the well documented changes in intracellular calcium levels consequent to FSH binding may be due, at least in part, to an influx of calcium through FSH receptor-regulated calcium channels.

  9. Complementation analysis of the murine scid cell line

    SciTech Connect

    Zdzienicka, M.Z. |; Priestly, A.; Jeggo, P.A.

    1995-09-01

    It has been shown that several X-ray-sensitive Chinese hamster cell mutants defective in repair of DNA double-strand breaks (DSBs) are also impaired in the process of V(D)J recombination. The hamster mutants with this phenotype represent three distinct complementation groups, represented by the xrs series, XR-1 and V-3. The murine scid cell line also shows the same phenotype, and therefore we examined whether the scid mutant represents a new complementation group or belongs to one of the existing groups. Scid cells were fused with hamster cell mutants representing the three complementation groups. Hybrids between V-3 and scid cells were only partially complemented for X-ray sensitivity, whereas hybrids derived from fusions with the other mutants were resistant to X rays. These results suggest that V-3 and scid cells are defective in the same gene. To confirm this finding, a single human chromosome 8, which is known to carry the scid gene, was introduced into V-3 cells by microcell-mediated chromosome transfer. Nine hybrid clones derived from V-3 and carrying human chromosome 8 were obtained, and seven were found to be partially complemented for X-ray sensitivity. When human chromosome 8 was introduced into scid cells, seven of eight hybrid clones became resistant to X rays. The results indicate that the defective genes in V-3 and scid are both localized on human chromosome 8. This supports the results from the fusion analysis that V-3 and scid cells are defective in the same gene. 53 refs., 4 figs., 1 tab.

  10. Induction of murine interleukin 1: stimuli and responsive primary cells.

    PubMed Central

    Koide, S; Steinman, R M

    1987-01-01

    An interleukin 1 alpha (IL-1 alpha) cDNA probe and an IL-1 responsive T-cell clone (D10.G4; half-maximal stimulation, 0.1-1 pM) have been used to study the production of IL-1 by primary murine cell populations, particularly macrophages and dendritic cells. Spleen and peritoneal macrophages produced IL-1 mRNA and released biologically active IL-1 when challenged with lipopolysaccharide (LPS). Induction of IL-1 was evident over a dose range of 0.01-10 micrograms of LPS per ml, and maximal mRNA levels were maintained from 4 to 20 hr. Several other stimuli did not induce IL-1 in cultured macrophages, including phorbol 12-myristate 13-acetate, gamma-interferon, Con A, macrophage colony-stimulating factor, IL-3, cachectin, and activated T cells. Activated T cells could markedly reduce the response of peritoneal macrophages to LPS. When other cell types were compared with macrophages, keratinocytes had high levels of IL-1 mRNA, apparently in response to endogenous LPS. However B and T lymphocytes did not yield detectable IL-1 during proliferative responses to LPS and Con A, respectively, while dendritic cells produced little or no IL-1 when challenged with a battery of stimuli. Therefore, IL-1 may not be required for the potent accessory function of dendritic cells in lymphocyte mitogenesis. The results indicate that macrophages and dendritic cells have different secretory capacities. The macrophage is the principal leukocyte that synthesizes IL-1, and select stimuli increase and decrease the levels of macrophage IL-1 mRNA. Images PMID:3495797

  11. Glycomics of Proteoglycan Biosynthesis in Murine Embryonic Stem Cell Differentiation

    PubMed Central

    Nairn, Alison V.; Kinoshita-Toyoda, Akiko; Toyoda, Hidenao; Xie, Jin; Harris, Kyle; Dalton, Stephen; Kulik, Michael; Pierce, J. Michael; Toida, Toshihiko; Moremen, Kelley W.; Linhardt, Robert J.

    2014-01-01

    Glycosaminoglycans (GAGs) play a critical role in binding and activation of growth factors involved in cell signaling critical for developmental biology. The biosynthetic pathways for GAGs have been elucidated over the past decade and now analytical methodology makes it possible to determine GAG composition in as few as 10 million cells. A glycomics approach was used to examine GAG content, composition, and the level of transcripts encoding for GAG biosynthetic enzymes as murine embryonic stem cells (mESCs) differentiate to embryoid bodies (EBs) and to extraembryonic endodermal cells (ExE) to better understand the role of GAGs in stem cell differentiation. Hyaluronan synthesis was enhanced by 13- and 24-fold, most likely due to increased expression of hyaluronan synthase-2. Chondroitin sulfate (CS)/dermatan sulfate (DS) synthesis was enhanced by 4- and 6-fold, and heparan sulfate (HS) synthesis was enhanced by 5- and 8-fold following the transition from mESC to EB and ExE. Transcripts associated with the synthesis of the early precursors were largely unaltered, suggesting other factors account for enhanced GAG synthesis. The composition of both CS/DS and HS also changed upon differentiation. Interestingly, CS type E and highly sulfated HS both increase as mESCs differentiate to EBs and ExE. Differentiation was also accompanied by enhanced 2-sulfation in both CS/DS and HS families. Transcript levels for core proteins generally showed increases or remained constant upon mESC differentiation. Finally, transcripts encoding selected enzymes and isoforms, including GlcNAc-4,6-O-sulfotransferase, C5-epimerases, and 3-O-sulfotransferases involved in late GAG biosynthesis, were also enriched. These biosynthetic enzymes are particularly important in introducing GAG fine structure, essential for intercellular communication, cell adhesion, and outside-in signaling. Knowing the changes in GAG fine structure should improve our understanding the biological properties of differentiated stem cells. PMID:17915907

  12. DNA repair in murine embryonic stem cells and differentiated cells

    SciTech Connect

    Tichy, Elisia D. Stambrook, Peter J.

    2008-06-10

    Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.

  13. B cell lymphoma and myeloma in murine Gaucher's disease.

    PubMed

    Pavlova, E V; Wang, S Z; Archer, J; Dekker, N; Aerts, J M F G; Karlsson, S; Cox, T M

    2013-09-01

    Multiple myeloma and B cell lymphoma are leading causes of death in Gaucher's disease but the nature of the stimulus driving the often noted clonal expansion of immunoglobulin-secreting B cells and cognate lymphoid malignancy is unknown. We investigated the long-term development of B cell malignancies in an authentic model of non-neuronopathic Gaucher's disease in mice: selective deficiency of β-glucocerebrosidase in haematopoietic cells [Gba(tm1Karl/tm1Karl)Tg(Mx1-cre)1Cgn/0, with excision of exons 9-11 of the murine GBA1 gene, is induced by poly[I:C]. Mice with Gaucher's disease showed visceral storage of β-glucosylceramide and greatly elevated plasma β-glucosylsphingosine [median 57.9 (range 19.8-159) nm; n = 39] compared with control mice from the same strain [median 0.56 (range 0.04-1.38) nm; n = 29] (p < 0.0001). Sporadic fatal B cell lymphomas developed in 11 of 21 GD mice (6-24 months) but only two of eight control animals developed tumours by age 24 months. Unexpectedly, most mice with overt lymphoma had absent or few Gaucher cells but local inflammatory macrophages were present. Eleven of 39 of Gaucher mice developed monoclonal gammopathy, but in the control group only one animal of 25 had clonal immunoglobulin abnormalities. Seven of 10 of the B cell lymphomas were found to secrete a monoclonal paraprotein and the lymphomas stained intensely for pan-B cell markers; reactive T lymphocytes were also present in tumour tissue. In the Gaucher mouse strain, it was notable that, as in patients with this disease, CD138(+) plasma cells frequently surrounded splenic macrophages engorged with glycosphingolipid. Our strain of mice, with inducible deficiency of β-glucocerebrosidase in haematopoietic cells and a high frequency of sporadic lethal B cell malignancies, faithfully recapitulates human Gaucher's disease: it serves as a tractable model to investigate the putative role of bioactive sphingolipids in the control of B cell proliferation and the pathogenesis of myelomatosis-the most prevalent human cancer associated with this disorder. PMID:23775597

  14. Characterization of iron uptake from transferrin by murine endothelial cells.

    PubMed

    Hallmann, R; Savigni, D L; Morgan, E H; Baker, E

    2000-01-01

    Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB. PMID:10865941

  15. Vitamin D controls murine and human plasmacytoid dendritic cell function.

    PubMed

    Karthaus, Nina; van Spriel, Annemiek B; Looman, Maaike W G; Chen, Shuo; Spilgies, Lisanne M; Lieben, Liesbet; Carmeliet, Geert; Ansems, Marleen; Adema, Gosse J

    2014-05-01

    Topical application of the vitamin D (VitD) analog calcipotriol is a highly effective standard treatment modality of psoriatic skin lesions. However, the immune modulatory effects of the treatment are incompletely understood. VitD is well known to induce tolerogenic responses in conventional dendritic cells (cDCs). Plasmacytoid DCs (pDCs) comprise a specialized, naturally occurring DC subset known to be important in autoimmune diseases including psoriasis. pDCs from the blood rapidly infiltrate psoriatic skin and are key to the initiation of the immune-mediated pathogenesis of the disease. We now demonstrate that pDCs express various proteins of the VitD receptor (VDR) pathway, including the VitD-metabolizing enzymes Cyp27B1 and Cyp24A1, and that VDR is transcriptionally active in pDCs. Moreover, VitD impairs the capacity of murine and human pDCs to induce T-cell proliferation and secretion of the T-helper 1 cytokine IFNγ. The inhibitory effect of VitD is dependent on the expression of the VDR in the DCs. This study demonstrates that VitD signaling can act as a natural inhibitory mechanism on both cDCs and pDCs, which may instigate the development of VitD-based therapeutic applications for psoriasis and other inflammatory skin diseases. PMID:24352045

  16. Generation of Murine Sympathoadrenergic Progenitor-Like Cells from Embryonic Stem Cells and Postnatal Adrenal Glands

    PubMed Central

    Saxena, Shobhit; Wahl, Joachim; Huber-Lang, Markus S.; Stadel, Dominic; Braubach, Peter; Debatin, Klaus-Michael; Beltinger, Christian

    2013-01-01

    Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS and for the genesis of neuroblastoma, the most common and often lethal extracranial solid tumor in childhood. However, it remains difficult to isolate sufficient numbers of SAPs for investigations. We therefore set out to improve generation of SAPs by using two complementary approaches, differentiation from murine embryonic stem cells (ESCs) and isolation from postnatal murine adrenal glands. We provide evidence that selecting for GD2 expression enriches for ESC-derived SAP-like cells and that proliferating SAP-like cells can be isolated from postnatal adrenal glands of mice. These advances may facilitate investigations about the development and malignant transformation of the sympathetic PNS. PMID:23675538

  17. Intravenous anesthetic propofol suppresses leukotriene production in murine dendritic cells.

    PubMed

    Inada, Takefumi; Ueshima, Hironobu; Shingu, Koh

    2013-01-01

    Leukotrienes, divided into cysteinyl leukotrienes (CysLTs), which are important mediators of asthmatic responses, and leukotriene B4 (LTB4), a chemotactic and chemokinetic agent for leukocytes, are potent lipid mediators generated from arachidonic acid by 5-lipoxygenase (5-LO). Leukotrienes are also considered to have immunoregulatory and pro-inflammatory actions. Propofol is an intravenous anesthetic widely used for anesthesia and sedation that is alleged to possess anti-inflammatory properties. The present study examined the effect of propofol on leukotriene production by dendritic cells (DC). In murine bone marrow-derived DC, propofol significantly suppressed CysLT and LTB4 production after short-term stimulation with zymosan. The protein levels of cytosolic phospholipase A2 and 5-LO, or arachidonic acid release from plasma membranes, were not affected by the presence of propofol. Although zymosan treatment induced or enhanced the phosphorylation of ERK1/2, p-38 MAPK, and JNK, which presumably up-regulates the activity of 5-LO, the presence of propofol had no additional effect on the phosphorylation status of any of these MAPKs. Similarly, zymosan significantly increased the concentration of intracellular calcium, which is the most crucial activator of 5-LO, but no additional concentration changes were observed with the addition of propofol. Lastly, in an in-vitro cell-free ferrous oxidation-xylenol orange assay, propofol significantly inhibited the 5-LO activity of purified human recombinant 5-LO enzyme with an IC50 of ~7.5 µM. Thus, propofol's inhibition of 5-LO is not likely restricted to the circumstances surrounding the production of leukotrienes from DC, but applicable to other types of immune and non-immune cells that produce leukotrienes. The 5-LO-inhibiting activity of propofol may, at least in part, contribute to the well-known anti-inflammatory activity of propofol. PMID:22953970

  18. Localization of Label-Retaining Cells in Murine Vocal Fold Epithelium

    ERIC Educational Resources Information Center

    Leydon, Ciara; Bartlett, Rebecca S.; Roenneburg, Drew A.; Thibeault, Susan L.

    2011-01-01

    Purpose: Epithelial homeostasis is critical for vocal fold health, yet little is known about the cells that support epithelial self-renewal. As a known characteristic of stem cells is that they are slow-cycling in vivo, the purpose of this prospective controlled study was to identify and quantify slow-cycling cells or putative stem cells in murine

  19. Effects of Common Pesticides on Prostaglandin D2 (PGD2) Inhibition in SC5 Mouse Sertoli Cells, Evidence of Binding at the COX-2 Active Site, and Implications for Endocrine Disruption

    PubMed Central

    Kugathas, Subramaniam; Audouze, Karine; Ermler, Sibylle; Orton, Frances; Rosivatz, Erika; Scholze, Martin; Kortenkamp, Andreas

    2015-01-01

    Background: There are concerns that diminished prostaglandin action in fetal life could increase the risk of congenital malformations. Many endocrine-disrupting chemicals have been found to suppress prostaglandin synthesis, but to our knowledge, pesticides have never been tested for these effects. Objectives: We assessed the ability of pesticides that are commonly used in the European Union to suppress prostaglandin D2 (PGD2) synthesis. Methods: Changes in PGD2 secretion in juvenile mouse Sertoli cells (SC5 cells) were measured using an ELISA. Coincubation with arachidonic acid (AA) was conducted to determine the site of action in the PGD2 synthetic pathway. Molecular modeling studies were performed to assess whether pesticides identified as PGD2-active could serve as ligands of the cyclooxygenase-2 (COX-2) binding pocket. Results: The pesticides boscalid, chlorpropham, cypermethrin, cyprodinil, fenhexamid, fludioxonil, imazalil (enilconazole), imidacloprid, iprodione, linuron, methiocarb, o-phenylphenol, pirimiphos-methyl, pyrimethanil, and tebuconazole suppressed PGD2 production. Strikingly, some of these substances—o-phenylphenol, cypermethrin, cyprodinil, linuron, and imazalil (enilconazole)—showed potencies (IC50) in the range between 175 and 1,500 nM, similar to those of analgesics intended to block COX enzymes. Supplementation with AA failed to reverse this effect, suggesting that the sites of action of these pesticides are COX enzymes. The molecular modeling studies revealed that the COX-2 binding pocket can accommodate most of the pesticides shown to suppress PGD2 synthesis. Some of these pesticides are also capable of antagonizing the androgen receptor. Conclusions: Chemicals with structural features more varied than previously thought can suppress PGD2 synthesis. Our findings signal a need for in vivo studies to establish the extent of endocrine-disrupting effects that might arise from simultaneous interference with PGD2 signaling and androgen action. Citation: Kugathas S, Audouze K, Ermler S, Orton F, Rosivatz E, Scholze M, Kortenkamp A. 2016. Effects of common pesticides on prostaglandin D2 (PGD2) inhibition in SC5 mouse Sertoli cells, evidence of binding at the COX-2 active site, and implications for endocrine disruption. Environ Health Perspect 124:452–459; http://dx.doi.org/10.1289/ehp.1409544 PMID:26359731

  20. Phenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells

    PubMed Central

    Reichert, Doreen; Friedrichs, Jens; Ritter, Steffi; Käubler, Theresa; Werner, Carsten; Bornhäuser, Martin; Corbeil, Denis

    2015-01-01

    Xenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34+ hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34+CD133– HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34+CD133+ and CD133+CD34– phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used. PMID:26498381

  1. Establishment of immortalized murine mesothelial cells and a novel mesothelioma cell line.

    PubMed

    Blum, Walter; Pecze, László; Felley-Bosco, Emanuela; Worthmüller-Rodriguez, Janine; Wu, Licun; Vrugt, Bart; de Perrot, Marc; Schwaller, Beat

    2015-08-01

    Mesothelial cells are susceptible to asbestos fiber-induced cytotoxicity and on longer time scales to transformation; the resulting mesothelioma is a highly aggressive neoplasm that is considered as incurable at the present time Zucali et al. (Cancer Treatment Reviews 37:543-558, 2011). Only few murine cell culture models of immortalized mesothelial cells and mesothelioma cell lines exist to date. We generated SV40-immortalized cell lines derived from wild-type (WT) and neurofibromatosis 2 (merlin) heterozygote (Nf2+/-) mice, both on a commonly used genetic background, C57Bl/6J. All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum. Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate. The tumor suppressor gene NF2 is one of the most frequently mutated genes in human mesothelioma, but its detailed function is still unknown. Thus, these genotypically distinct cell lines likely relevant for malignant mesothelioma formation are expected to serve as useful in vitro models, in particular to compare with in vivo studies in mice of the same genotype. Furthermore, we generated a novel murine mesothelioma cell line RN5 originating from an Nf2+/- mouse subjected to repeated crocidolite exposure. RN5 cells are highly tumorigenic. PMID:25877069

  2. TLX1 (HOX11) Immortalization of Embryonic Stem Cell-Derived and Primary Murine Hematopoietic Progenitors

    PubMed Central

    Hawley, Robert G.; Hawley, Teresa S.; Cantor, Alan B.

    2009-01-01

    The ability to generate genetically engineered cell lines is of great experimental value. They provide a renewable source of material that may be suitable for biochemical analyses, chromatin immunoprecipitation assays, structure-function studies, gene function assignment, and transcription factor target gene identification. This unit describes protocols for TLX1 (HOX11)-mediated immortalization of murine hematopoietic progenitors derived from in vitro differentiated murine embryonic stem cells, or from primary mouse fetal liver or bone marrow. A wide variety of hematopoietic cell types have been immortalized using these procedures including erythroid, megakaryocytic, monocytic, myelocytic, and multipotential cell types. These lines are typically cytokine dependent for their survival and growth. PMID:19085976

  3. In vitro differentiation of murine embryonic stem cells into keratinocyte-like cells.

    PubMed

    Haase, Ingo; Knaup, Renate; Wartenberg, Maria; Sauer, Heinrich; Hescheler, Jürgen; Mahrle, Gustav

    2007-12-01

    Embryonic stem (ES) cells are omnipotent; they can differentiate into every cell type of the body. The development of culture conditions that allow their differentiation has made it conceivable to produce large numbers of cells with lineage-specific characteristics in vitro. Here, we describe a method by which murine ES cells can be differentiated into cells with characteristics of epidermal keratinocytes. Keratinocyte-like cells were isolated from embryoid bodies and grown in culture. Potential applications of this method are the in vitro differentiation of cells of interest from ES cells of mice with lethal phenotypes during embryonic development and the production of genetically modified epidermal keratinocytes that could be used as temporary wound dressing or as carriers of genes of interest in gene therapeutic treatments. PMID:17716780

  4. Type I collagen gel protects murine fibrosarcoma L929 cells from TNFα-induced cell death

    SciTech Connect

    Wang, Hong-Ju; He, Wen-Qi; Chen, Ling; Liu, Wei-Wei; Xu, Qian; Xia, Ming-Yu; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-ichi; Onodera, Satoshi; Ikejima, Takashi

    2015-02-20

    Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however, were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death. - Highlights: • Collagen gel culture changed the morphology of L929 cells. • L929 cell cultured on collagen gel were resistant to TNFα-induced cell death. • Collagen gel culture inhibited TNFα-induced autophagy in L929 cells.

  5. Plasmacytoid dendritic cells promote rotavirus-induced human and murine B cell responses

    PubMed Central

    Deal, Emily M.; Lahl, Katharina; Narváez, Carlos F.; Butcher, Eugene C.; Greenberg, Harry B.

    2013-01-01

    B cell–dependent immunity to rotavirus, an important intestinal pathogen, plays a significant role in viral clearance and protects against reinfection. Human in vitro and murine in vivo models of rotavirus infection were used to delineate the role of primary plasmacytoid DCs (pDCs) in initiating B cell responses. Human pDCs were necessary and sufficient for B cell activation induced by rotavirus. Type I IFN recognition by B cells was essential for rotavirus-mediated B cell activation in vitro and murine pDCs and IFN-α/β–mediated B cell activation after in vivo intestinal rotavirus infection. Furthermore, rotavirus-specific serum and mucosal antibody responses were defective in mice lacking functional pDCs at the time of infection. These data demonstrate that optimal B cell activation and virus-specific antibody secretion following mucosal infection were a direct result of pDC-derived type I IFN. Importantly, viral shedding significantly increased in pDC-deficient mice, suggesting that pDC-dependent antibody production influences viral clearance. Thus, mucosal pDCs critically influence the course of rotavirus infection through rotavirus recognition and subsequent IFN production and display powerful adjuvant properties to initiate and enhance humoral immunity. PMID:23635775

  6. Characterization of the expression and gene promoter of CD22 in murine B cells.

    PubMed

    Andersson, K B; Draves, K E; Magaletti, D M; Fujioka, S; Holmes, K L; Law, C L; Clark, E A

    1996-12-01

    CD22 is a B cell-restricted surface molecule which may play an important role in interactions between B cells and other cells and in regulating signals through the B cell receptor (BCR) complex. Here we have examined whether the mouse is a suitable in vivo model for studying CD22 functions. In primary and secondary lymphoid organs of adult mice CD22 is on all mature B cells, including resting IgM+IgD+ B cells, IgG+ HSA(lo) memory B cells, syndecan+ plasma cells and CD5+ B cells, but it is not on immature IgM+IgD- B cells. Biochemical analysis revealed that murine CD22 is associated with the IgM receptor in some, but not all, CD22+ B leukemic and lymphoma cell lines; as with human CD22, murine CD22 is rapidly phosphorylated on tyrosine after ligation of the BCR. In the CD22- murine pro-B cell line, FEMCL, CD22 expression was inducible by treatment with phorbol 12-myristate 13-acetate. A genomic fragment of the cd22b allele containing 1.3 kb 5' of exon 1 was sequenced in order to identify potential DNA regulatory elements in the CD22 promoter region. Consensus sequences for transcription factor binding sites including PU.1, AP-1, AP-2, C/EBP and SP-1 were present, but no classical TATA elements or initiator motifs were evident at relevant positions. The 1.3-kb promoter fragment 5' of exon 1 was sufficient for directing basal promoter activity in B and T cells. There was no significant sequence similarity between the murine and human cd22 gene promoters, although both contain repetitive elements and Sp-1 and AP1 binding sites. Thus, murine CD22 shares a number of features with human CD22 and the mouse provides a suitable model system for elucidating the function of CD22 in vivo. PMID:8977319

  7. Murine trophoblast cells are not killed by tumor necrosis factor-alpha.

    PubMed

    Drake, B L; Head, J R

    1990-03-01

    Purified midgestation murine trophoblast cannot be killed by a variety of cell-mediated effector mechanisms, with the exception of highly lytic effectors such as lymphokine-activated killer cells. We now report that this trophoblast population is also resistant to tumor necrosis factor (TNF)-alpha. PMID:2109800

  8. Involvement of a chromatin modifier in response to mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury: Probably an indirect action via the regulation of NFκB/FasL circuitry

    SciTech Connect

    Chen, Shiwei; Dong, Yushu; Xu, Chun; Jiang, Liming; Chen, Yongjie; Jiang, Cheng; Hou, Wugang; Li, Wei

    2013-11-01

    Highlights: •MTA1 expression is upregulated in SCs upon MEHP treatment. •Knockdown of MTA1 in SCs impairs the MEHP-induced NFκB signaling activation. •Knockdown of MTA1 inhibits recruitment of NFκB onto FasL promoter in MEHP-treated SCs. -- Abstract: The Fas/FasL signaling pathway, controlled by nuclear factor-κB (NFκB) at the transcriptional level, is critical for triggering germ cell apoptosis in response to mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell (SC) injury, but the exact regulation mechanism remain unknown. Here, we discovered that expression level of Metastasis associated protein 1 (MTA1), a component of the Mi-2/nucleosome remodeling and deacetylase complex, was upregulated in SCs during the early recovery after MEHP exposure. This expression change was in line with the dynamic changes in germ cell apoptosis in response to MEHP treatment. Furthermore, a knockdown of MTA1 by RNAi in SCs was found to impair the MEHP-induced early activation of NFκB pathway and abolish the recruitment of NFκB onto FasL promoter, which consequently diminished the MEHP-triggered FasL induction. Considering that Fas/FasL is a well characterized apoptosis initiating signaling during SCs injury, our results point to a potential “switch on” effect of MTA1, which may govern the activation of NFκB/FasL cascade in MEHP-insulted SCs. Overall, the MTA1/NFκB/FasL circuit may serve as an important defensive/repairing mechanism to help to control the germ cell quality after SCs injury.

  9. Differential Effects of Mycobacterium bovis BCG on Macrophages and Dendritic Cells from Murine Spleen.

    PubMed

    Xu, Zhengzhong; Meng, Chuang; Qiang, Bin; Gu, Hongyan; Sun, Lin; Yin, Yuelan; Pan, Zhiming; Chen, Xiang; Jiao, Xinan

    2015-01-01

    Macrophages (MΦ) and dendritic cells (DCs) are both pivotal antigen presenting cells capable of inducing specific cellular responses to inhaled mycobacteria, and thus, they may be important in the initiation of early immune responses to mycobacterial infection. In this study, we evaluated and compared the roles of murine splenic DCs and MΦs in immunity against Mycobacterium bovis Bacillus Calmette-Guérin (M.bovis BCG). The number of internalized rBCG-GFP observed was obviously greater in murine splenic MΦs compared with DCs, and the intracellular reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels in MΦs were all higher than in DCs. DCs have a stronger capacity for presenting Ag85A peptide to specific T hybridoma and when the murine splenic MΦs were infected with BCG and rBCG::Ag85A, low level of antigen presenting activity was detected. These data suggest that murine splenic MΦs participate in mycobacteria uptake, killing and inducing inflammatory response, whereas the murine splenic DCs are primarily involved in specific antigen presentation and T cell activation. PMID:26473844

  10. Differential Effects of Mycobacterium bovis BCG on Macrophages and Dendritic Cells from Murine Spleen

    PubMed Central

    Xu, Zhengzhong; Meng, Chuang; Qiang, Bin; Gu, Hongyan; Sun, Lin; Yin, Yuelan; Pan, Zhiming; Chen, Xiang; Jiao, Xinan

    2015-01-01

    Macrophages (M?) and dendritic cells (DCs) are both pivotal antigen presenting cells capable of inducing specific cellular responses to inhaled mycobacteria, and thus, they may be important in the initiation of early immune responses to mycobacterial infection. In this study, we evaluated and compared the roles of murine splenic DCs and M?s in immunity against Mycobacterium bovis Bacillus Calmette-Gurin (M.bovis BCG). The number of internalized rBCG-GFP observed was obviously greater in murine splenic M?s compared with DCs, and the intracellular reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels in M?s were all higher than in DCs. DCs have a stronger capacity for presenting Ag85A peptide to specific T hybridoma and when the murine splenic M?s were infected with BCG and rBCG::Ag85A, low level of antigen presenting activity was detected. These data suggest that murine splenic M?s participate in mycobacteria uptake, killing and inducing inflammatory response, whereas the murine splenic DCs are primarily involved in specific antigen presentation and T cell activation. PMID:26473844

  11. Expression of Adrenergic and Cholinergic Receptors in Murine Renal Intercalated Cells

    PubMed Central

    JUN, Jin-Gon; MAEDA, Seishi; KUWAHARA-OTANI, Sachi; TANAKA, Koichi; HAYAKAWA, Tetsu; SEKI, Makoto

    2014-01-01

    ABSTRACT Neurons influence renal function and help to regulate fluid homeostasis, blood pressure and ion excretion. Intercalated cells (ICCs) are distributed throughout the renal collecting ducts and help regulate acid/base equilibration. Because ICCs are located among principal cells, it has been difficult to determine the effects that efferent nerve fibers have on this cell population. In this study, we examined the expression of neurotransmitter receptors on the murine renal epithelial M-1 cell line. We found that M-1 cells express a2 and b2 adrenergic receptor mRNA and the b2 receptor protein. Further, b2 receptor-positive cells in the murine cortical collecting ducts also express AQP6, indicating that these cells are ICCs. M-1 cells were found to express m1, m4 and m5 muscarinic receptor mRNAs and the m1 receptor protein. Cells in the collecting ducts also express the m1 receptor protein, and some m1-positive cells express AQP6. Acetylcholinesterase was detected in cortical collecting duct cells. Interestingly, acetylcholinesterase-positive cells neighbored AQP6-positive cells, suggesting that principal cells may regulate the availability of acetylcholine. In conclusion, our data suggest that ICCs in murine renal collecting ducts may be regulated by the adrenergic and cholinergic systems. PMID:25069412

  12. Butyric acid-induced apoptosis of murine thymocytes, splenic T cells, and human Jurkat T cells.

    PubMed Central

    Kurita-Ochiai, T; Fukushima, K; Ochiai, K

    1997-01-01

    The purpose of this study was to examine the effect of butyric acid, an extracellular metabolite from periodontopathic bacteria, on apoptosis induction in murine thymocytes, splenic T cells, and human Jurkat T cells. Butyric acid significantly suppressed T-cell viability in both a concentration- and time-dependent fashion. The results of DNA fragmentation assay indicated that butyric acid rapidly induced apoptosis in thymocytes (with 1.25 mM butyric acid and 6 h after treatment) and in splenic T cells and Jurkat cells (with 2.5 mM butyric acid and 16 h after treatment). Incubation of thymocytes or Jurkat cells with 5 mM butyric acid for 21 h resulted in the typical ladder pattern of DNA fragmentation. Furthermore, Jurkat cells treated with 5 mM butyric acid showed the characteristic pattern of apoptotic cells such as chromatin condensation and hypodiploid nuclei. Experiments with fractionated subpopulations of splenic T cells revealed that DNA fragmentation was predominantly observed in CD4+ T cells. Butyric acid-induced apoptosis of thymocytes was decreased by the protein kinase inhibitors H7 and staurosporine. These inhibitors were less effective with similarly treated splenic T cells and Jurkat cells. These data suggest that butyric acid, one of the volatile fatty acids produced by periodontopathic bacteria and one that easily penetrates the oral mucosa, can modulate the immunoregulatory cell population in periodontal tissue by inducing T-cell death through apoptosis. PMID:8975889

  13. Multispectral Imaging of T and B Cells in Murine Spleen and Tumor

    PubMed Central

    Feng, Zipei; Jensen, Shawn M.; Messenheimer, David J.; Farhad, Mohammed; Neuberger, Michael; Bifulco, Carlo B.

    2016-01-01

    Recent advances in multiplex immunohistochemistry techniques allow for quantitative, spatial identification of multiple immune parameters for enhanced diagnostic and prognostic insight. However, applying such techniques to murine fixed tissues, particularly sensitive epitopes, such as CD4, CD8α, and CD19, has been difficult. We compared different fixation protocols and Ag-retrieval techniques and validated the use of multiplex immunohistochemistry for detection of CD3+CD4+ and CD3+CD8+ T cell subsets in murine spleen and tumor. This allows for enumeration of these T cell subsets within immune environments, as well as the study of their spatial distribution. PMID:26994219

  14. Biological effects of insulin on murine melanoma cells and fish erythrophoroma cells: a comparative study.

    PubMed

    Luchs, Adriana; Sumida, Doris Hissako; Visconti, Maria Aparecida; Castrucci, Ana Maria de Lauro

    2008-04-01

    Insulin is the hormone that plays an essential role in metabolism and mitosis of normal and tumor cells, exerting its pleiotropic effects through binding to specific membrane receptors and promoting the phosphorylation of tyrosine residues of the receptor itself and of other components of the signaling pathway. The aim of this study was to investigate the effects of insulin on melanogenesis and cell growth in three different cell lines: the goldfish GEM-81 erythrophoroma cells (undifferentiated and differentiated with 1.5% dimethylsulfoxide-DMSO), and the murine B16F10 and Cloudman S91 melanoma cells. Undifferentiated GEM-81 and B16F10 cells responded to insulin with a small increase of cell proliferation, whereas S91 cells responded with a decrease of growth. In the two mammalian cell lines, and in DMSO-differentiated GEM-81 cells, the hormone strongly inhibited melanogenesis, by decreasing tyrosinase activity. In undifferentiated GEM-81 cells, insulin had no effect on tyrosinase activity. An increase in the tyrosine phosphorylation status of pp185 (insulin receptor substrate 1 and 2 -- IRS-1/2) phosphorylation degree was observed in S91 mouse melanoma and in differentiated GEM-81 erythrophoroma cells, suggesting that this specific protein was maintained during transformation process and participates in insulin signaling. Our results imply an ancient and diverse history of the insulin signaling system in vertebrate pigment cells. PMID:18329644

  15. Prevalence and Characterization of Murine Leukemia Virus Contamination in Human Cell Lines

    PubMed Central

    Uphoff, Cord C.; Lange, Sandra; Denkmann, Sabine A.; Garritsen, Henk S. P.; Drexler, Hans G.

    2015-01-01

    Contaminations of cell cultures with microbiological organisms are well documented and can be managed in cell culture laboratories applying reliable detection, elimination and prevention strategies. However, the presence of viral contaminations in cell cultures is still a matter of debate and cannot be determined with general detection methods. In the present study we screened 577 human cell lines for the presence of murine leukemia viruses (MLV). Nineteen cell lines were found to be contaminated with MLV, including 22RV1 which is contaminated with the xenotropic murine leukemia virus-related virus variant of MLV. Of these, 17 cell lines were shown to produce active retroviruses determined by product enhanced reverse transcriptase PCR assay for reverse transcriptase activity. The contaminated cell lines derive from various solid tumor types as well as from leukemia and lymphoma types. A contamination of primary human cells from healthy volunteers could not be substantiated. Sequence analyses of 17 MLV PCR products and five complete MLV genomes of different infected cell lines revealed at least three groups of related MLV genotypes. The viruses harvested from the supernatants of infected cell cultures were infectious to uninfected cell cultures. In the course of the study we found that contamination of human genomic DNA preparations with murine DNA can lead to false-positive results. Presumably, xenotransplantations of the human tumor cells into immune-deficient mice to determine the tumorigenicity of the cells are mainly responsible for the MLV contaminations. Furthermore, the use of murine feeder layer cells during the establishment of human cell lines and a cross-contamination with MLV from infected cultures might be sources of infection. A screening of cell cultures for MLV contamination is recommended given a contamination rate of 3.3%. PMID:25927683

  16. Transcriptional and post-transcriptional regulation of globin gene accumulation in murine erythroleukemia cells.

    PubMed Central

    Profous-Juchelka, H R; Reuben, R C; Marks, P A; Rifkind, R A

    1983-01-01

    The mechanism responsible for the accumulation of newly synthesized alpha- and beta-globin mRNA in the cytoplasm of induced murine erythroleukemia cells was examined by nuclear mRNA nascent chain elongation (run-off transcription). Hexamethylenebisacetimide, a potent inducer of murine erythroleukemia cell differention, induced high levels of both alpha- and beta-globin gene transcription within 48 to 72 h in culture. Butyric acid, a modest inducer of murine erythroleukemia cells, induced a somewhat lower level of globin gene transcription. With both inducers, alpha-globin transcriptional rates exceeded those of beta-globin. Hemin, on the other hand, showed no detectable increase over the basal rate observed in uninduced cells, even at a time (48 h) when newly synthesized globin mRNA was accumulating in the cytoplasm. These results suggest that there are at least two mechanisms responsible for regulating alpha- and beta-globin structural gene expression in induced murine erythroleukemia cells and that the mechanisms involved are inducer dependent. Hexamethylenebisacetimide and butyric acid increase the rate at which globin genes are transcribed, but hemin appears to allow constitutive levels of transcripts to accumulate. PMID:6572784

  17. Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages

    PubMed Central

    Alvarez, Mauricio; Casadevall, Arturo

    2007-01-01

    Background The interaction between macrophages and Cryptococcus neoformans (Cn) is critical for containing dissemination of this pathogenic yeast. However, Cn can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. Both events result in live extracellular yeasts capable of reproducing and disseminating in the extracellular milieu. Another method of exiting the intracellular confines of cells is through host cell-to-cell transfer of the pathogen, and this commonly occurs with the human immuno-deficiency virus (HIV) and CD4+ T cells and macrophages. In this report we have used time-lapse imaging to determine if this occurs with Cn. Results Live imaging of Cryptococcus neoformans interactions with murine macrophages revealed cell-to-cell spread of yeast cells from infected donor cells to uninfected cells. Although this phenomenon was relatively rare its occurrence documents a new capacity for this pathogen to infect adjacent cells without exiting the intracellular space. Cell-to-cell spread appeared to be an actin-dependent process. In addition, we noted that cryptococcal phagosomal extrusion was followed by the formation of massive vacuoles suggesting that intracellular residence is accompanied by long lasting damage to host cells. Conclusion C. neoformans can escape the intracellular confines of macrophages in an actin dependent manner by cell-to-cell transfer of the yeast leading to infection of adjacent cells. In addition, complete extrusion of internalized Cn cells can lead to the formation of a massive vacuole which may be a sign of damage to the host macrophage. These observations document new outcomes for the interaction of C. neoformans with host cells that provide precedents for cell biological effects that may contribute to the pathogenesis of cryptococcal infections. PMID:17705844

  18. Neonatal testicular cell transplantation restores murine spermatogenesis damaged in the course of herpes simplex virus-induced orchitis.

    PubMed

    Malolina, Ekaterina A; Kulibin, Andrey Yu; Kushch, Alla A

    2014-11-17

    Genital tract infection and inflammation may affect male fertility, causing germ and Sertoli cell loss. We determined if testicular cell transplantation is effective at repairing testicular injury induced by herpes simplex virus (HSV) orchitis. ROSA26 mice were used as donors and the recipients were C57BL/6 mice after HSV testicular inoculation; some of the recipients were treated with the antiviral drug acyclovir (ACV). ACV reduced the amount of HSV antigen in testes on Day 3 after transplantation and enhanced the efficacy of transplantation at Day 30. In recipient testes, donor Sertoli cells formed new seminiferous tubules; significantly more new tubules were observed in the testes of ACV-treated mice compared with mice not treated with ACV (17.8% vs 3.6%). Over half (50.4%) of new tubules in ACV-treated testes contained germ cells and round spermatids were detected in 14.2% of new tubules compared with 15.9% and 5.3% in testes not treated with ACV, respectively. At Day 150 the seminiferous epithelium was completely recovered in some donor tubules and elongated spermatids were observed inside it. Thus, our findings reveal the effectiveness of the combination of antiviral therapy with neonatal testis-cell transplantation for the restoration of spermatogenesis damaged by viral infection. PMID:25399480

  19. Cryoprotective effects of low-density lipoproteins, trehalose and soybean lecithin on murine spermatogonial stem cells.

    PubMed

    Wang, Peng; Li, Ying; Hu, Xiao-Chen; Cai, Xiao-Li; Hou, Li-Peng; Wang, Yan-Feng; Hu, Jian-Hong; Li, Qing-Wang; Suo, Li-Juan; Fan, Zhi-Guo; Zhang, Bo

    2014-05-01

    Spermatogonial stem cells (SSCs) have the ability to self-renew and offer a pathway for genetic engineering of the male germ line. Cryopreservation of SSCs has potential value for the treatment of male infertility, spermatogonial transplantation, and so on. In order to investigate the cryopreservation effects of different cryoprotectants on murine SSCs, 0.2 M of low-density lipoproteins (LDL), trehalose and soybean lecithin were added to the cryoprotective medium, respectively, and the murine SSCs were frozen at -80°C or -196°C. The results indicated that the optimal recovery rates of murine SSCs in the cryoprotective medium supplemented with LDL, trehalose and soybean lecithin were 92.53, 76.35 and 75.48% at -80°C, respectively. Compared with freezing at -196°C, the optimum temperature for improvement of recovery rates of frozen murine SSCs, cryopreservation in three different cryoprotectants at -80°C, were 17.11, 6.68 and 10.44% respectively. The recovery rates of murine SSCs in the cryoprotective medium supplemented with 0.2 M LDL were significantly higher than that of other cryoprotectants (P < 0.05). Moreover, the recovery rates were demonstrated to be greater at -80°C compared with at -196°C (P < 0.05). In conclusion, 0.2 M of LDL could significantly protect murine SSCs at -80°C. In the freezing-thawing process, LDL is responsible for the cryopreservation of murine SSCs because it can form a protective film at the surface of membranes. However, more research is needed to evaluate and understand the precise role of LDL during the freezing-thawing of SSCs. PMID:22974447

  20. Radiation survival of murine and human melanoma cells utilizing two assay systems: monolayer and soft agar.

    PubMed Central

    Yohem, K. H.; Slymen, D. J.; Bregman, M. D.; Meyskens, F. L.

    1988-01-01

    The radiation response of murine and human melanoma cells assayed in bilayer soft agar and monolayer was examined. Cells from the murine melanoma Cloudman S91 CCL 53.1 cell line and three human melanoma cell strains (C8146C, C8161, and R83-4) developed in our laboratory were irradiated by single dose X-rays and plated either in agar or on plastic. D0 values were the same within 95% confidence intervals for cells from the human melanoma cell strains C8146C, C8161, and R83-4 but were dissimilar for the murine cell line CCL 53.1 Dq values were different for all cells studied. The shape of the survival curve for all four melanomas was not identical for cells assayed in soft agar versus cells grown on plastic. This would indicate that apparent radiosensitivity was influenced by the method of assay although there were no apparent consistent differences between the curves generated by monolayer or bilayer soft agar assays. PMID:3348949

  1. Isolation of Murine Bone Marrow Derived Mesenchymal Stem Cells using Twist2 Cre Transgenic Mice

    PubMed Central

    Liu, Yaling; Wang, Liping; Fatahi, Reza; Kronenberg, Mark; Kalajzic, Ivo; Rowe, David; Li, Yingcui; Maye, Peter

    2010-01-01

    While human bone marrow derived mesenchymal stem cells (BMSCs) are of great interest for their potential therapeutic value, its murine equivalent remains an important basic research model that can provide critical insights into the biology of this progenitor cell population. Here we present a novel transgenic strategy that allowed for the selective identification and isolation of murine BMSCs at the early stages of stromal cell culture. This strategy involved crossing Twist2 –Cre mice with Cre reporter mice such as Z/EG or Ai9, which express EGFP or Tomato fluorescent protein, respectively, upon Cre mediated excision of a stop sequence. Using this approach, we identified an adherent fluorescent protein+ cell population (T2C+) that is present during the earliest stages of colony formation and by day 5 of culture represents ~20% of the total cell population. Cell surface profiling by flow cytometry showed that T2C+ cells are highly positive for SCA1 and CD29 and negative for CD45, CD117, TIE2, and TER119. Isolation of T2C+ cells by FACS selected for a cell population with skeletal potential that can be directed to differentiate into osteoblasts, adipocytes, or chondrocytes. We also demonstrated in a calvarial bone defect model that T2C+ cells retain a strong efficacy for osteogenic repair and can support a hematopoietic environment. Collectively, these studies provide evidence that the Twist2-Cre x Cre reporter breeding strategy can be used to positively identify and isolate multipotent murine BMSCs. PMID:20673822

  2. COMPARATIVE TOXICITY OF DIFFERENT EMISSION PARTICLES IN MURINE PULMONARY EPITHELIAL CELLS AND MACROPHAGES

    EPA Science Inventory

    Comparative Toxicity of Different Emission Particles in Murine Pulmonary Epithelial Cells and Macrophages. T Stevens1, M Daniels2, P Singh2, M I Gilmour2. 1 UNC, Chapel Hill 27599 2Experimental Toxicology Division, NHEERL, RTP, NC 27711

    Epidemiological studies have shown ...

  3. GPBAR1/TGR5 mediates bile acid-induced cytokine expression in murine Kupffer cells.

    PubMed

    Lou, Guiyu; Ma, Xiaoxiao; Fu, Xianghui; Meng, Zhipeng; Zhang, Wenyu; Wang, Yan-Dong; Van Ness, Carl; Yu, Donna; Xu, Rongzhen; Huang, Wendong

    2014-01-01

    GPBAR1/TGR5 is a novel plasma membrane-bound G protein-coupled bile acid (BA) receptor. BAs are known to induce the expression of inflammatory cytokines in the liver with unknown mechanism. Here we show that without other external stimuli, TGR5 activation alone induced the expression of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) in murine macrophage cell line RAW264.7 or murine Kupffer cells. The TGR5-mediated increase of pro-inflammatory cytokine expression was suppressed by JNK inhibition. Moreover, the induced pro-inflammatory cytokine expression in mouse liver by 1% cholic acid (CA) diet was blunted in JNK-/- mice. TGR5 activation by its ligands enhanced the phosphorylation levels, DNA-binding and trans-activities of c-Jun and ATF2 transcription factors. Finally, the induced pro-inflammatory cytokine expression in Kupffer cells by TGR5 activation correlated with the suppression of Cholesterol 7α-hydroxylase (Cyp7a1) expression in murine hepatocytes. These results suggest that TGR5 mediates the BA-induced pro-inflammatory cytokine production in murine Kupffer cells through JNK-dependent pathway. This novel role of TGR5 may correlate to the suppression of Cyp7a1 expression in hepatocytes and contribute to the delicate BA feedback regulation. PMID:24755711

  4. METHODOLOGICAL CONSIDERATIONS FOR THE MURINE HEPATOMA CELL BIOASSAY-DIRECTED ISOLATION OF CANCER CHEMOPREVENTIVE AGENTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The development of a murine hepatoma (Hepa 1c1c7) cell line over a decade ago for the isolation of phase II enzyme inducing agents by a group from Johns Hopkins University led to the identification of sulforaphane as a major cancer chemopreventive agent in broccoli. We have found that choices among...

  5. GPBAR1/TGR5 Mediates Bile Acid-Induced Cytokine Expression in Murine Kupffer Cells

    PubMed Central

    Lou, Guiyu; Ma, Xiaoxiao; Fu, Xianghui; Meng, Zhipeng; Zhang, Wenyu; Wang, Yan-Dong; Van Ness, Carl; Yu, Donna; Xu, Rongzhen; Huang, Wendong

    2014-01-01

    GPBAR1/TGR5 is a novel plasma membrane-bound G protein–coupled bile acid (BA) receptor. BAs are known to induce the expression of inflammatory cytokines in the liver with unknown mechanism. Here we show that without other external stimuli, TGR5 activation alone induced the expression of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) in murine macrophage cell line RAW264.7 or murine Kupffer cells. The TGR5-mediated increase of pro-inflammatory cytokine expression was suppressed by JNK inhibition. Moreover, the induced pro-inflammatory cytokine expression in mouse liver by 1% cholic acid (CA) diet was blunted in JNK−/− mice. TGR5 activation by its ligands enhanced the phosphorylation levels, DNA-binding and trans-activities of c-Jun and ATF2 transcription factors. Finally, the induced pro-inflammatory cytokine expression in Kupffer cells by TGR5 activation correlated with the suppression of Cholesterol 7α-hydroxylase (Cyp7a1) expression in murine hepatocytes. These results suggest that TGR5 mediates the BA-induced pro-inflammatory cytokine production in murine Kupffer cells through JNK-dependent pathway. This novel role of TGR5 may correlate to the suppression of Cyp7a1 expression in hepatocytes and contribute to the delicate BA feedback regulation. PMID:24755711

  6. Genetic and clonal dissection of murine small cell lung carcinoma progression by genome sequencing.

    PubMed

    McFadden, David G; Papagiannakopoulos, Thales; Taylor-Weiner, Amaro; Stewart, Chip; Carter, Scott L; Cibulskis, Kristian; Bhutkar, Arjun; McKenna, Aaron; Dooley, Alison; Vernon, Amanda; Sougnez, Carrie; Malstrom, Scott; Heimann, Megan; Park, Jennifer; Chen, Frances; Farago, Anna F; Dayton, Talya; Shefler, Erica; Gabriel, Stacey; Getz, Gad; Jacks, Tyler

    2014-03-13

    Small cell lung carcinoma (SCLC) is a highly lethal, smoking-associated cancer with few known targetable genetic alterations. Using genome sequencing, we characterized the somatic evolution of a genetically engineered mouse model (GEMM) of SCLC initiated by loss of Trp53 and Rb1. We identified alterations in DNA copy number and complex genomic rearrangements and demonstrated a low somatic point mutation frequency in the absence of tobacco mutagens. Alterations targeting the tumor suppressor Pten occurred in the majority of murine SCLC studied, and engineered Pten deletion accelerated murine SCLC and abrogated loss of Chr19 in Trp53; Rb1; Pten compound mutant tumors. Finally, we found evidence for polyclonal and sequential metastatic spread of murine SCLC by comparative sequencing of families of related primary tumors and metastases. We propose a temporal model of SCLC tumorigenesis with implications for human SCLC therapeutics and the nature of cancer-genome evolution in GEMMs. PMID:24630729

  7. Novel Murine Dendritic Cell Lines: A Powerful Auxiliary Tool for Dendritic Cell Research

    PubMed Central

    Fuertes Marraco, Silvia A.; Grosjean, Frédéric; Duval, Anaïs; Rosa, Muriel; Lavanchy, Christine; Ashok, Devika; Haller, Sergio; Otten, Luc A.; Steiner, Quynh-Giao; Descombes, Patrick; Luber, Christian A.; Meissner, Felix; Mann, Matthias; Szeles, Lajos; Reith, Walter; Acha-Orbea, Hans

    2012-01-01

    Research in vitro facilitates discovery, screening, and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC) tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines, and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research. PMID:23162549

  8. Novel murine dendritic cell lines: a powerful auxiliary tool for dendritic cell research.

    PubMed

    Fuertes Marraco, Silvia A; Grosjean, Frédéric; Duval, Anaïs; Rosa, Muriel; Lavanchy, Christine; Ashok, Devika; Haller, Sergio; Otten, Luc A; Steiner, Quynh-Giao; Descombes, Patrick; Luber, Christian A; Meissner, Felix; Mann, Matthias; Szeles, Lajos; Reith, Walter; Acha-Orbea, Hans

    2012-01-01

    Research in vitro facilitates discovery, screening, and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC) tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines, and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research. PMID:23162549

  9. Low pH reprograms somatic murine cells into pluripotent stem cells

    PubMed Central

    Williams, Jonathan S; Xiao, Ying; Brownell, Isaac

    2014-01-01

    Induced pluripotent stem cells (iPSCs) are somatic cells that are reprogrammed into a state resembling embryonic stem cells (ESCs). iPSCs represent a promising technology with applications in cancer research, yet current methods used to generate iPSCs limit their translation to clinical use. In a recent Nature article, Obokata et al. detail a novel technique to generate pluripotent murine cells called stimulus-triggered acquisition of pluripotency (STAP). STAP eliminates the need for exogenous expression of reprogramming factors used in previous iPSC technologies, instead transforming somatic cells to pluripotency using physical and chemical stimuli. The authors found that STAP cells are generated at a 10-fold higher efficiency than prior iPSC technologies. STAP cells display several features of pluripotency, namely the expression of pluripotency-related genes (Oct4, Nanog, Sox2, Ecat1, Esg1, and Dax1), the ability to form teratomas in vivo, and the ability to produce viable, fertile mice in blastocyst complementation assays. Here, we review these findings on STAP and contrast it to previous iPSC technologies, while noting the potential of this method to generate autologous anti-tumor immune cells for cancer therapy. PMID:24618709

  10. Cell differentiation mediated by co-culture of human umbilical cord blood stem cells with murine hepatic cells.

    PubMed

    Stecklum, Maria; Wulf-Goldenberg, Annika; Purfürst, Bettina; Siegert, Antje; Keil, Marlen; Eckert, Klaus; Fichtner, Iduna

    2015-02-01

    In the present study, purified human cord blood stem cells were co-cultivated with murine hepatic alpha mouse liver 12 (AML12) cells to compare the effect on endodermal stem cell differentiation by either direct cell-cell interaction or by soluble factors in conditioned hepatic cell medium. With that approach, we want to mimic in vitro the situation of preclinical transplantation experiments using human cells in mice. Cord blood stem cells, cultivated with hepatic conditioned medium, showed a low endodermal differentiation but an increased connexin 32 (Cx32) and Cx43, and cytokeratin 8 (CK8) and CK19 expression was monitored by reverse transcription polymerase chain reaction (RT-PCR). Microarray profiling indicated that in cultivated cord blood cells, 604 genes were upregulated 2-fold, with the highest expression for epithelial CK19 and epithelial cadherin (E-cadherin). On ultrastructural level, there were no major changes in the cellular morphology, except a higher presence of phago(ly)some-like structures observed. Direct co-culture of AML12 cells with cord blood cells led to less incisive differentiation with increased sex-determining region Y-box 17 (SOX17), Cx32 and Cx43, as well as epithelial CK8 and CK19 expressions. On ultrastructural level, tight cell contacts along the plasma membranes were revealed. FACS analysis in co-cultivated cells quantified dye exchange on low level, as also proved by time relapse video-imaging of labelled cells. Modulators of gap junction formation influenced dye transfer between the co-cultured cells, whereby retinoic acid increased and 3-heptanol reduced the dye transfer. The study indicated that the cell-co-cultured model of human umbilical cord blood cells and murine AML12 cells may be a suitable approach to study some aspects of endodermal/hepatic cell differentiation induction. PMID:25270685

  11. Human malignant mesothelioma is recapitulated in immunocompetent BALB/c mice injected with murine AB cells.

    PubMed

    Mezzapelle, Rosanna; Rrapaj, Eltjona; Gatti, Elena; Ceriotti, Chiara; Marchis, Francesco De; Preti, Alessandro; Spinelli, Antonello E; Perani, Laura; Venturini, Massimo; Valtorta, Silvia; Moresco, Rosa Maria; Pecciarini, Lorenza; Doglioni, Claudio; Frenquelli, Michela; Crippa, Luca; Recordati, Camilla; Scanziani, Eugenio; de Vries, Hilda; Berns, Anton; Frapolli, Roberta; Boldorini, Renzo; D'Incalci, Maurizio; Bianchi, Marco E; Crippa, Massimo P

    2016-01-01

    Malignant Mesothelioma is a highly aggressive cancer, which is difficult to diagnose and treat. Here we describe the molecular, cellular and morphological characterization of a syngeneic system consisting of murine AB1, AB12 and AB22 mesothelioma cells injected in immunocompetent BALB/c mice, which allows the study of the interplay of tumor cells with the immune system. Murine mesothelioma cells, like human ones, respond to exogenous High Mobility Group Box 1 protein, a Damage-Associated Molecular Pattern that acts as a chemoattractant for leukocytes and as a proinflammatory mediator. The tumors derived from AB cells are morphologically and histologically similar to human MM tumors, and respond to treatments used for MM patients. Our system largely recapitulates human mesothelioma, and we advocate its use for the study of MM development and treatment. PMID:26961782

  12. Human malignant mesothelioma is recapitulated in immunocompetent BALB/c mice injected with murine AB cells

    PubMed Central

    Mezzapelle, Rosanna; Rrapaj, Eltjona; Gatti, Elena; Ceriotti, Chiara; Marchis, Francesco De; Preti, Alessandro; Spinelli, Antonello E.; Perani, Laura; Venturini, Massimo; Valtorta, Silvia; Moresco, Rosa Maria; Pecciarini, Lorenza; Doglioni, Claudio; Frenquelli, Michela; Crippa, Luca; Recordati, Camilla; Scanziani, Eugenio; de Vries, Hilda; Berns, Anton; Frapolli, Roberta; Boldorini, Renzo; D’Incalci, Maurizio; Bianchi, Marco E.; Crippa, Massimo P.

    2016-01-01

    Malignant Mesothelioma is a highly aggressive cancer, which is difficult to diagnose and treat. Here we describe the molecular, cellular and morphological characterization of a syngeneic system consisting of murine AB1, AB12 and AB22 mesothelioma cells injected in immunocompetent BALB/c mice, which allows the study of the interplay of tumor cells with the immune system. Murine mesothelioma cells, like human ones, respond to exogenous High Mobility Group Box 1 protein, a Damage-Associated Molecular Pattern that acts as a chemoattractant for leukocytes and as a proinflammatory mediator. The tumors derived from AB cells are morphologically and histologically similar to human MM tumors, and respond to treatments used for MM patients. Our system largely recapitulates human mesothelioma, and we advocate its use for the study of MM development and treatment. PMID:26961782

  13. Cytotoxic activity of methanol extracts from Basidiomycete mushrooms on murine cancer cell lines.

    PubMed

    Tomasi, S; Lohézic-Le Dévéhat, F; Sauleau, P; Bézivin, C; Boustie, J

    2004-04-01

    Crude methanol extracts of 58 mushroom species were screened for their cytotoxic activities against two murine cancer cell lines, L1210 and 3LL, using the tetrazolium assay. A majority of extracts (74%) exhibited IC50 > 100 microg/ml against both cell lines. A most marked activity against one of the cell lines was noted for nine species (14% of the tested species). While Amanitales and Russulales tested were not found active, Polyporales and Boletales gave better results. Four species exhibited a significant cytotoxic activity (IC50 < or = 20 microg/ml) against at least one of the two murine cancer cell lines (Ganoderma lucidum, Meripilus giganteus, Suillus granulatus, S. luteus). The last one had never been investigated for its cytotoxic compounds before. PMID:15125575

  14. Influence of Murine Mesenchymal Stem Cells on Proliferation, Phenotype, Vitality, and Cytotoxicity of Murine Cytokine-Induced Killer Cells in Coculture

    PubMed Central

    Stolzing, Alexandra

    2014-01-01

    Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes. PMID:24516591

  15. Barriers in contribution of human mesenchymal stem cells to murine muscle regeneration

    PubMed Central

    de la Garza-Rodea, Anabel S; Boersma, Hester; Dambrot, Cheryl; de Vries, Antoine AF; van Bekkum, Dirk W; Knan-Shanzer, Shoshan

    2015-01-01

    AIM: To study regeneration of damaged human and murine muscle implants and the contribution of added xenogeneic mesenchymal stem cells (MSCs). METHODS: Minced human or mouse skeletal muscle tissues were implanted together with human or mouse MSCs subcutaneously on the back of non-obese diabetic/severe combined immunodeficient mice. The muscle tissues (both human and murine) were minced with scalpels into small pieces (< 1 mm3) and aliquoted in portions of 200 mm3. These portions were either cryopreserved in 10% dimethylsulfoxide or freshly implanted. Syngeneic or xenogeneic MSCs were added to the minced muscles directly before implantation. Implants were collected at 7, 14, 30 or 45 d after transplantation and processed for (immuno)histological analysis. The progression of muscle regeneration was assessed using a standard histological staining (hematoxylin-phloxin-saffron). Antibodies recognizing Pax7 and von Willebrand factor were used to detect the presence of satellite cells and blood vessels, respectively. To enable detection of the bone marrow-derived MSCs or their derivatives we used MSCs previously transduced with lentiviral vectors expressing a cytoplasmic LacZ gene. X-gal staining of the fixed tissues was used to detect ?-galactosidase-positive cells and myofibers. RESULTS: Myoregeneration in implants of fresh murine muscle was evident as early as day 7, and progressed with time to occupy 50% to 70% of the implants. Regeneration of fresh human muscle was slower. These observations of fresh muscle implants were in contrast to the regeneration of cryopreserved murine muscle that proceeded similarly to that of fresh tissue except for day 45 (P < 0.05). Cryopreserved human muscle showed minimal regeneration, suggesting that the freezing procedure was detrimental to human satellite cells. In fresh and cryopreserved mouse muscle supplemented with LacZ-tagged mouse MSCs, ?-galactosidase-positive myofibers were identified early after grafting at the well-vascularized periphery of the implants. The contribution of human MSCs to murine myofiber formation was, however, restricted to the cryopreserved mouse muscle implants. This suggests that fresh murine muscle tissue provides a suboptimal environment for maintenance of human MSCs. A detailed analysis of the histological sections of the various muscle implants revealed the presence of cellular structures with a deviating morphology. Additional stainings with alizarin red and alcian blue showed myofiber calcification in 50 of 66 human muscle implants, and encapsulated cartilage in 10 of 81 of murine muscle implants, respectively. CONCLUSION: In mouse models the engagement of human MSCs in myoregeneration might be underestimated. Furthermore, our model permits the dissection of species-specific factors in the microenvironment. PMID:25992329

  16. RANKL-mediated osteoclast formation from murine RAW 264.7 cells.

    PubMed

    Collin-Osdoby, Patricia; Osdoby, Philip

    2012-01-01

    Extensive research efforts over the years have provided us with great insights into how bone-resorbing osteoclasts (OCs) develop and function and, based on such work, valuable antiresorptive therapies have been developed to help combat the excessive bone loss that occurs in numerous skeletal disorders. The RAW 264.7 murine cell line has proven to be an important tool for in vitro studies of OC formation and function, having particular advantages over the use of OCs generated from primary bone marrow cell populations or directly isolated from murine bones. These include their ready access and availability, simple culture for this pure macrophage/pre-OC population, sensitive and rapid development into highly bone-resorptive OCs expressing hallmark OC characteristics following their RANKL stimulation, abundance of RAW cell-derived OCs that can be generated to provide large amounts of study material, relative ease of transfection for genetic and regulatory manipulation, and close correlation in characteristics, gene expression, signaling, and developmental or functional processes between RAW cell-derived OCs and OCs either directly isolated from murine bones or formed in vitro from primary bone marrow precursor cells. Here, we describe methods for the culture and RANKL-mediated differentiation of RAW cells into bone-resorptive OCs as well as procedures for their enrichment, characterization, and general use in diverse analytical assays. PMID:22130930

  17. Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones

    NASA Technical Reports Server (NTRS)

    Deepe, George S., Jr.; Smith, James G.; Denman, David; Bullock, Ward E.; Sonnenfeld, Gerald

    1986-01-01

    Several Histoplasma capsulatum-reactive murine cloned T-cell lines (TCLs) were isolated from spleens of C57BL/6 mice immunized with viable H. capsulatum yeast cells, using the methodology of Kimoto and Fathman (1980). These T-cells were characterized phenotypically as Thy-1.2(+) Lyt-1(+) L3T4(+) Lyt-2(-), that is, as the helper/inducer phenotype. The cloned T cells proliferate in response to histoplasmin and, in some cases, to heterologous fungal anigens. Upon injection of mice with the antigen, the T-cells mediate local delayed-type hypersensitivity responses and, after stimulation, release regulatory lymphokines.

  18. Lead and catechol hematotoxicity in vitro using human and murine hematopoietic progenitor cells.

    PubMed

    Van Den Heuvel, R L; Leppens, H; Schoeters, G E

    1999-04-01

    In vitro cloning assays for hematopoietic myeloid and erythroid precursor cells have been used as screening systems to investigate the hematotoxic potential of environmental chemicals in humans and mice. Granulocyte-monocyte progenitors (CFU-GM) from human umbilical cord blood and from mouse bone marrow (Balb/c and B6C3F1) were cultured in the presence of lead and the benzene metabolite catechol. Erythroid precursors (BFU-E) from human umbilical cord blood were cultured in the presence of lead. The in vitro exposure of the human and murine cells resulted in a dose-dependent depression of the colony numbers. The concentration effect relationship was studied. Results showed that: (1) Based on calculated IC50 values, human progenitors are more sensitive to lead and catechol than are murine progenitors. The dose that caused a 50% decrease in colony formation after catechol exposure was 6 times higher for murine cells (IC50 = 24 micromol/L) than for human cord blood cells (IC50 = 4 micromol/L). Lead was 10-15 times more toxic to human hematopoietic cells (IC50 = 61 micromol/L) than to murine bone marrow cells from both mice strains tested (Balb/c, IC50 = 1060 micromol/L; B6C3F1, IC50 = 536 micromol/L). (2) A lineage specificity was observed after exposure to lead. Human erythroid progenitors (hBFU-E) (IC50 = 3.31 micromol/L) were found to be 20 times more sensitive to the inhibitory effect of lead than were myeloid precursors (hCFU-GM) (IC50 = 63.58 micromol/L). (3) Individual differences in the susceptibility to the harmful effect of lead were seen among cord blood samples. (4) Toxicity of lead to progenitor cells occurred at environmentally relevant concentrations. PMID:10408357

  19. Stimulation of cell proliferation in the subventricular zone by synthetic murine pheromones

    PubMed Central

    Koyama, Sachiko; Soini, Helena A.; Foley, John; Novotny, Milos V.; Lai, Cary

    2013-01-01

    Adult neurogenesis in female mice is known to be enhanced by exposure to soiled bedding from males, although the identity of the relevant chemosignals has remained unknown. Here we show that the previously recognized male murine pheromones, the farnesenes and 2-sec-butyl-4,5-dihydrothiazole (SBT), strongly increase cell proliferation in the subventricular zone (SVZ) of adult female mice, but not younger female mice. In addition, we found that a unique female murine pheromone, 2,5-dimethylpyrazine, facilitates similar changes in males. SBT stimulated cell proliferation in the SVZ of only adult females and not in young adult or pre- and post-puberty females. Our study suggests that pheromonal communication between males and females is enhancing reproductive success by controlling the estrous cycle and by promoting cell proliferation in a reciprocal manner. PMID:23964214

  20. Inhibition of Murine Leukemia Virus Production in Chronically Infected AKR Cells: A Novel Effect of Interferon

    PubMed Central

    Friedman, Robert M.; Ramseur, Janet M.

    1974-01-01

    Treatment of AKR cells that had spontaneously become procedures of a murine leukemia virus with a partially purified mouse interferon (> 5 × 107 international mouse reference units per mg of protein) inhibited endogenous virus production. This inhibitory effect decreased over a 72-hr period in a manner similar to interferon-induced antiviral activity directed against vesicular stomatitis virus in AKR cells. Despite the inhibitory effect of interferon on infectious murine leukemia virus and viral reverse transcriptase (RNA-dependent DNA polymerase) titers in the culture fluids, intracellular levels of viral groups-specific antigens were significantly increased. These results suggest that interferon treatment in AKR cells inhibited the assembly or release of the virus. PMID:4139716

  1. Behaviour of four different B16 murine melanoma cell sublines: C57BL/6J skin

    PubMed Central

    Danciu, Corina; Oprean, Camelia; Coricovac, Dorina E; Andreea, Cioca; Cimpean, Anca; Radeke, Heinfried; Soica, Codruta; Dehelean, Cristina

    2015-01-01

    Transplantable murine melanomas are well-established models for the study of experimental cancer therapies. The aim of this study was to explore the behaviour of four different B16 murine melanoma cell sublines after inoculation into C57BL/6J mice; and, more specifically to analyse skin changes, with respect to two specific parameters: clinical (tumour volume, melanin amount, erythema) and histological (H & E, S100, VEGF expression). Both non-invasive and invasive analysis showed that B164A5 is the most aggressive melanoma cell line for C57BL/6J's skin, followed by B16F10 and then by diminished aggressive growth pattern by the B16GMCSF and B16FLT3 cell lines. PMID:25664478

  2. Diabetes, insulin-mediated glucose metabolism and Sertoli/blood-testis barrier function

    PubMed Central

    Alves, Marco G.; Martins, Ana D.; Cavaco, José E.; Socorro, Sílvia; Oliveira, Pedro F.

    2013-01-01

    Blood testis barrier (BTB) is one of the tightest blood-barriers controlling the entry of substances into the intratubular fluid. Diabetes Mellitus (DM) is an epidemic metabolic disease concurrent with falling fertility rates, which provokes severe detrimental BTB alterations. It induces testicular alterations, disrupting the metabolic cooperation between the cellular constituents of BTB, with dramatic consequences on sperm quality and fertility. As Sertoli cells are involved in the regulation of spermatogenesis, providing nutritional support for germ cells, any metabolic alteration in these cells derived from DM may be responsible for spermatogenesis disruption, playing a crucial role in fertility/subfertility associated with this pathology. These cells have a glucose sensing machinery that reacts to hormonal fluctuations and several mechanisms to counteract hyper/hypoglycemic events. The role of DM on Sertoli/BTB glucose metabolism dynamics and the metabolic molecular mechanisms through which DM and insulin deregulation alter its functioning, affecting male reproductive potential will be discussed. PMID:24665384

  3. Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells

    PubMed Central

    Phang, Wai-Mei; Tan, Aik-Aun; Gopinath, Subash C.B.; Hashim, Onn H.; Kiew, Lik Voon; Chen, Yeng

    2016-01-01

    Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer. PMID:27226773

  4. Differential gene expression in the murine gastric fundus lacking interstitial cells of Cajal

    PubMed Central

    Daigo, Yataro; Takayama, Ichiro; Ponder, Bruce AJ; Caldas, Carlos; Ward, Sean M; Sanders, Kenton M; Fujino, Masayuki A

    2003-01-01

    Background The muscle layers of murine gastric fundus have no interstitial cells of Cajal at the level of the myenteric plexus and only possess intramuscular interstitial cells and this tissue does not generate electric slow waves. The absence of intramuscular interstitial cells in W/WV mutants provides a unique opportunity to study the molecular changes that are associated with the loss of these intercalating cells. Method The gene expression profile of the gastric fundus of wild type and W/WV mice was assayed by murine microarray analysis displaying a total of 8734 elements. Queried genes from the microarray analysis were confirmed by semi-quantitative reverse transcription-polymerase chain reaction. Results Twenty-one genes were differentially expressed in wild type and W/WV mice. Eleven transcripts had 2.0–2.5 fold higher mRNA expression in W/WV gastric fundus when compared to wild type tissues. Ten transcripts had 2.1–3.9 fold lower expression in W/WV mutants in comparison with wild type animals. None of these genes have ever been implicated in any bowel motility function. Conclusions These data provides evidence that several important genes have significantly changed in the murine fundus of W/WV mutants that lack intramuscular interstitial cells of Cajal and have reduced enteric motor neurotransmission. PMID:12795813

  5. Murine and Human Myogenic Cells Identified by Elevated Aldehyde Dehydrogenase Activity: Implications for Muscle Regeneration and Repair

    PubMed Central

    Vella, Joseph B.; Thompson, Seth D.; Bucsek, Mark J.; Song, Minjung; Huard, Johnny

    2011-01-01

    Background Despite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. Murine muscle derived stem cells (MDSCs) isolated from slowly adhering cells (SACs) via the preplate technique, induce greater muscle regeneration than murine myoblasts, primarily due to improved post-transplantation survival, which is conferred by their increased stress resistance capacity. Aldehyde dehydrogenase (ALDH) represents a family of enzymes with important morphogenic as well as oxidative damage mitigating roles and has been found to be a marker of stem cells in both normal and malignant tissue. In this study, we hypothesized that elevated ALDH levels could identify murine and human muscle derived cell (hMDC) progenitors, endowed with enhanced stress resistance and muscle regeneration capacity. Methodology/Principal Findings Skeletal muscle progenitors were isolated from murine and human skeletal muscle by a modified preplate technique and unfractionated enzymatic digestion, respectively. ALDHhi subpopulations isolated by fluorescence activate cell sorting demonstrated increased proliferation and myogenic differentiation capacities compared to their ALDHlo counterparts when cultivated in oxidative and inflammatory stress media conditions. This behavior correlated with increased intracellular levels of reduced glutathione and superoxide dismutase. ALDHhi murine myoblasts were observed to exhibit an increased muscle regenerative potential compared to ALDHlo myoblasts, undergo multipotent differentiation (osteogenic and chondrogenic), and were found predominately in the SAC fraction, characteristics that are also observed in murine MDSCs. Likewise, human ALDHhi hMDCs demonstrated superior muscle regenerative capacity compared to ALDHlo hMDCs. Conclusions The methodology of isolating myogenic cells on the basis of elevated ALDH activity yielded cells with increased stress resistance, a behavior that conferred increased regenerative capacity of dystrophic murine skeletal muscle. This result demonstrates the critical role of stress resistance in myogenic cell therapy as well as confirms the role of ALDH as a marker for rapid isolation of murine and human myogenic progenitors for cell therapy. PMID:22195027

  6. Murine in vitro antigenic modification of tumor cells: effect on susceptibility to natural cell-mediated cytotoxicity.

    PubMed

    Langweiler, M; Weinhold, K J; Matthews, T J; Bolognesi, D P

    1985-03-01

    A 3-methylcholanthrene [(MCA) CAS: 56-49-5]-induced fibrosarcoma cell line and its Friend murine leukemia virus-infected counterpart were assessed for their susceptibility to lysis by so-called "natural" effector cells in a series of 51Cr release assays. Detailed functional and phenotypic analysis of lytic effector cell populations from normal C57BL/6 mouse spleens revealed an identity most closely associated with natural cytotoxic cells. Neither tumor cell line was found to be sensitive to natural killer-mediated lysis. Additionally, virus infection of the MCA-induced fibrosarcoma cell line did not affect susceptibility to natural cell-mediated cytotoxicity. PMID:2983140

  7. Localization of Label-Retaining Cells in Murine Vocal Fold Epithelium

    PubMed Central

    Leydon, Ciara; Bartlett, Rebecca S.; Roenneburg, Drew A.; Thibeault, Susan L.

    2011-01-01

    Purpose Epithelial homeostasis is critical for vocal fold health, yet little is known about the cells that support epithelial self-renewal. As a known characteristic of stem cells is that they are slow-cycling in vivo, the purpose of this prospective, controlled study was to identify and quantify slow-cycling cells or putative stem cells in murine vocal fold epithelium. Method Twelve mice were administered daily intraperitoneal injections of a nucleotide dye, bromodeoxyuridine (BrdU), over seven consecutive days. Under this pulse-chase paradigm, slow-cycling cells retain the dye (label-retaining cells; LRCs) while more rapidly cycling cells lose dye to dilution during multiple cell divisions. The percentage of label-retaining cells (%LRCs) was calculated following a chase period of two, four, and eight weeks post-injections. Results The %LRCs decreased significantly from 9.4% at two weeks to 3.1% at eight weeks following injections (p<.05). No statistically significant differences in the quantity of BrdU-positive cells were measured between the anterior, mid-membranous, or cartilaginous regions of the vocal fold (p>.05). Conclusions These findings are consistent with the presence and first report of a small population of putative stem cells along the length of murine vocal fold epithelium. PMID:21330647

  8. Isolation, cultivation, and characterization of adult murine prostate stem cells

    PubMed Central

    Lukacs, Rita U.; Goldstein, Andrew S.; Lawson, Devon A.; Cheng, Donghui; Witte, Owen N.

    2010-01-01

    ABSTRACT/SUMMARY The successful isolation and cultivation of prostate stem cells will allow us to study their unique biological properties and their application in therapeutic approaches. Here we provide step-by-step procedures on the basis of previous work in our laboratory for: the harvesting of primary prostate cells from adolescent male mice by a modified enzymatic procedure; the isolation of an enriched population of prostate stem cells through cell sorting; the cultivation of prostate stem cells in vitro; and characterization of these cells and their stem-like activity, including in vivo tubule regeneration. Normally it will take approximately 8 hours to harvest prostate cells, isolate the stem cell enriched population, and set up the in vitro sphere assay. It will take up to 8 weeks to analyze the unique properties of the stem cells, including their regenerative capacity in vivo. PMID:20360765

  9. Murine trophoblast can be killed by lymphokine-activated killer cells.

    PubMed

    Drake, B L; Head, J R

    1989-07-01

    The ability of fetal trophoblast cells in the placenta to resist cell-mediated lysis may be important for successful pregnancy. Previous studies in this laboratory demonstrated that cultured midterm mouse trophoblast cells are not susceptible to allospecific CTL generated by standard in vitro protocols, to antibody-dependent cell-mediated cytotoxicity, or to naive or IFN-activated NK cells, despite expressing the requisite target structures. However, we now report that murine trophoblast can be killed, in a non-MHC-specific manner, by LAK cells. Normal mouse spleen cells cultured for 4 days in IL-2-containing lymphokine preparations characteristically killed both NK-sensitive (YAC-1) and NK-resistant (EL4, P815) target cells, and mediated significant lysis of both cultured and freshly isolated trophoblast cells (35 to 55%, E/T 100/1). Pretreatment of the LAK cells with anti-ASGM1 antibody and C markedly reduced the lysis of trophoblast and YAC-1 targets, suggesting that the responsible cells belonged to the NK lineage. The ability of IL-2-activated NK cells to kill midterm murine trophoblast cells was confirmed using a population of highly lytic NK cells generated by culturing spleen cells from severe combined immunodeficiency mice in 500 U/ml rIL-2 for 5 days. These effector cells killed YAC-1, EL4 and P815 target cells at much lower E/T ratios than was achieved with the normal splenic LAK cells, and mediated significant lysis of both freshly isolated (45 to 50%, E/T 20/1) and cultured trophoblast cells (68 to 76%, E/T 20/1). The susceptibility of trophoblast to LAK cells and IL-2-activated NK cells supports the need for suppressor mechanisms regulating IL-2 activity at the maternal-fetal interface. PMID:2499634

  10. RHOF PROMOTES MURINE MARGINAL ZONE B CELL DEVELOPMENT

    PubMed Central

    KISHIMOTO, MAYUKO; MATSUDA, TAKENORI; YANASE, SHOUGO; KATSUMI, AKIRA; SUZUKI, NOBUAKI; IKEJIRI, MAKOTO; TAKAGI, AKIRA; IKAWA, MASAHITO; KOJIMA, TETSUHITO; KUNISHIMA, SHINJI; KIYOI, HITOSHI; NAOE, TOMOKI; MATSUSHITA, TADASHI; MARUYAMA, MITSUO

    2014-01-01

    ABSTRACT RhoF is a member of the Rho GTPase family that has been implicated in various cell functions including long filopodia formation, adhesion, and migration of cells. Although RhoF is expressed in lymphoid tissues, the roles of RhoF in B cell development remain largely unclear. On the other hand, other members of the Rho GTPase family, such as Cdc42, RhoA, and Rac, have been intensively studied and are known to be required for B cell development in the bone marrow and spleen. We hypothesized that RhoF is also involved in B cell development. To examine our hypothesis, we analyzed B cell development in RhoF knockout (KO) mice and found a significant reduction in marginal zone (MZ) B cells in the spleen, although T cell development in the thymus and spleen was not affected. Consistent with these results, the width of the MZ B cell region in the spleen was significantly reduced in the RhoF KO mice. However, the antigen-specific antibody titer of IgM and IgG3 after MZ B cell-specific antigen (T cell-independent antigen, type I) stimulation was not affected by RhoF deletion. Furthermore, we demonstrated that RhoF was dispensable for stromal cell-derived factor-1α- and B lymphocyte chemoattractant-induced B cell migration. These results suggest that RhoF promotes MZ B cell development in the spleen. PMID:25741038

  11. Potent Inhibition of Junín Virus Infection by Interferon in Murine Cells

    PubMed Central

    Huang, Cheng; Walker, Aida G.; Grant, Ashley M.; Kolokoltsova, Olga A.; Yun, Nadezhda E.; Seregin, Alexey V.; Paessler, Slobodan

    2014-01-01

    The new world arenavirus Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, a lethal human infectious disease. Adult laboratory mice are generally resistant to peripheral infection by JUNV. The mechanism underlying the mouse resistance to JUNV infection is largely unknown. We have reported that interferon receptor knockout mice succumb to JUNV infection, indicating the critical role of interferon in restricting JUNV infection in mice. Here we report that the pathogenic and vaccine strains of JUNV were highly sensitive to interferon in murine primary cells. Treatment with low concentrations of interferon abrogated viral NP protein expression in murine cells. The replication of both JUNVs was enhanced in IRF3/IRF7 deficient cells. In addition, the vaccine strain of JUNV displayed impaired growth in primary murine cells. Our data suggested a direct and potent role of host interferon response in restricting JUNV replication in mice. The defect in viral growth for vaccine JUNV might also partially explain its attenuation in mice. PMID:24901990

  12. Cell-intrinsic regulation of murine dendritic cell function and survival by prereceptor amplification of glucocorticoid.

    PubMed

    Soulier, Annelise; Blois, Sandra M; Sivakumaran, Shivajanani; Fallah-Arani, Farnaz; Henderson, Stephen; Flutter, Barry; Rabbitt, Elizabeth H; Stewart, Paul M; Lavery, Gareth G; Bennett, Clare; Curnow, S John; Chakraverty, Ronjon

    2013-11-01

    Although the inhibitory effects of therapeutic glucocorticoids (GCs) on dendritic cells (DCs) are well established, the roles of endogenous GCs in DC homeostasis are less clear. A critical element regulating endogenous GC concentrations involves local conversion of inactive substrates to active 11-hydroxyglucocorticoids, a reduction reaction catalyzed within the endoplasmic reticulum by an enzyme complex containing 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and hexose-6-phosphate dehydrogenase (H6PDH). In this study, we found that this GC amplification pathway operates both constitutively and maximally in steady state murine DC populations and is unaffected by additional inflammatory stimuli. Under physiologic conditions, 11βHSD1-H6PDH increases the sensitivity of plasmacytoid DCs (pDCs) to GC-induced apoptosis and restricts the survival of this population through a cell-intrinsic mechanism. Upon CpG activation, the effects of enzyme activity are overridden, with pDCs becoming resistant to GCs and fully competent to release type I interferon. CD8α(+) DCs are also highly proficient in amplifying GC levels, leading to impaired maturation following toll-like receptor-mediated signaling. Indeed, pharmacologic inhibition of 11βHSD1 synergized with CpG to enhance specific T-cell responses following vaccination targeted to CD8α(+) DCs. In conclusion, amplification of endogenous GCs is a critical cell-autonomous mechanism for regulating the survival and functions of DCs in vivo. PMID:24081658

  13. Immunosurveillance by gammadelta T cells: focus on the murine system.

    PubMed

    Girardi, Michael; Hayday, Adrian C

    2005-01-01

    Physiologic immune responses are an integration of the activities of several lymphoid cell types that include qualitatively distinct T cell subsets. The contributions that specific T cell subsets make during infection, inflammation, and carcinogenesis is becoming increasingly clear from a variety of mouse models and, importantly, their backcrossing onto different genetic backgrounds. This review considers what we have learned in the mouse about the crucial roles played by gammadelta T cells. We consider how the cells' associations with specific tissues have revealed that T cell responses are regulated locally as well as systemically, and we discuss the implications of this for understanding and enhancing immune surveillance in the clinical setting. PMID:15976492

  14. ?? T Cells Confer Protection against Murine Cytomegalovirus (MCMV)

    PubMed Central

    Villacreces, Arnaud; Juzan, Marina; Rousseau, Benot; Dulanto, Sara; Giese, Alban; Costet, Pierre; Praloran, Vincent; Moreau, Jean-Franois; Dubus, Pierre; Vermijlen, David

    2015-01-01

    Cytomegalovirus (CMV) is a leading infectious cause of morbidity in immune-compromised patients. ?? T cells have been involved in the response to CMV but their role in protection has not been firmly established and their dependency on other lymphocytes has not been addressed. Using C57BL/6 ?? and/or ?? T cell-deficient mice, we here show that ?? T cells are as competent as ?? T cells to protect mice from CMV-induced death. ?? T cell-mediated protection involved control of viral load and prevented organ damage. ?? T cell recovery by bone marrow transplant or adoptive transfer experiments rescued CD3??/? mice from CMV-induced death confirming the protective antiviral role of ?? T cells. As observed in humans, different ?? T cell subsets were induced upon CMV challenge, which differentiated into effector memory cells. This response was observed in the liver and lungs and implicated both CD27+ and CD27? ?? T cells. NK cells were the largely preponderant producers of IFN? and cytotoxic granules throughout the infection, suggesting that the protective role of ?? T cells did not principally rely on either of these two functions. Finally, ?? T cells were strikingly sufficient to fully protect Rag?/??c?/? mice from death, demonstrating that they can act in the absence of B and NK cells. Altogether our results uncover an autonomous protective antiviral function of ?? T cells, and open new perspectives for the characterization of a non classical mode of action which should foster the design of new ?? T cell based therapies, especially useful in ?? T cell compromised patients. PMID:25747674

  15. Conjugated linoleic acid induces apoptosis of murine mammary tumor cells via Bcl-2 loss

    PubMed Central

    Ou, Lihui; Ip, Clement; Lisafeld, Barbara; Ip, Margot M.

    2007-01-01

    Conjugated linoleic acid (CLA) is a powerful anticancer agent in a number of tumor model systems; however, its precise mechanism of action remains elusive. Here, we report that t10,c12 CLA, a component of synthetic CLA supplements, induced apoptosis and G1 arrest of p53 mutant TM4t murine mammary tumor cells. Furthermore, t10,c12-CLA induced a time- and concentration-dependent cleavage of caspases-3 and -9, and release of cytochrome c from mitochondria to cytosol. Levels of Bcl-2 protein were decreased both in total cellular lysates and in mitochondria after t10,c12-CLA treatment; however, there was no significant change in Bax or Bak. Overexpression of Bcl-2 attenuated apoptosis in response to t10,c12-CLA treatment. These results demonstrate that t10,c12-CLA triggers apoptosis of p53 mutant murine mammary tumor cells through the mitochondrial pathway by targeting Bcl-2. PMID:17400188

  16. Killed Candida albicans yeasts and hyphae inhibit gamma interferon release by murine natural killer cells.

    PubMed

    Murciano, Celia; Villamón, Eva; O'Connor, José-Enrique; Gozalbo, Daniel; Gil, M Luisa

    2006-02-01

    Killed yeasts and hyphae of Candida albicans inhibit gamma interferon secretion by highly purified murine NK cells in response to the Toll-like receptor ligands lipopolysaccharide and zymosan. This effect, which is also observed in the presence of NK-activating cytokines (interleukin-2 [IL-2], IL-12, and IL-15), may represent a novel mechanism of immune evasion that contributes to the virulence of C. albicans. PMID:16428793

  17. The Human Lung Adenocarcinoma Cell Line EKVX Produces an Infectious Xenotropic Murine Leukemia Virus

    PubMed Central

    Cmarik, Joan L.; Troxler, Jami A.; Hanson, Charlotte A.; Zhang, Xiang; Ruscetti, Sandra K.

    2011-01-01

    The cell lines of the NCI-60 panel represent different cancer types and have been widely utilized for drug screening and molecular target identification. Screening these cell lines for envelope proteins or gene sequences related to xenotropic murine leukemia viruses (X-MLVs) revealed that one cell line, EKVX, was a candidate for production of an infectious gammaretrovirus. The presence of a retrovirus infectious to human cells was confirmed by the cell-free transmission of infection to the human prostate cancer cell line LNCaP. Amplification and sequencing of additional proviral sequences from EKVX confirmed a high degree of similarity to X-MLV. The cell line EKVX was established following passage of the original tumor cells through nude mice, providing a possible source of the X-MLV found in the EKVX cells. PMID:22355448

  18. The human lung adenocarcinoma cell line EKVX produces an infectious xenotropic murine leukemia virus.

    PubMed

    Cmarik, Joan L; Troxler, Jami A; Hanson, Charlotte A; Zhang, Xiang; Ruscetti, Sandra K

    2011-12-01

    The cell lines of the NCI-60 panel represent different cancer types and have been widely utilized for drug screening and molecular target identification. Screening these cell lines for envelope proteins or gene sequences related to xenotropic murine leukemia viruses (X-MLVs) revealed that one cell line, EKVX, was a candidate for production of an infectious gammaretrovirus. The presence of a retrovirus infectious to human cells was confirmed by the cell-free transmission of infection to the human prostate cancer cell line LNCaP. Amplification and sequencing of additional proviral sequences from EKVX confirmed a high degree of similarity to X-MLV. The cell line EKVX was established following passage of the original tumor cells through nude mice, providing a possible source of the X-MLV found in the EKVX cells. PMID:22355448

  19. Timing is everything: dendritic cell subsets in murine Leishmania infection.

    PubMed

    Ashok, Devika; Acha-Orbea, Hans

    2014-10-01

    Mouse models of Leishmania major infection have shown that a predominant CD4(+) T helper type 1 cell (Th1) response leads to protection, while T helper type 2 cell (Th2) predominance confers susceptibility. Dendritic cells (DCs) are antigen-presenting cells that orchestrate the T cell response. The immune response to L. major involves direct antigen presentation by migrating DCs or transfer of antigens to resident DCs to prime T cells. In this review, we discuss the timing and consequences of antigen presentation by DC subsets and how this affects Leishmania susceptibility. We propose a model where dermal DCs and Langerhans cells play a role early in infection, followed by inflammatory monocyte-derived DC and lymph node (LN)-resident DCs at later time points of infection to establish the resistant Th1 response. PMID:25190685

  20. No attenuation of the ATM-dependent DNA damage response in murine telomerase-deficient cells

    PubMed Central

    Erdmann, Natalie; Harrington, Lea A.

    2009-01-01

    Inactivation of mammalian telomerase leads to telomere attrition, eventually culminating in uncapped telomeres, which elicit a DNA damage response and cell cycle arrest or death. In some instances, telomerase modulation evokes a response not obviously attributable to changes in telomere length. One such example is the suppression of the DNA damage response (DDR) and changes in histone modification that occur upon repression of the telomerase reverse transcriptase, TERT, in human primary cells [1]. Here, we evaluate the contribution of TERT to the DDR in murine Tert−/− cells without critically shortened telomeres. We treated mTert−/− embryonic stem (ES) cells and murine embryonic fibroblasts (MEFs) with etoposide and irradiation, and assessed the status of p53pS15, 53BP1, ATMpS1981, SMC1pS957, and γH2AX by indirect immunofluorescence or western blotting. In four independently derived mTert−/− ES cell lines, there was no significant difference in the induction of γH2AX, 53BP1 foci formation, or the phosphorylation of ATM targets (ATM, SMC1, p53) between wildtype and mTert−/− ES cells and MEFs. A slight difference in post-translational modification of histones H3 and H4 was observed in a subset of mTert−/− ES cells, however this difference was reflected in the cellular levels of H3 and H4. Thus, in contrast to previous studies in human cells, the absence of Tert does not overtly affect the ATM-dependent response to DNA damage in murine cells. PMID:19071232

  1. SuperSILAC Quantitative Proteome Profiling of Murine Middle Ear Epithelial Cell Remodeling with NTHi

    PubMed Central

    Val, Stéphanie; Burgett, Katelyn; Brown, Kristy J.; Preciado, Diego

    2016-01-01

    Background Chronic Otitis Media with effusion (COME) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Non-typeable Haemophilus influenzae (NTHi), the most common acute Otitis Media (OM) pathogen, is postulated to promote middle ear epithelial remodeling in the progression of OM from acute to chronic. The goals of this study were to examine histopathological and quantitative proteomic epithelial effects of NTHi challenge in a murine middle ear epithelial cell line. Methods NTHi lysates were generated and used to stimulate murine epithelial cells (mMEEC) cultured at air-liquid interface over 48 hours– 1 week. Conditional quantitative Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) of cell lysates was performed to interrogate the global protein production in the cells, using the SuperSILAC technique. Histology of the epithelium over time was done to measure bacterial dependent remodeling. Results Mass spectrometry analysis identified 2,565 proteins across samples, of which 74 exhibited differential enrichment or depletion in cell lysates (+/-2.0 fold-change; p value<0.05). The key molecular functions regulated by NTHi lysates exposure were related to cell proliferation, death, migration, adhesion and inflammation. Finally, chronic exposure induced significant epithelial thickening of cells grown at air liquid interface. Conclusions NTHi lysates drive pathways responsible of cell remodeling in murine middle ear epithelium which likely contributes to observed epithelial hyperplasia in vitro. Further elucidation of these mediators will be critical in understanding the progression of OM from acute to chronic at the molecular level. PMID:26859300

  2. Cytoplasmic superoxide dismutase and catalase activity and resistance to radiation lethality in murine tumor cells

    SciTech Connect

    Davy, C.A.; Tesfay, Z.; Jones, J.; Rosenberg, R.C.; McCarthy, C.; Rosenberg, S.O.

    1986-05-01

    Reduced species of molecular oxygen are produced by the interaction of ionizing radiation with aqueous solutions containing molecular oxygen. The enzymes catalase and superoxide dismutase (SOD) are thought to function in vivo as scavengers of metabolically produced peroxide and superoxide respectively. SOD has been shown to protect against the lethal effects of ionizing radiation in vitro and in vivo. The authors have investigated the relationship between the cytosolic SOD catalase content and the sensitivity to radiation lethality of a number of murine cell lines (402AX, EL-4, MB-2T3, MB-4, MEL, P-815, SAI, SP-2, and SV-3T3). K/sub i/(CN/sup -/) for murine Cu-Zn-SOD was determined to be 6.8 x 10/sup -6/ M. No cytosolic Mn-SOD activity was found in any of the cell lines studied. No correlation was found between the cytosolic Cu-Zn-SOD or cytosolic catalase activity and the resistance to radiation lethality or the murine cell lines studied.

  3. Effect of cooling rate on human and murine hemopoietic precursor cell recovery

    SciTech Connect

    Niskanen, E.; Pirsch, G.

    1983-08-01

    The effect of cooling rate on recovery of human and murine hemopoietic precursor cells was studied. In the presence of 10% Me2SO, a cooling rate of 7 degrees C/min from -4 to -30 degrees C was optimal for recovery of both human and murine precursor cells which give rise to colonies in diffusion chambers implanted in mice (CFU-DG). Cooling of human marrow at a rate between 3 and 7 degrees C/min resulted in the best CFU-C recovery, although no good correlation between the cooling rate and murine CFU-C recovery was demonstrated. These data suggest that recovery of the primitive hemopoietic precursor cells can be improved by changing the standard cryopreservation programs used presently. However, improved recovery of CFU-DG does not necessarily translate into faster reconstitution of hemopoiesis. No significant difference was observed in overall recovery of bone marrow cellularity in lethally irradiated mice following injection of untreated marrow and marrow cooled at a rate of 1 and 7 degrees C/min.

  4. Cell-surface receptor for ecotropic murine retroviruses is a basic amino-acid transporter.

    PubMed

    Wang, H; Kavanaugh, M P; North, R A; Kabat, D

    1991-08-22

    The complementary DNA sequence encoding the cell-surface receptor for ecotropic host-range murine retroviruses (ecoR) shows that it contains 622 amino acids and 14 hydrophobic potentially membrane-spanning sequences. Because this receptor occurs on many or all murine cells and is probably essential for viability of cultured fibroblasts, its normal function might be to transport an essential metabolite. We expressed ecoR in Xenopus laevis oocytes by injecting RNA transcribed from the cloned cDNA. These oocytes specifically bound the gp70 envelope glycoprotein from an ecotropic murine leukaemia virus. An inward current was recorded electrophysiologically when a mixture of amino-acids was applied: this resulted from a stereoselective, saturable uptake of lysine, arginine and ornithine; it was independent of sodium and not substantially altered by gp70. Cysteine and homoserine were also taken up, but sodium was necessary for their transport. These properties of ecoR correspond to those of the y+ amino-acid transporter. Our results demonstrate the subversion of a ubiquitous cell membrane protein, in this case a basic amino acid transporter, for use as a retroviral receptor. PMID:1908564

  5. Targeting Syk-activated B cells in murine and human chronic graft-versus-host disease.

    PubMed

    Flynn, Ryan; Allen, Jessica L; Luznik, Leo; MacDonald, Kelli P; Paz, Katelyn; Alexander, Kylie A; Vulic, Ante; Du, Jing; Panoskaltsis-Mortari, Angela; Taylor, Patricia A; Poe, Jonathan C; Serody, Jonathan S; Murphy, William J; Hill, Geoffrey R; Maillard, Ivan; Koreth, John; Cutler, Corey S; Soiffer, Robert J; Antin, Joseph H; Ritz, Jerome; Chao, Nelson J; Clynes, Raphael A; Sarantopoulos, Stefanie; Blazar, Bruce R

    2015-06-25

    Novel therapies for chronic graft-versus-host disease (cGVHD) are needed. Aberrant B-cell activation has been demonstrated in mice and humans with cGVHD. Having previously found that human cGVHD B cells are activated and primed for survival, we sought to further evaluate the role of the spleen tyrosine kinase (Syk) in cGVHD in multiple murine models and human peripheral blood cells. In a murine model of multiorgan system, nonsclerodermatous disease with bronchiolitis obliterans where cGVHD is dependent on antibody and germinal center (GC) B cells, we found that activation of Syk was necessary in donor B cells, but not T cells, for disease progression. Bone marrow-specific Syk deletion in vivo was effective in treating established cGVHD, as was a small-molecule inhibitor of Syk, fostamatinib, which normalized GC formation and decreased activated CD80/86(+) dendritic cells. In multiple distinct models of sclerodermatous cGVHD, clinical and pathological disease manifestations were not eliminated when mice were therapeutically treated with fostamatinib, though both clinical and immunologic effects could be observed in one of these scleroderma models. We further demonstrated that Syk inhibition was effective at inducing apoptosis of human cGVHD B cells. Together, these data demonstrate a therapeutic potential of targeting B-cell Syk signaling in cGVHD. PMID:25852057

  6. Activation of normal murine B cells by Echinococcus granulosus.

    PubMed Central

    Cox, D A; Marshall-Clarke, S; Dixon, J B

    1989-01-01

    Echinococcus granulosus protoscolex (PSC) infection of BALB/c mice led, after 4 days, to raised numbers of cells forming plaques with trinitrophenyl-treated sheep red cells and bromelain-treated mouse red cells. The findings were similar in athymic and euthymic CBA mice. Activation of B cells was accompanied by secretion of immunoglobulin, as indicated by the reverse plaque technique. In addition, co-culture of PSC with the 7OZ/3 pre-B-cell led to the induction of differentiation, resulting in the expression of surface immunoglobulin (Ig). It is concluded that E. granulosus is a polyclonal activator of B cells inducing both transformation and differentiation, and that the effect is thymus-independent. PMID:2661414

  7. Regulation of Glycan Structures in Murine Embryonic Stem Cells

    PubMed Central

    Nairn, Alison V.; Aoki, Kazuhiro; dela Rosa, Mitche; Porterfield, Mindy; Lim, Jae-Min; Kulik, Michael; Pierce, J. Michael; Wells, Lance; Dalton, Stephen; Tiemeyer, Michael; Moremen, Kelley W.

    2012-01-01

    The abundance and structural diversity of glycans on glycoproteins and glycolipids are highly regulated and play important roles during vertebrate development. Because of the challenges associated with studying glycan regulation in vertebrate embryos, we have chosen to study mouse embryonic stem (ES) cells as they differentiate into embryoid bodies (EBs) or into extraembryonic endodermal (ExE) cells as a model for cellular differentiation. We profiled N- and O-glycan structures isolated from these cell populations and examined transcripts encoding the corresponding enzymatic machinery for glycan biosynthesis in an effort to probe the mechanisms that drive the regulation of glycan diversity. During differentiation from mouse ES cells to either EBs or ExE cells, general trends were detected. The predominance of high mannose N-glycans in ES cells shifted to an equal abundance of complex and high mannose structures, increased sialylation, and increased α-Gal termination in the differentiated cell populations. Whereas core 1 O-glycan structures predominated in all three cell populations, increased sialylation and increased core diversity characterized the O-glycans of both differentiated cell types. Increased polysialylation was also found in both differentiated cell types. Differences between the two differentiated cell types included greater sialylation of N-glycans in EBs, whereas α-Gal-capped structures were more prevalent in ExE cells. Changes in glycan structures generally, but not uniformly, correlated with alterations in transcript abundance for the corresponding biosynthetic enzymes, suggesting that transcriptional regulation contributes significantly to the regulation of glycan expression. Knowledge of glycan structural diversity and transcript regulation should provide greater understanding of the roles of protein glycosylation in vertebrate development. PMID:22988249

  8. Characterization studies of suppressor cells in murine bone marrow chimaeras.

    PubMed Central

    Imamura, M; Fujimoto, H; Fukuhara, T; Kobayashi, M; Kasai, M; Sakurada, K; Miyazaki, T

    1987-01-01

    When BALB/c bone marrow cells were transferred to lethally X-irradiated C3H/He mice either intrasplenically (i.s.) or intravenously (i.v.), suppressor cells were detected by means of MLR coculture assays in the spleen of i.s. and i.v. chimaeras. Some but not all of the suppressor cells expressed a Thy 1.2 antigen, indicating that suppressor cells either sensitive or insensitive to anti-Thy 1.2 antibody plus complement treatment were generated in the spleen of i.s. and i.v. chimaeras. According to the examination of Lyt alloantigen expression on suppressor T cells, Lyt 1+2-, Lyt 1-2+, and Lyt 1+2+ suppressor T cells appeared to be present. The culture supernatants from several T-cell clones showed the suppressor activity against alloreactive MLR. Cell surface markers of these clones were composed of Lyt 1+2-, Lyt 1+2+ and Lyt 1-2+. In addition, since Carrageenan treatment abrogated the suppressor activity of plastic dish adherent cells, we conclude that some of them were composed mainly of macrophages. PMID:2950049

  9. Murine Lung Cancer Induces Generalized T Cell Exhaustion

    PubMed Central

    Mittal, Rohit; Chen, Ching-Wen; Lyons, John D; Margoles, Lindsay M; Liang, Zhe; Coopersmith, Craig M; Ford, Mandy L

    2015-01-01

    Background Cancer is known to modulate tumor-specific immune responses by establishing a micro-environment that leads to the upregulation of T cell inhibitory receptors, resulting in the progressive loss of function and eventual death of tumor-specific T cells. However, the ability of cancer to impact the functionality of the immune system on a systemic level is much less well characterized. Because cancer is known to predispose patients to infectious complications including sepsis, we hypothesized that the presence of cancer alters pathogen-directed immune responses on a systemic level. Materials and Methods We assessed systemic T cell coinhibitory receptor expression, cytokine production, and apoptosis in mice with established subcutaneous lung cancer tumors and in unmanipulated mice without cancer. Results Results indicated that the frequencies of PD-1+, BTLA+, and 2B4+ cells in both the CD4+ and CD8+ T cell compartments were increased in mice with localized cancer relative to non-cancer controls, and the frequencies of both CD4+ and CD8+ T cells expressing multiple different inhibitory receptors was increased in cancer animals relative to non-cancer controls. Additionally, 2B4+CD8+ T cells in cancer mice exhibited reduced IL-2 and IFN-γ, while BTLA+CD8+ T cells in cancer mice exhibited reduced IL-2 and TNF. Conversely, CD4+ T cells in cancer animals demonstrated an increase in the frequency of Annexin V+ apoptotic cells. Conclusion Taken together, these data suggest that the presence of cancer induces systemic T cell exhaustion and generalized immune suppression. PMID:25748104

  10. AUGMENTATION OF MURINE NATURAL KILLER CELL ACTIVITY BY MANGANESE CHLORIDE

    EPA Science Inventory

    Natural Killer (NK) cell activity of spleen cells from male CBA/J mice was augmented by a single parenteral injection of MnCl2 administered 1 day prior to testing by in vitro and in vivo isotope release assays. Increased cytotoxic activity was observed in vitro against both NK-se...

  11. The effects of simulated hypogravity on murine bone marrow cells

    NASA Technical Reports Server (NTRS)

    Lawless, Desales

    1989-01-01

    Mouse bone marrow cells grown in complete medium at unit gravity were compared with a similar population cultured in conditions that mimic some aspects of microgravity. After the cells adjusted to the conditions that simulated microgravity, they proliferated as fetal or oncogenic populations; their numbers doubled in twelve hour periods. Differentiated subpopulations were depleted from the heterogeneous mixture with time and the undifferentiated hematopoietic stem cells increased in numbers. The cells in the control groups in unit gravity and those in the bioreactors in conditions of microgravity were monitored under a number of parameters. Each were phenotyped as to cell surface antigens using a panel of monoclonal antibodies and flow cytometry. Other parameters compared included: pH, glucose uptake, oxygen consumption and carbon-dioxide production. Nuclear DNA was monitored by flow cytometry. Functional responses were studied by mitogenic stimulation by various lectins. The importance of these findings should have relevance to the space program. Cells should behave predictably in zero gravity; specific populations can be eliminated from diverse populations and other populations isolated. The availability of stem cell populations will enhance both bone marrow and gene transplant programs. Stem cells will permit developmental biologists study the paths of hematopoiesis.

  12. AN IN VITRO MODEL FOR MURINE URETERIC EPITHELIAL CELLS

    EPA Science Inventory

    This report presents a model developed to study growth and differentiation of primary cultures of ureteric epithelial cells from embryonic C57BL/6N mouse urinary tracts. Single cells were resuspended in medium and plated onto transwells coated with collagen IV and laminin. Basa...

  13. Characterization of Long-Term Cultured Murine Submandibular Gland Epithelial Cells

    PubMed Central

    Tsunoda, Kazuyuki; Nakagawa, Taneaki; Tsubota, Kazuo

    2016-01-01

    Purpose Human and rat salivary gland cell lines derived from tumors or genetic modification are currently available for research. Here, we attempted to culture and characterize long-term cultured cells spontaneously derived from wild type murine submandibular glands (SGs). Methods SGs were removed from 3-week-old C57B/6J female mice and dissociated by collagenase type 1 and hyaluronidase digestion. Isolated SG epithelial cells were cultured in low calcium, serum-free growth media in the presence of cholera toxin (CT) during early passages. Single-cell colonies were isolated by limiting dilution culture after 25 passages. Early- and late-stage cell cultures were characterized for keratin 14, keratin 18, α-smooth muscle actin, and p63 by immunostaining and quantitative real-time PCR analysis. Results SG epithelial cells cultured in optimized media maintained their proliferative ability and morphology for over 80 passages. Long-term cultured cells expressed keratin 14, keratin 18, and p63, indicative of an epithelial phenotype. Conclusions Epithelial cells originating from wild type murine SGs could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium. PMID:26800086

  14. Generation of murine induced pluripotent stem cells by using high-density distributed electrodes network.

    PubMed

    Lu, Ming-Yu; Li, Zhihong; Hwang, Shiaw-Min; Linju Yen, B; Lee, Gwo-Bin

    2015-09-01

    This study reports a robust method of gene transfection in a murine primary cell model by using a high-density electrodes network (HDEN). By demonstrating high cell viability after gene transfection and successful expression of transgenes including fluorescent proteins, the HDEN device shows great promise as a solution in which reprogramming efficiency using non-viral induction for generation of murine induced pluripotent stem cells (iPSCs) is optimized. High and steady transgene expression levels in host cells of iPSCs can be demonstrated using this method. Moreover, the HDEN device achieved successful gene transfection with a low voltage of less than 180 V while requiring relatively low cell numbers (less than 1.5 × 10(4) cells). The results are comparable to current conventional methods, demonstrating a reasonable fluorescent-plasmid transfection rate (42.4% in single transfection and 24.5% in triple transfection) and high cell viability of over 95%. The gene expression levels of each iPSC factor was measured to be over 10-fold higher than that reported in previous studies using a single mouse embryonic fibroblast cell. Our results demonstrate that the generation of iPSCs using HDEN transfection of plasmid DNA may be a feasible and safe alternative to using viral transfection methods in the near future. PMID:26396661

  15. Study on the therapeutic effect of neural progenitor cells in mice of a glioma murine model

    PubMed Central

    XU, GUOZHENG; LIU, YING; ZHANG, YI; YANG, QIAN; DIAO, BO

    2016-01-01

    Glioma is a common malignacy of the brain that affects elderly patients in particular. Despite treatment, however, the survival rate is 12 months. The aim of the present study was to examine the therapeutic effect of neural progenitor cells (NPCs) on a glioma murine model, and to determine the possible mechanism of action. A glioma murine model was constructed and the tumor volume and tumor growth rate were measured. The therapeutic effect of cell injection on the glioma mouse model mice was confirmed. The quantitative polymerase chain reaction method was used to detect the expression of proto-oncogene and tumor suppressor gene. Intracranial injection of NPCs was performed to determine cell apoptosis. Preliminary results showed the mechanism of cell therapy effect at the transcription and cellular level. Compared with the model group, the tumor volume of the mice of the cell therapy group was significantly reduced from the 6th to 8th week, and the tumor growth rate was downregulated. The mechanism of action identified that NPCs regulate gene expression in tumor tissues, increase the expression of tumor suppressor gene, downregulate the gene expression of tumor cells, and reverse the proto-oncogene and imbalance of gene expression in gliomas. In conclusion, the new type of cell injection method can regulate the proto-oncogene of tumor tissue and tumor suppressor gene, improve the function phenotype of the cell, and effectively improve the clinical symptoms of mice with gliomas.

  16. Pontin is essential for murine hematopoietic stem cell survival

    PubMed Central

    Bereshchenko, Oxana; Mancini, Elena; Luciani, Luisa; Gambardella, Adriana; Riccardi, Carlo; Nerlov, Claus

    2012-01-01

    Pontin is a highly conserved DNA helicase/ATPase which is a component of several macromolecular complexes with functions that include DNA repair, telomere maintenance and tumor suppression. While Pontin is known to be essential in yeast, fruit flies and frogs, its physiological role in mammalian organisms remains to be determined. We here find that Pontin is highly expressed in embryonic stem cells and hematopoietic tissues. Through germline inactivation of Ruvbl1, the gene encoding Pontin, we found it to be essential for early embryogenesis, as Ruvbl1 null embryos could not be recovered beyond the blastocyst stage where proliferation of the pluripotent inner cell mass was impaired. Conditional ablation of Ruvbl1 in hematopoietic tissues led to bone marrow failure. Competitive repopulation experiments showed that this included the loss of hematopoietic stem cells through apopotosis. Pontin is, therefore, essential for the function of both embryonic pluripotent cells and adult hematopoietic stem cells. PMID:22371176

  17. Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues

    PubMed Central

    Bjorkdahl, Olle; Barber, Karen A; Brett, Sara J; Daly, Maria G; Plumpton, Christopher; Elshourbagy, Nabil A; Tite, John P; Thomsen, Lindy L

    2003-01-01

    Expression of the lymph node homing and CC-chemokine receptor 7 (CCR7), with L-selectin (CD62L), has been shown to divide human memory T cells into two functionally distinct subsets. We generated a polyclonal antibody against murine CCR7 and used this antibody to study CCR7 expression on murine T-cell subsets. Using flow cytometric staining of T cells for visualisation expression of CCR7 in association with CD62L and CD44, a major population of CD4 or CD8 T cells expressing CCR7 were found to be CD62Lhigh CD44low, which would suggest a naïve cell phenotype. By analogy with human studies, memory cells could be subdivided into CCR7high CD62Lhigh CD44high (central memory) and CCR7low CD62Llow CD44high (effector memory). The proportions of these populations were different in lymph node, blood and spleen. Functional, short-term in vitro polyclonal stimulation of blood, spleen and lymph node cells from naive mice demonstrated that CCR7high CD4 T cells produced predominantly interleukin (IL)-2, whereas CCR7low CD4 T cells produced both IL-2 and interferon-γ (IFN-γ). However, in contrast to previously published reports, the CCR7high CD8 T-cell subpopulation produced both IFN-γ and IL-2. Analysis of effector T cells, induced by immunization in vivo, showed that a proportion of activated naïve CD4 T cells down-regulated CCR7 only after multiple cell divisions, and this coincided with the down-regulation of CD62L and production of IL-4 and IFN-γ. Finally, analysis of effector T cells during the phase of maximal clonal expansion of secondary immune responses in vivo indicated that the vast majority of both IL-2- and IFN-γ-producing cells are CCR7low, while few cytokine-expressing CCR7high T cells were detected. Our results support the hypothesis, developed from studies with human cells, that CCR7 may separate functionally different murine memory T-cell subpopulations, but indicate additional complexity in that CCR7high CD8 T cells also may produce IFN-γ. PMID:14511230

  18. Identification and Analysis of Natural Killer Cells in Murine Nasal Passages

    PubMed Central

    Okada, Kazunari; Sato, Shintaro; Sato, Ayuko; Mandelboim, Ofer; Yamasoba, Tatsuya; Kiyono, Hiroshi

    2015-01-01

    Background Natural killer (NK) cells in the upper respiratory airways are not well characterized. In the current study, we sought to characterize and functionally assess murine nasal NK cells. Methods Using immunohistochemistry and flow cytometry, we compared the nasal NK cells of Ncr1GFP/+ knock-in mice, whose NK cells produced green fluorescent protein, with their splenic and pulmonary counterparts. In addition, we functionally analyzed the nasal NK cells of these mice in vitro. To assess the in vivo functions of nasal NK cells, C57BL/6 mice depleted of NK cells after treatment with PK136 antibody were nasally infected with influenza virus PR8. Results Immunohistochemical analysis confirmed the presence of NK cells in the lamina propria of nasal mucosa, and flow cytometry showed that these cells were of NK cell lineage. The expression patterns of Ly49 receptor, CD11b/CD27, CD62L and CD69 revealed that nasal NK cells had an immature and activated phenotype compared with that of their splenic and pulmonary counterparts. Effector functions including degranulation and IFN(interferon)-γ production after in vitro stimulation with phorbol 12-myristate-13-acetate plus ionomycin or IL(interleukin)-12 plus IL-18 were dampened in nasal NK cells, and the depletion of NK cells led to an increased influenza virus titer in nasal passages. Conclusions The NK cells of the murine nasal passage belong to the conventional NK cell linage and characteristically demonstrate an immature and activated phenotype. Despite their hyporesponsiveness in vitro, nasal NK cells play important roles in the host defense against nasal influenza virus infection. PMID:26575399

  19. Murine mechanical ventilation stimulates alveolar epithelial cell proliferation.

    PubMed

    Chess, Patricia Rose; Benson, Randi Potter; Maniscalco, William M; Wright, Terry W; O'Reilly, Michael A; Johnston, Carl J

    2010-08-01

    High tidal volume mechanical ventilation can cause inflammation and lung damage. Mechanical strain is also necessary for normal lung growth. The current work was performed to determine if mechanical ventilation with clinically utilized tidal volumes stimulates a proliferative response in the lung. Six- to 8-week-old C57/Bl6 mice, anesthetized with ketamine/xylozine, were ventilated for 6 hours with 10 mL/kg tidal volume, positive end-expiratory pressure (PEEP) 3cm H(2)O. Pulmonary function testing demonstrated decreased compliance within 3 hours of ventilation. Assessment of bronchoalveolar lavage (BAL) demonstrated no significant increase in lactate dehydrogenase, total lavagable cell number, or total protein after ventilation. There was evidence of inflammation in the lungs of ventilated mice, with an increased percentage of lymphocytes and neutrophils in BAL, and an increase in macrophage inflammatory protein (MIP)-2 and interleukin (IL)-1beta message in lung tissue. Immunohistochemistry of inflation-fixed lungs demonstrated increased alveolar cell proliferation, as measured by both proliferating cell nuclear antigen and Ki67 staining. Dual staining confirmed that proliferating cells labeled with proSP-B, demonstrating that ventilation induces proliferation of alveolar type II cells. Ventilation did not increase apoptosis in alveolar type II cells, as measured by TUNEL staining. Ventilation at low tidal volumes leads to a mild inflammatory response and alveolar epithelial cell proliferation. PMID:20653468

  20. Distinct Murine Mucosal Langerhans Cell Subsets Develop from Pre-dendritic Cells and Monocytes.

    PubMed

    Capucha, Tal; Mizraji, Gabriel; Segev, Hadas; Blecher-Gonen, Ronnie; Winter, Deborah; Khalaileh, Abed; Tabib, Yaara; Attal, Tsipora; Nassar, Maria; Zelentsova, Katya; Kisos, Hen; Zenke, Martin; Seré, Kristin; Hieronymus, Thomas; Burstyn-Cohen, Tal; Amit, Ido; Wilensky, Asaf; Hovav, Avi-Hai

    2015-08-18

    Langerhans cells (LCs) populate the mucosal epithelium, a major entry portal for pathogens, yet their ontogeny remains unclear. We found that, in contrast to skin LCs originating from self-renewing radioresistant embryonic precursors, oral mucosal LCs derive from circulating radiosensitive precursors. Mucosal LCs can be segregated into CD103(+)CD11b(lo) (CD103(+)) and CD11b(+)CD103(-) (CD11b(+)) subsets. We further demonstrated that similar to non-lymphoid dendritic cells (DCs), CD103(+) LCs originate from pre-DCs, whereas CD11b(+) LCs differentiate from both pre-DCs and monocytic precursors. Despite this ontogenetic discrepancy between skin and mucosal LCs, the transcriptomic signature and immunological function of oral LCs highly resemble those of skin LCs but not DCs. These findings, along with the epithelial position, morphology, and expression of the LC-associated phenotype strongly suggest that oral mucosal LCs are genuine LCs. Collectively, in a tissue-dependent manner, murine LCs differentiate from at least three distinct precursors (embryonic, pre-DC, and monocytic) in steady state. PMID:26231115

  1. MN1-Fli1 oncofusion transforms murine hematopoietic progenitor cells into acute megakaryoblastic leukemia cells.

    PubMed

    Wenge, D V; Felipe-Fumero, E; Angenendt, L; Schliemann, C; Schmidt, E; Schmidt, L H; Thiede, C; Ehninger, G; Berdel, W E; Arteaga, M-F; Mikesch, J-H

    2015-01-01

    Long-term outcome of acute megakaryoblastic leukemia (AMKL) patients without Down's syndrome remains poor. Founding mutations and chimeric oncogenes characterize various AMKL subtypes. However, for around one third of all cases the underlying mechanisms of AMKL leukemogenesis are still largely unknown. Recently, an in-frame fusion of meningeoma 1-friend leukemia virus integration 1 (MN1-Fli1) gene was detected in a child with AMKL. We intended to investigate the potential role of this oncofusion in leukemogenesis of acute myeloid leukemia. Strikingly, expression of MN1-Fli1 in murine hematopoietic progenitor cells was sufficient to induce leukemic transformation generating immature myeloid cells with cytomorphology and expression of surface markers typical for AMKL. Systematic structure function analyses revealed FLS and 3'ETS domains of Fli1 as decisive domains for the AMKL phenotype. Our data highlight an important role of MN1-Fli1 in AMKL leukemogenesis and provide a basis for research assessing the value of this oncofusion as a future diagnostic marker and/or therapeutic target in AMKL patients. PMID:26690545

  2. Murine CMV Infection Induces the Continuous Production of Mucosal Resident T Cells.

    PubMed

    Smith, Corinne J; Caldeira-Dantas, Sofia; Turula, Holly; Snyder, Christopher M

    2015-11-10

    Cytomegalovirus (CMV) is a herpesvirus that persists for life and maintains extremely large numbers of T cells with select specificities in circulation. However, it is unknown how viral persistence impacts T cell populations in mucosal sites. We found that many murine (M)CMV-specific CD8s in mucosal tissues became resident memory T cells (TRM). These cells adopted an intraepithelial localization in the salivary gland that correlated with, but did not depend on, expression of the integrin CD103. MCMV-specific TRM cells formed early after infection, and spleen-localized cells had reduced capacities to become TRM at late times. Surprisingly, however, small numbers of new TRM cells were formed from the circulating pool throughout infection, favoring populations maintained at high levels in the blood and shifting the immunodominance within the TRM populations over time. These data show that mucosal TRM populations can be dynamically maintained by a persistent infection. PMID:26526996

  3. Murine CMV infection induces the continuous production of mucosal resident T cells

    PubMed Central

    Smith, Corinne J.; Caldeira-Dantas, Sofia; Turula, Holly; Snyder, Christopher M.

    2015-01-01

    Summary Cytomegalovirus (CMV) is a herpesvirus that persists for life and maintains extremely large numbers of T cells with select specificities in circulation. However, it is unknown how viral persistence impacts T cell populations in mucosal sites. We found that many murine (M)CMV-specific CD8s in mucosal tissues became resident memory T cells (TRM). These cells adopted an intraepithelial localization in the salivary gland that correlated with, but did not depend on, expression of the integrin CD103. MCMV-specific TRM cells formed early after infection and spleen-localized cells had reduced capacities to become TRM at late times. Surprisingly however, small numbers of new TRM cells were formed from the circulating pool throughout infection, favoring populations maintained at high levels in the blood and shifting the immunodominance within the TRM populations over time. These data show that mucosal TRM populations can be dynamically maintained by a persistent infection. PMID:26526996

  4. Direct demonstration of murine thymus-dependent cell surface endogenous immunoglobin.

    PubMed Central

    Szenberg, A; Marchalonis, J J; Warner, N L

    1977-01-01

    Antisera raised in mammals to murine immunoglobulin (Ig) do not detect surface Ig on thymus-dependent (T) lymphoma cells as assessed by immunofluorescence analysis. In contrast, chicken antibodies, produced against the (Fab)2 fragment of normal mouse IgG and purified by binding to and elution from IgG-Sepharose 4B, give strong indirect fluorescence with murine T cells and cultured T lymphoma cells. The surface Ig caps, is shed, and reappears, indicating that it is of endogenous origin. Nonlymphoid tumor cells of various myeloid types do not bind this reagent, even though they bear avid Fc receptors. The capacity of chicken antibodies to bind to both bone-marrow-dependent and T cell lymphomas was abolished by adsorption with myeloma-derived kappa chains coupled to Sepharose. The kappa antigenic determinant recognized by the chicken antibodies may thus be different from that seen by mammalian antibodies, and the degree of exposure of Ig on the T lymphoma surface might also affect ease of detectability with these reagents. These data provide direct evidence that T lymphocytes and T lymphoma cells express and synthesize a surface Ig containing determinants that at least 'crossreact with bone-marrow-cell-derived kappa chains. Images PMID:405673

  5. Sublethal oxidative stress inhibits tumor cell adhesion and enhances experimental metastasis of murine mammary carcinoma.

    PubMed

    Kundu, N; Zhang, S; Fulton, A M

    1995-01-01

    We have postulated that murine mammary tumor progression is fueled, in part, by tumor-associated macrophages that deliver sub-lethal oxidative stress to tumor cells. In the present study, we determined whether oxidative stress would affect murine mammary tumor cell attachment to laminin and fibronectin, critical functions in the metastatic process. Sublethal oxidative stress generated by exposure of cells to hydrogen peroxide (H2O2, 1-1000 microM/L) inhibited tumor cell attachment to immobilized laminin or fibronectin. This oxidant effect was blocked in the presence of catalase which removes H2O2. The inhibitory effect on attachment was rapid, with significant inhibition occurring at 5 min; total inhibition was achieved at 60 min with 1 mM H2O2. The oxidative stress effect was partially reversible at 20 h post-treatment and occurred at concentrations of H2O2 that do not adversely affect cell viability or growth. Pretreatment of tumor cells with H2O2 or hypoxanthanine and xanthine oxidase (to generate superoxide radical and H2O2) prior to intravenous injection, enhanced experimental lung tumor colony formation. The enhancement of experimental metastatic potential with enzyme-generated oxidative stress was completely reversed by catalase; the H2O2-mediated enhancement was only partially reversed with catalase. Thus, treatments that inhibit tumor cell attachment to extracellular matrix proteins in vitro enhance experimental metastasis in vivo. PMID:7820952

  6. 1.8 Astroms Structure of Murine GITR Ligand Dimer Expressed in Drosophila Melanogaster S2 Cells

    SciTech Connect

    Chattopadhyay, K.; Ramagopal, U; Nathenson, S; Almo, S

    2009-01-01

    Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique 'strand-exchanged' dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 {angstrom} resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical 'strand-exchanged' dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.

  7. Specific uptake of serotonin by murine lymphoid cells

    SciTech Connect

    Jackson, J.C.; Walker, R.F.; Brooks, W.H.; Roszman, T.L.

    1986-03-01

    Recently the authors confirmed and extended earlier observations that serotonin (5-hydroxytryptamine, 5HT) can influence immune function. Both 5HT and its precursor, 5-hydroxytryptophan inhibit the primary, in vivo antibody response to sheep red blood cells, in mice. Here, the authors report specific in vitro association of this amine with mouse splenocytes. Spleen cells from 6-8 week old CBA/J mice incorporated /sup 3/H-5HT(10/sup -8/ to 2.5 x 10/sup -6/M) in a saturable manner, at 37/sup 0/C. Specificity of uptake was indicated by competition with excess (10/sup -5/M) unlabelled 5HT and with 10/sup -5/M fluoxetine, a selective inhibitor of active 5HT reuptake in rat brain. The 5HT receptor antagonists, methysergide and cyproheptadine, also blocked 5HT uptake. Cell lysis and displacement studies revealed largely intracellular accumulation of /sup 3/H-5HT with little membrane association, in splenocytes. Hofstee analysis of uptake kinetics yielded an apparent Km of 0.82 +/- 0.22 x 10/sup -7/M and Vmax of 501 +/- 108 pM/3 x 10/sup 6/ cells/10 min. Spleen cells fractionated on Sephadex G10 showed virtually no specific 5HT uptake while peritoneal exudate cells from thioglycollate treated mice displayed 5HT uptake kinetics similar to those of splenocytes. The site of specific /sup 3/H-5HT incorporation within a population of spleen cells and the functional significance of this phenomenon to immunomodulation by 5HT remain to be elucidated.

  8. Dielectrophoresis and electrorotation of neurospora slime and murine myeloma cells.

    PubMed

    Gimsa, J; Marszalek, P; Loewe, U; Tsong, T Y

    1991-10-01

    Dielectrophoresis and electrorotation are commonly used to measure dielectric properties and membrane electrical parameters of biological cells. We have derived quantitative relationships for several critical points, defined in Fig. A 1, which characterize the dielectrophoretic spectrum and the electrorotational spectrum of a cell, based on the single-shell model (Pauly, H., and H.P. Schwan, 1959. Z. Naturforsch. 14b:125-131; Sauer, F.A. 1985. Interactions between Electromagnetic Field and Cells. A. Chiabrera, C. Nicolini, and H.P. Schwan, editors. Plenum Publishing Corp., New York. 181-202). To test these equations and to obtain membrane electrical parameters, a technique which allowed simultaneous measurements of the dielectrophoresis and the electrorotation of single cells in the same chamber, was developed and applied to the study of Neurospora slime and the Myeloma Tib9 cell line. Membrane electrical parameters were determined by the dependence of the first critical frequency of dielectrophoresis (fct1) and the first characteristic frequency of electrorotation (fc1) on the conductivity of the suspending medium. Membrane conductances of Neurospora slime and Myeloma also were found to be 500 and 380 S m-2, respectively. Several observations indicate that these cells are more complex than that described by the single-shell model. First, the membrane capacities from fct1 (0.81 x 10(-2) and 1.55 x 10(-2) F m-2 for neurospora slime and Myeloma, respectively) were at least twice those derived from fc1. Second, the electrorotation spectrum of Myeloma cells deviated from the single-shell like behavior. These discrepancies could be eliminated by adapting a three-shell model (Furhr, G., J. Gimsa, and R. Glaser. 1985. Stud. Biophys. 108:149-164). Apparently, there was more than one membrane relaxation process which could influence the lower frequency region of the beta-dispersion. fct1 of Myeloma in a medium of given external conductivity were found to be similar for most cells, but for some a dramatically increased fct1 was recorded. Model analysis suggested that a decrease in the cytoplasmatic conductivity due to a drastic ion loss in a cell could cause this increase in fct1. Model analysis also suggested that the electrorotation spectrum in the counter-field rotation range and fc1 would be more sensitive to conductivity changes of the cytoplasmic fluid and to the influence of internal membranes than would fct1, although the latter would be sensitive to changes in capacitance of the cytoplasmic membranes. PMID:1835890

  9. Engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation.

    PubMed

    van der Schaft, Daisy W J; van Spreeuwel, Ariane C C; Boonen, Kristel J M; Langelaan, Marloes L P; Bouten, Carlijn V C; Baaijens, Frank P T

    2013-01-01

    Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative (1). The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues (2,3). Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts (4), neonatal muscle derived progenitor cells (5), cells derived from adult muscle tissues from other species such as human (6) or even induced pluripotent stem cells (iPS cells) (7). Cell contractility causes alignment of the cells along the long axis of the construct (8,9) and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent (8). Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary. PMID:23542531

  10. Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

    PubMed Central

    van der Schaft, Daisy W. J.; van Spreeuwel, Ariane C. C.; Boonen, Kristel J. M.; Langelaan, Marloes L. P.; Bouten, Carlijn V. C.; Baaijens, Frank P. T.

    2013-01-01

    Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary. PMID:23542531

  11. Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro

    SciTech Connect

    Srivastava, Anand S.; Kaushal, Sharmeela; Mishra, Rangnath; Lane, Thomas A.; Carrier, Ewa . E-mail: assrivastava@ucsd.edu

    2006-07-28

    Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, and direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes ({alpha}-globin, {beta}H-1 globin, {beta}-major globin, {epsilon} -globin, and {zeta}-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, {epsilon}-globin, {gamma}-globin, {beta}H1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the ES cells of secondary EB origin cultured in media containing dexamethasone showed a down-regulation of GATA-3 and an up-regulation of GATA-1, Flk-1, and Epo-R in comparison to the two other cytokines and growth factor combinations containing media. The secondary differentiation also showed an enhanced production of erythrocytic precursors in dexamethasone containing media in comparison to that in the control media. Our results indicate that dexamethasone can prove to be an effective agent which can be employed to enhance differentiation towards erythrocytic progenitors from ES cells.

  12. Granzyme D is a novel murine mast cell protease that is highly induced by multiple pathways of mast cell activation.

    PubMed

    Rönnberg, Elin; Calounova, Gabriela; Guss, Bengt; Lundequist, Anders; Pejler, Gunnar

    2013-06-01

    Granzymes are serine proteases known mostly for their role in the induction of apoptosis. Granzymes A and B have been extensively studied, but relatively little is known about granzymes C to G and K to M. T cells, lymphohematopoietic stromal cells, and granulated metrial gland cells express granzyme D, but the function of granzyme D is unknown. Here we show that granzyme D is expressed by murine mast cells and that its level of expression correlates positively with the extent of mast cell maturation. Coculture of mast cells with live, Gram-positive bacteria caused a profound, Toll-like receptor 2 (TLR2)-dependent induction of granzyme D expression. Granzyme D expression was also induced by isolated bacterial cell wall components, including lipopolysaccharide (LPS) and peptidoglycan, and by stem cell factor, IgE receptor cross-linking, and calcium ionophore stimulation. Granzyme D was released into the medium in response to mast cell activation. Granzyme D induction was dependent on protein kinase C and nuclear factor of activated T cells (NFAT). Together, these findings identify granzyme D as a novel murine mast cell protease and implicate granzyme D in settings where mast cells are activated, such as bacterial infection and allergy. PMID:23529614

  13. Characterization of tumor cell lines derived from murine gammaherpesvirus-68-infected mice.

    PubMed Central

    Usherwood, E J; Stewart, J P; Nash, A A

    1996-01-01

    Cell lines were derived from mice with murine gammaherpesvirus-68 (MHV-68)-associated lymphoproliferative disease. Four were of an ambiguous phenotype and were MHV-68 negative. One, S11, was a B lymphocyte that contained MHV-68 genomes in both linear and episomal forms and released virus. The line was clonable and grew into tumors in nude mice. This is the first naturally occurring MHV-68-positive B-cell line to be generated, and it will be an invaluable tool for the study of MHV-68 latency. PMID:8709292

  14. Modeling the pathways of energy balance using the N1E-115 murine neuroblastoma cell line.

    PubMed

    Roth, Jonathan D; Yee, Daniel K; Kisley, Lori R; Fluharty, Steven J

    2002-06-30

    A good in vitro model within which to investigate molecular interactions between feeding relevant neuropeptide systems has been lacking. Consequently, we began using reverse transcriptase-polymerase chain reaction (RT-PCR) to screen various neuronal cell lines for the presence of feeding relevant neuropeptides and receptors. N1E-115 murine neuroblastoma cells have emerged as an attractive candidate for further analysis because they contain mRNA for a variety of key systems implicated in the regulation of energy homeostasis. PMID:12106700

  15. Disruption of canonical TGFβ-signaling in murine coronary progenitor cells by low level arsenic

    SciTech Connect

    Allison, Patrick; Huang, Tianfang; Broka, Derrick; Parker, Patti; Barnett, Joey V.; Camenisch, Todd D.

    2013-10-01

    Exposure to arsenic results in several types of cancers as well as heart disease. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types including smooth muscle cells which contribute to the coronary vessels. The TGFβ family of ligands and receptors is essential for developmental cardiac epithelial to mesenchymal transition (EMT) and differentiation into coronary smooth muscle cells. In this in vitro study, 18 hour exposure to 1.34 μM arsenite disrupted developmental EMT programming in murine epicardial cells causing a deficit in cardiac mesenchyme. The expression of EMT genes including TGFβ2, TGFβ receptor-3, Snail, and Has-2 are decreased in a dose-dependent manner following exposure to arsenite. TGFβ2 cell signaling is abrogated as detected by decreases in phosphorylated Smad2/3 when cells are exposed to 1.34 μM arsenite. There is also loss of nuclear accumulation pSmad due to arsenite exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenite does not block TGFβ2 mediated smooth muscle cell differentiation by epicardial cells. Overall these results show that arsenic exposure blocks developmental EMT gene programming in murine coronary progenitor cells by disrupting TGFβ2 signals and Smad activation, and that smooth muscle cell differentiation is refractory to this arsenic toxicity. - Highlights: • Arsenic blocks TGFβ2 induced expression of EMT genes. • Arsenic blocks TGFβ2 triggered Smad2/3 phosphorylation and nuclear translocation. • Arsenic blocks epicardial cell differentiation into cardiac mesenchyme. • Arsenic does not block TGFβ2 induced smooth muscle cell differentiation.

  16. Estrogen and progesterone together expand murine endometrial epithelial progenitor cells

    PubMed Central

    Janzen, DM; Cheng, D; Schafenacker, AM; Paik, DY; Goldstein, AS; Witte, ON; Jaroszewicz, A; Pellegrini, M; Memarzadeh, S

    2013-01-01

    Synchronous with massive shifts in reproductive hormones, the uterus and its lining the endometrium expand to accommodate a growing fetus during pregnancy. In the absence of an embryo the endometrium, composed of epithelium and stroma, undergoes numerous hormonally regulated cycles of breakdown and regeneration. The hormonally mediated regenerative capacity of the endometrium suggests that signals that govern the growth of endometrial progenitors must be regulated by estrogen and progesterone. Here we report an antigenic profile for isolation of mouse endometrial epithelial progenitors. These cells are EpCAM+CD44+ITGA6hiThy1−PECAM1−PTPRC−Ter119−, comprise a minor subpopulation of total endometrial epithelia and possess a gene expression profile that is unique and different from other cells of the endometrium. The epithelial progenitors of the endometrium could regenerate in vivo, undergo multi-lineage differentiation and proliferate. We show that the number of endometrial epithelial progenitors is regulated by reproductive hormones. Co-administration of estrogen and progesterone dramatically expanded the endometrial epithelial progenitor cell pool. This effect was not observed when estrogen or progesterone was administered alone. Despite the remarkable sensitivity to hormonal signals, endometrial epithelial progenitors do not express estrogen or progesterone receptors. Therefore their hormonal regulation must be mediated through paracrine signals resulting from binding of steroid hormones to the progenitor cell niche. Discovery of signaling defects in endometrial epithelial progenitors or their niche can lead to development of better therapies in diseases of the endometrium. PMID:23341289

  17. Combined chemotherapy and ALA-based photodynamic therapy in leukemic murine cells.

    PubMed

    Diez, Berenice; Ernst, Glenda; Teijo, María Julieta; Batlle, Alcira; Hajos, Silvia; Fukuda, Haydée

    2012-09-01

    The effects of combined administration of doxorubicin (DOX) and vincristine (VCR), with 5-aminolevulinic acid photodynamic treatment (ALA-PDT), were analyzed in sensitive murine leukemic cell lines (LBR-) and DOX and VCR chemoresistant LBR-D160 and LBR-V160 cell lines. Low doses of DOX and VCR increased anti-cancer effect of ALA-PDT in LBR-cells. Decrease in cell survival was higher when the combination VCR+ALA-PDT was used compared to DOX+ALA-PDT. Resistant cell lines LBR-D160 and LBR-V160 were sensitive to ALA-PDT; however, no changes occured when combining therapies. Thus, ALA-PDT can overcome drug resistance and is a good candidate for using treating multidrug resistant (MDR) cells. PMID:22613191

  18. Control of beta-interferon expression in murine embryonal carcinoma F9 cells.

    PubMed Central

    Francis, M K; Lehman, J M

    1989-01-01

    Murine embryonal carcinoma F9 cells, a tissue culture model for early embryonic development, do not produce interferon (IFN) in response to poly(I-C), as determined by an antiviral assay. RNase protection analyses were used to examine total RNA extracted from the cells for the presence of beta-IFN RNA. Whereas F9 cells differentiated in vitro with retinoic acid produced a biologically active protein as well as beta-IFN RNA in response to poly(I-C), undifferentiated F9 cells produced no detectable beta-IFN RNA even in the presence of cycloheximide, an IFN-superinducing agent. These results show that undifferentiated embryonal carcinoma cells do not accumulate beta-IFN RNA in response to an IFN-inducing agent, suggesting a transcriptional regulatory mechanism. However, this control mechanism is altered upon differentiation, since the gene can be transcriptionally activated in retinoic acid-differentiated cells. Images PMID:2796997

  19. Oatp-associated uptake and toxicity of microcystins in primary murine whole brain cells

    SciTech Connect

    Feurstein, D.; Holst, K.; Fischer, A.; Dietrich, D.R.

    2009-01-15

    Microcystins (MCs) are naturally occurring cyclic heptapeptides that exhibit hepato-, nephro- and possibly neurotoxic effects in mammals. Organic anion transporting polypeptides (rodent Oatp/human OATP) appear to be specifically required for active uptake of MCs into hepatocytes and kidney epithelial cells. Based on symptoms of neurotoxicity in MC-intoxicated patients and the presence of Oatp/OATP at the blood-brain-barrier (BBB) and blood-cerebrospinal-fluid-barrier (BCFB) it is hypothesized that MCs can be transported across the BBB/BCFB in an Oatp/OATP-dependent manner and can induce toxicity in brain cells via inhibition of protein phosphatase (PP). To test these hypotheses, the presence of murine Oatp (mOatp) in primary murine whole brain cells (mWBC) was investigated at the mRNA and protein level. MC transport was tested by exposing mWBCs to three different MC-congeners (MC-LR, -LW, -LF) with/without co-incubation with the OATP/Oatp-substrates taurocholate (TC) and bromosulfophthalein (BSP). Uptake of MCs and cytotoxicity was demonstrated via MC-Western blot analysis, immunocytochemistry, cell viability and PP inhibition assays. All MC congeners bound covalently and inhibited mWBC PP. MC-LF was the most cytotoxic congener followed by -LW and -LR. The lowest toxin concentration significantly reducing mWBC viability after 48 h exposure was 400 nM (MC-LF). Uptake of MCs into mWBCs was inhibited via co-incubation with excess TC (50 and 500 {mu}M) and BSP (50 {mu}M). MC-Western blot analysis demonstrated a concentration-dependent accumulation of MCs. In conclusion, the in vitro data support the assumed MC-congener-dependent uptake in a mOatp-associated manner and cytotoxicity of MCs in primary murine whole brain cells.

  20. Murine lipid phosphate phosphohydrolase-3 acts as a cell-associated integrin ligand

    SciTech Connect

    Humtsoe, Joseph O.; Bowling, Rodney A.; Feng, Shu; Wary, Kishore K. . E-mail: kwary@ibt.tamhsc.edu

    2005-09-30

    Lipid phosphate phosphohydrolase-3 (LPP3) is a cell surface protein that exhibits ectoenzyme activity. Previously, we identified human LPP3 in a functional assay of angiogenesis and showed that the Arg-Gly-Asp (RGD) motif in the proposed second extracellular domain interacts with a subset of integrins to mediate cell-cell adhesion. In contrast to the RGD domain of human LPP3, murine Lpp3 contains a variant sequence, Arg-Gly-Glu (RGE). Whether the RGE motif of murine Lpp3 mediates cell-cell interaction has not been studied. In this report, we test the hypothesis that the cell adhesion function of the LPP3 protein is conserved across mouse and human. A glutathione S-transferase (GST) fusion protein of the proposed second extracellular loop of the murine Lpp3 sequence (GST-mLpp3-RGE) promoted attachment of cells in a long-term cell adhesion assay. GST-mLpp3-RGE interacted with {alpha}{sub 5}{beta}{sub 1} and {alpha}{sub v}{beta}{sub 3} integrins in a solid-phase ELISA, while a mutant control, GST-hLPP3-RAD, did not. Long-term adhesion of endothelial cells to GST-mLpp3-RGE induced phosphorylation of FAK, SHC, and CAS, whereas adhesion to GST-hLPP3-RAD failed to do so. Upon long-term adhesion both the GST-hLPP3-RGD and GST-mLpp3-RGE substrates bound to the {alpha}{sub 5}{beta}{sub 1} integrin of FRT-{alpha}{sub 5}(+) cells, an interaction that was inhibited by an anti-{alpha}{sub 5} integrin antibody. In addition, a cell aggregation assay showed that the intact mLpp3-RGE protein interacts with {alpha}{sub 5}{beta}{sub 1} and {alpha}{sub v}{beta}{sub 3} integrins expressed by adjacent cells, an interaction that can be blocked by GRGDSP peptides and anti-LPP3-RGD antibodies. These data, together with the known importance of integrins in angiogenesis, provide a mechanism for the function of LPP3 in cell-cell interactions in both human and mouse.

  1. Murine CD83-positive T cells mediate suppressor functions in vitro and in vivo.

    PubMed

    Kreiser, Simon; Eckhardt, Jenny; Kuhnt, Christine; Stein, Marcello; Krzyzak, Lena; Seitz, Christine; Tucher, Christine; Knippertz, Ilka; Becker, Christoph; Günther, Claudia; Steinkasserer, Alexander; Lechmann, Matthias

    2015-02-01

    The CD83 molecule (CD83) is a well-known surface marker present on mature dendritic cells (mDC). In this study, we show that CD83 is also expressed on a subset of T cells which mediate regulatory T cell (Treg)-like suppressor functions in vitro and in vivo. Treg-associated molecules including CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4), glucocorticoid-induced TNFR family-related gene (GITR), Helios and neuropilin-1 (NRP-1) as well as forkhead box protein 3 (FOXP3) were specifically expressed by these CD83(+) T cells. In contrast, CD83(-) T cells showed a naive T cell phenotype with effector T cell properties upon activation. Noteworthy, CD83(-) T cells were not able to upregulate CD83 despite activation. Furthermore, CD83(+) T cells suppressed the proliferation and inflammatory cytokine release of CD83(-) T cells in vitro. Strikingly, stimulated CD83(+) T cells released soluble CD83 (sCD83), which has been reported to possess immunosuppressive properties. In vivo, using the murine transfer colitis model we could show that CD83(+) T cells were able to suppress colitis symptoms while CD83(-) T cells possessed effector functions. In addition, this CD83 expression is also conserved on expanded human Treg. Thus, from these studies we conclude that CD83(+) T cells share important features with regulatory T cells, identifying CD83 as a novel lineage marker to discriminate between different T cell populations. PMID:25151500

  2. Zinc deprivation impairs growth factor-stimulated calcium influx into murine 3T3 cells associated with decreased cell proliferation.

    PubMed

    O'Dell, Boyd L; Browning, Jimmy D

    2011-06-01

    Zinc plays a critical role in growth, a process that depends primarily on cell proliferation. Murine fibroblasts, Swiss 3T3 cells, were used to explore the hypothesis that a critical role of zinc in cell proliferation relates to its function in calcium influx. Cells were deprived of zinc by an impermeant chelator, diethylenetriaminepentaacetate (0.6 mmol/L), and low-calcium status was achieved by using a low- (<5 μmol/L) calcium medium. Cells were stimulated by a composite of growth factors (GF): platelet-derived GF, insulin-like GF-I, and epidermal GF. GF stimulation of cell proliferation was assessed by the incorporation of tritiated thymidine and calcium influx by the increase in fluorescence of cells loaded with Fluo-4. Proliferation was dependent on both zinc and calcium and they interacted in this process. GF stimulated an immediate sharp increase in intracellular calcium, indicative of internal calcium release, which peaked within 1 min and decreased to an elevated plateau, a pattern typical of a store-operated calcium channel. The sustained calcium influx of zinc-deprived cells was markedly lower than that of supplemented cells. Verapamil, a calcium channel blocker, also depressed both cell proliferation and calcium influx. In summary, zinc deficiency impaired GF-stimulated calcium influx into murine fibroblasts in association with decreased cell proliferation. PMID:21508206

  3. Retrovirally transduced murine T lymphocytes expressing FasL mediate effective killing of prostate cancer cells

    PubMed Central

    Symes, JC; Siatskas, C; Fowler, DH; Medin, JA

    2010-01-01

    Adoptively transferred T cells possess anticancer activities partially mediated by T-cell FasL engagement of Fas tumor targets. However, antigen-induced T-cell activation and clonal expansion, which stimulates FasL activity, is often inefficient in tumors. As a gene therapy approach to overcome this obstacle, we have created oncoretroviral vectors to overexpress FasL or non-cleavable FasL (ncFasL) on murine T cells of a diverse T-cell receptor repertoire. Expression of c-FLIP was also engineered to prevent apoptosis of transduced cells. Retroviral transduction of murine T lymphocytes has historically been problematic, and we describe optimized T-cell transduction protocols involving CD3/CD28 co-stimulation of T cells, transduction on ice using concentrated oncoretrovirus, and culture with IL-15. Genetically modified T cells home to established prostate cancer tumors in vivo. Co-stimulated T cells expressing FasL, ncFasL and ncFasL/c-FLIP each mediated cytotoxicity in vitro against RM-1 and LNCaP prostate cancer cells. To evaluate the compatibility of this approach with current prostate cancer therapies, we exposed RM-1, LNCaP, and TRAMP-C1 cells to radiation, mitoxantrone, or docetaxel. Fas and H-2b expression were upregulated by these methods. We have developed a novel FasL-based immuno-gene therapy for prostate cancer that warrants further investigation given the apparent constitutive and inducible Fas pathway expression in this malignancy. PMID:19096446

  4. Differential effects of protoporphyrin and uroporphyrin on murine mast cells

    SciTech Connect

    Lim, H.W.; Gigli, I.; Wasserman, S.I.

    1987-03-01

    To investigate the mechanisms responsible for the distinct cutaneous manifestations of erythropoietic protoporphyria and porphyria cutanea tarda, the effects of protoporphyrin (PP) and uroporphyrin (URO), the predominant porphyrins in the respective disease, on mast cells were examined. Release of preformed and generated mediators was assessed by the release of radioactivity from cells labeled with (/sup 3/H)serotonin and (/sup 14/C)arachidonic acid, respectively. Clinically relevant doses of PP (25-500 ng/ml) and 396-407 nm irradiation (3-16 X 10(2)J/m2) induced maximal net release of preformed mediators ,f 44.52 +/- 6.6 to 58.01 +/- 4.0% (mean +/- SE). In contrast, irradiation in the presence of URO (50-5000 ng/ml) resulted in less than 5% net release. (3H)Serotonin release induced by PP and irradiation was calcium-independent, and was not enhanced by phorbol 12-myristate 13-acetate, a known activator of protein kinase C. This release was suppressed by catalase, a scavenger of hydrogen peroxide. Furthermore, irradiation in the presence of PP, but not in the presence of URO, resulted in perturbation of cell membrane. Irradiation in the presence of PP also resulted in a maximal net release of generated mediators of 9.98 +/- 3.5% (mean +/- SE), whereas similar treatment in the presence of URO induced less than 0.5% net release. These results suggested that the burning, stinging, erythema, and edema experienced by patients with erythropoietic protoporphyria following sun exposure, and the lack of such findings in patients with porphyria cutanea tarda, may be explained, at least in part, by the differential effects of PP and URO on mast cells.

  5. Direct Induction of Trophoblast Stem Cells from Murine Fibroblasts.

    PubMed

    Kubaczka, Caroline; Senner, Claire E; Cierlitza, Monika; Araúzo-Bravo, Marcos J; Kuckenberg, Peter; Peitz, Michael; Hemberger, Myriam; Schorle, Hubert

    2015-11-01

    Trophoblast stem cells (TSCs) arise from the first cell fate decision in the developing embryo and generate extra-embryonic lineages, giving rise to the fetal portion of the placenta. Mouse embryonic and extra-embryonic lineages are strictly separated by a distinct epigenetic barrier, which is not fully overcome following expression of TSC-determining factors in embryonic stem cells. Here, we show that transient expression of Tfap2c, Gata3, Eomes, and Ets2 is sufficient to reprogram mouse embryonic fibroblasts and post-natal tail-tip-derived fibroblasts into induced TSCs (iTSCs) and surmount the epigenetic barrier separating somatic from extra-embryonic lineages. iTSCs share nearly identical morphological characteristics, gene expression profiles, and DNA methylation patterns with blastocyst-derived TSCs. Furthermore, iTSCs display transgene-independent self-renewal, differentiate along extra-embryonic lineages, and chimerize host placentas following blastocyst injection. These findings provide insights into the transcription factor networks governing TSC identity and opportunities for studying the epigenetic barriers underlying embryonic and extra-embryonic lineage segregation. PMID:26412560

  6. Label-Retaining Cells in the Adult Murine Salivary Glands Possess Characteristics of Adult Progenitor Cells

    PubMed Central

    Chibly, Alejandro M.; Querin, Lauren; Harris, Zoey; Limesand, Kirsten H.

    2014-01-01

    Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 500,000 annual cases worldwide. Dysfunction of the salivary glands and associated conditions like xerostomia and dysphagia are often developed by these patients, greatly diminishing their life quality. Current preventative and palliative care fail to deliver an improvement in the quality of life, thus accentuating the need for regenerative therapies. In this study, a model of label retaining cells (LRCs) in murine salivary glands was developed, in which LRCs demonstrated proliferative potential and possessed markers of putative salivary progenitors. Mice were labeled with 5-Ethynyl-2′-deoxyuridine (EdU) at postnatal day 10 and chased for 8 weeks. Tissue sections from salivary glands obtained at the end of chase demonstrated co-localization between LRCs and the salivary progenitor markers keratin 5 and keratin 14, as well as kit mRNA, indicating that LRCs encompass a heterogeneous population of salivary progenitors. Proliferative potential of LRCs was demonstrated by a sphere assay, in which LRCs were found in primary and secondary spheres and they co-localized with the proliferation marker Ki67 throughout sphere formation. Surprisingly, LRCs were shown to be radio-resistant and evade apoptosis following radiation treatment. The clinical significance of these findings lie in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction. PMID:25238060

  7. An ES-Like Pluripotent State in FGF-Dependent Murine iPS cells

    PubMed Central

    Ungaro, Federica; Prigione, Alessandro; Chen, Hsu-Hsin; Welling, Maaike; Eijpe, Maureen; Mostoslavsky, Gustavo; Tesar, Paul; Adjaye, James; Geijsen, Niels; Broccoli, Vania

    2010-01-01

    Recent data demonstrates that stem cells can exist in two morphologically, molecularly and functionally distinct pluripotent states; a naïve LIF-dependent pluripotent state which is represented by murine embryonic stem cells (mESCs) and an FGF-dependent primed pluripotent state represented by murine and rat epiblast stem cells (EpiSCs). We find that derivation of induced pluripotent stem cells (iPSCs) under EpiSC culture conditions yields FGF-dependent iPSCs from hereon called FGF-iPSCs) which, unexpectedly, display naïve ES-like/ICM properties. FGF-iPSCs display X-chromosome activation, multi-lineage differentiation, teratoma competence and chimera contribution in vivo. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions. Characterization of the key molecular signalling pathways revealed FGF-iPSCs to depend on the Activin/Nodal and FGF pathways, while signalling through the JAK-STAT pathway is not required for FGF-iPS cell maintenance. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions. PMID:21209851

  8. Index sorting resolves heterogeneous murine hematopoietic stem cell populations

    PubMed Central

    Schulte, Reiner; Wilson, Nicola K.; Prick, Janine C.M.; Cossetti, Chiara; Maj, Michal K.; Gottgens, Berthold; Kent, David G.

    2015-01-01

    Recent advances in the cellular and molecular biology of single stem cells have uncovered significant heterogeneity in the functional properties of stem cell populations. This has prompted the development of approaches to study single cells in isolation, often performed using multiparameter flow cytometry. However, many stem cell populations are too rare to test all possible cell surface marker combinations, and virtually nothing is known about functional differences associated with varying intensities of such markers. Here we describe the use of index sorting for further resolution of the flow cytometric isolation of single murine hematopoietic stem cells (HSCs). Specifically, we associate single-cell functional assay outcomes with distinct cell surface marker expression intensities. High levels of both CD150 and EPCR associate with delayed kinetics of cell division and low levels of differentiation. Moreover, cells that do not form single HSC-derived clones appear in the 7AADdim fraction, suggesting that even low levels of 7AAD staining are indicative of less healthy cell populations. These data indicate that when used in combination with single-cell functional assays, index sorting is a powerful tool for refining cell isolation strategies. This approach can be broadly applied to other single-cell systems, both to improve isolation and to acquire additional cell surface marker information. PMID:26051918

  9. Magnetite- and maghemite-induced different toxicity in murine alveolar macrophage cells.

    PubMed

    Park, Eun-Jung; Umh, Ha Nee; Choi, Dong-Hyuk; Cho, Myung Haing; Choi, Wookhee; Kim, Sang-Wook; Kim, Younghun; Kim, Jae-Ho

    2014-08-01

    The unique properties of nanoparticles and biological systems are important factors affecting the biological response following nanoparticle exposure. Iron oxide nanoparticles are classified mainly as magnetite (M-FeNPs) and maghemite (NM-FeNPs). In our previous study, NM-FeNPs induced autophagic cell death in RAW264.7, a murine peritoneal macrophage cell line, which has excellent lysosomal activity. In this study, we compared the toxicity of M-FeNPs and NM-FeNPs in MH-S, a murine alveolar macrophage cell line, which has relatively low lysosomal activity. At 24 h post-exposure, M-FeNPs decreased cell viability and ATP production, and elevated the levels of reactive oxygen species, nitric oxide, and pro-inflammatory cytokines to a higher extent than NM-FeNPs. Damage of mitochondria and the endoplasmic reticulum and the down-regulation of mitochondrial function and transcription-related genes were also higher in cells exposed to M-FeNPs than in cells exposed to NM-FeNPs (50 μg/ml). In addition, cells exposed to M-FeNPs (50 μg/ml) showed an increase in the number of autophagosome-like vacuoles, whereas cells exposed to NM-FeNPs formed large vacuoles in the cytosol. However, an autophagy-related molecular response was not induced by exposure to either FeNPs, unlike the results seen in our previous study with RAW264.7 cells. We suggest that M-FeNPs induced higher toxicity compared to NM-FeNPs in MH-S cells, and lysosomal activity plays an important role in determining cell death pathway. PMID:24525745

  10. FimH Can Directly Activate Human and Murine Natural Killer Cells via TLR4

    PubMed Central

    Mian, M Firoz; Lauzon, Nicole M; Andrews, David W; Lichty, Brian D; Ashkar, Ali A

    2010-01-01

    Although the importance of natural killer (NK) cells in innate immune responses against tumors or viral infections are well documented, their ability to directly recognize pathogens is less well defined. We have recently reported FimH, a bacterial fimbrial protein, as a novel Toll-like receptor (TLR)4 ligand that potently induces antiviral responses. Here, we investigated whether FimH either directly or indirectly can activate human and murine NK cells. We demonstrate that FimH potently activates both human and murine NK cells in vitro to induce cytokines [interferon (IFN)-γ and tumor necrosis factor (TNF)-α] and cytotoxicity. Importantly, NK cells directly recognize FimH-expressing pathogens as FimH+, but not FimH−, bacteria were able to activate human NK cells. FimH activation of NK cells required TLR4 and MyD88 signaling, as NK cells from both TLR4−/− and MyD88−/− mice as well as human NK-92 cells, which lack TLR4, were all unresponsive to FimH. In addition, TLR4 neutralization significantly abrogated the response of human NK cells to FimH. Activation of purified NK cells by FimH was independent of lipopolysaccharide (LPS) or other bacterial contaminations. These data demonstrate for the first time that highly purified NK cells directly recognize and respond to FimH via TLR4–MyD88 pathways to aid innate protection against cancer or microbial infections. PMID:20442710

  11. Lipid derivatives activate GPR119 and trigger GLP-1 secretion in primary murine L-cells

    PubMed Central

    Moss, Catherine E.; Glass, Leslie L.; Diakogiannaki, Eleftheria; Pais, Ramona; Lenaghan, Carol; Smith, David M.; Wedin, Marianne; Bohlooly-Y, Mohammad; Gribble, Fiona M.; Reimann, Frank

    2016-01-01

    Aims/hypothesis Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from proglucagon, which is released from intestinal L-cells and increases insulin secretion in a glucose dependent manner. GPR119 is a lipid derivative receptor present in L-cells, believed to play a role in the detection of dietary fat. This study aimed to characterize the responses of primary murine L-cells to GPR119 agonism and assess the importance of GPR119 for the detection of ingested lipid. Methods GLP-1 secretion was measured from murine primary cell cultures stimulated with a panel of GPR119 ligands. Plasma GLP-1 levels were measured in mice lacking GPR119 in proglucagon-expressing cells and controls after lipid gavage. Intracellular cAMP responses to GPR119 agonists were measured in single primary L-cells using transgenic mice expressing a cAMP FRET sensor driven by the proglucagon promoter. Results L-cell specific knockout of GPR119 dramatically decreased plasma GLP-1 levels after a lipid gavage. GPR119 ligands triggered GLP-1 secretion in a GPR119 dependent manner in primary epithelial cultures from the colon, but were less effective in the upper small intestine. GPR119 agonists elevated cAMP in ∼70% of colonic L-cells and 50% of small intestinal L-cells. Conclusions/interpretation GPR119 ligands strongly enhanced GLP-1 release from colonic cultures, reflecting the high proportion of colonic L-cells that exhibited cAMP responses to GPR119 agonists. Less GPR119-dependence could be demonstrated in the upper small intestine. In vivo, GPR119 in L-cells plays a key role in oral lipid-triggered GLP-1 secretion. PMID:26144594

  12. An In Vitro Murine Model of Vascular Smooth Muscle Cell Mineralization.

    PubMed

    Kelynack, Kristen J; Holt, Stephen G

    2016-01-01

    Vascular calcification (VC) is seen ubiquitously in aging blood vessels and prematurely in disease states like renal failure. It is thought to be driven by a number of systemic and local factors that lead to extra-osseous deposition of mineral in the vascular wall and valves as a common endpoint. The response of resident vascular smooth muscle cell to these dystrophic signals appears to be important in this process. Whilst in vivo models allow the observation of global changes in a pro-calcific environment, identifying the specific cells and mechanisms involved has been largely garnered from in vitro experiments, which provide added benefits in terms of reproducibility, cost, and convenience. Here we describe a 7-21 day cell culture model of calcification developed using immortalized murine vascular smooth muscle cells (MOVAS-1). This model provides a method by which vascular smooth muscle cell involvement and manipulation within a mineralizing domain can be studied. PMID:26676135

  13. Slow and steady cell shrinkage reduces osmotic stress in bovine and murine oocyte and zygote vitrification

    PubMed Central

    Lai, D.; Ding, J.; Smith, G.W.; Smith, G.D.; Takayama, S.

    2015-01-01

    STUDY QUESTION Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal cryodamage? SUMMARY ANSWER The use of a new CPA exchange protocol made possible by automated microfluidics improved oocyte and zygote vitrification with superior morphology as indicated by a smoother cell surface, higher sphericity, higher cytoplasmic lipid retention, less cytoplasmic leakage and higher developmental competence compared with conventional methods. WHAT IS KNOWN ALREADY The use of more ‘steps’ of CPA exposure during the vitrification protocol increases cryosurvival and development in the bovine model. However, such an attempt to eliminate osmotic stress is limited by the practicality of performing numerous precise pipetting steps in a short amount of time. STUDY DESIGN, SIZE, DURATION Murine meiotically competent germinal vesicle intact oocytes and zygotes were harvested from the antral follicles in ovaries and ampulla, respectively. Bovine ovaries were obtained from a local abattoir at random stages of the estrous cycle. A total of 110 murine oocytes, 802 murine zygotes and 52 bovine oocytes were used in this study. PARTICIPANTS/MATERIALS, SETTING, METHODS Microfluidic devices were fabricated using conventional photo- and soft-lithography. CPAs used were 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for equilibration solution and 15% EG, 15% DMSO and 0.5 M sucrose for vitrification solution. End-point analyses include mathematical modeling using Kedem–Katchalsky equations, morphometrics assessed by conventional and confocal microscopy, cytoplasmic lipid quantification by nile red staining, cytoplasmic leakage quantification by fluorescent dextran intercalation and developmental competence analysis by 96 h embryo culture and blastomere quantification. MAIN RESULTS AND THE ROLE OF CHANCE The automated microfluidics protocol decreased the shrinkage rate of the oocyte and zygote by 13.8 times over its manual pipetting alternative. Oocytes and zygotes with a lower shrinkage rate during CPA exposure experienced less osmotic stress resulting in better morphology, higher cell quality and improved developmental competence. This microfluidic procedure resulted in murine zygotes with a significantly smoother cell surface (P < 0.001), more spherical cellular morphology (P < 0.001), increased cytoplasmic lipid retention in vitrified and warmed bovine oocytes (P < 0.01), decreased membrane perforations and cytoplasmic leakage in CPA-exposed murine zygotes (P < 0.05) and improved developmental competence of vitrified and warmed murine zygotes (P < 0.05) than CPA exposure using the current clinically used manual pipetting method. LIMITATIONS, REASONS FOR CAUTION It is necessary to design the microfluidic device to be more user-friendly for widespread use. WIDER IMPLICATIONS OF THE FINDINGS The theory and approach of eliminating osmotic stress by decreasing shrinkage rate is complementary to the prevalent osmotic stress theory in cryobiology which focuses on a minimum cell volume at which the cells shrink. The auto-microfluidic protocol described here has immediate applications for improving animal and human oocyte, zygote and embryo cryopreservation. On a fundamental level, the clear demonstration that at the same minimum cell volume, cell shrinkage rate affects sublethal damage should be broadly useful for cryobiology. STUDY FUNDING/COMPETING INTEREST(S) This project was funded by the National Institutes of Health and the University of Michigan Reproductive Sciences Program. The authors declare no conflicts of interest. PMID:25355589

  14. Isolation, Expansion and Transplantation of Postnatal Murine Progenitor Cells of the Enteric Nervous System

    PubMed Central

    Dettmann, Heike Monika; Zhang, Ying; Wronna, Nadine; Kraushaar, Udo; Guenther, Elke; Mohr, Roland; Neckel, Peter Helmut; Mack, Andreas; Fuchs, Joerg; Just, Lothar; Obermayr, Florian

    2014-01-01

    Neural stem or progenitor cells have been proposed to restore gastrointestinal function in patients suffering from congenital or acquired defects of the enteric nervous system. Various, mainly embryonic cell sources have been identified for this purpose. However, immunological and ethical issues make a postnatal cell based therapy desirable. We therefore evaluated and quantified the potential of progenitor cells of the postnatal murine enteric nervous system to give rise to neurons and glial cells in vitro. Electrophysiological analysis and BrdU uptake studies provided direct evidence that generated neurons derive from expanded cells in vitro. Transplantation of isolated and expanded postnatal progenitor cells into the distal colon of adult mice demonstrated cell survival for 12 weeks (end of study). Implanted cells migrated within the gut wall and differentiated into neurons and glial cells, both of which were shown to derive from proliferated cells by BrdU uptake. This study indicates that progenitor cells isolated from the postnatal enteric nervous system might have the potential to serve as a source for a cell based therapy for neurogastrointestinal motility disorders. However, further studies are necessary to provide evidence that the generated cells are capable to positively influence the motility of the diseased gastrointestinal tract. PMID:24871092

  15. Murine and Human Model Systems for the Study of Dendritic Cell Immunobiology.

    PubMed

    Hargadon, Kristian M

    2016-03-01

    Dendritic cells are a population of innate immune cells that possess their own effector functions as well as numerous regulatory properties that shape the activity of other innate and adaptive cells of the immune system. Following their development from either lymphoid or myeloid progenitors, the function of dendritic cells is tightly linked to their maturation and activation status. Differentiation into specialized subsets of dendritic cells also contributes to the diverse immunologic functions of these cells. Because of the key role played by dendritic cells in the regulation of both immune tolerance and activation, significant efforts have been focused on understanding dendritic cell biology. This review highlights the model systems currently available to study dendritic cell immunobiology and emphasizes the advantages and disadvantages to each system in both murine and human settings. In particular, in vitro cell culture systems involving immortalized dendritic cell lines, ex vivo systems for differentiating and expanding dendritic cells from their precursor populations, and systems for expanding, ablating, and manipulating dendritic cells in vivo are discussed. Emphasis is placed on the contribution of these systems to our current understanding of the development, function, and immunotherapeutic applications of dendritic cells, and insights into how these models might be extended in the future to answer remaining questions in the field are discussed. PMID:25203775

  16. Nitric oxide production by murine spleen cells stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans.

    PubMed

    Sosroseno, Wihaskoro; Herminajeng, Endang; Susilowati, Heni; Budiarti, Sri

    2002-12-01

    The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could induce murine spleen cells to produce nitric oxide (NO). Spleen cells derived from Balb/c mice were stimulated with LPS-A. actinomycetemcomitans or LPS from Escherichia coli for 4 days. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B, and cytokines (IFN-gamma and IL-4) on the production of NO were also assessed. The NO production from the carrageenan-treated spleen cells stimulated with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma was determined. The carrageenan-treated mice were transferred with splenic macrophages and the NO production was assessed from the spleen cells stimulated with LPS-A. actinomycetemcomitans or LPS-A. actinomycetemcomitans and IFN-gamma. The results showed that NO production was detectable in the cultures of spleen cells stimulated with LPS-A. actinomycetemcomitans in a dose-dependent fashion, but was lower than in the cells stimulated with LPS from E. coli. The NO production was blocked by NMMA and polymyxin B. IFN-gamma up-regulated but IL-4 suppressed the production of NO by the spleen cells stimulated with LPS-A. actinomycetemcomitans. The carrageenan-treated spleen cells failed to produce NO after stimulation with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma. Adoptive transfer of splenic macrophages to the carrageenan-treated mice could restore the ability of the spleen cells to produce NO. The results of the present study suggest that LPS-A. actinomycetemcomitans under the regulatory control of cytokines induces murine spleen cells to produce NO and that splenic macrophages are the cellular source of the NO production. Therefore, these results may support the view that NO production by LPS-A. actinomycetemcomitans-stimulated macrophages may play a role in the course of periodontal diseases. PMID:16887678

  17. Generation of a murine hepatic angiosarcoma cell line and reproducible mouse tumor model.

    PubMed

    Rothweiler, Sonja; Dill, Michael T; Terracciano, Luigi; Makowska, Zuzanna; Quagliata, Luca; Hlushchuk, Ruslan; Djonov, Valentin; Heim, Markus H; Semela, David

    2015-03-01

    Hepatic angiosarcoma (AS) is a rare and highly aggressive tumor of endothelial origin with dismal prognosis. Studies of the molecular biology of AS and treatment options are limited as animal models are rare. We have previously shown that inducible knockout of Notch1 in mice leads to spontaneous formation of hepatic AS. The aims of this study were to: (1) establish and characterize a cell line derived from this murine AS, (2) identify molecular pathways involved in the pathogenesis and potential therapeutic targets, and (3) generate a tumor transplantation model. AS cells retained specific endothelial properties such as tube formation activity, as well as expression of CD31 and Von Willebrand factor. However, electron microscopy analysis revealed signs of dedifferentiation with loss of fenestrae and loss of contact inhibition. Microarray and pathway analysis showed substantial changes in gene expression and revealed activation of the Myc pathway. Exposing the AS cells to sorafenib reduced migration, filopodia dynamics, and cell proliferation but did not induce apoptosis. In addition, sorafenib suppressed ERK phosphorylation and expression of cyclin D2. Injection of AS cells into NOD/SCID mice resulted in formation of undifferentiated tumors, confirming the tumorigenic potential of these cells. In summary, we established and characterized a murine model of spontaneous AS formation and hepatic AS cell lines as a useful in vitro tool. Our data demonstrate antitumor activity of sorafenib in AS cells with potent inhibition of migration, filopodia formation, and cell proliferation, supporting further evaluation of sorafenib as a novel treatment strategy. In addition, AS cell transplantation provides a subcutaneous tumor model useful for in vivo preclinical drug testing. PMID:25418579

  18. Murine Hematopoietic Stem Cells and Multipotent Progenitors Express Truncated Intracellular Form of c-kit Receptor

    PubMed Central

    ZAYAS, JENNIFER; SPASSOV, DANISLAV S.; NACHTMAN, RONALD G.; JURECIC, ROLAND

    2011-01-01

    The c-kit receptor plays a vital role in self-renewal and differentiation of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). We have discovered that besides c-kit, the murine multipotent HSC/MPP-like cell line EML expresses the transcript and protein for a truncated intracellular form of c-kit receptor, called tr-kit. Notably, the tr-kit transcript and protein levels were down-regulated during cytokine-induced differentiation of the HSC/MPP-like cell line EML into myeloerythroid lineages. These findings prompted us to analyze tr-kit expression in purified murine fetal liver and bone marrow cell populations containing long-term repopulating (LTR) HSCs, short-term repopulating (STR) HSCs, MPPs, lineage-committed progenitors, and immature blood cells. Remarkably, these studies have revealed that in contrast to more widespread expression of c-kit, tr-kit is transcribed solely in cell populations enriched for LTR-HSCs, STR-HSCs, and MPPs. On the other hand, cell populations in which HSCs and MPPs are either present at a much lower frequency or are absent altogether, cells representing more advanced stages of differentiation into lymphoid and myeloid lineages do not express tr-kit. The observation that tr-kit is co-expressed with c-kit only in more primitive HSC- and MPP-enriched cell populations raises an exciting possibility that tr-kit functions either as a new component of the stem cell factor (SCF)/c-kit pathway or is involved in a novel signaling pathway, present exclusively in HSC and MPPs. Taken together, these findings necessitate functional characterization of tr-kit and analysis of its potential role in the self-renewal, proliferation, and/or differentiation of HSC and multipotent progenitors. PMID:18447649

  19. Murine hematopoietic stem cells and multipotent progenitors express truncated intracellular form of c-kit receptor.

    PubMed

    Zayas, Jennifer; Spassov, Danislav S; Nachtman, Ronald G; Jurecic, Roland

    2008-04-01

    The c-kit receptor plays a vital role in self-renewal and differentiation of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). We have discovered that besides c-kit, the murine multipotent HSC/MPP-like cell line EML expresses the transcript and protein for a truncated intracellular form of c-kit receptor, called tr-kit. Notably, the tr-kit transcript and protein levels were down-regulated during cytokine-induced differentiation of the HSC/MPP-like cell line EML into myeloerythroid lineages. These findings prompted us to analyze tr-kit expression in purified murine fetal liver and bone marrow cell populations containing long-term repopulating (LTR) HSCs, short-term repopulating (STR) HSCs, MPPs, lineage-committed progenitors, and immature blood cells. Remarkably, these studies have revealed that in contrast to more widespread expression of c-kit, tr-kit is transcribed solely in cell populations enriched for LTR-HSCs, STR-HSCs, and MPPs. On the other hand, cell populations in which HSCs and MPPs are either present at a much lower frequency or are absent altogether, cells representing more advanced stages of differentiation into lymphoid and myeloid lineages do not express tr-kit. The observation that tr-kit is co-expressed with c-kit only in more primitive HSC- and MPP-enriched cell populations raises an exciting possibility that tr-kit functions either as a new component of the stem cell factor (SCF)/c-kit pathway or is involved in a novel signaling pathway, present exclusively in HSC and MPPs. Taken together, these findings necessitate functional characterization of tr-kit and analysis of its potential role in the self-renewal, proliferation, and/or differentiation of HSC and multipotent progenitors. PMID:18447649

  20. Stratified epithelial sheets engineered from a single adult murine corneal/limbal progenitor cell

    PubMed Central

    Kawakita, Tetsuya; Shimmura, Shigeto; Hornia, Armand; Higa, Kazunari; Tseng, Scheffer C G

    2008-01-01

    The limbal region of the adult cornea contains stem cells which are ultimately responsible for regeneration of the corneal epithelium during wound repair. However, primarily-isolated murine corneal/limbal epithelial cells rapidly senesce on plastic in a serum-free low [Ca2+] medium, suggesting only transit amplifying cells are promoted. We developed a novel expansion method by seeding at a low cell density (<500 cells/cm2) and prolonging each culture time beyond the lifespan of transit amplifying cells (4 weeks). Expanded cells were uniformly small, negative to K12 keratin, but positive for p63 nuclear staining, and could be subcultured beyond 100 passages. After limiting dilution, one clone (TKE2) was selected that exhibited single cell clonal expansion with a doubling time of 34.2 hrs, and had normal karyotyping, but no anchorage-independent growth. A single cell could be continually expanded to a confluent monolayer on denuded amniotic membrane and became stratified by exposing to the air-medium interface. The resultant stratified epithelium expressed K14 keratin, involucrin, connexin 43 and p63, but not K12 keratin or Pax 6. However, expression of K12 could be up-regulated by increasing extracellular calcium concentration and addition of foetal bovine serum (FBS) at P12, but less so at P85. Therefore, this murine lim-bal/corneal epithelium-derived progenitor cell line still retained the plasticity for adopting corneal lineage differentiation, could be useful for investigating limbal niche cues that may promote corneal epithelial fate decision. PMID:18318692

  1. Modulation of prostaglandin biosynthesis in murine mammary adenocarcinoma tumor cells

    SciTech Connect

    Shalinsky, D.R.

    1988-01-01

    In efforts to exploit the differential oxygen levels within the subcompartments of solid neoplasms, this project has focused on modulating prostaglandin (PG) biosynthesis under aerobic and hypoxic conditions. Mammary adenocarcinoma tumor cells (Line 4526), either intact or sonicated, were incubated with either 2.0 uM {sup 14}C-arachidonic acid (AA) or 20.0 uM {sup 14}C-PGH{sub 2}, respectively. Following metabolism, products were extracted, separated by thin layer chromatography and analyzed by radiochromatographic scan. PGE{sub 2} was predominantly formed with minimal amounts of PGF{sub 2a} or PGD{sub 2}. Indomethacin and ibuprofen inhibited the PGE{sub 2} formation from AA with an IC{sub 50} value of 6.3 {times} 10{sup {minus}8} and 9.6 {times} 10{sup {minus}5}M, respectively. Suspended cells in glass vials were made hypoxic by flushing with N{sub 2} for varying time intervals to study AA metabolism. A time-dependent inhibition of PG biosynthesis was observed under hypoxia, and by 30 min, the PGE{sub 2} synthesis was reduced by 50% which was further inhibited by indomethacin. Misonidazole, a 2-nitroimidazole analogue, partially reversed the inhibition of PGE{sub 2} synthesis under hypoxia by 49% at 100 uM. However, misonidazole did not affect PG biosynthesis under aerobic conditions. The stimulation of PGE{sub 2} biosynthesis by misonidazole under hypoxia was blocked by indomethacin, suggesting that misonidazole can not act independently of the cyclooxygenase.

  2. IL-33 promotes MHC class II expression in murine mast cells

    PubMed Central

    Ito, Tomonobu; Egusa, Chizu; Maeda, Tatsuo; Numata, Takafumi; Nakano, Nobuhiro; Nishiyama, Chiharu; Tsuboi, Ryoji

    2015-01-01

    Mast cells (MCs), recognized as tissue-resident cells of hematopoietic origin, are involved in cellular and pathological manifestations of allergic disorders including atopic dermatitis. IL-33, a member of the IL-1 cytokine family, activates Th2-type immune responses, and promotes the degranulation and maturation of MCs. However, it is uncertain whether IL-33 treatment induces mature mast cells to acquire the characteristics of the monocyte-dendritic cell lineage.We investigated the effect of IL-33 on the MHC class II expression and function of murine mast cells. IL-33-treated mature murine bone marrow-derived mast cells (BMMCs) were analyzed by FACS, real-time PCR, chromatin immunoprecipitation (ChIP) assay, and Western blotting. The morphology and degranulation activity of BMMCs and T-cell activation by BMMCs were also examined. BMMCs treated with IL-33 for 10 days induced cell surface expression of the MHC class II protein, whereas the expression of FcεRI and c-kit was not affected by IL-33. The expression of CIITA, driven from pIII and pIV, was up-regulated in IL-33-treated BMMCs. The amount of PU.1 mRNA and protein significantly increased in IL-33-treated BMMCs. The ChIP assay showed PU.1 binding to CIITA pIII, and enhanced histone acetylation due to IL-33 treatment. Syngeneic T cells were activated by co-culture with IL-33-treated BMMCs, although the expression of the co-stimulatory molecules, CD40, CD80, CD86, and PDL-1, was not detected. Mast cells express MHC class II after prolonged exposure to IL-33, probably due to enhanced recruitment of PU.1 to CIITA pIII, resulting in transactivation of CIITA and MHC class II. IL-33 is an important cytokine in allergic disorders. Mast cells have the ability to express MHC class II after prolonged exposure to IL-33 in a murine model. IL-33 holds a key to understanding the etiology of atopic dermatitis. PMID:26417437

  3. IL-33 promotes MHC class II expression in murine mast cells.

    PubMed

    Ito, Tomonobu; Egusa, Chizu; Maeda, Tatsuo; Numata, Takafumi; Nakano, Nobuhiro; Nishiyama, Chiharu; Tsuboi, Ryoji

    2015-09-01

    Mast cells (MCs), recognized as tissue-resident cells of hematopoietic origin, are involved in cellular and pathological manifestations of allergic disorders including atopic dermatitis. IL-33, a member of the IL-1 cytokine family, activates Th2-type immune responses, and promotes the degranulation and maturation of MCs. However, it is uncertain whether IL-33 treatment induces mature mast cells to acquire the characteristics of the monocyte-dendritic cell lineage.We investigated the effect of IL-33 on the MHC class II expression and function of murine mast cells. IL-33-treated mature murine bone marrow-derived mast cells (BMMCs) were analyzed by FACS, real-time PCR, chromatin immunoprecipitation (ChIP) assay, and Western blotting. The morphology and degranulation activity of BMMCs and T-cell activation by BMMCs were also examined. BMMCs treated with IL-33 for 10 days induced cell surface expression of the MHC class II protein, whereas the expression of Fc?RI and c-kit was not affected by IL-33. The expression of CIITA, driven from pIII and pIV, was up-regulated in IL-33-treated BMMCs. The amount of PU.1 mRNA and protein significantly increased in IL-33-treated BMMCs. The ChIP assay showed PU.1 binding to CIITA pIII, and enhanced histone acetylation due to IL-33 treatment. Syngeneic T cells were activated by co-culture with IL-33-treated BMMCs, although the expression of the co-stimulatory molecules, CD40, CD80, CD86, and PDL-1, was not detected. Mast cells express MHC class II after prolonged exposure to IL-33, probably due to enhanced recruitment of PU.1 to CIITA pIII, resulting in transactivation of CIITA and MHC class II. IL-33 is an important cytokine in allergic disorders. Mast cells have the ability to express MHC class II after prolonged exposure to IL-33 in a murine model. IL-33 holds a key to understanding the etiology of atopic dermatitis. PMID:26417437

  4. Biosynthesis of eicosanoids and transcellular metabolism of leukotrienes in murine bone marrow cells.

    PubMed

    Gijón, Miguel A; Zarini, Simona; Murphy, Robert C

    2007-03-01

    Leukotriene B(4) (LTB(4)) biosynthesis by polymorphonuclear leukocytes (PMNs) is an important factor of inflammatory responses. PMNs also release LTA(4), an unstable intermediate that can be taken up by neighboring cells and metabolized into LTC(4). Most studies of LT synthesis have been carried out using human PMNs, but very little information is available about mouse PMNs. Mouse bone marrow PMNs were found to synthesize eicosanoids upon stimulation with A23187, fMLP, or zymosan. The major eicosanoids produced are LTB(4) and 5-hydroxyeicosatetraenoic acid, with some nonenzymatic products of LTA(4) hydrolysis. No cysteinyl leukotrienes were produced, in contrast to what was observed with human blood neutrophil preparations. Human megakaryoblast-like MEG-01 cells synthesized thromboxane B(2) and prostaglandin E(2) in response to A23187 but produced no 5-lipoxygenase (5-LO)-derived eicosanoids. When mouse bone marrow cells (mBMCs) and MEG-01 cells were stimulated during coincubation, LTC(4) and LTD(4) were produced. Mouse peritoneal macrophages from 5-LO-deficient mice were able to synthesize LTC(4) when incubated with mBMCs from wild-type mice, demonstrating transcellular exchange of LTA(4) from mBMCs into murine peritoneal macrophages. These data demonstrate that murine bone marrow PMNs are a valid model for the study of LT biosynthesis, which now offers the possibility to investigate specific biochemical pathways through the use of transgenic mice. PMID:17179116

  5. Proliferating mesodermal cells in murine embryos exhibiting macrophage and lymphendothelial characteristics

    PubMed Central

    Buttler, Kerstin; Ezaki, Taichi; Wilting, Jörg

    2008-01-01

    Background The data on the embryonic origin of lymphatic endothelial cells (LECs) from either deep embryonic veins or mesenchymal (or circulating) lymphangioblasts presently available remain inconsistent. In various vertebrates, markers for LECs are first expressed in specific segments of embryonic veins arguing for a venous origin of lymph vessels. Very recently, studies on the mouse have strongly supported this view. However, in the chick, we have observed a dual origin of LECs from veins and from mesodermal lymphangioblasts. Additionally, in murine embryos we have detected mesenchymal cells that co-express LEC markers and the pan-leukocyte marker CD45. Here, we have characterized the mesoderm of murine embryos with LEC markers Prox1, Lyve-1 and LA102 in combination with macrophage markers CD11b and F4/80. Results We observed cells co-expressing both types of markers (e.g. Prox1 – Lyve-1 – F4/80 triple-positive) located in the mesoderm, immediately adjacent to, and within lymph vessels. Our proliferation studies with Ki-67 antibodies showed high proliferative capacities of both the Lyve-1-positive LECs of lymph sacs/lymphatic sprouts and the Lyve-1-positive mesenchymal cells. Conclusion Our data argue for a dual origin of LECs in the mouse, although the primary source of embryonic LECs may reside in specific embryonic veins and mesenchymal lymphangioblasts integrated secondarily into lymph vessels. The impact of a dual source of LECs for ontogenetic, phylogenetic and pathological lymphangiogenesis is discussed. PMID:18430230

  6. Effects of Wnt-10b on proliferation and differentiation of murine melanoma cells

    SciTech Connect

    Misu, Masayasu; Ouji, Yukiteru; Kawai, Norikazu; Nishimura, Fumihiko; Nakamura-Uchiyama, Fukumi; Yoshikawa, Masahide

    2015-08-07

    In spite of the strong expression of Wnt-10b in melanomas, its role in melanoma cells has not been elucidated. In the present study, the biological effects of Wnt-10b on murine B16F10 (B16) melanoma cells were investigated using conditioned medium from Wnt-10b-producing COS cells (Wnt-CM). After 2 days of culture in the presence of Wnt-CM, proliferation of B16 melanoma cells was inhibited, whereas tyrosinase activity was increased. An in vitro wound healing assay demonstrated that migration of melanoma cells to the wound area was inhibited with the addition of Wnt-CM. Furthermore, evaluation of cellular senescence revealed prominent induction of SA-β-gal-positive senescent cells in cultures with Wnt-CM. Finally, the growth of B16 melanoma cell aggregates in collagen 3D-gel cultures was markedly suppressed in the presence of Wnt-CM. These results suggest that Wnt-10b represses tumor cell properties, such as proliferation and migration of B16 melanoma cells, driving them toward a more differentiated state along a melanocyte lineage. - Highlights: • Wnt-10b inhibited proliferation and migration of melanoma cells. • Wnt-10b induced tyrosinase activity and senescence of melanoma cells. • Wnt-10b suppressed growth of cell aggregates in collagen 3D-gel cultures. • Wnt-10b represses tumor cell properties, driving them toward a more differentiated state along a melanocyte lineage.

  7. Toll-like receptor ligand activation of murine bone marrow-derived dendritic cells.

    PubMed

    Dearman, Rebecca J; Cumberbatch, Marie; Maxwell, Gavin; Basketter, David A; Kimber, Ian

    2009-04-01

    Dendritic cells (DCs) are required for the initiation of primary immune responses. The pattern of Toll-like receptor (TLR) expression on various subsets of these cells has been shown to differ, suggestive of distinct roles in influencing immune responses. We have examined here the responses of immature DCs derived from murine bone marrow (BMDCs) to a range of TLR ligands. BMDCs cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor were stimulated for 24 hr with ligands to TLR1-2 [Pam(3)Cys-Ser-(Lys)(4) (PAM)], TLR2-6 (macrophage-activating lipopeptide-2 (MALP-2); zymosan or peptidoglycan (PG)], TLR3 (polyinosinic-polycytidylic acid), TLR4 [lipopolysaccharide R515 (LPS)], TLR5 (flagellin), TLR7 (polyuridylic acid) and TLR9 [CpG ODN2395 (CpG)]. DC activation was monitored using membrane marker expression and analysis of culture supernatants for cytokine/chemokine release. Ligands to TLR3 and TLR7 failed to activate BMDCs. All other TLR ligands caused elevated expression of membrane markers. PAM, MALP-2 and LPS induced high-level expression of proinflammatory cytokines and chemokines. Treatment with CpG was associated with a preferential type 1 cytokine and chemokine profile. Zymosan and PG were proinflammatory but also skewed towards a type 2 pattern of cytokines and chemokines. In contrast, flagellin did not cause marked secretion by BMDCs of cytokines or chemokines. These data for BMDCs are largely consistent with the reported TLR repertoire of freshly isolated murine Langerhans cells. In addition, murine BMDCs show selective responses to TLR ligands with respect to general activation, with differentiated cytokine patterns suggestive of potential priming for divergent immune responses. PMID:18778283

  8. Murine retroviral vector producer cells survival and toxicity in the dog liver.

    PubMed

    Link, C J; Seregina, T; Levy, J P; Martin, M; Ackermann, M; Moorman, D W

    2000-01-01

    To develop a safe method to target gene delivery into intrahepatic tumors, we examined the toxicity of intrahepatic (IH) injection of retroviral vector producer cells (VPC) into the canine liver. VPC have been demonstrated to effectively transfer genes in vivo. To evaluate for adverse effects form xenogeneic cell transplantation, mongrel dogs were injected IH with 1 x 10(9) murine LTKOSN.2 VPC divided into three aliquots. The animals were then monitored for acute toxicity induced by the VPC. The intraoperative IH injections of the cells were tolerated without difficulty. Starting 7 days after IH injection, the dogs then received intravenous ganciclovir (GCV) twice daily (5 mg/kg) for 7 days. GCV treatment did not cause significant toxicities. Dogs underwent serial blood tests to evaluate bone marrow, renal, liver and immunological function. Complete blood counts, electrolytes, liver function and renal function tests remained normal except for mild elevations of alkaline phosphatase. Histologic examination of liver tissues from the IH injection site revealed no apparent normal tissue destruction induced by the VPC. Two of the four treated dogs underwent liver biopsy on day 3. These biopsy specimens were cultured and persistent, viable VPC were recovered. The dogs mounted an antibody response to the murine VPC that was first demonstrated 5 days post injection. PCR analysis demonstrated low level gene transfer into dog liver tissue. Overall, our results demonstrate that IH xenogeneic VPC injections are not accompanied by significant adverse effects over a 1 month period following administration into the canine liver. These data support the safety aspects of using murine VPC in Phase I clinical trials. PMID:11212842

  9. Cinnamon extract reduces symptoms, inflammatory mediators and mast cell markers in murine IL-10(-/-) colitis.

    PubMed

    Hagenlocher, Yvonne; Hösel, Angela; Bischoff, Stephan C; Lorentz, Axel

    2016-04-01

    Inflammatory bowel disease (IBD) shows an increasing prevalence and harm in western countries. Conventional therapies are associated with bad compliance and adverse side effects. Natural substances like cinnamon extract (CE) could be an additional therapy. We found recently that CE acts anti-inflammatory on mast cells - discussed of being relevant in IBD. Here, we analysed the effects of CE on murine IL-10(-/-) colitis as model for IBD. Mice were treated 12 weeks with or without CE in drinking water. Clinical scores and disease activity index were assessed. Colonic tissue samples were analysed for infiltration, tissue damage, bowel wall thickness, expression of pro-inflammatory mediators, mast cell proteases, tight junction proteins, and NF-κB signaling. Following treatment with CE, symptoms of murine colitis as well as increased infiltration of immune cells, tissue damage and bowel wall thickness in colon tissue of IL-10(-/-) mice were diminished significantly. MIP-2, TNF, IFNγ, CCL2, CCL3, CCL4 and IL-1β as well as MC-CPA, MCP-1 and MCP-4 were strongly upregulated in IL-10(-/-) mice compared to WT, but noteworthy not in CE group. Expression of tight junction proteins was not influenced by CE. Phosphorylation of IκB was slightly down-regulated in CE treated IL-10(-/-) mice compared to IL-10(-/-) controls. In summary, CE decreases inflammatory symptoms and expression of inflammatory markers in murine IL-10(-/-) colitis. CE has no influence on tight junction proteins, but seems acting via reducing pro-inflammatory mediators and recruitment of neutrophil granulocytes probably by inhibiting NF-κB signaling. PMID:27012624

  10. Viral vector tropism for supporting cells in the developing murine cochlea.

    PubMed

    Sheffield, Abraham M; Gubbels, Samuel P; Hildebrand, Michael S; Newton, Stephen S; Chiorini, John A; Di Pasquale, Giovanni; Smith, Richard J H

    2011-07-01

    Gene-based therapeutics are being developed as novel treatments for genetic hearing loss. One roadblock to effective gene therapy is the identification of vectors which will safely deliver therapeutics to targeted cells. The cellular heterogeneity that exists within the cochlea makes viral tropism a vital consideration for effective inner ear gene therapy. There are compelling reasons to identify a viral vector with tropism for organ of Corti supporting cells. Supporting cells are the primary expression site of connexin 26 gap junction proteins that are mutated in the most common form of congenital genetic deafness (DFNB1). Supporting cells are also primary targets for inducing hair cell regeneration. Since many genetic forms of deafness are congenital it is necessary to administer gene transfer-based therapeutics prior to the onset of significant hearing loss. We have used transuterine microinjection of the fetal murine otocyst to investigate viral tropism in the developing inner ear. For the first time we have characterized viral tropism for supporting cells following in utero delivery to their progenitors. We report the inner ear tropism and potential ototoxicity of three previously untested vectors: early-generation adenovirus (Ad5.CMV.GFP), advanced-generation adenovirus (Adf.11D) and bovine adeno-associated virus (BAAV.CMV.GFP). Adenovirus showed robust tropism for organ of Corti supporting cells throughout the cochlea but induced increased ABR thresholds indicating ototoxicity. BAAV also showed tropism for organ of Corti supporting cells, with preferential transduction toward the cochlear apex. Additionally, BAAV readily transduced spiral ganglion neurons. Importantly, the BAAV-injected ears exhibited normal hearing at 5 weeks of age when compared to non-injected ears. Our results support the use of BAAV for safe and efficient targeting of supporting cell progenitors in the developing murine inner ear. PMID:21530627

  11. Suppression of properties associated with malignancy in murine melanoma-melanocyte hybrid cells.

    PubMed Central

    Wakeling, W. F.; Greetham, J.; Devlin, L. M.; Bennett, D. C.

    1992-01-01

    Murine and human melanoma cells differ relatively reliably from non-tumorigenic melanocytes in certain biological properties. When cultured at low pH, melanocytes tend to be pigmented and melanoma cells unpigmented. The growth of virtually all metastatic melanoma cells is inhibited by phorbol esters such as TPA (12-O-tetradecanoyl phorbol-13-acetate), which stimulate melanocyte growth. Melanocytes fail to grow in suspension culture or produce tumours when implanted in animals, while many melanoma lines can do both. Here we studied which of these properties were dominant in hybrid cells formed by fusion of drug-resistant murine B16-F10RR melanoma cells to melanocytes of the albino and brown lines, melan-c and melan-b. The albino melanocytes are unpigmented but well-differentiated, the brown melanocytes produce pale brown pigment and the melanoma cells are unpigmented under the conditions used. All hybrid colonies observed produced black pigment, except some melan-b/melanoma hybrids when growing sparsely with TPA. Thus pigmentation was generally dominant. 14/15 hybrid lines showed stimulation of proliferation by TPA, as do melanocytes. Most hybrid lines showed no or reduced capacity for growth in suspension, though some grew better in suspension when TPA was present. There was marked suppression of the tumorigenicity of the parental melanoma cells in 4/8 hybrids examined, and tumorigenicity was reduced in the others, despite considerable chromosome loss by the passage level tested. Thus most properties of the non-tumorigenic pigment cells were dominant, as often observed for other cell lineages, and providing further evidence for gene loss in the genesis of malignant melanoma. Images Figure 1 Figure 3 PMID:1562463

  12. Leukocyte-dependent antibody in sheep immunized with murine mastocytoma cells.

    PubMed

    Grant, C K; Adams, E; Miller, H R

    1975-05-01

    Sheep serum removed after hyperimmunization with murine P-815 mastocytoma cells was fractionated and two types of cytotoxic antibody were isolated. Complement-dependent antibody (CDA) was detected in the IgM and IgG1 fractions whereas "leukocyte" -dependent antibody (LDA) was found in the IgG1 and IgG2 fractions. Effector cells that mediated LDA cytotoxicity were isolated from sheep blood but not from sheep lymph, showing that recirculating lymphocytes do not have LDA effector function. Removal of adherent cells from blood leukocyte suspensions reduced antibody-mediated cytotoxicity, suggesting that LDA effector leukocytes may be a heterogenous population containing both adherent and nonadherent cells. PMID:824135

  13. Accessing the Genomic Effects of Naked Nanoceria in Murine Neuronal Cells

    PubMed Central

    Lee, Tin-Lap; Raitano, Joan M.; Rennert, Owen M; Chan, Siu-Wai; Chan, Wai-Yee

    2011-01-01

    Cerium oxide nanoparticles (nanoceria) are versatile engineered nanoparticles (ENPs) due to their unique redox properties. We and others have previously demonstrated naked nanoceria could act as antioxidants to protect cells against oxidative damage. While the redox properties may be beneficial, the genome-wide effects of nanoceria on gene transcription and associated biological processes remain elusive. Here we applied functional genomic approach to examine the genome-wide effects of nanoceria on global gene transcription and cellular functions in mouse neuronal cells. Importantly, we demonstrated nanoceria induced chemical- and size-specific changes in the murine neuronal cell transcriptome. The nanoceria specially contributed more than 83% of uniquely altered gene population and associate with a unique spectrum of genes related to neurological disease, cell cycle control and growth. These observations suggest an in-depth assessment of potential health effects of naked nanoceria and other naked nanoparticles is both necessary and imminent. PMID:21889474

  14. Potentiated cytotoxic effects of statins and ajoene in murine melanoma cells.

    PubMed

    Ledezma, Eliades; Wittig, Olga; Alonso, Jose; Cardier, Jose E

    2009-04-01

    Because statins and ajoene inhibit the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, we evaluated the hypothesis that the cytotoxic effect of these compounds may be potentiated when both are used in combination on tumor cells. We showed that cotreatment of the murine melanoma B16F10 cell with statins (atorvastatin and pravastatin) and ajoene, all at nontoxic doses, dramatically increased their cytotoxicity. B16F10 cell death induced by statins, but not by ajoene, was prevented by mevalonate and geranylgeranylpyrophosphate. To our knowledge, this is the first report that the combination of statins and ajoene, which alters the mevalonate pathway, might potentiate their cytotoxic effects on tumor cells. PMID:19276863

  15. Sca-1 Identifies a Distinct Androgen-Independent Murine Prostatic Luminal Cell Lineage with Bipotent Potential

    PubMed Central

    Kwon, Oh-Joon; Zhang, Li; Xin, Li

    2016-01-01

    Recent lineage tracing studies support the existence of prostate luminal progenitors that possess extensive regenerative capacity, but their identity remains unknown. We show that Sca-1 (Stem Cell Antigen-1) identifies a small population of murine prostate luminal cells that reside in the proximal prostatic ducts adjacent to the urethra. Sca-1+ luminal cells do not express Nkx3.1. They do not carry the secretory function, although they express the androgen receptor. These cells are enriched in the prostates of castrated mice. In the in vitro prostate organoid assay, a small fraction of the Sca-1+ luminal cells are capable of generating budding organoids that are morphologically distinct from those derived from other cell lineages. Histologically, this type of organoid is composed of multiple inner layers of luminal cells surrounded by multiple outer layers of basal cells. When passaged, these organoids retain their morphological and histological features. Finally, the Sca-1+ luminal cells are capable of forming small prostate glands containing both basal and luminal cells in an in vivo prostate regeneration assay. Collectively, our study establishes the androgen-independent and bipotent organoid-forming Sca-1+ luminal cells as a functionally distinct cellular entity. These cells may represent a putative luminal progenitor population and serve as a cellular origin for castration resistant prostate cancer. PMID:26418304

  16. Simultaneous Isolation of Three Different Stem Cell Populations from Murine Skin

    PubMed Central

    Forni, Maria Fernanda; Ramos Maia Lobba, Aline; Pereira Ferreira, Alexandre Hamilton; Sogayar, Mari Cleide

    2015-01-01

    The skin is a rich source of readily accessible stem cells. The level of plasticity afforded by these cells is becoming increasingly important as the potential of stem cells in Cell Therapy and Regenerative Medicine continues to be explored. Several protocols described single type stem cell isolation from skin; however, none of them afforded simultaneous isolation of more than one population. Herein, we describe the simultaneous isolation and characterization of three stem cell populations from the dermis and epidermis of murine skin, namely Epidermal Stem Cells (EpiSCs), Skin-derived Precursors (SKPs) and Mesenchymal Stem Cells (MSCs). The simultaneous isolation was possible through a simple protocol based on culture selection techniques. These cell populations are shown to be capable of generating chondrocytes, adipocytes, osteocytes, terminally differentiated keratinocytes, neurons and glia, rendering this protocol suitable for the isolation of cells for tissue replenishment and cell based therapies. The advantages of this procedure are far-reaching since the skin is not only the largest organ in the body, but also provides an easily accessible source of stem cells for autologous graft. PMID:26462205

  17. Cell cycle progression in denV-transfected murine fibroblasts exposed to ultraviolet radiation.

    PubMed

    Kusewitt, D F; Budge, C L; Nolla, H A; Edwards, B S; Ley, R D

    1992-09-01

    Repair-proficient murine fibroblasts transfected with the denV gene of bacteriophage T4 repaired 70-80% of pyrimidine dimers within 24 h after exposure to 150 J/m2 ultraviolet radiation (UVR) from an FS-40 sunlamp. Under the same conditions, control cells repaired only about 20% of UVR-induced pyrimidine dimers. After UVR exposure, both control and denV-transfected cells exhibited some degree of DNA-synthesis inhibition, as determined by flow cytometric analysis of cell-cycle kinetics in propidium iodide-stained cells. DenV-transfected cells had a longer and more profound S phase arrest than control cells, but both control and denV-transfected cells had largely recovered from UVR effects on cell-cycle kinetics by 48 h after UVR exposure. Inhibition of DNA synthesis by UVR was also measured by determining post-UVR incorporation of bromodeoxyuridine (BrdU). The amount of BrdU incorporated was quantitated by determining with flow cytometry the quenching of Hoechst dye 33342 by BrdU incorporated in cellular DNA. DenV-transfected cells showed more marked inhibition of BrdU incorporation after low fluences of UVR than control cells. Differences between denV-transfected and control cells in cell-cycle kinetics following UVR exposure may be related to differences in mechanisms of repair when excision repair of pyrimidine dimers is initiated by endonuclease V instead of cellular repair enzymes. PMID:1380650

  18. Aneuploidy, oncogene amplification and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells

    PubMed Central

    Padilla-Nash, Hesed M.; McNeil, Nicole E.

    2013-01-01

    Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research. PMID:23619298

  19. Dichloroacetic acid accelerates initial development of 2-cell murine embryos in vitro.

    PubMed

    Penzias, A S; Rossi, G; Gutmann, J N; Haj-Hassan, L; Leykin, L; Diamond, M P

    1993-09-01

    Preimplantation embryos up to the 8-cell stage of development use lactate and pyruvate but not glucose or Krebs cycle intermediates to support growth, development, and cleavage. The dominant effect of dichloroacetic acid (DCA) is the irreversible stimulation of pyruvate dehydrogenase (PDH) activity, thus accelerating the oxidative metabolism of pyruvate and lactate. To test the hypothesis that early induction of oxidative metabolism in 2-cell murine embryos accelerates preimplantation embryo cleavage rates, female B6C3F1 mice at 6 to 8 weeks of age were superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) and mated. All 2-cell stage embryos were randomly assigned to culture media with or without 130 micrograms/mL DCA. The developmental stage of all embryos was then noted every 24 hours for a total of 72 hours. Chi-square analysis and the method of average rank sum were used to compare the distribution of embryos at each observation point. At 24 hours, DCA-exposed embryos had achieved an advanced stage of growth and development relative to controls (average rank sum, P = .026; chi-square distribution, P = .047). Subsequently, at 48 and 72 hours, neither the average rank sum nor the chi-square distribution was different. Our data suggest that DCA accelerates early growth and development of murine embryos before implantation, possibly through the early induction of oxidative metabolism. PMID:8412755

  20. Identification and enrichment of colony-forming cells from the adult murine pituitary

    SciTech Connect

    Lepore, D.A.; Roeszler, K.; Wagner, J.; Ross, S.A.; Bauer, K.; Thomas, P.Q. , E-Mail: paul.thomas@mcri.edu.au

    2005-08-01

    Stem and progenitor cells have been identified in many adult tissues including bone marrow, the central nervous system, and skin. While there is direct evidence to indicate the activity of a progenitor cell population in the pituitary gland, this putative subpopulation has not yet been identified. Herein we describe the isolation and characterization of a novel clonogenic cell type in the adult murine pituitary, which we have termed Pituitary Colony-Forming Cells (PCFCs). PCFCs constitute 0.2% of pituitary cells, and generate heterogeneous colonies from single cells. PCFCs exhibit variable proliferative potential, and may exceed 11 population doublings in 14 days. Enrichment of PCFCs to 61.5-fold with 100% recovery can be obtained through the active uptake of the fluorescent dipeptide, {beta}-Ala-Lys-N{epsilon}-AMCA. PCFCs are mostly contained within the large, agranular subpopulation of AMCA{sup +} cells, and constitute 28% of this fraction, corresponding to 140.5-fold enrichment. Interestingly, the AMCA{sup +} population contains rare cells that are GH{sup +} or PRL{sup +}. GH{sup +} cells were also identified in PCFC single cell colonies, suggesting that PCFCs have the potential to differentiate into GH{sup +} cells. Together, these data show that the pituitary contains a rare clonogenic population which may correspond to the somatotrope/lactotrope progenitors suggested by previous experiments.

  1. Metabolic pathways that correlate with post-transfusion circulation of stored murine red blood cells.

    PubMed

    de Wolski, Karen; Fu, Xiaoyoun; Dumont, Larry J; Roback, John D; Waterman, Hayley; Odem-Davis, Katherine; Howie, Heather L; Zimring, James C

    2016-05-01

    Transfusion of red blood cells is a very common inpatient procedure, with more than 1 in 70 people in the USA receiving a red blood cell transfusion annually. However, stored red blood cells are a non-uniform product, based upon donor-to-donor variation in red blood cell storage biology. While thousands of biological parameters change in red blood cells over storage, it has remained unclear which changes correlate with function of the red blood cells, as opposed to being co-incidental changes. In the current report, a murine model of red blood cell storage/transfusion is applied across 13 genetically distinct mouse strains and combined with high resolution metabolomics to identify metabolic changes that correlated with red blood cell circulation post storage. Oxidation in general, and peroxidation of lipids in particular, emerged as changes that correlated with extreme statistical significance, including generation of dicarboxylic acids and monohydroxy fatty acids. In addition, differences in anti-oxidant pathways known to regulate oxidative stress on lipid membranes were identified. Finally, metabolites were identified that differed at the time the blood was harvested, and predict how the red blood cells perform after storage, allowing the potential to screen donors at time of collection. Together, these findings map out a new landscape in understanding metabolic changes during red blood cell storage as they relate to red blood cell circulation. PMID:26921359

  2. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    SciTech Connect

    Morizane, Ryuji; Monkawa, Toshiaki; Itoh, Hiroshi

    2009-12-25

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  3. Murinization of Internalin Extends Its Receptor Repertoire, Altering Listeria monocytogenes Cell Tropism and Host Responses

    PubMed Central

    Tsai, Yu-Huan; Disson, Olivier; Bierne, Hlne; Lecuit, Marc

    2013-01-01

    Listeria monocytogenes (Lm) is an invasive foodborne pathogen that leads to severe central nervous system and maternal-fetal infections. Lm ability to actively cross the intestinal barrier is one of its key pathogenic properties. Lm crosses the intestinal epithelium upon the interaction of its surface protein internalin (InlA) with its host receptor E-cadherin (Ecad). InlA-Ecad interaction is species-specific, does not occur in wild-type mice, but does in transgenic mice expressing human Ecad and knock-in mice expressing humanized mouse Ecad. To study listeriosis in wild-type mice, InlA has been murinized to interact with mouse Ecad. Here, we demonstrate that, unexpectedly, murinized InlA (InlAm) mediates not only Ecad-dependent internalization, but also N-cadherin-dependent internalization. Consequently, InlAm-expressing Lm targets not only goblet cells expressing luminally-accessible Ecad, as does Lm in humanized mice, but also targets villous M cells, which express luminally-accessible N-cadherin. This aberrant Lm portal of entry results in enhanced innate immune responses and intestinal barrier damage, both of which are not observed in wild-type Lm-infected humanized mice. Murinization of InlA therefore not only extends the host range of Lm, but also broadens its receptor repertoire, providing Lm with artifactual pathogenic properties. These results challenge the relevance of using InlAm-expressing Lm to study human listeriosis and in vivo host responses to this human pathogen. PMID:23737746

  4. Injury of neoplastic cells by murine macrophages leads to inhibition of mitochondrial respiration.

    PubMed Central

    Granger, D L; Taintor, R R; Cook, J L; Hibbs, J B

    1980-01-01

    Cytotoxic activated macrophages (CM) inhibited the growth of neoplastic L1210 cells in vitro but L1210 cell death was minimal to nonexistent. L1210 cells injured by CM were separated from macrophages and studied in an isolated system. CM-injured L1210 cells had an absolute requirement for glucose or another glycolyzable hexose (mannose or fructose) for at least 40 h after removal from macrophages. If the culture medium lacked sufficient concentration of one of these sugars, CM-injured L1210 cells died within 4 h. Uninjured L1210 cells cultured alone or with peptone-stimulated macrophages had no such requirement and maintained complete viability in hexoseless medium. The hexose requirement of CM-injured L1210 cells could not be fulfilled by other naturally occurring monosaccharides, glucose or mannose derivatives, or substrates that can be oxidized by mitochondria. The concentration requirements for glucose, mannose, and fructose by CM-injured L1210 cells correlated with the concentrations required to support maximal glycolysis of these sugars by other murine ascites cells. A concentration of 2-deoxy-D-glucose which completely inhibited L1210 cell glycolysis also complete prevented the ability of glucose or mannose to maintain viability of CM-injured L1210 cells. Interaction with CM led to inhibition of L1210 cell mitochondrial oxidative phosphorylation. This was supported by the findings that: (a) CM-injured L1210 cells had no Pasteur effect; their rate of aerobic glycolysis was the same as the rate of anaerobic glycolysis of uninjured L1210 cells, (b) Endogenous respiration of CM-injured L1210 cells was 15% of normal. Maximal inhibition of uninjured L1210 cell respiration by a specific mitochondrial poison (oligomycin) was nearly the same (13% of normal). It followed that CM-injured L1210 cells required hexose for chemical energy production via the glycolytic pathway. CM-induced mitochondrial injury occurred in five other neoplastic cell lines tested. Images PMID:7356685

  5. Identification of molecular markers of bipolar cells in the murine retina

    PubMed Central

    Kim, Douglas S; Ross, Sarah E; Trimarchi, Jeffrey M; Aach, John; Greenberg, Michael E; Cepko, Constance L

    2008-01-01

    Retinal bipolar neurons serve as relay interneurons that connect rod and cone photoreceptor cells to amacrine and ganglion cells. They exhibit diverse morphologies essential for correct routing of photoreceptor cell signals to specific postsynaptic amacrine and ganglion cells. The development and physiology of these interneurons have not been completely defined molecularly. Despite previous identification of genes expressed in several bipolar cell subtypes, molecules that mark each bipolar cell type still await discovery. In this report, novel genetic markers of murine bipolar cells were found. Candidates were initially generated by using microarray analysis of single bipolar cells and mining of retinal serial analysis of gene expression (SAGE) data. These candidates were subsequently tested for expression in bipolar cells by RNA in situ hybridization. Ten new molecular markers were identified, five of which are highly enriched in their expression in bipolar cells within the adult retina. Double-labeling experiments using probes for previously characterized subsets of bipolar cells were performed to identify the subtypes of bipolar cells that express the novel markers. Additionally, the expression of bipolar cell genes was analyzed in Bhlhb4 knockout retinas, in which rod bipolar cells degenerate postnatally, to delineate further the identity of bipolar cells in which novel markers are found. From the analysis of Bhlhb4 mutant retinas, cone bipolar cell gene expression appears to be relatively unaffected by the degeneration of rod bipolar cells. Identification of molecular markers for the various subtypes of bipolar cells will lead to greater insights into the development and function of these diverse interneurons. PMID:18260140

  6. Effects of ethanol on cAMP production in murine embryonic palate mesenchymal cells

    SciTech Connect

    Weston, W.M.; Greene, R.M. )

    1991-01-01

    Ethanol affected the ability of murine embryonic palate mesenchymal (MEPM) cells to produce cAMP in response to hormone treatment. Acute exposure to ethanol resulted in an increase in hormone-stimulated cAMP levels, while chronic ethanol treatment led to decreased sensitivity to hormone. Forskolin-stimulated cAMP levels were decreased by both acute and chronic ethanol treatment, while the cells' response to cholera toxin was unchanged by ethanol treatment. The lack of sensitivity of the cholera toxin response to ethanol suggests that,in contrast to what has been observed in other systems, ethanol does not affect the production or activity of G{alpha}s in MEPM cells. These results suggest a possible explanation for the molecular basis for the craniofacial abnormalities observed in the fetal alcohol syndrome.

  7. Immunoglobulin heavy chain gene rearrangement and transcription in murine T cell hybrids and T lymphomas.

    PubMed Central

    Ziga, M C; D'Eustachio, P; Ruddle, N H

    1982-01-01

    We have examined the arrangement of immunoglobulin heavy chain constant (CH) and joining (JH) region genes in murine T cell hybrid lines and in T lymphomas. CH genes derived from both parental cell types were present in all hybrids for which polymorphism in sequences flanking CH genes permitted us to distinguish parental CH genes. All T lymphomas and T cell hybrids retained the C alpha gene in germ-line configuration and all but one cell line had germ-line C mu genes. Novel DNA fragments reactive with JH probes were observed in six of nine T cell hybrids, as well as in two T lymphomas, WEHI7.1 and YAC-1, but not in the fusion parent, BW5147. No RNA homologous to C gamma 2b, C alpha, or lambda genes was detected in any of the T cell lines. T cell lines contained poly(A)+ RNA homologous to a C mu cDNA probe. More importantly, in several cell lines the C mu RNAs were associated with membrane-bound polyribosomes. These results suggest that both JH rearrangements and C mu RNA production occur in at least some mature, antigen-specific T cells. They may therefore reflect events in normal T cell development and function related to those involved in the generation of the T receptor for antigen. Images PMID:6806823

  8. Perforin Gene Transfer Into Hematopoietic Stem Cells Improves Immune Dysregulation in Murine Models of Perforin Deficiency

    PubMed Central

    Carmo, Marlene; Risma, Kimberly A; Arumugam, Paritha; Tiwari, Swati; Hontz, Adrianne E; Montiel-Equihua, Claudia A; Alonso-Ferrero, Maria E; Blundell, Michael P; Schambach, Axel; Baum, Christopher; Malik, Punam; Thrasher, Adrian J; Jordan, Michael B; Gaspar, H Bobby

    2015-01-01

    Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8+ T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8+ lymphoblasts had reduced interferon-γ secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL. PMID:25523759

  9. Rhesus rotavirus VP4 sequence-specific activation of mononuclear cells is associated with cholangiopathy in murine biliary atresia.

    PubMed

    Walther, Ashley; Mohanty, Sujit K; Donnelly, Bryan; Coots, Abigail; Lages, Celine S; Lobeck, Inna; Dupree, Phylicia; Meller, Jaroslaw; McNeal, Monica; Sestak, Karol; Tiao, Greg

    2015-09-15

    Biliary atresia (BA), a neonatal obstructive cholangiopathy, remains the most common indication for pediatric liver transplantation in the United States. In the murine model of BA, Rhesus rotavirus (RRV) VP4 surface protein determines biliary duct tropism. In this study, we investigated how VP4 governs induction of murine BA. Newborn mice were injected with 16 strains of rotavirus and observed for clinical symptoms of BA and mortality. Cholangiograms were performed to confirm bile duct obstruction. Livers and bile ducts were harvested 7 days postinfection for virus titers and histology. Flow cytometry assessed mononuclear cell activation in harvested cell populations from the liver. Cytotoxic NK cell activity was determined by the ability of NK cells to kill noninfected cholangiocytes. Of the 16 strains investigated, the 6 with the highest homology to the RRV VP4 (>87%) were capable of infecting bile ducts in vivo. Although the strain Ro1845 replicated to a titer similar to RRV in vivo, it caused no symptoms or mortality. A Ro1845 reassortant containing the RRV VP4 induced all BA symptoms, with a mortality rate of 89%. Flow cytometry revealed that NK cell activation was significantly increased in the disease-inducing strains and these NK cells demonstrated a significantly higher percentage of cytotoxicity against noninfected cholangiocytes. Rotavirus strains with >87% homology to RRV's VP4 were capable of infecting murine bile ducts in vivo. Development of murine BA was mediated by RRV VP4-specific activation of mononuclear cells, independent of viral titers. PMID:26206856

  10. Interleukin 21 Signaling in B Cells Is Required for Efficient Establishment of Murine Gammaherpesvirus Latency

    PubMed Central

    Collins, Christopher M.; Speck, Samuel H.

    2015-01-01

    The human gammaherpesviruses take advantage of normal B cell differentiation pathways to establish life-long infection in memory B cells. Murine gammaherpesvirus 68 (MHV68) infection of laboratory strains of mice also leads to life-long infection in memory B cells. To gain access to the memory B cell population, MHV68 infected B cells pass through the germinal center reaction during the onset of latency and require signals from T follicular helper (TFH) cells for proliferation. Interleukin 21 (IL-21), one of the secreted factors produced by TFH cells, plays an important role in both the maintenance of the germinal center response as well as in the generation of long-lived plasma cells. Using IL-21R deficient mice, we show that IL-21 signaling is required for efficient establishment of MHV68 infection. In the absence of IL-21 signaling, fewer infected splenocytes are able to gain access to either the germinal center B cell population or the plasma cell population the latter being a major site of MHV68 reactivation. Furthermore, the germinal center B cell population in IL-21R-/- mice is skewed towards the non-proliferating centrocyte phenotype, resulting in reduced expansion of infected B cells. Additionally, the reduced frequency of infected plasma cells results in a significant reduction in the frequency of splenocytes capable of reactivating virus. This defect in establishment of MHV68 infection is intrinsic to B cells, as MHV68 preferentially establishes infection in IL-21R sufficient B cells in mixed bone marrow chimeric mice. Taken together, these data indicate that IL-21 signaling plays multiple roles during establishment of MHV68 infection, and identify IL-21 as a critical TFH cell-derived factor for efficient establishment of gammaherpesvirus B cell latency. PMID:25875847

  11. Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

    PubMed Central

    Fricke, Stephan; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

    2014-01-01

    Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies. PMID:25546418

  12. Activation-dependent apoptosis in CD4+ T cells during murine AIDS.

    PubMed

    Cohen, D A; Fitzpatrick, E A; Barve, S S; Guthridge, J M; Jacob, R J; Simmerman, L; Kaplan, A M

    1993-10-15

    The mechanism by which CD4+ T cells are depleted during HIV infection remains a matter of controversy. Recent reports have suggested that activation-induced apoptosis of antigen-specific CD4+ T cells may lead ultimately to depletion of this T cell subset during HIV infection. The murine retroviral model of AIDS (MAIDS) also displays progressive immunodeficiency, but depletion of the CD4+ T cell subset is not characteristic of the disease. We report that a fraction of splenic CD4+ T cells from 8- to 14-week MAIDS-infected C57B1/6 mice, but not normal mice, was undergoing apoptosis at the time of cell isolation. Typical apoptotic morphology and internucleosomal DNA fragmentation was seen in CD4+ T cells only from infected mice. Moreover, injection of anti-CD3 mAb enhanced DNA fragmentation in CD4+ T cells from infected but not normal mice, suggesting that the apoptosis in vivo in CD4+ T cells during MAIDS may be dependent on cell activation. Induction of apoptosis was associated with defective signaling through the TcR complex, since anti-CD3 stimulation in vitro of CD4+ T cells from infected mice caused a diminished calcium response, yet no cellular proliferation. Despite the occurrence of apoptosis in vivo in CD4+ T cells from MAIDS-infected mice, CD4+ T cells were not depleted during the course of disease. Thus, while apoptosis in CD4+ T cells is a characteristic of MAIDS immunodeficiency disease as well as HIV infections in humans, CD4+ T cell depletion is only observed in HIV infections. In view of the extensive lymphocyte expansion which occurs in vivo in MAIDS, the balance between activation-induced apoptosis and chronic cell proliferation may determine whether cell depletion is a characteristic feature of retrovirus-induced immunodeficiencies. PMID:8104712

  13. Loss of interstitial cells of Cajal and development of electrical dysfunction in murine small bowel obstruction

    PubMed Central

    Chang, In-Youb; Glasgow, Nichola J; Takayama, Ichiro; Horiguchi, Kazuhide; Sanders, Kenton M; Ward, Sean M

    2001-01-01

    Partial obstruction of the murine ileum led to changes in the gross morphology and ultrastructure of the tunica muscularis. Populations of interstitial cells of Cajal (ICC) decreased oral, but not aboral, to the site of obstruction. Since ICC generate and propagate electrical slow waves in gastrointestinal muscles, we investigated whether the loss of ICC leads to loss of function in partial bowel obstruction. Changes in ICC networks and electrical activity were monitored in the obstructed murine intestine using immunohistochemistry, electron microscopy and intracellular electrophysiological techniques. Two weeks following the onset of a partial obstruction, the bowel increased in diameter and hypertrophy of the tunica muscularis was observed oral to the obstruction site. ICC networks were disrupted oral to the obstruction, and this disruption was accompanied by the loss of electrical slow waves and responses to enteric nerve stimulation. These defects were not observed aboral to the obstruction. Ultrastructural analysis revealed no evidence of cell death in regions where the lesion in ICC networks was developing. Cells with a morphology intermediate between smooth muscle cells and fibroblasts were found in locations that are typically populated by ICC. These cells may have been the redifferentiated remnants of ICC networks. Removal of the obstruction led to the redevelopment of ICC networks and recovery of slow wave activity within 30 days. Neural responses were partially restored in 30 days. These data describe the plasticity of ICC networks in response to partial obstruction. After obstruction the ICC phenotype was lost, but these cells regenerated when the obstruction was removed. This model may be an important tool for evaluating the cellular/molecular factors responsible for the regulation and maintenance of the ICC phenotype. PMID:11600689

  14. Dendritic Cell-Based Vaccination in Cancer: Therapeutic Implications Emerging from Murine Models

    PubMed Central

    Mac Keon, Soledad; Ruiz, María Sol; Gazzaniga, Silvina; Wainstok, Rosa

    2015-01-01

    Dendritic cells (DCs) play a pivotal role in the orchestration of immune responses, and are thus key targets in cancer vaccine design. Since the 2010 FDA approval of the first cancer DC-based vaccine (Sipuleucel-T), there has been a surge of interest in exploiting these cells as a therapeutic option for the treatment of tumors of diverse origin. In spite of the encouraging results obtained in the clinic, many elements of DC-based vaccination strategies need to be optimized. In this context, the use of experimental cancer models can help direct efforts toward an effective vaccine design. This paper reviews recent findings in murine models regarding the antitumoral mechanisms of DC-based vaccination, covering issues related to antigen sources, the use of adjuvants and maturing agents, and the role of DC subsets and their interaction in the initiation of antitumoral immune responses. The summary of such diverse aspects will highlight advantages and drawbacks in the use of murine models, and contribute to the design of successful DC-based translational approaches for cancer treatment. PMID:26042126

  15. Protein Tyrosine Phosphatase PTPRS Is an Inhibitory Receptor on Human and Murine Plasmacytoid Dendritic Cells.

    PubMed

    Bunin, Anna; Sisirak, Vanja; Ghosh, Hiyaa S; Grajkowska, Lucja T; Hou, Z Esther; Miron, Michelle; Yang, Cliff; Ceribelli, Michele; Uetani, Noriko; Chaperot, Laurence; Plumas, Joel; Hendriks, Wiljan; Tremblay, Michel L; Häcker, Hans; Staudt, Louis M; Green, Peter H; Bhagat, Govind; Reizis, Boris

    2015-08-18

    Plasmacytoid dendritic cells (pDCs) are primary producers of type I interferon (IFN) in response to viruses. The IFN-producing capacity of pDCs is regulated by specific inhibitory receptors, yet none of the known receptors are conserved in evolution. We report that within the human immune system, receptor protein tyrosine phosphatase sigma (PTPRS) is expressed specifically on pDCs. Surface PTPRS was rapidly downregulated after pDC activation, and only PTPRS(-) pDCs produced IFN-α. Antibody-mediated PTPRS crosslinking inhibited pDC activation, whereas PTPRS knockdown enhanced IFN response in a pDC cell line. Similarly, murine Ptprs and the homologous receptor phosphatase Ptprf were specifically co-expressed in murine pDCs. Haplodeficiency or DC-specific deletion of Ptprs on Ptprf-deficient background were associated with enhanced IFN response of pDCs, leukocyte infiltration in the intestine and mild colitis. Thus, PTPRS represents an evolutionarily conserved pDC-specific inhibitory receptor, and is required to prevent spontaneous IFN production and immune-mediated intestinal inflammation. PMID:26231120

  16. Isolation and (111)In-Oxine Labeling of Murine NK Cells for Assessment of Cell Trafficking in Orthotopic Lung Tumor Model.

    PubMed

    Malviya, Gaurav; Nayak, Tapan; Gerdes, Christian; Dierckx, Rudi A J O; Signore, Alberto; de Vries, Erik F J

    2016-04-01

    A noninvasive in vivo imaging method for NK cell trafficking is essential to gain further understanding of the pathogenesis of NK cell mediated immune response to the novel cancer treatment strategies, and to discover the homing sites and physiological distribution of NK cells. Although human NK cells can be labeled for in vivo imaging, little is known about the murine NK cell labeling and its application in animal models. This study describes the isolation and ex vivo radiolabeling of murine NK cells for the evaluation of cell trafficking in an orthotopic model of human lung cancer in mice. Scid-Tg(FCGR3A)Blt transgenic SCID mice were used to isolate NK cells from mouse splenocytes using the CD49b (DX5) MicroBeads positive selection method. The purity and viability of the isolated NK cells were confirmed by FACS analysis. Different labeling buffers and incubation times were evaluated to optimize (111)In-oxine labeling conditions. Functionality of the radiolabeled NK cell was assessed by (51)Cr-release assay. We evaluated physiological distribution of (111)In-oxine labeled murine NK cells in normal SCID mice and biodistribution in irradiated and nonirradiated SCID mice with orthotopic A549 human lung tumor lesions. Imaging findings were confirmed by histology. Results showed that incubation with 0.011 MBq of (111)In-oxine per million murine NK cells in PBS (pH 7.4) for 20 min is the best condition that provides optimum labeling efficiency without affecting cell viability and functionality. Physiological distribution in normal SCID mice demonstrated NK cells homing mainly in the spleen, while (111)In released from NK cells was excreted via kidneys into urine. Biodistribution studies demonstrated a higher lung uptake in orthotopic lung tumor-bearing mice than control mice. In irradiated mice, lung tumor uptake of radiolabeled murine NK cells decreased between 24 h and 72 h postinjection (p.i.), which was accompanied by tumor regression, while in nonirradiated mice, radiolabeled NK cells were retained in the lung tumor lesions up to 72 h p.i. without tumor regression. In tumor-bearing mice that were only irradiated but did not receive radiolabeled murine NK cells, a high tumor burden was observed at 72 h p.i., which indicates that irradiation in combination with murine NK cell allocation, but not irradiation alone, induced a remarkable antitumor effect in the orthotopic A549 lung tumor bearing mouse model. In conclusion, we describe a method to evaluate murine NK cell trafficking and biodistribution, which can be used to determine potential effects of immune-mediated therapeutic agents on NK cell biodistribution. PMID:26962716

  17. Murine mesenchymal progenitor cells from different tissues differentiated via mesenchymal microspheres into the mesodermal direction

    PubMed Central

    2009-01-01

    Background Because specific marker molecules for phenotypical identification of mesenchymal stem and progenitor cells are missing, the assessment of the in vitro-differentiation capacity is a prerequisite to characterize these cells. However, classical differentiation protocols are often cell-consuming and time intensive. Therefore, the establishment of novel strategies for differentiation is one topic of current efforts in stem cell biology. The goal of this study was to demonstrate the practicability of a new differentiation test using plastic adherent cell isolates from different tissues. Results We introduced the mesenchymal microsphere method as a feasible time- and cell saving screening method to analyse multilineage differentiation properties of adult progenitor cells in a three-dimensional system. For this purpose we isolated, characterized and analyzed new sources of adult murine mesenchymal progenitor cells from perirenal adipose tissue and mediastinal stromal tissue in comparison to bone marrow progenitor cells. The proliferation capacity of the cells was demonstrated by determination of the daily doubling index. Although the flow cytometry analysis of undifferentiated cells revealed differences in the expression of CD marker molecules, all isolates have the capacity for multilineage differentiation following the mesenchymal microsphere protocol as well as the classical "micro mass body" protocol for chondrogenic and the monolayer cultivation protocol for osteogenic and adipogenic differentiation. Differentiation was characterized using histochemical and immunhistochemical staining as well as RT-PCR. Conclusions We were able to show that the mesenchymal microsphere method is an efficient test system for chondro-, osteo- and adipogenic differentiation of adult progenitor cells. The advantage of this system in comparison to classical protocols is that approximately 7 times lower cell numbers are necessary. Since classical culture procedures are time intensive because high cell numbers have to be obtained, the new differentiation method may also save cells and time in future clinical applications using human mesenchymal stromal cells. PMID:20021685

  18. Macrophage matrix metalloproteinase-9 mediates epithelial-mesenchymal transition in vitro in murine renal tubular cells.

    PubMed

    Tan, Thian Kui; Zheng, Guoping; Hsu, Tzu-Ting; Wang, Ying; Lee, Vincent W S; Tian, Xinrui; Wang, Yiping; Cao, Qi; Wang, Ya; Harris, David C H

    2010-03-01

    As a rich source of pro-fibrogenic growth factors and matrix metalloproteinases (MMPs), macrophages are well-placed to play an important role in renal fibrosis. However, the exact underlying mechanisms and the extent of macrophage involvement are unclear. Tubular cell epithelial-mesenchymal transition (EMT) is an important contributor to renal fibrosis and MMPs to induction of tubular cell EMT. The aim of this study was to investigate the contribution of macrophages and MMPs to induction of tubular cell EMT. The murine C1.1 tubular epithelial cell line and primary tubular epithelial cells were cultured in activated macrophage-conditioned medium (AMCM) derived from lipopolysaccharide-activated J774 macrophages. MMP-9, but not MMP-2 activity was detected in AMCM. AMCM-induced tubular cell EMT in C1.1 cells was inhibited by broad-spectrum MMP inhibitor (GM6001), MMP-2/9 inhibitor, and in AMCM after MMP-9 removal by monoclonal Ab against MMP-9. AMCM-induced EMT in primary tubular epithelial cells was inhibited by MMP-2/9 inhibitor. MMP-9 induced tubular cell EMT in both C1.1 cells and primary tubular epithelial cells. Furthermore, MMP-9 induced tubular cell EMT in C1.1 cells to an extent similar to transforming growth factor-beta. Transforming growth factor-beta-induced tubular cell EMT in C1.1 cells was inhibited by MMP-2/9 inhibitor. Our in vitro study provides evidence that MMPs, specifically MMP-9, secreted by effector macrophages can induce tubular cell EMT and thereby contribute to renal fibrosis. PMID:20075196

  19. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    SciTech Connect

    Chattopadhyay, Kausik; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.

    2009-05-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.

  20. Effects of ionizing radiation on bone cell differentiation in an experimental murine bone cell model

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, Christa; Lau, Patrick; Hellweg, Christine; Reitz, Guenther

    During long-term space travel astronauts are exposed to a complex mixture of different radiation types under conditions of dramatically reduced weight-bearing activity. It has been validated that astronauts loose a considerable amount of bone mass at a rate up to one to two percent each month in space. Therapeutic doses of ionizing radiation cause bone damage and increase fracture risks after treatment for head-and-neck cancer and in pelvic irradiation. For low radiation doses, the possibility of a disturbed healing potential of bone was described. Radiation induced damage has been discussed to inflict mainly on immature and healing bone. Little is known about radiation effects on bone remodelling and even less on the combined action of microgravity and radiation. Bone remodelling is a life-long process performed by balanced action of cells from the osteoblast and osteoclast lineages. While osteoblasts differentiate either into bone-lining cells or into osteocytes and play a crucial role in bone matrix synthesis, osteoclasts are responsible for bone resorption. We hypothesize that the balance between bone matrix assembly by osteocytes and bone degradation by osteoclasts is modulated by microgravity as well as by ionizing radiation. To address this, a cell model consisting of murine cell lines with the potential to differentiate into bone-forming osteoblasts (OCT-1, MC3T3-E1 S24, and MC3T3-E1 S4) was used for studying radiation response after exposure to simulated components of cosmic radiation. Cells were exposed to graded doses of 150 kV X-rays, α particles (0.525 MeV/u, 160 keV/µm; PTB, Braunschweig, Germany) and accelerated heavy ions (75 MeV/u carbon, 29 keV/µm; 95 MeV/u argon, 230 keV/µm; GANIL, Caen, France). Cell survival was measured as colony forming ability; cell cycle progression was analyzed via fluorescence-activated cell scanning (FACS) by measurement of the content of propidium iodide-stained DNA, DNA damage was visualized by γH2AX-immunostaining. Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (von-Kossa staining, Alizarin Red staining). During the process of osteoblastic cell differentiation, the expression of the bone specific marker genes osteocalcin (OCN) and osteopontin (OPN) were recorded by quantitative real time reverse transcription PCR (qRT-PCR). Compared with standard culture conditions, the osteogenic marker genes OCN and OPN were highly expressed during the differentiation process induced either by osteo-inductive media additives (50 µg/ml ascorbic acid, 10 mmol/l β-glycero phosphate) or by sparsely ionizing radiation (X-rays). After 21 days of postirradiation incubation sparsely ionizing radiation could be shown to induce the formation of bone-like nodules (von-Kossa staining) for OCT-1 and MC3T3-E1 S4 cells but nor for MC3T3- E1 S24 cells. Ionizing radiation leads to a cell cycle arrest which is resolved in a dose and time dependent way. This was accompanied by a dose dependent regulation of the cyclin kinase inhibitor CDKN1A (p21/WAF) and transforming growth factor beta 1 (TGF-β1). TGF-β1 is known to affect osteoblast differentiation, matrix formation and mineralization. Modulation of its expression could influence the expression of main osteogenic transcription factors. For exposure with high LET radiation a pronounced cell cycle block was evident. The expression of the osteogenic marker genes OCN and Osterix (OSX) was increased in the OCT-1 cells with differentiation potential for exposure to α particles and accelerated carbon and argon ions. The results on the expression of differentiation markers during radiation-induced premature differentiation of bone cells of the osteoblast lineage show that densely ionizing radiation results in expression of proteins essential for bone formation and consequently in an increase in bone volume. Such an effect has been observed in in-vivo carbon ion irradiated rats. As radiation dependent permanent cell cycle blocks lead to a depletion of proliferation-competent cells from the osteoblastic precursor pool in the body, a gradual decrease of bone mass in weightlessness may be attributed to synergistic effects of radiation and weightlessness.

  1. Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells.

    PubMed

    Kochenderfer, James N; Yu, Zhiya; Frasheri, Dorina; Restifo, Nicholas P; Rosenberg, Steven A

    2010-11-11

    Adoptive T-cell therapy with anti-CD19 chimeric antigen receptor (CAR)-expressing T cells is a new approach for treating advanced B-cell malignancies. To evaluate anti-CD19-CAR-transduced T cells in a murine model of adoptive T-cell therapy, we developed a CAR that specifically recognized murine CD19. We used T cells that were retrovirally transduced with this CAR to treat mice bearing a syngeneic lymphoma that naturally expressed the self-antigen murine CD19. One infusion of anti-CD19-CAR-transduced T cells completely eliminated normal B cells from mice for at least 143 days. Anti-CD19-CAR-transduced T cells eradicated intraperitoneally injected lymphoma cells and large subcutaneous lymphoma masses. The antilymphoma efficacy of anti-CD19-CAR-transduced T cells was critically dependent on irradiation of mice before anti-CD19-CAR-transduced T-cell infusion. Anti-CD19-CAR-transduced T cells had superior antilymphoma efficacy compared with the anti-CD19 monoclonal antibody from which the anti-CD19 CAR was derived. Our results demonstrated impressive antilymphoma activity and profound destruction of normal B cells caused by anti-CD19-CAR-transduced T cells in a clinically relevant murine model. PMID:20631379

  2. Anti-inflammatory effects of natural product formulations on murine dendritic cells.

    PubMed

    Miller, Andrea K; Benson, Jenna M; Muanza, Dave N; Smith, Jerry R; Shepherd, David M

    2011-03-01

    The popularity and availability of herbal extracts has increased dramatically over the last decade, providing an inexpensive manner of self-medication. Although the efficacy of individual extracts is currently being studied intensively, research regarding complex mixtures is limited. Therefore, we evaluated the effects of three complex formulations, including BRC-301, a polyherbal extract; BRC-304, a mixture of vitamins, minerals, antioxidant enzymes, botanical extracts, and carotenoids; and BRC-306, a proprietary blend of Uncaria tomentosa (cat's claw) and Phytolens(®) on murine dendritic cells (DCs). We hypothesized that these formulations would decrease the inflammatory responsiveness and innate function of DCs. In order to address this hypothesis, we evaluated the effects of BRC-301, BRC-304, and BRC-306 on DC2.4 cells and assessed the effects of BRC-301 on bone marrow-derived DCs (bmDCs). Lipopolysaccharide (LPS) stimulation of DC2.4 cells and bmDCs induced production of nitric oxide (NO), TNF-α, and IL-6, a response that was modulated by concomitant treatment with non-cytotoxic concentrations of BRC-301. In contrast, only the production of NOor IL-6 by LPS-activated DC2.4 cells was affected by BRC-304 or BRC-306, respectively. Flow cytometric evaluation following concurrent BRC-301 and LPS treatment revealed an increased relative expression of CD11c, CD86, and CD54 on bmDCs and an increased frequency of bmDCs expressing MHC II. Finally, BRC-301 enhanced the uptake of fluorescein isothiocyanate-conjugated ovalbumin by bmDCs. Taken together, these results suggest that these commercially available formulations modulate the innate responsiveness of murine DCs and may enhance their ability to initiate T cell-mediated immunity. PMID:21399725

  3. Anti-inflammatory effects of natural product formulations on murine dendritic cells

    PubMed Central

    Miller, Andrea K.; Benson, Jenna M.; Muanza, Dave N.; Smith, Jerry R.; Shepherd, David M.

    2011-01-01

    The popularity and availability of herbal extracts has increased dramatically over the last decade, providing an inexpensive manner of self-medication. Although the efficacy of individual extracts is currently being intensively studied, research regarding complex mixtures is limited. Therefore, we evaluated the effects of three complex formulations including BRC-301, a polyherbal extract; BRC-304, a mixture of vitamins, minerals, antioxidant enzymes, botanical extracts and carotenoids; and BRC-306, a proprietary blend of Uncaria tomentosa (cats claw) and Phytolens on murine dendritic cells (DCs). We hypothesized that these formulations would decrease the inflammatory responsiveness and innate function of DCs. To address this hypothesis, we evaluated the effects of BRC-301, 304, and 306 on DC2.4 cells and further assessed the effects of BRC-301 on bone marrow-derived DCs (bmDCs). LPS stimulation of DC2.4 cells and bmDCs induced production of NO, TNF-?, and IL-6, a response that was modulated by concomitant treatment with non-cytotoxic concentrations of BRC-301. In contrast, only the production of NO or IL-6 by LPS-activated DC2.4 cells was affected by BRC-304 or BRC-306, respectively. Flow cytometric evaluation following concurrent BRC-301 and LPS treatment revealed an increased relative expression of CD11c, CD86, and CD54 on bmDCs and an increased frequency of bmDCs expressing MHC II. Finally, BRC-301 enhanced the uptake of FITC-conjugated ovalbumin by bmDCs. Taken together, these results suggest that these commercially available formulations modulate the innate responsiveness of murine DCs and may enhance their ability to initiate T cell-mediated immunity. PMID:21399725

  4. Dynamic Expression of BCL6 in Murine Conventional Dendritic Cells during In Vivo Development and Activation

    PubMed Central

    Zhang, Ting-ting; Liu, Dong; Calabro, Samuele; Eisenbarth, Stephanie C.; Cattoretti, Giorgio; Haberman, Ann M.

    2014-01-01

    The transcriptional repressor BCL6 plays an essential role in the development of germinal center B cells and follicular helper T cells. However, much less is known about the expression and function of BCL6 in other cell types. Here we report that during murine dendritic cell (DC) ontogeny in vivo, BCL6 is not expressed in bone marrow hematopoietic stem cells, common DC precursors and committed precursors of conventional DCs (pre-cDCs), but is elevated in peripheral pre-cDCs. BCL6 protein levels rise as pre-cDCs differentiate into cDCs in secondary lymphoid organs. Elevated protein levels of Bcl6 are observed in all cDC subsets, with CD8α+ cDCs displaying the greatest levels. Co-staining of Ki-67 revealed BCL6hi cDCs to be more proliferative than BCL6lo cDCs. After adjuvant inoculation, BCL6 levels are significantly reduced in the CD11cint MHC class IIhi CD86hi cDCs. Activation-induced BCL6 reduction correlated with reduced proliferation. A LPS injection study further confirmed that, in response to microbial stimuli, BCL6 levels are dynamically regulated during the maturation of CD11cint MHC class IIhi splenic cDCs. This reduction of BCL6 levels in cDCs does not occur after LPS injection in MyD88−/− TRIF−/− mice. Thus, regulation of Bcl6 protein levels is dynamic in murine cDCs during development, maturation and activation in vivo. PMID:24979752

  5. Dynamic expression of BCL6 in murine conventional dendritic cells during in vivo development and activation.

    PubMed

    Zhang, Ting-ting; Liu, Dong; Calabro, Samuele; Eisenbarth, Stephanie C; Cattoretti, Giorgio; Haberman, Ann M

    2014-01-01

    The transcriptional repressor BCL6 plays an essential role in the development of germinal center B cells and follicular helper T cells. However, much less is known about the expression and function of BCL6 in other cell types. Here we report that during murine dendritic cell (DC) ontogeny in vivo, BCL6 is not expressed in bone marrow hematopoietic stem cells, common DC precursors and committed precursors of conventional DCs (pre-cDCs), but is elevated in peripheral pre-cDCs. BCL6 protein levels rise as pre-cDCs differentiate into cDCs in secondary lymphoid organs. Elevated protein levels of Bcl6 are observed in all cDC subsets, with CD8α+ cDCs displaying the greatest levels. Co-staining of Ki-67 revealed BCL6hi cDCs to be more proliferative than BCL6lo cDCs. After adjuvant inoculation, BCL6 levels are significantly reduced in the CD11cint MHC class IIhi CD86hi cDCs. Activation-induced BCL6 reduction correlated with reduced proliferation. A LPS injection study further confirmed that, in response to microbial stimuli, BCL6 levels are dynamically regulated during the maturation of CD11cint MHC class IIhi splenic cDCs. This reduction of BCL6 levels in cDCs does not occur after LPS injection in MyD88-/- TRIF-/- mice. Thus, regu