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Luteinizing hormone (LH) stimulates testosterone production from Leydig cells. Both LH and testosterone play important roles in spermatogenesis and male fertility. To identify LH - and testosterone - responsive transporter genes that play key roles in spermatogenesis, we performed large-scale gene expression analyses on testes obtained from adult control and Lhb knockout mice. We found a significant reduction in cystine/glutamate transporter encoding Slc7a11 mRNA in testes of Lhb null mice. We observed that Slc7a11/SLC7A11 expression was initiated pre-pubertally and developmentally regulated in mouse testis. Immunolocalization studies confirmed that SLC7A11 was mostly expressed in Sertoli cells in testes of control and germ cell-deficient mice. Western blot analyses indicated that SLC7A11 was significantly reduced in testes of mutant mice lacking either LH or androgen receptor selectively in Sertoli cells. Genetic and pharmacological rescue of Lhb knockout mice restored the testicular expression of Slc7a11 comparable to that observed in controls. Additionally, Slc7a11 mRNA was significantly suppressed upon Sertoli cell/testicular damage induced in mice by cadmium treatment. Knockdown of Slc7a11 in vitro in TM4 Sertoli cells or treatment of mice with sulfasalazine, a SLC7A11 inhibitor caused a significant reduction in intracellular cysteine and glutathione levels but glutamate content remained unchanged as determined by metabolomic analysis. Knockdown of Slc7a11 resulted in compensatory upregulation of other glutamate transporters belonging to the Slc1a family presumably to maintain intracellular glutamate levels. Collectively, our studies identified that SLC7A11 is an LH/testosterone-regulated transporter that is required for cysteine/glutathione but not glutamate homeostasis in mouse Sertoli cells.

The synergistic actions of Testosterone (T) and FSH via testicular Sertoli cells (Sc) regulate male fertility. We have previously reported that the actions of these hormones (T and FSH) in infant monkey testes are restricted only to the expansion of Sc and spermatogonial cells. The robust differentiation of male Germ cells (Gc) occurs after pubertal maturation of testis. The present study was aimed to investigate the molecular basis of the synergy between T and FSH action in pubertal primate (Macaca mulatta) Sc. Using primary Sc culture, we here have demonstrated that T (but not FSH) downregulated AMH and Inhibin-β-B (INHBB) mRNAs in pubertal Sc. We also found that, prolonged stimulation of T in pubertal Sc significantly elevated the expression of genes involved in FSH signaling pathway like FSH-Receptor (FSHR), GNAS and RIC8B, and this was associated with a rise in cAMP production. T also augmented FSH induced expression of genes like SCF, GDNF, ABP and Transferrin (TF) in pubertal Sc. We therefore conclude that T acts in synergy with FSH signaling in pubertal Sc. Such a coordinated network of hormonal signaling in Sc may facilitate the timely onset of the first spermatogenic wave in pubertal primates and is responsible for quantitatively and qualitatively normal spermatogenesis.

Testicular aromatase catalyzes the synthesis of estradiol, which contributes to regulation of porcine Sertoli cell proliferation and postpubertal maintenance of Sertoli cell numbers. Although aromatase enzymatic activity decreases with age and is persistently reprogrammed by prepubertal treatment with the aromatase inhibitor letrozole, the molecular bases for regulation have not been identified. DNA methylation was examined as a potential regulatory mechanism using DNA from Leydig cells isolated from 16-, 40-, and 68-week-old boars and from 68- week-old littermates treated with the aromatase inhibitor, letrozole. Methylation levels of individual CpG dinucleotides located in the distal untranslated exon 1 of the relevant aromatase encoding gene, CYP19A3, were quite high in Leydig cell DNA, and increased further with maturity of boar (P

Although the follicle-stimulating hormone (FSH) plays a vital role in male reproduction, the molecular relationships among FSH, autophagy, and the secretory function of Sertoli cells remain largely undetermined. In this study, we sought to investigate the effects of FSH on dairy goat Sertoli cell autophagy and the role of autophagy in protein clearance. FSH treatment of primary Sertoli cells was found to enhance the expression level of LC3-II, reduce p62 degradation and the number of lysosomes, and downregulate the levels of LAMP2 protein and lysosomal gene mRNAs. Further analyses revealed that starvation-induced autophagy promotes the translocation of transcription factor EB (TFEB) from the cytoplasm to nucleus and its binding to the promoter region of LAMP2, whereas FSH suppresses the nuclear translocation of TFEB. Moreover, we found that the FSH-mediated inhibition of autophagy extends the biological half-lives of androgen-binding protein (ABP), glial-derived neurotrophic factor (GDNF), and stem cell factor (SCF) and promotes the secretion of these proteins. Collectively, these observations indicate that FSH inhibits autophagy by reducing lysosomal biogenesis, which is associated with the suppression of TFEB nuclear translocation via activation of the PI3K/Akt/mTOR pathway, thereby extending the biological half-lives and enhancing the expression of ABP, GDNF, and SCF in dairy goat Sertoli cells.

The expression of the zona pellucida glycoprotein 3 (ZP3), originally thought to be specific for oocytes, was recently extended to ovarian, prostate, colorectal and lung cancers. Earlier successful ZP3 immunization of a transgenic mouse model carrying a ZP3 positive ovarian tumor emphasized the suitability of ZP3 for cancer immunotherapy. This study was carried out to determine whether any other normal tissues besides the ovary in healthy human and mouse tissues may express ZP3, considered important to exclude off-target effects of ZP3 cancer immunotherapy. Strong ZP3 expression was found in normal human and mouse testis. ZP3 protein and mRNA transcripts were localized in spermatogonia, spermatocytes and round and elongated spermatids of both human and mouse testis, as well as in a mouse spermatogonial cell line, but absent in testicular Sertoli, Leydig, spermatogonial stem and progenitor cells. All other normal human and mouse tissues were ZP3 negative. This surprising testicular ZP3 expression has implications for the development of ZP3 cancer immunotherapies, and it also alludes to the potential of using ZP3 as a target for the development of a male immunocontraceptive.

Epitestosterone is the 17α-epimer of testosterone and has been described as an anti-androgen, since it inhibits the effects produced by testosterone and dihydrotestosterone via the nuclear androgen receptor (nAR). However, epitestosterone also displays an effect which is similar to the non-classical effect of testosterone, depolarizing the membrane potential of Sertoli cells and inducing a rapid Ca2+ uptake. This study aimed to investigate the effects of a treatment with epitestosterone on developmental parameters of immature rats. Animals were chemically castrated by using the gonadotropin-releasing hormone (GnRH) antagonist cetrorelix and then received a replacement of 7 days with epitestosterone or testosterone. Replacement with either epitestosterone or testosterone restored the anogenital distance (AGD) and testicular weight which had been reduced by chemical castration. The immunocontent of nAR and the nAR-immunoreactivity were reduced by epitestosterone treatment in the testis of both castrated and non-castrated animals. Furthermore, testosterone was unable of changing the membrane potential of Sertoli cells through its non-classical action in the group of animals castrated and replaced with epitestosterone. In conclusion, in relation to the level of protein expression of nAR epitestosterone acts as an anti-androgen. However, it acts in the same way as testosterone when genital development parameters are evaluated. Moreover, in castrated rats epitestosterone suppressed the non-classical response of testosterone, changing the pattern of testosterone signalling via a membrane mechanism in Sertoli cells.

FSH mediated cyclic AMP (cAMP) signaling is crucial for function of testicular Sertoli cells (Sc) during puberty. Yes-kinase Associated Protein (YAP), a transcriptional co-activator, regulates cell proliferation and differentiation. However, its role in testicular function is not known. In present study, we have identified YAP as an important regulator of cAMP signaling in Sc, in-vitro. Verteporfin, a YAP-inhibitor, down regulated the expression of cAMP responsive genes necessary for spermatogenesis in Sc. Action of forskolin, which acts via cAMP, was also antagonized by verteporfin, limiting expression of these genes. Assessment of cAMP-responsive-element-binding-protein (CREB) phosphorylation revealed that verteporfin augmented the phosphorylation of CREB at Ser133 residue. This effect of verteporfin on CREB phosphorylation was attenuated by H-89, the PKA inhibitor. This clearly suggested involvement of PKA in verteporfin mediated CREB phosphorylation. We provided evidence for the first time that YAP modulates cAMP signaling in Sc which may be critical for testicular function.

The molecular mechanisms regulating undifferentiated spermatogonial cell proliferation and differentiation are still not fully understood. Irisin is an exercise-induced hormone, which is a cleaved and secreted fragment of the fibronectin type III repeat containing 5 (FNDC5) transmembrane protein. Recent studies have demonstrated the role of irisin in cell proliferation and differentiation in various tissues. However, testicular irisin expression and its potential action have not been analyzed. Here, we demonstrate expression of irisin in undifferentiated spermatogonia of primates and in the tree shrew, a bridging species between primates and insectivores. Rhesus monkeys are seasonal breeders with annual phases of high and low testicular activity and germ cell proliferation. Interestingly, expression of both FNDC5 mRNA and irisin is altered between breeding (high spermatogenesis) and nonbreeding seasons (low spermatogenesis). Organotypic testis culture in the presence of irisin increased the expression levels of the Sertoli cell (GDNF) and spermatogonial transcripts Kruppel-like factor 4 (KLF4), Inhibitor of differentiation 4 (ID4), Cluster of differentiation 117 (cKIT), and SALL4, compared to untreated controls, while irisin suppressed its own FNDC5 mRNA. Our data suggest that irisin is a novel endocrine factor involved in the regulation of spermatogonial activities in the testes of primates.

The testis is a temperature-sensitive organ that needs to be maintained 2–7 °C below core body temperature to ensure the production of normal sperm. Failure to maintain testicular temperature in mammals impairs spermatogenesis and leads to low sperm counts, poor sperm motility and abnormal sperm morphology in the ejaculate. This review discusses the recent knowledge on the response of testicular somatic cells to heat stress and, specifically, regarding the relevant contributions of heat, germ cell depletion and inflammatory reactions on the functions of Sertoli and Leydig cells. It also outlines mechanisms of testicular thermoregulation, as well as the thermogenic factors that impact testicular function.

Porcine Sertoli cell number including number present at puberty is increased if testicular estradiol synthesis is reduced during the neonatal interval. Evaluating the changes in gene expression during the crucial interval of suppressed estradiol that leads to the increased Sertoli cell population will increase our understanding of Sertoli cell biology but this evaluation first required a more precise determination of the critical interval for treatment and timing of a detectable response. Previously, reduced testicular estrogens from 1 week of age were accompanied by increased Sertoli cell number at 6.5 weeks of age but the age at which Sertoli cell numbers were initially increased was unknown, one of the current objectives. Additional experiments were designed to further delineate the essential timing of treatment for the Sertoli cell response. Finally, changes in gene expression induced by the reduced estradiol synthesis were evaluated to elucidate molecular mechanisms. Experimental design typically consisted of one member of littermate pairs of boars treated with the aromatase inhibitor, letrozole, beginning at 1 week of age and the remaining member treated with canola oil vehicle. Weekly treatments continued through 5 weeks of age or tissue collection, whichever came first. Increases in Sertoli cell numbers were not detectable prior to 6.5 weeks of age and persistent treatment through 5 weeks of age was required to induce the increase in Sertoli cell numbers. This increase resulted from prolonging the first interval of Sertoli cell proliferation in the treated animals. Few genes exhibited dramatically altered transcription and similarities in pathway analysis or principal modified genes were quite limited in 2, 3, and 5-week-old boars. The critical timing and prolonged treatment required and the sequential changes in gene expression suggest a complex mechanism is involved in this model of increased proliferation of Sertoli cells.

Previous studies have shown that bisphenol A (BPA) is a potential endocrine disruptor and testicular toxicant. The present study focused on exploring the impact of exposure to low dose of BPA on male reproductive development during the early embryo stage and the underlying mechanisms. BPA (20 μg/kg/day) was orally administered to female mice on days 1-5 of gestation. The male offspring were euthanized at PND10, 20, 24, 35 or PND50. We found that the mice exposed to BPA before implantation (BPA-mice) displayed retardation of testicular development with reduction of testosterone level. The diameter and epithelium height of seminiferous tubules were reduced in BPA-mice at PND35. The numbers of spermatogenic cells at different stages were significantly reduced in BPA-mice at PND50. BPA-mice showed a persistent reduction in serum and testicular testosterone levels starting from PND24, whereas GnRH mRNA was significantly increased at PND35 and PND50. The expressions of testicular StAR and P450scc in BPA-mice also decreased relative to those of the controls at PND35 and PND50. Further analysis found that the levels of histone H3 and H3K14 acetylation (Ac-H3 and H3K14ac) in the promoter of StAR were decreased relative to those of control mice, whereas the level of Ac-H3 in the promoter of P450scc was not significantly different between the groups. These results provide evidence that exposure to BPA in preimplantation embryo retards the development of testes by reducing histone acetylation of the StAR promoter to disrupt the testicular testosterone synthesis.

Src kinases are non-receptor tyrosine kinases that phosphorylate diverse substrates, which control processes such as cell proliferation, differentiation and survival; cell adhesion; and cell motility. c-Src, the prototypical member of this protein family, is widely expressed by several organs that include the testis. In the seminiferous epithelium of the adult rat testis, c-Src is highest at the tubule lumen during the release of mature spermatids. Other studies show that testosterone regulates spermatid adhesion to Sertoli cells via c-Src, indicating Src phosphorylates key substrates that prompt the disassembly of Sertoli cell-spermatid junctions. A more recent in vitro study reveals that c-Src participates in the internalization of proteins that constitute the blood-testis barrier, which is present between Sertoli cells, suggesting a similar mechanism of junction disassembly is at play during spermiation. In this review, we discuss recent findings on c-Src, with an emphasis on its role in spermatogenesis in the mammalian testis.

Di-n-Butyl (DBP) and Di-(2-EthylHexyl) (DEHP) phthalates can leach from daily-use products resulting in environmental exposure. In male rodents, phthalate exposure results in reproductive effects. To evaluate effects on the immature primate testis, testis fragments from 6-month-old rhesus macaques were grafted subcutaneously to immune-deficient mice, which were exposed to 0, 10, or 500 mg/kg of DBP or DEHP for 14 weeks or 28 weeks (DBP only). DBP exposure reduced the expression of key steroidogenic genes, indicating that Leydig cell function was compromised. Exposure to 500 mg/kg impaired tubule formation and germ cell differentiation and reduced numbers of spermatogonia. Exposure to 10 mg/kg did not affect development, but reduced Sertoli cell number and resulted in increased expression of inhibin B. Exposure to DEHP for 14 week also affected steroidogenic genes expression. Therefore, long-term exposure to phthalate esters affected development and function of the primate testis in a time and dosage dependent manner.

Spermatogenesis is a highly complicated biological process that occurs in the epithelium of the seminiferous tubules. It is regulated by a complex network of endocrine and paracrine factors and by juxtacrine testicular cross-talk. Sertoli cells (SC) play a key role in spermatogenesis due to their production of trophic, differentiation and immune-modulating factors, but many of the molecular pathways of SC action remain controversial and unclear. Over the last two decades, research has focused on extracellular vesicles as an important mechanism of intercellular communication. The aim of this study was to investigate the presence of extracellular vesicles (EVs) in SC and the modulation of their content in SC after FSH and testosterone stimulation. Highly purified porcine pre-pubertal Sertoli cells were isolated according to previously established methods. After 48 h of culture with FSH or FSH + testosterone stimulation, we identified sertolian EVs containing specific mRNAs. Proteomic analysis of EVs content identified 29 proteins under non-stimulatory conditions, most of which were related to receptor binding activity. FSH stimulation induced increases in inhibin-alpha, inhibin-beta, plakoglobin, haptoglobin, D-3-phosphoglycerate dehydrogenase and sodium/potassium-transporting ATPase in sertolian EVs. Testosterone stimulation enhanced the abundance of inhibin-alpha, inhibin-beta, tissue-type plasminogen activator, epidermal growth factor-like protein 8, elongating factor 1-gamma and D-3-phosphoglycerate dehydrogenase. These results are likely to help determine the unknown molecular secretion of Sertoli cells.

Mature Sertoli cells (SC) are critical mediators of androgen regulation of spermatogenesis, via the androgen receptor (AR) signaling. Available immortalized SC lines loose AR expression or androgen responsiveness, hampering the study of endogenous AR regulation in SC. We have established and characterized a novel clonal mouse immortalized SC line, ST38c. These cells express some SC specific genes (sox9, wt1, tjp1, clu, abp, inhbb), but not fshr, yet more importantly, maintain substantial expression of endogenous AR as determined by PCR, immunocytochemistry, testosterone binding assays and Western blots. Microarrays allowed identification of some (146) but not all (rhox5, spinlw1), androgen-dependent, SC expressed target genes. Quantitative Real-Time PCR validated regulation of five up-regulated and two down-regulated genes. We show that AR undergoes androgen-dependent transcriptional activation as well as agonist-dependent posttranslational stabilization in ST38c cells. This cell line constitutes a useful experimental tool for future investigations on the molecular and cellular mechanisms of androgen receptor signaling in SC function.

The purpose of this study was to evaluate the existence of a possible simultaneous regulation of fatty acid (FA) metabolism and lactate production by PPAR α and PPAR β/δ activation in Sertoli cells (SC). SC cultures obtained from 20-day-old rats were incubated with WY14643 or GW0742-pharmacological activators of PPAR α and PPAR β/δ respectively. The fatty acid transporter CD36, carnitine palmitoyltransferase 1, long- and medium-chain 3-hydroxyacyl-CoA dehydrogenases mRNA levels were analyzed. An increase in the above-mentioned genes in response to activation of both nuclear receptors was observed. Additionally, PPAR β/δ activation increased lactate production as a consequence of increased pyruvate availability by inhibiting the Pyruvate Dehydrogenase Complex. Altogether, these results suggest that in SC, PPAR α activation participates in the regulation of FA metabolism. On the other hand, PPAR β/δ activation regulates FA metabolism and lactate production ensuring simultaneously the energetic metabolism for SC and germ cells.

Testicular inflammation, including noninfectious inflammatory responses in the testis, may impair male fertility. Mechanisms underlying the initiation of noninfectious testicular inflammation are poorly understood. In the current study, we demonstrate that damaged spermatogenic cell products (DSCPs) induce expression of various inflammatory mediators, including TNF-α, IL-1β, IL-6, and macrophage chemotactic protein 1 (MCP-1), in Sertoli cells. Notably, the DSCP-induced inflammatory gene expression was significantly reduced by knockout Toll-like receptor (TLR)2 or TLR4, and abolished by double knockout TLR2 and TLR4 (TLR2−/−TLR4−/−). MCP-1 secreted by Sertoli cells after stimulation with DSCPs promotes macrophage migration. We also provide evidence that busulfan-induced spermatogenic cell damages in vivo upregulate TNF-α and MCP-1 expression in Sertoli cells, and facilitate macrophage infiltration into the testis in wild-type mice. These phenomena were not observed in TLR2−/−TLR4−/− mice. Data indicate that DSCPs induce inflammatory gene expression in Sertoli cells via the activation of TLR2 and TLR4, which may initiate noninfectious inflammatory responses in the testis. The results provide novel insights into the mechanisms underlying damaged spermatogenic cell-induced testicular inflammation.

Ghrelin is a growth hormone-releasing peptide that has been suggested to interfere with spermatogenesis, though the underling mechanisms remain unknown. We studied the effect of ghrelin in human Sertoli cells (hSCs) metabolic phenotype. For that, hSCs were exposed to increasing concentrations of ghrelin (20, 100 and 500 pM) mimicking the levels reported in obese, normal weight, and severely undernourished individuals. The metabolite production/consumption was determined. The protein levels of key glycolysis-related transporters and enzymes were assessed. The lactate dehydrogenase (LDH) activity was measured. Mitochondrial complexes protein levels and mitochondria membrane potential were also measured. We showed that hSCs express the growth hormone secretagogue receptor. At the concentration present in the plasma of normal weight men, ghrelin caused a decrease of glucose consumption and mitochondrial membrane potential in hSCs, though LDH activity and lactate production remained unchanged, illustrating an alteration of glycolytic flux efficiency. Exposure of hSCs to levels of ghrelin found in the plasma of severely undernourished individuals decreased pyruvate consumption and mitochondrial complex III protein expression. All concentrations of ghrelin decreased alanine and acetate production by hSCs. Notably, the effects of ghrelin levels found in severely undernourished individuals were more pronounced in hSCs metabolic phenotype highlighting the importance of a proper eating behavior to maintain male reproductive potential. In conclusion, ghrelin acts as an energy status sensor for hSCs in a dose-dependent manner, showing an inverse association with the production of lactate, thus controlling the nutritional support of spermatogenesis.

Protection of the spermatogenic cells from the host immune response is fundamental to male fertility. Significantly, this protection extends to the tolerance of foreign tissue grafts placed within the testicular environment, a phenomenon that is called 'immune privilege'. This privilege of the testis appears to involve several levels of immune control, encompassing the normal mechanisms of immune tolerance, antigen sequestration behind the blood-testis barrier, reduced immune activation, localised immunosuppression and antigen-specific immunoregulation. Central to these regulatory processes are the somatic cells of the testis, particularly the Sertoli cells, and testicular secretions, including androgens, cytokines, peptides and bioactive lipids. Failure of these protective mechanisms, which may be precipitated by trauma, inflammation or infection, or as the consequence of genetic factors, can lead to androgen deficiency, infertility and autoimmunity.

Spermatogenesis is a complex biochemical event, involving the participation of the hypothalamus and the pituitary gland via the secretion of the hypothalamus hormone GnRH, and two pituitary hormones FSH and LH. Thus, the hypothalamus-pituitary-testicular axis is a crucial regulatory axis for testicular function. Recent studies have shown that in the microenvironment of the seminiferous epithelium, wherein each Sertoli cell is supporting ~30-50 germ cells at different stages of their development, locally produced autocrine and paracrine factors are also involved in spermatogenesis, in particular at the level of cell junctions. These cell junctions at the Sertoli-Sertoli and Sertoli-germ cell interface are crucial to coordinate different events of spermatogenesis by sending signals back-and-forth between Sertoli and germ cells, in order to precisely regulate spermatogonial cells renewal by mitosis, cell cycle progression, meiosis, spermiogenesis, germ cell movement across the epithelium, spermiation and germ cell apoptosis. In this minireview, we provide an update on these latest findings for an emerging new concept regarding the presence of a local “apical ectoplasmic specialization – blood-testis barrier – hemidesmosome/basement membrane” functional axis that regulates the events of spermiation and blood-testis barrier (BTB) restructuring via paracrine/autocrine factors and polarity proteins produced locally in the seminiferous epithelium. These findings provide a new window of research for investigators in the field to tackle the functional regulation of spermatogenesis.

Bacterial lipopolysaccharide increased the production of interleukin 1alpha and activin A, and reduced production of inhibin B, in Sertoli cells from immature male rats measured by enzyme-linked immunosorbent assay (ELISA). The majority of immunoreactive interleukin 1alpha remained within the Sertoli cell, while both activin A and inhibin B were secreted. Lipopolysaccharide-stimulated expression of two interleukin 1alpha mRNA transcripts, measured by quantitative RT-PCR, but the levels of bioactive interleukin 1alpha in Sertoli cell extracts and medium, measured by in vitro bioassay, were comparatively low to undetectable. A specific antagonist of interleukin 1alpha had no effect on lipopolysaccharide-stimulated activin A or inhibin B responses. These data indicate that, in contrast to Sertoli cells from adult rats, lipopolysaccharide-induced regulation of activin A and inhibin B by prepubertal Sertoli cells does not involve secreted interleukin 1alpha. The data highlight the possibility of a role for intracellular interleukin 1alpha in the Sertoli cell response to inflammation, particularly in the immature testis.

Development and maintenance of the male phenotype and establishment of fertility are all dependent upon the activity of the Sertoli cells and Leydig cells of the testis. This review examines the regulation and function of these cell during fetal and post-natal development. Fetal Leydig cells are sensitive to both luteinising hormone (LH) and adrenocorticotrophic hormone (ACTH) but Leydig cell function appears normal in fetal mice lacking both hormones or their receptors. Post-natally, the Sertoli cells and Leydig cells are reliant upon the pituitary gonadotrophins. Leydig cells are critically dependent on LH but follicle-stimulating hormone (FSH), presumably acting through the Sertoli cell, can also affect Leydig cell function. Testosterone secreted by the Leydig cells acts with FSH to stimulate Sertoli cell activity and spermatogenesis. Study of animals lacking FSH-receptors and androgen-receptors shows that both hormones can act to maintain the meiotic germ cell population but that androgens are critical for completion of meiosis.

It is well known that exposure to cis-diaminedichloroplatinum (CDDP) results in impairment of spermatogenesis; however, little is known about the signal mechanisms involved in CDDP regulation of Sertoli cell (SC) function. This study was designed to evaluate how CDDP regulates SC signal molecules and mechanisms. Purified rat SC was cultured under serum-free conditions and treated with CDDP (10 ng/ml) at various time points. Western blot analysis was used to determine the activation of extracellular signal-related kinases 1 and 2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), cJun-N-terminal kinase (JNK), cyclooxygenase (COX)-1 and COX-2, inducible and endothelial nitric oxide synthase (iNOS and eNOS). The levels of transferrin (TF) and prostaglandin (PG)E2, PGF2alpha, PGD2, carbaprostacyclin (cPGI2 analog) in culture medium were quantified by ELISA. Nitrite (NO2(-)) and nitrate (NO3(-)) in culture medium were also quantified by Griess assay. Interleukin (IL)-1beta and IL-6 mRNAs were measured by quantitative real-time PCR (QRT-PCR) analysis. CDDP activated the phosphorylation of ERK1/2 (p-ERK1/2) in the early phase (within 5 min) and the level of transferrin (TF) fell significantly. In addition, CDDP significantly increased the level of COX-2, PGs, and ILs in the late phase (within 24h). When ERK activity inhibitor (PD98059, 10 microM) or COX-2 activity inhibitor (NS-398, 10 microM) was used, CDDP reduction of TF and induction of PG and IL expression were prevented, suggesting that the detrimental effects on spermatogenesis through the impairment of SC induced by CDDP are mediated by the activation of ERK1/2 and COX-2 pathways in SC.

The sex determining region Y (SRY/Sry) gene is necessary and sufficient for testis determination and differentiation in mammals. SRY/Sry encodes a putative transcription factor with a high mobility group (HMG) DNA-binding domain. The spatiotemporal regulation of Sry expression suggests that a brief action of SRY in a limited number of progenitor cells (pre-Sertoli cells) before the onset of default ovarian differentiation is sufficient to switch on testicular differentiation. Recent identification and characterization of the Krüppel-associated box only (KRAB-O) protein as an SRY-interacting protein have provided experimental evidence supporting an interesting model for SRY function. In this model, SRY recruits the KRAB-KAP1 (KRAB-associating protein 1) complex as a chromatin modulator, which provides a molecular mechanism of SRY as a transcription factor. Moreover, the sufficiency of a brief action of SRY for testis differentiation can be partly explained by the heritability of KRAB-mediated chromatin remodeling. Although it is currently uncertain whether KRAB-O is the only KRAB protein with which SRY interacts, we hypothesize that KRAB-O or yet-to-be identified KRAB-containing proteins might play various roles in sex determination and gonadal differentiation.

Sertoli cells support the development of germ cells by providing a microenvironment in the seminiferous tubules. FSH stimulates Sertoli cell proliferation only during neonatal period till day 18 in the immature rat whereas FSH regulates only functional parameters in the adult rat Sertoli cells. This suggests that FSH exerts differential action in immature and adult Sertoli cells. In an attempt to elucidate the mechanism by which FSH exerts the differential effects, we have carried out both in vivo and in vitro studies using Sertoli cells isolated from immature (7-10 days old) and adult (90 days old) rats. The differential role of FSH was studied at the receptor as well as at the signaling level. Monitoring the level of expression of FSH receptor by RTPCR and northern blot analysis revealed that the expression was more in immature Sertoli cells. Furthermore, it was found that FSH up (1.8-fold) regulates its receptor level only in the immature Sertoli cells and not in the adult. Results also revealed that PKIbeta and calcium, which are the downstream signaling molecules, are involved in FSH regulated Sertoli cells proliferation. It was also observed that FSH up (1.4-fold) regulates the levels of expression of IL-6 mRNA only in the immature rat Sertoli cells suggesting the possibility of its involvement in FSH regulated Sertoli cell proliferation.

We investigated the involvement of protein synthesis in the stimulatory action of thyroid hormones on amino acid accumulation and characterized K(+) currents involved in the hyperpolarizing effect of thyroxine (T(4)) on Sertoli cells. Immature rat testes were incubated in Krebs Ringer-bicarbonate buffer (KRb) in the presence of [(14)C]methylaminoisobutyric acid with and without T(4), 3,5,3'-l-triiodothyronine (T(3)) and/or cycloheximide. Sertoli cells were monitored by intracellular recording in a chamber perfused with KRb with and without T(4), T(3) and/or blockers, and the membrane potential was monitored. T(4) and T(3) stimulated amino acid accumulation and protein synthesis. Treatment with cycloheximide diminished T(3) stimulatory actions on amino acid accumulation but had no effect on T(4) action. Both hormones elicited a hyperpolarization of the Sertoli cell membrane potential which involved K(+) channels, since TEA and apamin abolished this effect. These findings on rapid membrane actions of thyroid hormone in the testis suggest that some effects of T(4) are modulated by non-genomic mechanisms.

In the mammalian postnatal testis, the biochemical and structural features of Sertoli cells change, depending on developmental stage and spermatogenic cycle, to support efficient spermatogenesis. Consequently, basic transcription factors that determine fundamental properties should be strictly maintained in postnatal Sertoli cells. We have confirmed that GATA-4 expression is kept at a constant level in mouse Sertoli cells during postnatal development, and is also maintained at a constant level in primary cultures, independent of treatment with hormones or the addition of germ cell fractions. In transient transfection assays with the testicular cell line TM3, established from Leydig cells, GATA-4 induced several Sertoli cell-specific genes. In the Sertoli cell line TM4, and in Sertoli cells in primary culture, GATA-4 slightly up-regulated these genes. These results suggest that GATA-4 plays an important role in the regulation of Sertoli cell function, and is exactly regulated in these cells.

The pituitary gonadodotropins are the main regulators of testicular hormonal secretion in humans. Hypogonadotropic hypogonadism (HH), characterized by the absence of secretion of endogenous gonadotropins is therefore a convenient model to asses the respective effects of luteinizing hormone (LH) (or human chorionic gonadotropin (hCG)), exogenous testosterone (T) and FSH on gonadal function. In order to investigate the hormonal control of AMH secretion in man, serum AMH levels were measured in adult patients with congenital HH (CHH) and with post-pubertal acquired HH (AHH) either untreated, during hCG or T therapy. In untreated CHH patients, serum AMH levels were significantly higher than in normal men and similar to those previously reported in prepubertal boys indicating the absence of pubertal maturation of Sertoli cells. In men with AHH, serum AMH levels were also significantly increased when compared to healthy men, but less than in CHH because a persistent testicular T secretion in these patients with less complete gonadotropin deficiency. The high AMH levels in AHH suggest that the post-pubertal suppression of AMH is a reversible phenomenon. In HH patients, hCG treatment induced an increase of plasma T associated with a dramatic decrease of serum AMH, whereas the similar increase in plasma T levels obtained with exogenous T induced only a partial decrease of serum AMH. This dissociation was related to the higher intratesticular T induced by hCG. Taken together, our results confirms the clinical relevance of previous data obtained in rodent models concerning the hormonal regulation of AMH secretion.

In vitro studies were designed to determine whether Sertoli cell-delivered ABP could act on spermatogenetic events, whether such an action could occur via a paracrine or a juxtacrine pathway and whether sex steroids could be involved in this action. ABP delivery to germ cells was achieved using an in vitro model based on recombinant rat ABP-producing mouse Sertoli cells cocultivated with rat spermatids. Using semi-quantitative RT-PCR, the expression of the Tnp 1 gene encoding the Transition Protein 1, involved in the histone to protamine replacement during spermatid nuclear transformation, was analyzed. Our results provide clear evidence that Sertoli cell-derived ABP acts on spermatids by modifying the TP1 mRNA level. This outcome, strictly requiring juxtacrine conditions, is obtained in the absence of sex steroid hormones. To our knowledge this is the first evidence of an effect of ABP itself on male germ cells.

Testes from rats of different maturational ages were explored for presence of paracrine sertoli cell growth factors. Pubertal and adult testes contained a 17 kDa protein, with potent stimulatory effect on immature Sertoli cell multiplication in vitro. The bioactivity of this protein was mimicked by rat interleukin-1 (IL-1) and neutralized by IL-1 receptor antagonist. A receptor-mediated action was further supported by the demonstration of IL-1 receptor type I mRNA and protein expression in the cultured sertoli cells and in intact immature rat testes. IL-1alpha showed higher efficacy in stimulating proliferation than IL-1beta and follicle-stimulating hormone (FSH), and displayed synergistic action in combination with FSH. As IL-1alpha is constitutively produced by the rat testis and IL-1beta readily inducible by proinflammatory stimuli, our results suggest that IL-1 may serve as a growth factor for Sertoli cells under physiological and pathophysiological conditions.

The interaction of ouabain with the sodium pump induces signalling cascades resembling those triggered by hormone/receptor interactions. In the rat Sertoli cell line 93RS2, ouabain at low concentrations stimulates the c-Src/c-Raf/Erk1/2 signalling cascade via its interaction with the α4 isoform of the sodium pump expressed in these cells, leading to the activation of the transcription factor CREB. As a result of this signalling sequence, ouabain stimulates expression of claudin-1 and claudin-11, which are also controlled by a CRE promoter. Both of these proteins are known to be essential constituents of tight junctions (TJ) between Sertoli cells, and as a result of the ouabain-induced signalling TJ formation between neighbouring Sertoli cells is significantly enhanced by the steroid. Thus, ouabain-treated cell monolayers display higher transepithelial resistance and reduced free diffusion of FITC-coupled dextran in tracer diffusion assays. Taking into consideration that the formation of TJ is indispensable for the maintenance of the blood-testis barrier (BTB) and therefore for male fertility, the actions of ouabain described here and the fact that this and other related cardiotonic steroids (CTS) are produced endogenously suggest a direct influence of ouabain/sodium pump interactions on the maintenance of the BTB and thereby an effect on male fertility. Since claudin-1 and claudin-11 are also present in other blood-tissue barriers, one can speculate that ouabain and perhaps other CTS influence the dynamics of these barriers as well.

beta-estradiol 3-benzoate (E(2)B) (10, 16, 20, 40, 80 and 160 microg/kg body weight) was administered daily to experimental groups of adult mice for the following periods; 2, 3 days, 1, 2, 4, and 8 weeks. Morphological changes in the testes were observed by both light and electron microscopy. Exfoliation of the germ cells was observed in the lumen of the seminiferous tubule. The spermatogenic cycle, especially stage XII, was disordered. Spermatids older than step 6 were severely affected. Detected abnormalities in the spermatids were deformation of the nucleus and acrosome. Partial deletion in the Sertoli-spermatid ectoplasmic specialization was also observed. Germ cells younger than step 7 spermatids were not affected morphologically. These abnormalities were not detected in the mice treated with the chemical at less than 16 microg/kg body weight. It is concluded that the chemical seems to affect round spermatids metabolically, but the morphological effect can be detected only from the spermatids older than step 6. The effects of the chemical on adult mice were reversible.

The role of estrogens produced by the testis may involve negative feedback regulation of androgen biosynthesis. Estrogens are also associated with contractile processes of seminiferous tubules, and might have mitogenic effects on Sertoli and Leydig cells. To investigate the location of aromatase (estrogen synthetase) in the testes, tissue from normal human subjects, aged 3 months to 72 years were studied using immunocytochemistry. In mature testes, aromatase immunostain was always associated with Leydig cells and was absent from Sertoli cells. Aromatase activity ranged from 0.014-0.55 pmol estrogen per mg/h and was significantly correlated with the immunostain intensity (P0.02). Activity and immunostain intensity did not correlate with increasing age. Rather, the highest levels were measured in four of six testes of men aged 18-20 years, three of whom also had the strongest immunostain in larger and more prominent Leydig cell clusters than those in the other specimens. A low level of aromatase activity but no immunostain was detected in prepubertal testes. However, in several prepubertal patients with Peutz-Jegher's Syndrome (PJS) with bilateral multifocal sex cord tumors and enlarged seminiferous tubules and Sertoli cells, aromatase was expressed in these Sertoli cells, but absent from normal Sertoli and Leydig cells. Increased aromatase expression in these tissues involved activation of upstream regulatory elements of the gonadal P II promoter of P-450(arom). In a prepubertal boy with gynecomastia but without PJS, aromatase excess appeared to be due to increased aromatization in skin fibroblasts and lymphocytes. Several members of the patient's family including his sister also expressed high levels of aromatase. This condition appears to be inherited in an autosomal dominant manner.

The cytochrome P450 aromatase (P450arom) is a key enzyme responsible for the formation of estrogens from androgens and is present in the endoplasmic reticulum of various tissues. P450arom has been immunolocalized in Leydig cells of numerous species as well as in germ cells of mouse, bank vole and brown bear. Aromatase activity has been measured in vitro in immature and mature rat Leydig cells and Sertoli cells, whereas in pig, ram and humans the enzyme activity is only present in Leydig cells. In the mature rat testis we have used complementary approaches to demonstrate that not only somatic cells but also germ cells represent a new source of estrogens. In pachytene spermatocytes and Leydig cells, the amount of P450arom mRNA measured by a quantitative competitive RT-PCR method is 10-fold higher than in Sertoli cells. According to the stage of the germ cell maturation, the amount of aromatase transcripts decreases, being more elevated in younger than in mature rat germ cells. By contrast, the aromatase activity in the microsomal fractions is two- to four-fold greater in spermatozoa when compared to the two other enriched germ cell preparations used. We have immunolocalized the P450arom in elongated spermatids and spermatozoa. Moreover, we described the existence of alternative splicing events of P450arom mRNA in pachytene spermatocytes and round spermatids that are not likely to encode functional aromatase molecules. Therefore, the aromatase gene expression and its transduction in a fully active protein in rat germ cells evidences an additional site for estrogen production within the testis of some mammals. Taking into account the large distribution of estrogen receptors in the testicular cells, we begin to understand the physiological role of these female hormones in the male gonad.

Aromatase activity has recently been assumed as a Sertoli cell functional maturation marker since it is maximally expressed in prepuberal age then it dramatically decreases at puberty and is virtually absent in adult age. Neonatal hypothyroidism is associated with a prolonged proliferation of Sertoli cells. This immature stage persists concomitantly with a dramatic enhancement of aromatase activity reversed by triiodothyronine (T3) either in vivo or in vitro administration. Therefore, in the present study, after immunolocalisation of aromatase in the cytoplasm of cultured Sertoli cells as well as in testis section, we investigate the regulatory effects of T3 in the same cells just at the age when aromatase activity is reported to be maximally expressed. In this aim, the effects of thyroid hormone have been evaluated in 2-weeks-old rats, in basal condition and upon stimulation with dibutyryl cyclic AMP [(Bu)(2)cAMP] by simultaneously analysing three functional levels of aromatase, mRNA expression; protein content; enzymatic activity. Western-blot analysis of Sertoli cell extracts revealed a protein, which co-migrated with a 55 kDa protein detected in human placenta used a positive control. The presence of a functional P450 aromatase protein in purified Sertoli cells was confirmed by the measurement [3H]H(2)O released after incubation with [1beta-(3)H]androst-4-3,17-dione. At the dose used, T3 down-regulates basal aromatase activity, while aromatase mRNA expression was apparently not inhibited. It is noteworthy that aromatase content pattern evaluated by Western blot analysis did not tightly parallel the aromatase activity pattern which clearly displays the inhibitory effects of T3, in basal condition ad upon (Bu)(2)cAMP stimulation, simulating FSH stimulation. The detection of mRNA altered transcript coding for putative protein lacking both aromatic and heme-binding regions upon T3 treatment and unable to convert androgens into estrogens, provides a reasonable explanation for the observed discrepancies between aromatase protein pattern, P450arom mRNA levels and aromatase activity. The authors conclude that although the altered transcript induced by prolonged exposure to T3 is a mechanism by which T3 may down regulate aromatase activity, it cannot be ruled out a direct effect of this hormone at the transcription levels since a recognisable emisite for potential TR(s) binding is located in the promoter region of aromatase gene. Thus a further investigation on T3 modulator role on aromatase gene promoter should be pursued even utilising higher doses of T3.

Androgens are important for the structural and functional integrity of the testis and the prostate and this may in part be mediated by the aromatisation of testosterone to oestradiol. The aim of the present study was to establish an in vivo model that would allow the identification of genes, the expression of which was regulated acutely by androgen and/or oestrogen in the male reproductive system. In rats in which the Leydig cells were ablated by administration of ethane dimethane sulfonate (EDS) 6 days earlier, testosterone esters (T) were administered from day 0 (To), and additional animals were administered either T, 17β-oestradiol benzoate (EB) or diethylstilbestrol (DES) for 1 or 4 h on day 6 after EDS-treatment. Nuclear immunoexpression of the androgen receptor (AR) was reduced or absent from the testis but unaffected in the ventral prostate following these treatments. ERβ immunoexpression in these tissues was unchanged. Northern blot analysis showed that EB and DES as well as T administration 4 h earlier could modulate mRNA expression of two androgen-responsive genes, C3 and SGP-2, in the prostate. The co-administration of T or EB with the AR antagonist, flutamide, or with the ER antagonist, ICI 182,780 (ICI), did not block the suppression of SGP-2 mRNA expression by T or EB. In contrast, the upregulation of C3 mRNA expression by T was successfully antagonised by both flutamide and by ICI. A preliminary evaluation of the expression of three Sertoli cell and five germ cell mRNAs revealed that their expression was not steroid regulated. Our results support the hypothesis that the action of testosterone in the male reproductive system may in part be mediated by its conversion to oestradiol. This in vivo model should prove of value in future studies to identify androgen and oestrogen regulated genes in the male reproductive system.

The action of follicle-stimulating hormone (FSH) in spermatogenesis is regulated at a fundamental level by controlling the number of competent receptors present at the surface of Sertoli cells. By controlling the number of receptors, the cell is able to modulate the timing and magnitude of subsequent signal transduction in response to FSH. One mechanism of control is the down-regulation of the steady state levels of the FSH receptor gene after exposure to FSH or agents that stimulate or prolong the cAMP signal transduction cascade (homologous down-regulation) in Sertoli cells. The goals of this study were to examine possible mechanisms involved in the down-regulation of mRNA levels of this gene. Analysis of transcription and processing by a PCR-based assay showed that treatment of Sertoli cells with FSH caused at least a 50% reduction of hnRNA for the FSH receptor gene. Reporter genes controlled by 5′ flanking sequences of the FSH receptor gene that were transiently transfected into Sertoli cells were not down-regulated. In electrophoretic mobility shift assays (EMSA), cAMP-inducible nuclear protein complex containing c-Fos formed on the activator protein-1/cAMP responsive element-like site located at −216 to −210 in the promoter of the rat FSH receptor gene. We concluded from this study that there was no evidence for the putative role of ICER in the down-regulation of the FSH receptor promoter. In addition, the FSH-induced down-regulation of the transcription of the FSH receptor gene in Sertoli cells was prevented by the treatment of Sertoli cells with trichostatin A prior to the addition of FSH. This experiment coupled with other observations suggested that the down-regulation may be mediated by changes in chromatin structure.

Development of germ cells during spermatogenesis is characterized by a complex series of differentiation events finally leading to the production of spermatozoa. Beside the main hormonal regulators, paracrine interactions are thought to play a major role in this process. Mitochondria in germ cells pass through unique alterations ranging from the ‘typical’ cristae-rich mitochondria found in spermatogonia to the ‘condensed’ form in pachytene spermatocytes. This study provides further support that paracrine factors produced by Sertoli cells, most likely activin A, are involved in germ cell differentiation as monitored by the maintenance of the physiological ‘condensed’ mitochondrial phenotype in primary spennatocytes. Culture of primary spermatocytes in Sertoli cell conditioned medium (SCCM) for 18 h resulted in the maintenance of a high percentage of ‘condensed-type’ mitochondria in comparison to cells cultured in Dulbecco's minimum essential medium (DMEM). Activin A, a product of Sertoli cells, showed at subnanogram concentrations a similar ability to SCCM to maintain a high percentage of spematocyte mitochondria in the ‘condensed’ state, while inhibin had no effect. The addition of an antiserum specific for activin A resulted in a neutralization of the effect caused by activin A or SCCM. This strongly suggested that the active substance in SCCM was activin A. Taken together these data indicate that activin A is the first Sertoli cell product that has been identified to influence differentiation of male meiotic germ cells.

Sertoli cells regulate the spermatogenic process mainly through the secretion of a complex fluid into the lumen of the seminiferous tubules behind the blood–testis barrier, containing many of the essential proteins necessary for maintenance and maturation of male germ cells. Thus, the study of Sertoli cell secretory processes is strictly correlated with the understanding of the regulatory mechanisms of spermatogenesis. In this work the authors have explored the voltage-sensitive calcium channel variety in the immature rat testis, their localisation and distribution within the seminiferous epithelium and peritubular and interstitial tissues as well as the possible role in the control of Sertoli cell secretion. The results reported in this paper, obtained by in situ hybridisation, immunohistology of rat testicular sections and Western blot analysis of Sertoli cell plasma membranes, show that mammalian Sertoli cells express mRNA encoding for several voltage-operated calcium channel subunits and express such proteins on their surface. Experiments performed on Sertoli cell monolayers cultured in the presence of specific toxins indicate that both N and P/Q-type Ca2+ channels are involved in the regulation of protein secretion.

In the present study, we examined the in vitro regulation of 20-day-old rat Sertoli cell transferrin expression by tumor necrosis factor alpha (TNFalpha), a paracrine factor produced by germ cells. Addition of TNFalpha to highly purified cultured Sertoli cells resulted in a dose and time-dependent enhancement in the levels of transferrin mRNA (Northern-blot) and protein (RadioImmunoAssay) with an ED50 of 120 pM. Co-treatment of Sertoli cells with the optimal dose of retinoic acid (RA, a potent inducer of transferrin) and TNFalpha induced a stimulation of transferrin that was significantly higher than the FIRT combination, a well known mixture of transferrin activators. Actinomycin D inhibited the effects of TNFalpha and of RA, suggesting that ongoing RNA synthesis was required to enhance transferrin. We next demonstrated that RA and TNFalpha exerted additive effects on transferrin expression as assessed by dose-response and kinetics studies. Moreover pre-treatment with RA, while greatly increasing the amount of transferrin produced, did not modify Sertoli cell responsiveness to TNFalpha. Together these results show that TNFalpha and RA are likely to act independently, additively and at least at the transcriptional level to increase transferrin expression.

A plasmid expressing the rat FSH receptor (R) cDNA under the Simian virus (SV) 40 promoter/enhancer was stably transfected into a mouse Sertoli cell (SC) line (MSC-1) established from transgenic mice carrying a fusion gene of the human anti-Müllerian hormone (AMH) promoter sequences linked to the SV40 T-antigen gene (Peschon et al., 1992). The original cell line has numerous SC characteristics, but it was reported not to express the inhibin-alpha and follicle-stimulating hormone (FSH)R genes. The new FSHR expressing cell line possessed approximately 2000 per cell with equilibrium association constant (Ka) of 1.5 x 10(9) l/mol. In Northern blots, an FSHR mRNA species of 2.6 kb was found. The cells responded to recombinant human FSH (recFSH) and pertussis toxin (PT) with stimulated cAMP production. Moreover, PT enhanced the FSH-stimulated cAMP production in these cells, indicating the presence of a functional Gi protein. 12-O-tetradecanoylphorbol-13-acetate (TPA) suppressed the FSH-stimulated cAMP production of the cells, which effect was similar to that observed previously upon protein kinase C (PKC) activation in rat seminiferous tubules in vitro. Hence, the FSHR signalling, and its modulatory pathways, were intact in the FSHR expressing MSC-1 cell line. RT-PCR with inhibin-alpha specific oligonucleotide primers. followed by Southern hybridization, indicated that, unlike previously shown, the original and the FSHR expressing MSC-1 cells do express the inhibin alpha gene. FSH stimulation of the cells decreased their proliferation and, unexpectedly, the inhibin-alpha mRNA levels. The cells have functional features both from neonatal and mature SC. A feature of the former cells is the lack of FSH-stimulated up-regulation of inhibin-alpha expression; in fact FSH decreased this message. The antiproliferative, and apparently differentiating, effect of FSH on these cells resembled mature SC functions. Since adult SC do not proliferate in vitro, the new FSHR expressing and proliferating cell line provides a useful in vitro model for studying some facets of SC functions, though keeping in mind that these transformed cells do not behave identically with adult SC in vivo. The constitutive expression of FSHR in these cells allows the study of posttranscriptional events in the FSHR regulation.

Anti-Mullerian hormone (AMH) induces the regression of Mullerian ducts in the male foetus; it is secreted by prepubertal testicular Sertoli cells and repressed at puberty. Using an AMH promoter/Simian virus 40 (SV40) oncogene fusion gene, we generated transgenic mouse lines exhibiting heritable Sertoli cell tumorigenesis. One cell line, derived from an adult male, expressed mRNAs characteristic of mature Sertoli cells, but no AMH. Two other cell lines were obtained from pretumoral testes at 6.5 days. One was cloned to yield SMAT1, whose expression pattern was characteristic of prepubertal Sertoli cells, namely no transferrin and high SF-1 and AMH expression. SMAT1 also secretes AMH protein into the culture medium and expresses the AMH receptor. To the best of our knowledge, this is the first Sertoli cell line stably expressing AMH and its receptor. Our results show that, in targeted oncogenesis, the timing of cell line derivation plays a critical role even when using a developmentally regulated promoter.

Interleukin-1 (IL-1) and IL-6 are pleiotropic cytokines produced by a large variety of cell types. In the testis, Sertoli cells produce IL-1alpha and IL-6. Previous studies have demonstrated that, in vitro, Sertoli cell IL-1alpha production is stimulated by some inducers of macrophage IL-1, as well as by phagocytosis of residual bodies. Furthermore, we have also shown that IL-1alpha is able to enhance Sertoli cell IL-6 production by an autocrine action. The aim of the present study was to further investigate the regulation of Sertoli cell IL-1 and IL-6 production. Three categories of potential regulators were tested; the lipopolysaccharide (LPS) and the yeast extract zymosan; follicle stimulating hormone (FSH), testosterone and dexamethasone; tumor necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma) and the nerve growth factor beta (NGFbeta). It was found that zymosan (400-800 microg/ml) and LPS (20 microg/ml) stimulated Sertoli cell IL-1 and IL-6 production. FSH (1 x 10(-2)-1 microg/ml) and NGF (25-200 ug/ml) stimulated Sertoli cell IL-6 levels in a dose-dependent manner but had no effect on IL-1. The effect of testosterone on Sertoli cell IL-1 and IL-6 secretion was biphasic: dramatic increased secretion with low concentrations (0.01-1 nM) and no effect with the higher concentration tested (100 nM). Dexamethasone reduced LPS-induced IL-1 and IL-6 production in a concentration-responsive manner (0.04-0.4 and 0.4-40 ng/ml, respectively). Addition of TNFalpha to Sertoli cells resulted in a dose-dependent increase of both cytokines (50-100 U/ml for IL-1, 100-200 U/ml for IL-6). In the case of IFNgamma, intermediate concentrations (50-100 U/ml) stimulated IL-1alpha, whereas the highest concentrations (200-400 U/ml) inhibited IL-6. It is concluded that regulation of Sertoli cell IL-1 and IL-6 is very complex as it involves factors as different as hormones, paracrine factors and activators of macrophages. The latter agents may be mimicking the action of pathogens or the action of intratesticular agents whose nature remains to be elucidated.

Activation of cyclic AMP-dependent protein kinases (protein kinase A, PKA) by gonadotropins and cyclic AMP (cAMP) plays an important role in the regulation of testicular functions. A regulatory subunit, RII β , of PKA is transcriptionally induced in rat Sertoli cells in response to treatment with cAMP. The present study addresses regulatory mechanisms leading to increased transcription of the rat RII β gene. We have localized a footprint which overlaps one of the major transcription initiation sites in the basal promoter (−293 to −123). One of the proteins binding this sequence belongs to the NF-1 family of transcription factors. We also observed binding to a basic helix-loop-helix (bHLH) response element. Furthermore, transfection studies of various 5′-deletions of the rat RII β gene in primary cultures of rat Sertoli cells and in peritubular cells revealed the presence of an upstream region (−723 to −395, cAMP-responsive region) inhibiting basal expression from the rat RII β gene only in Sertoli cells. This region was found to enhance cAMP responsiveness in Sertoli cells but not in peritubular cells. Interactions with downstream elements seemed to be important for the function of the cAMP-responsive region. Although some short stretches reveal homology to the cAMP-responsive regions of other slowly cAMP-responding genes, and an AP-1-like element is present, no strong resemblance to any known regulatory element responsive to cAMP is found.