Proliferation Of Immature Sertoli Cells Research Articles

TLR4 signalling protects Sertoli cells from cell stress via reprogramming inflammasome and autophagy pathways in MAPK1/ERK2 dependent way

Sertoli cells constitutively maintain the spermatogonial stem cell niche and blood-testis barrier via impenetrable tight junctions under hormonal, growth factor and cytokine control. Immature Sertoli cell proliferation and survival benefit from FSH, NF-κB, and IL-1α-dependent signalling effective via the MAPK family of pathways and related to p38 MAPK dependent Ca2+ and K+ fluxes. Little is known about how the NF-κB and MAPK axis interact with pro-inflammatory NOD1/TLR4 and different voltage-gated channels. Silencing MAPK1, we found that MAPK1/ERK2 has a synergistic role with TLR4 in restricting the pro-inflammatory NF-κB/Caspase-1/IL-1β axis while having an antagonistic effect on TLR4 in promoting adult 15 P-1 Sertoli cell line proliferation and migration. In MAPK1/ERK2 absence, there was an unexpected TLR4-NF-κB behaviour, showing a strong NF-κB transactivation, assessed using a pNIFTY-SEAP reporter plasmid, accompanied by significantly activated cell migration and proliferation, as demonstrated by the wound healing assay. Voltage-gated potassium flux, most likely via Kv1.3, was significantly increased after TLR4 and NOD1 challenge in ATP DAMP signal presence, associated with inflammasome activation. TLR4 challenge also activated macroautophagy while inhibiting mitophagy. Thus, TLR4 activation restricted the NF-κB and Caspase-1/IL-1β pro-inflammatory axis, cell migration and proliferation, voltage-gated potassium efflux, and shifted mitophagy to macroautophagy (towards an inflammasome pathway-related cell death), promoting cell survival. MAPK1/ERK2 had a differential effect, as in its absence, TLR4 activation had the opposite effect on NF-κB transactivation and cell migration and proliferation. Thus, TLR4/MAPK1/NF-κB/Kv1.3 interaction provides optimal Sertoli cell maintenance conditions and allows cell death only in extreme circumstances of cumulative DAMP/PAMP signalling stimulation.

FSH promotes immature porcine Sertoli cell proliferation by activating the CCR7/Ras-ERK signaling axis.

The appropriate growth and functions of Sertoli cells are crucial to testis development and spermatogenesis in mammals. This study reveals a novel mechanism of follicle-stimulating hormone in immature porcine Sertoli cell proliferation. Follicle-stimulating hormone (FSH) is a major Sertoli cell mitogen that binds to the FSH receptor. Sertoli cells are indispensable for testis development and spermatogenesis. However, the regulatory mechanisms of FSH in immature Sertoli cell proliferation have not been determined, particularly in domestic animals. In the present study, we identified the regulatory mechanisms of FSH during immature porcine Sertoli cell proliferation. Transcriptome analysis revealed 114 differentially expressed genes that were induced by FSH treatment, which contains 68 upregulated and 46 downregulated genes. These differentially expressed genes were enriched in multiple pathways, including the Ras signaling pathway. Knockdown of the CC-chemokine receptor 7 (CCR7) gene, which was upregulated by FSH, inhibited cell cycle progression by arresting cells in the G1 phase and reduced the cell proliferation and ERK1/2 phosphorylation. In addition, Kobe0065 inhibited Ras signaling in a similar manner as CCR7 knockdown. Furthermore, FSH abolished the effects of Ras signaling pathway inhibition and CCR7 knockdown. Collectively, FSH promotes immature porcine Sertoli cell proliferation by activating the CCR7/Ras-ERK signaling axis. Our results provide novel insights into the regulatory mechanism of FSH in porcine testis development and spermatogenesis by deciding the fate of immature porcine Sertoli cells.

TNFα stimulates the proliferation of immature Sertoli cells by attenuating UPS-degradation of cyclin D1 and leads to the delay of BTB maturation in pubertal rats.

The Sertoli cell that plays a vital role during spermatogenesis is a known target of physiological and pathological factors affecting testicular development. Tumor necrosis factor alpha (TNFα) participates in the blood-testis barrier reconstruction, cell apoptosis, and inflammatory response by recognizing receptors on Sertoli cell. TNFα has also been shown to induce the proliferation of immature Sertoli cell in vitro, yet the mechanism still remains unclarified. This study was designed to investigate the effect of TNFα on blood-testis barrier development during puberty and the underlying mechanisms of TNFα-induced immature Sertoli cell proliferation. Immature male Sprague-Dawley rats of postnatal day 12 were intraperitoneally injected with TNFα. Biotin-labeled method was used to detect permeability of the developing blood-testis barrier after TNFα treatment, and the distribution of occludin and junctional adhesion molecule-A (JAM-A) were detected by immunofluorescence. Sertoli cells isolated from Sprague-Dawley rats of postnatal day 10 were cultured in vitro and treated with TNFα. Cell proliferation rate was reflected by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assay. Immunoblot and quantitative polymerase chain reaction were used to detect the expression of proliferating cell nuclear antigen, Fbxo4, and cyclin D1. Immunoprecipitation was used to detect the ubiquitination of cyclin D1 and the interaction between Fbxo4 and cyclin D1. Ammonium pyrrolidinedithiocarbamate (PDTC) was applied to detect the effect of nuclear factor kappaB (NFκB) activity inhibition on TNFα-induced Sertoli cell proliferation. The adenoviral recombinant plasmid containing rat Fbxo4 gene was constructed to investigate the effect of Fbxo4 overexpression on Sertoli cell proliferation promoted by TNFα. The in vivo experiment revealed a significant delay of blood-testis barrier maturation in pubertal rats caused by exogenous TNFα. TNFα (10ng/ml) treatment in vitro was found to promote the proliferation of immature Sertoli cells, accompanied with increased NFκB activity and cyclin D1 protein level. The level of Fbxo4 and ubiquitination of cyclin D1 were decreased after TNFα treatment. Inhibitor of NFκB or overexpression of Fbxo4 could both reverse the TNFα-induced proliferation of immature Sertoli cells, meanwhile restore the ubiquitin-proteasome system-dependent degradation of cyclin D1. Overexpression of Fbxo4 could not affect the activation of NFκB caused by TNFα. These results indicate that TNFα inhibits the ubiquitination and degradation of cyclin D1 through the NFκB pathway, thereby promoting the proliferation of immature Sertoli cell in vitro and inducing the delay of blood-testis barrier maturation in pubertal rats.

AMH Regulation by Steroids in the Mammalian Testis: Underlying Mechanisms and Clinical Implications

Anti-Müllerian hormone (AMH) is a distinctive biomarker of the immature Sertoli cell. AMH expression, triggered by specific transcription factors upon fetal Sertoli cells differentiation independently of gonadotropins or sex steroids, drives Müllerian duct regression in the male, preventing the development of the uterus and Fallopian tubes. AMH continues to be highly expressed by Sertoli until the onset of puberty, when it is downregulated to low adult levels. FSH increases testicular AMH output by promoting immature Sertoli cell proliferation and individual cell expression. AMH secretion also showcases a differential regulation exerted by intratesticular levels of androgens and estrogens. In the fetus and the newborn, Sertoli cells do not express the androgen receptor, and the high androgen concentrations do not affect AMH expression. Conversely, estrogens can stimulate AMH production because estrogen receptors are present in Sertoli cells and aromatase is stimulated by FSH. During childhood, sex steroids levels are very low and do not play a physiological role on AMH production. However, hyperestrogenic states upregulate AMH expression. During puberty, testosterone inhibition of AMH expression overrides stimulation by estrogens and FSH. The direct effects of sex steroids on AMH transcription are mediated by androgen receptor and estrogen receptor α action on AMH promoter sequences. A modest estrogen action is also mediated by the membrane G-coupled estrogen receptor GPER. The understanding of these complex regulatory mechanisms helps in the interpretation of serum AMH levels found in physiological or pathological conditions, which underscores the importance of serum AMH as a biomarker of intratesticular steroid concentrations.

MiR-191 promotes the porcine immature Sertoli cell proliferation by targeting the BDNF gene through activating the PI3K/AKT signaling pathway.

The number of Sertoli cells in the testis is a major regulator on the sperm production capacity. MicroRNAs (miRNAs) participate in regulating the proliferation and apoptosis of porcine immature Sertoli cells. However, the functions and mechanisms of action of most identified miRNAs in porcine Sertoli cells remain largely unknown. In the present study, based on our previous results from an EdU-based high-content screening assay, we further studied the mechanism of action of miR-191 on the proliferation and apoptosis of porcine immature Sertoli cells through flow cytometry, Western blotting, and dual-luciferase activity analyses. The results demonstrated that overexpression of miR-191 promoted cell cycle progression from G1 phase to the S and G2 phases, enhanced cell proliferation, and inhibited apoptosis in the porcine immature Sertoli cells, whereasmiR-191 inhibition resulted in the opposite effects. The results from a luciferase reporter assay showed that miR-191 directly targeted the 3'-UTR of theBDNF gene. BDNF knockdown also promoted cell cycle progression to the S phase, cell proliferation and inhibited cell apoptosis, which were consistent with the effects of the miR-191overexpression. A co-transfection experiment showed that BDNF knockdown abolished the effects of miR-191 inhibition. Furthermore, both miR-191 overexpression and BDNFinhibition elevated the phosphorylation of PI3K and AKT, the key components of the PI3K/AKT signaling pathway, whereas BDNFinhibition offset the effects of the miR-191 knockdown. Overall, these data indicated that miR-191 promotes cell proliferation and inhibits apoptosis in porcine immature Sertoli cells by targeting theBDNF gene through activating the PI3K/AKT signaling pathway. This study provides a novel scientific basis for further investigation on the biological functions of miR-191 on porcine spermatogenesis.

MiR‐130a promotes immature porcine Sertoli cell growth by activating SMAD5 through the TGF‐β‐PI3K/AKT signaling pathway

Sertoli cells play vital roles in normal spermatogenesis, and microRNAs (miRNAs) participate in regulating Sertoli cell development. However, the functions and mechanisms of action of most identified miRNAs in porcine Sertoli cells remain largely unknown. Herein, we primarily explored the regulatory roles of miR-130a in immature porcine Sertoli cells using EdU-based high-content screening assay. The results demonstrated that 27 miRNAs have potential roles in the promotion of immature porcine Sertoli cell proliferation, and miR-130a was identified as a promising candidate. miR-130a promoted cell cycle progression and cell proliferation, whereas it impeded cell apoptosis in immature porcine Sertoli cells. It also contributed to Sertoli cell proliferation and testis development in vivo. A TMT-based proteomics approach revealed that miR-130a regulated the expression of 91 proteins and multiple pathways, including the TGF-β and PI3K/AKT signaling. miR-130a did not directly target the 3'-UTR of SMAD5; however, it increased SMAD5 phosphorylation. Moreover, miR-130a enhanced TGF-β signaling by activating SMAD5 protein, and TGF-β signaling further activated the PI3K/AKT signaling pathway to promote cell proliferation and inhibit cell apoptosis in porcine immature Sertoli cells. Collectively, miR-130a promoted immature porcine Sertoli cell growth by activating SMAD5 through the TGF-β-PI3K/AKT signaling pathway. This study, therefore, provides novel insights into the effects of miR-130a on porcine spermatogenesis through the regulation of immature Sertoli cell proliferation and apoptosis.

MiR-362 knock-down promotes proliferation and inhibits apoptosis in porcine immature Sertoli cells by targeting the RMI1 gene.

Immature Sertoli cell proliferation determines the total number of mature Sertoli cells and further regulates normal spermatogenesis. Accumulating evidence demonstrates that microRNAs (miRNAs) play regulatory roles in immature Sertoli cell proliferation, while the functions and mechanisms of the Sertoli cells of domestic animals are poorly understood. In the present study, we aimed to investigate the roles of miR-362 in cell proliferation and apoptosis of porcine immature Sertoli cells. The results showed that miR-362 inhibition promoted the entrance of cells into the S phase and increased the expressions of cell cycle-related genes c-MYC, CNNE1, CCND1 and CDK4. Knock-down of miR-362 also promoted cell proliferation and inhibited apoptosis, which was demonstrated by the results from cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) and Annexin V-FITC/PI staining assays. The recQ-mediated genome instability protein 1 (RMI1) gene was identified as a potential target gene of miR-362 via luciferase reporter assay, and miR-362 repressed the protein expression of RMI1 in porcine immature Sertoli cells. siRNA-induced RMI1 knock-down further abolished the effects of miR-362 inhibition on porcine immature Sertoli cells. Collectively, we concluded that miR-362 knock-down promotes proliferation and inhibits apoptosis in porcine immature Sertoli cells by targeting the RMI1 gene, which indicates that miR-362 determines the fate of immature Sertoli cells.

MiR-499 promotes immature porcine Sertoli cell growth through the PI3K/AKT pathway by targeting the PTEN gene.

Sertoli cells are indispensable for normal spermatogenesis, and increasing evidence has shown that miRNAs participate in the regulation of Sertoli cell growth. However, the functions and regulatory mechanisms of miRNAs in Sertoli cells of domestic animals have not been fully investigated. In the present study, we mainly investigated the regulatory roles of miR-499 in immature porcine Sertoli cells. The results showed that miR-499 was mainly located in the basement section of seminiferous tubules of prepubertal porcine testicular tissue. Overexpression of miR-499 promoted cell proliferation and inhibited apoptosis, whereas miR-499 inhibition resulted in the opposite effect. The PTEN gene was directly targeted by miR-499, and the expression of mRNA and protein was also negatively regulated by miR-499 in immature porcine Sertoli cells. siRNA-induced PTEN knockdown resulted in a similar effect as an overexpression of miR-499 and abolished the effects of miR-499 inhibition on immature porcine Sertoli cells. Moreover, both miR-499 overexpression and the PTEN knockdown activated the PI3K/AKT signaling pathway, whereas inhibition of the PI3K/AKT signaling pathway caused immature porcine Sertoli cell apoptosis and inhibited cell proliferation. Overall, miR-499 promotes proliferation and inhibits apoptosis in immature porcine Sertoli cells through the PI3K/AKT pathway by targeting the PTEN gene. This study provides novel insights into the effects of miR-499 in spermatogenesis through the regulation of immature Sertoli cell proliferation and apoptosis.

MiR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene

MicroRNAs (miRNAs) have been widely identified in porcine testicular tissues and implicated as crucial regulators of proliferation, apoptosis, and differentiation in porcine spermatogenesis related cells. However, the function roles of most of the miRNAs that have been identified in Sertoli cells are poorly understood. In the present study, six experiments were conducted to study the regulatory role of miR-10b in porcine immature Sertoli cells. In experiment 1, the results showed that the relative mRNA expression level of miR-10b in porcine testicular tissues decreased quadratically (P<0.001) with increasing age, while the relative mRNA expression level of DAZAP1 gene increased (P<0.001). In addition, the mRNA expression of miR-10b was negatively (P<0.01) correlated with DAZAP1 mRNA expression (r=−0.550). In experiment 2, the results from the bioinformatic analysis and a luciferase reporter assay demonstrated that miR-10b directly targeted the DAZAP1 gene in porcine immature Sertoli cells. DAZAP1 mRNA and protein expressions were both regulated (P<0.05) by miR-10b. In experiments 3 to 5, the over-expression of miR-10b or the siRNA-mediated knockdown of the DAZAP1 gene promoted (P<0.05) porcine immature Sertoli cell proliferation, as determined by the Cell Counting Kit-8 (CCK-8) assay and the 5-Ethynyl-2'-deoxyuridine (EdU) assay. However, an annexin V-FITC/PI staining assay and the expression of cell survival-related genes indicated that over-expression of miR-10b or knockdown of DAZAP1 had no effect (P>0.05) on porcine immature Sertoli cell apoptosis. In experiment 6, the co-transfection treatment results showed that miR-10b promoted (P<0.05) porcine immature Sertoli cell proliferation by targeting DAZAP1 gene. Overall, these experiments demonstrated that miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene.

Tumor Suppressor PTEN Regulates Negatively Sertoli Cell Proliferation, Testis Size, and Sperm Production In Vivo.

The IGFs are the major intratesticular factors regulating immature Sertoli cell proliferation and are, therefore, critical to establish the magnitude of sperm production. However, the intratesticular source of IGF production and the downstream signaling pathway mediating IGF-dependent Sertoli cell proliferation remain unclear. Single-cell RNA sequencing on mouse embryonic testis revealed a robust expression of Igf1 and Igf2 in interstitial steroidogenic progenitors, suggesting that IGFs exert paracrine actions on immature Sertoli cells. To elucidate the intracellular signaling mechanism that underlies the proliferative effects of IGFs on immature Sertoli cells, we have generated mice with Sertoli cell-specific deletion of the Pten gene, a negative regulator of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway, alone or together with the insulin receptor (Insr) and the IGF1 receptor (Igf1r). Although ablation of Pten appears dispensable for Sertoli cell proliferation and spermatogenesis, inactivation of Pten in the absence of Insr and Igf1r rescued the Sertoli cell proliferation rate during late fetal development, testis size, and sperm production. Overall, these findings suggest that IGFs secreted by interstitial progenitor cells act in a paracrine fashion to promote the proliferation of immature Sertoli cells through the IGF/PTEN/PI3K pathway.

MiR-196a Promotes Proliferation and Inhibits Apoptosis of Immature Porcine Sertoli Cells.

Our previous study showed that the expression of miR-196a was significantly higher in immature porcine testes than in mature porcine testes. However, the role of miR-196a in immature Sertoli cells remains unclear. In this study, miR-196a mimics, miR-196a inhibitor, and microRNA-small hairpin negative control (miRNA-ShNC) were transfected into immature Sertoli cells, respectively. Subsequently, the cell cycle and apoptosis rate of the immature Sertoli cells were measured by flow cytometry, and the viability of the Sertoli cells was measured by the MTS assay. Furthermore, the candidate target genes of miR-196a were analyzed by bioinformatics, and the target genes were validated by dual luciferase reporter assays, then the expression of target genes was detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assays. The results showed that miR-196a promotes the proliferation and inhibits the apoptosis of immature Sertoli cells. miR-196a directly binds the 3' untranslated region (3' UTR) of RCC2 and ABCB9. The expression of miR-196a was shown to be negatively correlated with the messenger RNA and protein levels of the RCC2 and ABCB9 genes. The study demonstrates that miR-196a regulates immature Sertoli cell proliferation and apoptosis and inhibits the expression of RCC2 and ABCB9.

Effect of resveratrol on Sertoli cell proliferation.

Resveratrol (RSV), a polyphenolic compound largely found in red grape skin, has been used as a nutritional supplement as it exhibits beneficial health effects, such as anticancer, cardioprotective, antiaging, and anti-inflammatory. Particularly, it has been shown that it participates in the mechanisms involved in cell proliferation. Sirtuin 1 (SIRT1) is considered a well-known RSV effector. Noteworthy, Sirt1-knockout animals are infertile. The aim of this study was, first, to determine whether RSV has any effect on Sertoli cell proliferation and, second, whether SIRT1, a putative target of RSV, is present in immature Sertoli cells. Sertoli cell cultures obtained from 8-day-old rats, which actively proliferate, were treated with RSV (10 and 50 µM) under basal and follicle-stimulating hormone (FSH)-stimulated conditions. Bromodeoxyuridine (BrdU) incorporation and the expression of cyclins D1, D2, D3, E1, and E2 and the Cip/Kip cell cycle inhibitors p21Cip and p27Kip were analyzed. RSV decreased BrdU incorporation and cyclins D1, D2, E1, and E2 expression and increased p21Cip and p27Kip messenger RNA (mRNA) levels. RSV also decreased FSH-stimulated BrdU incorporation and cyclins D1 and D2 mRNA levels. The effect of RSV on cMYC was also analyzed. RSV treatment did not modify basal and FSH-stimulated cMyc expression; however, it inhibited basal and FSH-stimulated cMYC transcriptional activity, suggesting a role of cMYC in RSV effects. Additionally, Sirt1 was detected in immature Sertoli cells. Altogether, these results suggest that RSV possibly, by activating SIRT1 and regulating cMYC transcriptional activity, participates in the regulation of immature Sertoli cell proliferation.

MiR-362 regulates the proliferation and apoptosis of porcine immature Sertoli cells through targeting the ZNF644 gene

In testicular tissue, immature Sertoli cell proliferation ability determines the size of mature Sertoli cell populations, which further regulates the spermatogenesis in the adult male animals. Studies have demonstrated that microRNAs (miRNAs) participate in the regulation of the immature Sertoli cell proliferation and apoptosis, but the functions of most identified miRNAs remain unclear. In this study, based on previous RNA-seq results, we analyzed the regulatory role (s) of miR-362 in proliferation and apoptosis of porcine immature Sertoli cells. The ZFN644 gene was predicted to be a target gene of miR-362 using bioinformatics methods. The expression levels of miR-362 and ZNF644 gene were measured using qRT-PCR assay in developing porcine testicular tissues and in immature Sertoli cells transfected with either miR-362 mimic or miR-362 inhibitor. The dual luciferase reporter assay was used to determine the regulatory relationship between miR-362 and ZNF644. The results showed that a putative target site of miR-362 was located in the 3'UTR of ZNF644. The expression of miR-362 was significantly and negatively correlated with ZNF644 expression in the developing porcine testicular tissues. Co-transfection of miR-362 and psiCHECK2-ZNF644-WT 3'UTR luciferase vector significantly suppressed luciferase activity. The ZNF644 gene expression level was significantly regulated by miR-362, demonstrating that miR-362 targets ZNF644 gene and inhibits its expression in porcine immature Sertoli cells. Flow cytometry, CCK8, and EdU assays were used to measure the effects of over-expression of miR-362 or knockdown of ZNF644 on porcine immature Sertoli cell proliferation; Annexin V-FITC/PI staining assays and qRT-PCR technology were used to test the apoptosis and the expression levels of cell survival-related genes, respectively. Over-expression of miR-362 and knockdown of ZNF644 arrested the porcine immature Sertoli cells in G1 and G2 phases of the cell cycle, respectively, and inhibited proliferation, enhanced apoptosis in the porcine immature Sertoli cells, and significantly regulated the expression levels of cell survival-related genes. Taken together, these data indicate that miR-362 inhibits proliferation and promotes apoptosis in porcine immature Sertoli cells by targeting the ZNF644 gene, thereby providing the scientific basis for further study on the function(s) of miR-362 in the porcine spermatogenesis.

Crosstalk between FSH and relaxin at the end of the proliferative stage of rat Sertoli cells.

Follicle-stimulating hormone (FSH) stimulates the proliferation of immature Sertoli cells through the activation of PI3K/AKT/mTORC1 and MEK/ERK1/2 pathways. Mature Sertoli cells stop proliferating and respond to FSH by stimulating cAMP production. To gain insight into possible mechanisms involved in this switch as well as the impact of paracrine factors that stimulate cell proliferation, we analyzed the effects of FSH and relaxin on intracellular signaling pathways involved with proliferation and differentiation in Sertoli cells from 15-day-old rats, which are close to the transition between the two stages. FSH stimulated 3H-thymidine incorporation and cyclin D1 expression, changes associated with proliferation. In contrast, FSH inhibited AKT and ERK1/2 phosphorylation, activated cAMP production and induced changes in several cell cycle genes that were compatible with differentiation. Relaxin also stimulated 3H-thymidine incorporation but increased phosphorylation of ERK1/2 and AKT. When both hormones were added simultaneously, relaxin attenuated FSH-mediated inhibition of ERK1/2 and AKT phosphorylation and FSH-mediated activation of cAMP production. FSH but not relaxin increased CREB phosphorylation, and relaxin but not FSH shifted NF-κB expression from the cytoplasm to the nucleus. Relaxin did not inhibit the effects of FSH on inhibin α and Bcl2 expression. We propose that at this time of Sertoli cell development, FSH starts to direct cells to differentiation through activation of cAMP/CREB and inhibition of ERK1/2 and AKT pathways. Relaxin counteracts FSH signaling through the inhibition of cAMP and activation of ERK1/2, AKT and NF-κB, but does not block the differentiation process triggered by FSH.

GDNF stimulates the proliferation of cultured mouse immature Sertoli cells via its receptor subunit NCAM and ERK1/2 signaling pathway

BackgroundThe proliferation and final density of Sertoli cells in the testis are regulated by hormones and local factors. Glial cell line-derived neurotrophic factor (GDNF), a distantly related member of the transforming growth factor-β superfamily, and its receptor subunits GDNF family receptor alpha 1 (GFRα1), RET tyrosine kinase, and neural cell adhesion molecule (NCAM) have been reported to be expressed in the testis and involved in the regulation of proliferation of immature Sertoli cells (ISCs). However, the expression patterns of these receptor subunits and the downstream signaling pathways have not been addressed in ISCs.ResultsIn the present study, we have reported that the proliferation of cultured ISCs was significantly enhanced by GDNF. The receptor subunits GFRα1 and NCAM but not RET were expressed in ISCs, and the stimulatory effect of GDNF on the proliferation of ISCs was significantly reduced by anti-NCAM antibody blocking or siRNA that specifically targets NCAM mRNA. Additionally, the ERK1/2 inhibitor, PD98059, completely abolished the mitogenic effect of GDNF on ISCs.ConclusionsGDNF stimulates the proliferation of ISCs via its receptor subunit NCAM and the consequent activation of the ERK1/2 signaling pathway.

149. Smad3 DOSAGE INFLUENCES TESTICULAR MATURATION

Activin A, a TGF-beta superfamily ligand which signals via Smad2 and Smad3, is critical for normal mouse testis development and quantitatively normal sperm production. Whereas activin enhances immature Sertoli cell proliferation (1), excessive activin production causes Sertoli cell tumours (2); this is alleviated when mice lack Smad3 (3). Sertoli cells exhibit developmentally regulated Smad utilization in activin signalling. Immature Sertoli cells signal via Smad3 while the onset of Smad2-mediated signal transduction correlates with Sertoli cell maturation (4). This change coincides with decreased testicular Smad3 production at puberty and a shift in follicle stimulating hormone (FSH)-induced Smad transcription, from Smad3 in 6 dpp (days post partum) Sertoli cells to Smad2 in 15 dpp cells. These findings suggest that Smad3 is more important for testis development than adult spermatogenesis. To test this hypothesis, we examined testis development in Smad3+/– and Smad3–/– mice. At 7 dpp, testis weight and cord diameter were reduced in Smad3–/–mice, indicating impaired Sertoli cell proliferation. Levels of FSH, a potent Sertoli cell mitogen, were unaltered. Histological analysis revealed advanced spermatogenesis in heterozygous mice, with round spermatids already present at 16 dpp. Quantitative PCR also identified advanced Sertoli and germ cell maturation in Smad3+/– mice, while Leydig cell maturation appeared unaltered. Adult Smad3+/– and Smad3–/– mice were fertile, but had smaller testes. This is the first study relating Smad3 levels to puberty onset and identifies the Smad3+/– mouse as a model of peripheral precocious puberty with otherwise normal physiological status, i.e. no gonadal tumours and normal FSH levels. These results demonstrate that FSH influences testis growth and maturation by regulating Smad3 expression and highlights the importance of testing whether environmental factors, toxicants and endocrine disruptors affect Smad3 expression, thereby leading to altered testis development.

Effects of an Early Postnatal Treatment of Hypogonadotropic Hypogonadism with a Continuous Subcutaneous Infusion of Recombinant Follicle-Stimulating Hormone and Luteinizing Hormone

The neonatal-midinfancy surge in pulsatile gonadotropin secretion is attributable to an increase in GnRH pulse amplitude and is associated with a rapid expansion of Leydig and Sertoli cell populations with concomitant surges in testosterone, inhibin, and anti-Mullerian hormone production as well as an increase in testicular volume. Boys with congenital hypogonadotropic hypogonadism (HH) do not activate these processes. A potential cause for azoospermia and infertility in adult life is deficient proliferation of immature Sertoli cells before and during puberty due to the absence of FSH. The objective of the study was to investigate whether early postnatal continuous sc infusion of gonadotropins could mimic the physiological growth of testes and to evaluate responses of the Leydig and Sertoli cells to early gonadotropin replacement. Two neonates (P1 with hypotuitarism and P2 with HH) with micropenis and microorchidism were treated for 6 months with high doses of recombinant LH and FSH (a gift of Luveris and Gonal-F from Serono, Lyon, France) delivered sc with an insulin pump. Gonadotropin continuous sc infusion increased mean serum LH and FSH to normal or supranormal levels. Mean testosterone increased from undetectable levels to 7.6 and 5.2 nmol/liter, respectively, in P1 and P2. Inhibin B and anti-Müllerian hormone increased to normal levels. Mean testicular volume increased from 0.45 to 0.57 ml at birth to 2.10 ml at 7 months. Stretched penile length increased from 8 to 30 mm (P1) and 12 to 48 mm (P2). The present regimen induced physiological postnatal testes growth and high-normal activation of Leydig and Sertoli cells.

17Beta-Estradiol Induces the Translocation of the Estrogen Receptors ESR1 and ESR2 to the Cell Membrane, MAPK3/1 Phosphorylation and Proliferation of Cultured Immature Rat Sertoli Cells1

The aim of the present study was to determine the mechanisms involved in estrogen actions in cultured rat Sertoli cells. RT-PCR detected transcripts for the estrogen receptors ESR1 and ESR2 in cultured immature Sertoli cells and in the testis of 15-, 28-, and 120-day-old rats. The expression of ESR1 and ESR2 was confirmed in Sertoli cells by immunofluorescence and Western blot. Immunohistochemistry with cryosections of testes from immature and adult rats revealed that ESR1 is present in Sertoli, Leydig, and some peritubular myoid cells, and ESR2 is present in multiple cell types, including germ cells. Treatment of Sertoli cells with 17beta-estradiol (E(2)) induced a translocation of ESR1 and ESR2 to the plasma membrane and a concomitant phosphorylation of MAPK3/1. Both effects reached a maximum after 10 min and were blocked by PP2, an inhibitor of the SRC family of protein tyrosine kinases, and by the antiestrogen ICI 182,780 (ICI). MAPK3/1 phosphorylation was also decreased in the presence of AG 1478, an inhibitor of the epidermal growth factor receptor (EGFR) kinase, and in the presence of MAP2K1/2 inhibitor UO126. Treatment with E(2) for 24 h increased the incorporation of [methyl-(3)H]thymidine, which was blocked by ICI. These results indicate that E(2) activates an SRC-mediated translocation of estrogen receptors to the plasma membrane, which results in the activation of EGFR and the mitogen-activated protein kinase signaling pathway. In addition, activation of ESR1 and/or ESR2 by E(2) is involved in proliferation of immature Sertoli cells. The estrogen actions in Sertoli cells might be a key step mediating cellular events important for spermatogenesis and fertility.

Treatment of gonadotropin-deficient boys with recombinant human FSH: long-term observation and outcome

Boys with prepubertal onset of hypogonadotropic hypogonadism (HH) are at a risk of poor testis growth and impaired spermatogenesis. One potential cause for this is deficient proliferation of immature Sertoli cells before and during puberty due to the absence of FSH. To evaluate the effects of recombinant human FSH (r-hFSH) and human chorionicgonadotropin (hCG) on testicular function and pubertal development in boys with prepubertal onset of HH. Retrospective clinical study. Two university central hospitals, pediatric referral endocrinology outpatient clinics. Fourteen boys (aged, 9.9-17.7 years) with prepubertal (testicular volume (TV) <3 ml) onset of HH (idiopathic HH, n=2; Kallman syndrome, n=2; idiopathic panhypopituitarism, n=4; organic panhypopituitarism, n=6). Treatment with r-hFSH alone (2 mo-2.8 years) prior to induction of puberty with the combination of FSH and hCG. Progression of puberty, change in serum inhibin B, spermatogenesis. r-hFSH alone increased testicular volume twofold, from 0.9+/-0.6 ml (mean+/-s.d.) to 1.8 +/- 1.1 ml (P<0.005), and serum inhibin B threefold, from 27+/-14 to 80+/-57 pg/ml (P<0.01). Three boys with an apparent absence of postnatal hypothalamic-pituitary-testicular axis activation displayed attenuated inhibin B responses to long-term (>or=1 year) r-hFSH (P<0.01). Further significant increase in both TVand inhibin B occurred with induction of puberty with FSH and hCG (P<0.001). Seven boys provided semen samples: one had azoospermia, and others displayed a maximal sperm count range from 2.9 to 92 million/ml (median 8.5 million/ml). (i) r-hFSH induces prepubertal testis growth and increases circulating inhibin B levels, findings suggesting proliferation of immature Sertoli cells. (ii) Puberty was successfully induced with hCG and r-hFSH following r-hFSH priming. (iii) Inhibin B appears useful for monitoring spermatogenetic activity in boys treated with hCG. (iv) Despite the extremely small initial testis volume, six out of seven patients (86%) primed with r-hFSH displayed sperm in the ejaculate suggesting beneficial effect of r-hFSH priming on testicular function later in life.

Reduced Mitotic Activity and Increased Apoptosis of Fetal Sertoli Cells in Rat Hypogonadic (hgn/hgn) Testes

Sterility in male hypogonadic (hgn/hgn) rats results from congenital testicular dysplasia caused by a single recessive gene hgn on rat chromosome 10. We recently identified an insertion mutation in the Spag5/astrin gene of hgn/hgn rats that may cause defective proliferation of immature Sertoli cells in the postnatal hgn/hgn testis. Since the pathological alterations were present in the testes at birth, we examined the involvement of defective mitosis and apoptotic cell death in embryonic development of hgn/hgn testes. Testicular hypoplasia was apparent at embryonic day (ED) 18.5. Immunostaining of hgn/hgn testes at ED 21.5 with antibody to GATA-4, which is specific for fetal Sertoli cells in the seminiferous cords, showed that the significant decrease in the number of fetal Sertoli cells was accompanied by a two fold increase in their mitotic index and abnormal mitosis and apoptosis. Prior to this, we observed a decrease in the number of BrdU-labeled cells, an increase in the number of TUNEL-positive apoptotic cells, and presence of MIS-positive apoptotic cells in hgn/hgn testes on ED 17.5 and 18.5. These results suggest that the Spag5 mutation may cause a reduction in mitotic activity and an increase in apoptosis of fetal Sertoli cells in hgn/hgn testes.