9-cis-retinoic acid signaling in Sertoli cells regulates their immunomodulatory function to control lymphocyte physiology and Treg differentiation.

BACKGROUND: Testis is an immune privileged organ, which prevents the immune response against sperm antigens and inflammation. Testicular cells responsible for immune tolerance are mainly Sertoli cells, which form the blood-testis barrier and produce immunosuppressive factors. Sertoli cells prevent inflammation in the testis and maintain immune tolerance by inhibiting proliferation and inducing lymphocyte apoptosis. It has been shown that 9-cis-retinoic acid (9cRA) blocks ex vivo apoptosis of peripheral blood lymphocytes and promotes the differentiation of Treg cells in the gut. However, the role of retinoid signaling in regulating the immune privilege of the testes remains unknown. OBJECTIVE: The aim of this study was to determine whether 9cRA, acting via the retinoic acid receptors (RAR) and the retinoic X receptors (RXR), controls the immunomodulatory functions of Sertoli cells by influencing the secretion of anti-inflammatory/pro-inflammatory factors, lymphocyte physiology and Treg cell differentiation. METHODS: Experiments were performed using in vitro model of co-cultures of murine Sertoli cells and T lymphocytes. Agonists and antagonists of retinoic acid receptors were used to inhibit/stimulate retinoid signaling in Sertoli cells. RESULTS: Our results have demonstrated that 9cRA inhibits the expression of immunosuppressive genes and enhances the expression of pro-inflammatory factors in Sertoli cells and lymphocytes, increases lymphocyte viability and decreases apoptosis rate. Moreover, we have found that 9cRA blocks lymphocyte apoptosis acting through both RAR and RXR and inhibiting FasL/Fas/Caspase 8 and Bax/Bcl-2/Caspase 9 pathways. Finally, we have shown that 9cRA signaling in Sertoli cells inhibits Treg differentiation. CONCLUSION: Collectively, our results indicate that retinoid signaling negatively regulates immunologically privileged functions of Sertoli cells, crucial for ensuring male fertility. 9cRA inhibits lymphocyte apoptosis, which can be related to the development of autoimmunity, inflammation, and, in consequence, infertility.

Androgens and Notch signaling cooperate in seminiferous epithelium to regulate genes related to germ cell development and apoptosis.

It was reported previously that in adult males disruption of both androgen and Notch signaling impairs spermatid development and germ cell survival in rodent seminiferous epithelium. To explain the molecular mechanisms of these effects, we focused on the interaction between Notch signaling and androgen receptor (AR) in Sertoli cells and investigate its role in the control of proteins involved in apical ectoplasmic specializations, actin remodeling during spermiogenesis, and induction of germ cell apoptosis. First, it was revealed that in rat testicular explants ex vivo both testosterone and Notch signaling modulate AR expression and cooperate in the regulation of spermiogenesis-related genes (Nectin2, Afdn, Arp2, Eps8) and apoptosis-related genes (Fasl, Fas, Bax, Bcl2). Further, altered expression of these genes was found following exposure of Sertoli cells (TM4 cell line) and germ cells (GC-2 cell line) to ligands for Notch receptors (Delta-like1, Delta-like4, and Jagged1) and/or Notch pathway inhibition. Finally, direct interactions of Notch effector, Hairy/enhancer-of-split related with YRPW motif protein 1, and the promoter of Ar gene or AR protein were revealed in TM4 Sertoli cells. In conclusion, Notch pathway activity in Sertoli and germ cells regulates genes related to germ cell development and apoptosis acting both directly and indirectly by influencing androgen signaling in Sertoli cells.

Follicle-stimulating hormone regulates Notch signalling in the seminiferous epithelium of continuously and seasonally breeding rodents.

CONTEXT: Juxtacrine (contact-dependent) communication between the cells of seminiferous epithelium mediated by Notch signalling is of importance for the proper course of spermatogenesis in mammals. AIMS: The present study was designed to evaluate the role of follicle-stimulating hormone (FSH) in the regulation of Notch signalling in rodent seminiferous epithelium. METHODS: We explored the effects (1) of pharmacological inhibition of the hypothalamus-pituitary-gonadal (HPG) axis and FSH replacement in pubertal rats, and (2) of photoinhibition of HPG axis followed by FSH substitution in seasonally breeding rodents, bank voles, on Notch pathway activity. Experiments on isolated rat Sertoli cells exposed to FSH were also performed. Gene and protein expressions of Notch pathway components were analysed using RT-qPCR, western blot and immunohistochemistry/immunofluorescence. KEY RESULTS: Distribution patterns of Notch pathway proteins in bank vole and rat seminiferous epithelium were comparable; however, levels of activated Notch1 and Notch3, hairy/enhancer of split 1 (HES1) and hairy/enhancer of split-related with YRPW motif 1 (HEY1) in bank voles were dependent on the length of the photoperiod. In response to FSH similar changes in these proteins were found in both species, indicating that FSH is a negative regulator of Notch pathway activity in seminiferous epithelium. CONCLUSIONS: Our results support a common mechanism of FSH action on Notch pathway during onset and recrudescence of spermatogenesis in rodents. IMPLICATIONS: Interaction between FSH signalling and Notch pathway in Sertoli cells may be involved in spermatogenic activity changes of the testes occurring during puberty or photoperiod shift in continuously and seasonally breeding rodents, respectively.

Nuclear and Membrane Receptors for Sex Steroids Are Involved in the Regulation of Delta/Serrate/LAG-2 Proteins in Rodent Sertoli Cells.

Delta/Serrate/LAG-2 (DSL) proteins, which serve as ligands for Notch receptors, mediate direct cell-cell interactions involved in the determination of cell fate and functioning. The present study aimed to explore the role of androgens and estrogens, and their receptors in the regulation of DSL proteins in Sertoli cells. To this end, primary rat Sertoli cells and TM4 Sertoli cell line were treated with either testosterone or 17ß-estradiol and antagonists of their receptors. To confirm the role of particular receptors, knockdown experiments were performed. mRNA and protein expressions of Jagged1 (JAG1), Delta-like1 (DLL1), and Delta-like4 (DLL4) were analyzed using RT-qPCR, Western blot, and immunofluorescence. Testosterone caused downregulation of JAG1 and DLL1 expression, acting through membrane androgen receptor ZRT- and Irt-like protein 9 (ZIP9) or nuclear androgen receptor (AR), respectively. DLL4 was stimulated by testosterone in the manner independent of AR and ZIP9 in Sertoli cells. The expression of all studied DSL proteins was upregulated by 17ß-estradiol. Estrogen action on JAG1 and DLL1 was mediated chiefly via estrogen receptor α (ERα), while DLL4 was controlled via estrogen receptor ß (ERß) and membrane G-protein-coupled estrogen receptor (GPER). To summarize, the co-operation of nuclear and membrane receptors for sex steroids controls DSL proteins in Sertoli cells, contributing to balanced Notch signaling activity in seminiferous epithelium.