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

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

Relaxin and Sertoli cell proliferation.

Immature Sertoli cells proliferate and several factors affect their number, including the follicle stimulating hormone (FSH), testosterone, estradiol and several paracrine growth factors. Using a primary culture of Sertoli cells isolated from 15-day old Wistar rats we have shown that relaxin stimulates Sertoli cell proliferation through the activation of MEK/ERK1/2 and PI3K/AKT pathways. In contrast, FSH inhibited both ERK1/2 and AKT phosphorylation. Furthermore, FSH strongly increased cAMP production, whereas relaxin inhibited basal cAMP production. Our results indicate that in rat Sertoli cells from 15-day old rats relaxin and FSH affect the same signaling pathways in opposite directions. Interplay between both hormones may be important to control the proliferation and differentiation of Sertoli cells.

Effects of relaxin in a co-culture of Sertoli and germ cells.

Spermatogenesis is controlled by FSH, testosterone and paracrine factors produced by Sertoli cells. The knockout of relaxin decreases sperm maturation in mice. Studies from our laboratory have shown that relaxin and its receptor RXFP1 are expressed in rat Sertoli cells, and exogenous relaxin stimulates Sertoli cell proliferation. Relaxin receptors are also detected in the rat germ cells at specific stages of development. Relaxin could therefore affect spermatogenesis either indirectly, by stimulating Sertoli cell proliferation, or directly, by affecting germ cells. The aim of the present study was to explore a role of relaxin at specific stages of spermatogenesis using a co-culture of rat Sertoli and germ cells. Relaxin seems to increase the number of pre-meiotic and meiotic cells.

17ß-estradiol signaling and regulation of Sertoli cell function.

In this review, we will present an overview of estrogen actions in the testis from immature and adult animals, with special emphasis on signaling mechanisms involved in the 17ß-estradiol regulation of Sertoli cell function in immature rats. 17ß-estradiol activates Sertoli cell proliferation in immature rats by a mechanism that involves the translocation of the estrogen receptors ESR1 and ESR2 to the plasma membrane, phosphorylation of epidermal growth factor receptor and activation of mitogen-activated protein kinase 3/1. Activation of the G protein-coupled estrogen receptor (GPER) also induces phosphorylation of mitogen-activated protein kinase 3/1 via epidermal growth factor receptor transactivation, which in turn increases expression of the antiapoptotic protein BCL2 and decreases the expression of proapoptotic protein BAX, indicating an antiapoptotic role of E2-GPER in immature rat Sertoli cells. In conclusion, ESRs and GPER can mediate rapid 17ß-estradiol signaling in Sertoli cells, and modulate transcriptional events important for Sertoli cell function and maintenance of normal testis development and homeostasis. Our findings are important to clarify the role of estrogen in a critical period of testicular development and to direct further studies, which may contribute to better understand the causes of male infertility.

Relaxin family peptide receptors Rxfp1 and Rxfp2: mapping of the mRNA and protein distribution in the reproductive tract of the male rat.

BACKGROUND: Relaxin is the endogenous ligand of the G-protein coupled receptor RXFP1, previously known as LGR7. In humans relaxin can also activate, but with lower affinity, the closely related receptor for the insulin-like peptide from Leydig cells, RXFP2, previously known as LGR8. The lack of relaxin impairs male fertility but the precise distribution and the function of relaxin receptors in the male reproductive tract is not known. We investigated the distribution of Rxfp1 and Rxfp2 in the reproductive tract of the male rat and the function of relaxin in the vas deferens, a tissue with high expression of both receptors. METHODS: The presence of mRNA for Rxfp1 and Rxfp2 was investigated in testes, cultured Sertoli cells, epididymis, vas deferens, seminal vesicle, prostate, and spermatozoa by RT-PCR and Southern blot. Protein expression in the testis, vas deferens, primary culture of Sertoli cells, and spermatozoa was assessed by immunohistochemistry and immunofluorescence. The role of relaxin in the vas deferens was evaluated by contractility studies and radioimmunoassay of cAMP production. The effect of relaxin on mRNA levels for metalloproteinase-7 was measured by Northern blot. RESULTS: Transcripts for Rxfp1 and Rxfp2 were present in almost all parts of the male reproductive tract, with high levels in testis and vas deferens. Both receptors were immunolocalized in late stage germ cells but not in mature spermatozoa, although mRNAs for both receptors were also present in mature spermatozoa. Rxfp1 but not Rxfp2 was detected in cultured Sertoli cells. Strong immunostaining for Rxfp1 and Rxfp2 was seen in muscular and epithelial layers of the vas deferens and in arteriolar walls. Relaxin did not affect contractility and cyclic AMP production of the vas deferens, but increased the levels of mRNA for metalloproteinase-7. CONCLUSION: Rxfp1 and Rxfp2 are widely and similarly distributed throughout the male reproductive tract. Our results suggest that Rxfp1 on spermatids and Sertoli cells may be important in spermatogenesis. Relaxin in the vas deferens does not affect contractility, but may affect vascular compliance and collagen and matrix remodeling.

Molecular cloning, sequence analysis and expression of the snake follicle-stimulating hormone receptor.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) control gonadal function in mammalian and many non-mammalian vertebrates through the interaction with their receptors, FSHR and LHR. Although the same is true for some reptilian species, in Squamata (lizards and snakes) there is no definitive evidence for the presence of either two distinct gonadotropins or two distinct gonadotropin receptors. Our aim was to characterize the gonadotropin receptor(s) of the Bothrops jararaca snake. Using a cDNA library from snake testis and amplification of the 5'-cDNA ending, we cloned a cDNA related to FSHR. Attempts to clone a cDNA more closely related to LHR were unsuccessful. Expression of FSHR mRNA was restricted to gonadal tissues. The snake FSHR is a G protein-coupled receptor with 673 amino acids, and the aminoterminal domain with 346 amino acids consists of a nine leucine-rich repeat-containing subdomain (LRR) flanked by two cysteine-rich subdomains. The beta-strands in the LRR are conserved with exception of the third, a region that may be important for FSH binding. In contrast with mammalian, avian and amphibian FSHRs, the snake FSHR presents amino acid deletions in the carboxyterminal region of the extracellular domain which are also seen in fish and lizard FSHRs. cAMP assays with the recombinant protein transiently expressed in HEK-293 cells showed that the snake FSHR is more sensitive to human FSH (hFSH) than to human chorionic gonadotropin. Phylogenetic analysis indicated that the squamate FSHRs group separately from mammalian FSHRs. Our data are consistent with the apparently unique gonadotropin-receptor system in Squamata reptilian subgroup. Knowledge about the snake FSHR structure may help identify structural determinants for receptor function.