Regulation of Spermatogenesis by Non-Classical Testosterone Signaling.

Abstract

Testosterone signaling through the androgen receptor in Sertoli cells in the testis is essential for maintaining spermatogenesis and male fertility. It is known that testosterone causes the androgen receptor to translocate from the cytoplasm to the nucleus to regulate gene expression in Sertoli cells. However, testosterone-mediated gene expression requires more than 40 min to be initiated and relatively few genes essential for fertility are known to be regulated by this classical pathway of testosterone signaling. We have characterized an alternative mechanism of T action (the non-classical pathway) in which testosterone stimulation causes the rapid (within 1 min) and sustained (>12 h) activation of kinases and transcription factors including Src, ERK and CREB that are known to regulate cellular processes and gene expression required for spermatogenesis. Our overall goal is to identify the factors and molecular mechanisms by which testosterone acts via the non-classical pathway in Sertoli cells to support spermatogenesis and male fertility. We have obtained evidence that rapid kinase activation occurs due to testosterone signaling in Sertoli cells in vivo. Adult rats were treated with the GnRH antagonist cetrorelix to decrease testosterone levels and then injected with testosterone to rapidly increase testicular testosterone levels. We found that the phosphorylation of ERK was decreased to 34% of control levels after cetrorelix treatment and then restored to 103% of controls within 1 h of testosterone injection. Immunofluorescence studies confirmed that the levels of ERK phoshorylation in Sertoli cells associated with the blood testis barrier (BTB) and germ cell attachment sites rapidly decreased and increased with testosterone levels. We have also determined that peptide and peptidomimetic inhibitors of non-classical signaling disrupt tight junction formation between cultured Sertoli cells required to maintain the BTB and block the release of sperm from cultured seminiferous tubule fragments. We have identified at least two downstream targets of non-classical kinase activity (N-cadherin and β-catenin) that regulate cell-cell adhesion and are rapidly phosphorylated (within 15 min) by testosterone. Treatment of explants from adult rat testes with a peptidomimetic non-classical inhibitor decreased CREB phosphorylation in Sertoli cells in testosterone sensitive stage VII seminiferous tubule cross sections. Finally, injection of mouse seminiferous tubules with an adenovirus expressing a peptide inhibitor of the non-classical pathway resulted in expression of the inhibitor only in Sertoli cells and the disruption of spermatogenesis (loss of spermatocytes). These data support the hypothesis that testosterone acts via the non-classical pathway in Sertoli cells in vivo and that the non-classical pathway is required to maintain spermatogenesis. Furthermore, we have characterized inhibitors of non-classical signaling that may be used to identify the genes and proteins that are regulated by the pathway. This information may be used for the development of clinical assays to better diagnose the causes of male infertility and provide potential therapies. Our results will promote the development of contraceptive drugs that inhibit specific testosterone-regulated genes and proteins in Sertoli cells while retaining other T-mediated functions. This research was supported by NIH-HD043143 (WW).