Non-classical Testosterone Signaling Research Articles

Tetrapeptides Modelled to the Androgen Binding Site of ZIP9 Stimulate Expression of Tight Junction Proteins and Tight Junction Formation in Sertoli Cells.

Simple SummaryAndrogens such as testosterone act either through the nuclear androgen receptor or through the plasma membrane-bound androgen receptor ZIP9. Androgens are crucial for male fertility. In the testis they stimulate the formation of tight junctions between Sertoli cells, thereby creating an immune-privileged environment for the development of fertile sperm known as the blood–testis barrier. Androgens are also absolutely required for spermatogenesis. Nevertheless, when low testosterone is supplemented by exogenous testosterone (e.g., in testosterone replacement therapy), it triggers a loss of sperm, and thus and infertility, by a negative feed-back mechanism based on the hypothalamic-pituitary-gonadal axis. In order to investigate this discrepancy, the effects of small peptides that fit within the androgen binding site of ZIP9 were tested for their effects on the expression of proteins involved in the formation of tight junctions. Our results provide evidence that all three peptides tested act through ZIP9 as androgens and promote tight junction protein expression and tight junction formation. They can therefore act as surrogates of testosterone for the formation and maintenance of the blood–testis barrier.Androgens stimulate the expression of tight junction (TJ) proteins and the formation of the blood–testis barrier (BTB). Interactions of testosterone with the zinc transporter ZIP9 stimulate the expression of TJ-forming proteins and promote TJ formation in Sertoli cells. In order to investigate androgenic effects mediated by ZIP9 but not by the nuclear androgen receptor (AR), the effects of three tetrapeptides fitting the androgen binding site of ZIP9 were compared with those induced by testosterone in a Sertoli cell line expressing ZIP9 but not the AR. Three tetrapeptides and testosterone displaced testosterone-BSA-FITC from the surface of 93RS2 cells and stimulated the non-classical testosterone signaling pathway that includes the activation of Erk1/2 kinases and transcription factors CREB and ATF-1. The expression of the TJ-associated proteins ZO-1 and claudin-5 was triggered as was the re-distribution of claudin-1 from the cytosol to the membrane and nucleus. Furthermore, TJ formation was stimulated, indicated by increased transepithelial electrical resistance. Silencing ZIP9 expression by siRNA prevented all of these responses. These results are consistent with an alternative pathway for testosterone action at the BTB that does not involve the nuclear AR and highlight the significant role of ZIP9 as a cell-surface androgen receptor that stimulates TJ formation.

Androgen Actions in the Testis and the Regulation of Spermatogenesis.

Testosterone is essential for spermatogenesis and male fertility. In this review, topics related to testosterone control of spermatogenesis are covered including testosterone production and levels in the testis, classical and nonclassical testosterone signaling pathways, cell- and temporal-specific expression of the androgen receptor in the testis and autocrine and paracrine signaling of testis cells in the testis. Also discussed are the contributions of testosterone to testis descent, the blood-testis barrier, control of gonocyte numbers and spermatogonia expansion, completion of meiosis and attachment and release of elongaed spermatids. Testosterone-regulated genes identified in various mouse models of idsrupted Androgen receptor expression are discussed. Finally, examples of synergism and antagonism between androgen and follicle-stimulating hormone signaling pathways are summarized.

Non-classical testosterone signaling in the testis

Non-classical testosterone signaling in the testis

Non-classical testosterone signaling mediated through ZIP9 stimulates claudin expression and tight junction formation in Sertoli cells

In the classical signaling pathway, testosterone regulates gene expression by activating the cytosolic/nuclear androgen receptor. In the non-classical pathway, testosterone activates cytosolic signaling cascades that are normally triggered by growth factors. The nature of the receptor involved in this signaling pathway is a source of controversy.In the Sertoli cell line 93RS2, which lacks the classical AR, we determined that testosterone stimulates the non-classical signaling pathway, characterized by the phosphorylation of Erk1/2 and transcription factors CREB and ATF-1. We also demonstrated that testosterone increases the expression of the tight junction (TJ) proteins claudin-1 and claudin-5. Both of these proteins are known to be essential constituents of TJs between Sertoli cells, and as a consequence of their increased expression transepithelial resistance across Sertoli cell monolayers is increased. ZIP9 is a Zn2+transporter that was recently shown to be a membrane-bound testosterone receptor. Silencing its expression in 93RS2 Sertoli cells by siRNA completely prevents Erk1/2, CREB, and ATF-1 phosphorylation as well the stimulation of claudin-1 and -5 expression and TJ formation between neighboring cells.The study presented here demonstrates for the first time that in Sertoli cells testosterone acts through the receptor ZIP9 to trigger the non-classical signaling cascade, resulting in increased claudin expression and TJ formation. Since TJ formation is a prerequisite for the maintenance of the blood-testis barrier, the testosterone/ZIP9 effects might be significant for male physiology. Further assessment of these interactions will help to supplement our knowledge concerning the mechanism by which testosterone plays a role in male fertility.

Mouse Spermatogenesis Requires Classical and Nonclassical Testosterone Signaling.

Testosterone acts though the androgen receptor in Sertoli cells to support germ cell development (spermatogenesis) and male fertility, but the molecular and cellular mechanisms by which testosterone acts are not well understood. Previously, we found that in addition to acting through androgen receptor to directly regulate gene expression (classical testosterone signaling pathway), testosterone acts through a nonclassical pathway via the androgen receptor to rapidly activate kinases that are known to regulate spermatogenesis. In this study, we provide the first evidence that nonclassical testosterone signaling occurs in vivo as the MAP kinase cascade is rapidly activated in Sertoli cells within the testis by increasing testosterone levels in the rat. We find that either classical or nonclassical signaling regulates testosterone-mediated Rhox5 gene expression in Sertoli cells within testis explants. The selective activation of classical or nonclassical signaling pathways in Sertoli cells within testis explants also resulted in the differential activation of the Zbtb16 and c-Kit genes in adjacent spermatogonia germ cells. Delivery of an inhibitor of either pathway to Sertoli cells of mouse testes disrupted the blood-testis barrier that is essential for spermatogenesis. Furthermore, an inhibitor of nonclassical testosterone signaling blocked meiosis in pubertal mice and caused the loss of meiotic and postmeiotic germ cells in adult mouse testes. An inhibitor of the classical pathway caused the premature release of immature germ cells. Collectively, these observations indicate that classical and nonclassical testosterone signaling regulate overlapping and distinct functions that are required for the maintenance of spermatogenesis and male fertility.

Non-classical testosterone signaling in spermatogenic GC-2 cells is mediated through ZIP9 interacting with Gnα11

Although classical and non-classical signaling of testosterone has been documented in several investigations, the nature of the receptor involved in the non-classical pathway remains a source of controversy. While some investigators favor the exclusive participation of the cytosolic/nuclear androgen receptor (AR) in both pathways, others propose a membrane-bound receptor as the mediator of the non-classical testosterone signaling. Evidence is provided here that in the spermatogenic cell line GC-2 the non-classical signaling pathway of testosterone, characterized through the activation of Erk1/2 and transcription factors like CREB or ATF-1, is not mediated through the classical nuclear androgen receptor (AR) but rather by a membrane-associated receptor. This receptor is ZIP9, a Zn2+ transporter from the family of the ZRT, IRT-like proteins (ZRT=zinc-regulated transporter; IRT=iron-regulated transporter), which directly interacts with the G-protein Gnα11. siRNA-induced abrogation of the expression of either of these two proteins, whose close contacts are demonstrated by an in situ proximity assay, completely prevents all non-classical signaling effects of testosterone addressed. In contrast, silencing of AR expression does not influence the same signaling events. The identification of ZIP9/Gnα11 interactions as the mediators of the non-classical testosterone signaling cascade in spermatogenic GC-2 cells might help to supplement our knowledge concerning the role of testosterone in male fertility and reproduction.

Non-classical testosterone signaling is mediated by a G-protein-coupled receptor interacting with Gnα11

Testosterone is known to mediate its effects by two different mechanisms of action. In the so-called “classical” pathway testosterone binds to cytosolic androgen receptors (AR), which essentially function as ligand-activated transcription factors. Once activated, these receptors bind to DNA and activate the expression of target genes. In the “non-classical” pathway, the steroid hormone binds to receptors associated with the plasma membrane and induces signaling cascades mediated through activation of Erk1/2. The precise nature of the membrane-associated AR, however, remains controversial. Although some assume that the membrane and cytosolic AR are identical, others propose that the AR of the membrane is a G-protein-coupled receptor (GPCR). To evaluate these two possibilities we first searched for testosterone-induced signaling cascades in the spermatogenic cell line GC-2. Testosterone was found to cause phosphorylation (activation) of Erk1/2, CREB, and ATF-1, consistent with its non-classical mechanism of action. Silencing of AR expression by means of siRNA did not influence testosterone-induced activation of Erk1/2, CREB, or ATF-1, indicating that this pathway is not activated by the classical cytosolic/nuclear AR. In contrast, when the expression of the G-protein Gnα11 is suppressed, the activation of these signaling molecules is abolished, suggesting that these responses are elicited through a membrane-bound GPCR. The results presented here and the identification of the testosterone-specific GPCR in future investigations will help to reveal and characterize new testosterone-mediated mechanisms associated not only with fertility and reproduction but perhaps also with other physiological processes.

Regulation of Spermatogenesis by Non-Classical Testosterone Signaling.

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).

Cardiotonic steroids trigger non-classical testosterone signaling in Sertoli cells via the α4 isoform of the sodium pump

The α4 isoform of the Na+,K+-ATPase (sodium pump) is known to be expressed in spermatozoa and to be critical for their motility. In the investigation presented here, we find that the rat-derived Sertoli cell line 93RS2 also expresses considerable amounts of the α4 isoform in addition to the α1 isoform. Since Sertoli cells are not motile, one can assume that the function of the α4 isoform in these cells must differ from that in spermatozoa. Thus, we assessed a potential involvement of this isoform in signaling pathways that are activated by the cardiotonic steroid (CTS) ouabain, a highly specific sodium pump ligand. Treatment of 93RS2 cells with ouabain leads to activation of the c-Src/c-Raf/Erk1/2 signaling cascade. Furthermore, we show for the first time that the activation of this cascade by ouabain results in phosphorylation and activation of the transcription factor CREB. This signaling cascade is induced at low nanomolar concentrations of ouabain, consistent with the involvement of the α4 isoform. This is further supported by experiments involving siRNA: silencing of α4 expression entirely blocks ouabain-induced activation of Erk1/2 whereas silencing of α1 has no effect. The findings of this study unveil new aspects in CTS/sodium pump interactions by demonstrating for the first time ouabain-induced signaling through the α4 isoform. The c-Src/c-Raf/Erk1/2/CREB cascade activated by ouabain is identical to the so-called non-classical signaling cascade that is normally triggered in Sertoli cells by testosterone. Taking into consideration that CTS are produced endogenously, our results may help to gain new insights into the physiological mechanisms associated with male fertility and reproduction.

Testosterone signaling and the regulation of spermatogenesis

Spermatogenesis and male fertility are dependent upon the presence of testosterone in the testis. In the absence of testosterone or the androgen receptor, spermatogenesis does not proceed beyond the meiosis stage. The major cellular target and translator of testosterone signals to developing germ cells is the Sertoli cell. In the Sertoli cell, testosterone signals can be translated directly to changes in gene expression (the classical pathway) or testosterone can activate kinases that may regulate processes required to maintain spermatogenesis (the non-classical pathway). Contributions of the classical and non-classical testosterone signaling pathways to the maintenance of spermatogenesis are discussed. Studies that may further elaborate the mechanisms by with the pathways support spermatogenesis are proposed.

Regulation of Sertoli-Germ Cell Adhesion and Sperm Release by FSH and Nonclassical Testosterone Signaling

Regulation of Sertoli-Germ Cell Adhesion and Sperm Release by FSH and Nonclassical Testosterone Signaling

Regulation of Sertoli-Germ Cell Adhesion and Sperm Release by FSH and Nonclassical Testosterone Signaling

Testosterone and FSH act in synergy to produce the factors required to maximize the production of spermatozoa and male fertility. However, the molecular mechanisms by which these hormones support spermatogenesis are not well established. Recently, we identified a nonclassical mechanism of testosterone signaling in cultured rat Sertoli cells. We found that testosterone binding to the androgen receptor recruits and activates Src tyrosine kinase. Src then causes the activation of the epidermal growth factor receptor, which results in the phosphorylation and activation of the ERK MAPK and the cAMP response element-binding protein transcription factor. In this report, we find that FSH inhibits testosterone-mediated activation of ERK and the MAPK pathway in Sertoli cells via the protein kinase A-mediated inhibition of Raf kinase. In addition, FSH, as well as inhibitors of Src and ERK kinase activity, reduced germ cell attachment to Sertoli cells in culture. Using pathway-specific androgen receptor mutants we found that the nonclassical pathway is required for testosterone-mediated increases in germ cell attachment to Sertoli cells. Studies of seminiferous tubule explants determined that Src kinase, but not ERK kinase, activity is required for the release of sperm from seminiferous tubule explants. These findings suggest the nonclassical testosterone-signaling pathway acts via Src and ERK kinases to facilitate the adhesion of immature germ cells to Sertoli cells and through Src to permit the release of mature spermatozoa. In contrast, FSH acts to limit testosterone-mediated ERK kinase activity and germ cell attachment.

Non-Classical Testosterone Signaling in Sertoli Cells and the Regulation of Male Fertility.William H. Walker, Ph.D.

Non-Classical Testosterone Signaling in Sertoli Cells and the Regulation of Male Fertility.William H. Walker, Ph.D.