Nonclassical Testosterone Signaling: A New Pathway Controlling Spermatogenesis?

Testosterone is the essential regulator of spermatogenesis [1]. Produced by the testicular Leydig cells in response to pituitary Luteinizing Hormone (LH) stimulation, testosterone mediates its effects by binding to its cognate receptor, the ligand-activated transcription factor NR3C4, also known as the androgen receptor (AR). The role of testosterone in promoting spermatogenesis was established in experiments using systemic blockade of AR signaling or suppression of testosterone production [2], either of which leads to complete arrest of spermatogenesis during meiosis. It is also now commonly accepted that AR is widely expressed in the somatic cells but not the germ cells of the adult testis. However, it is only in the past decade, with the development and wide exploitation of cell-specific AR knockout (ARKO) mice that we have begun to unravel the specific intracellular mechanisms involved in vivo. Several groups have now independently shown that testosterone signaling through AR specifically within Sertoli cells is essential for post-meiotic germ cell development [3-6]; so our understanding is complete, yes?Well no, it would appear we still have much to learn. Because of the well-established role of AR as a transcription factor, almost all interpretations of testosterone impacts have been via modulation of the transcription of AR target genes, many of which have been identified by scanning for the presence of androgen response elements (ARE) in their non-coding regions. This well-established and extremely well studied pathway, which can take hours to show a response, has become known as the classical androgen-signaling pathway (Figure 1).However, back in 2004, Sertoli cells isolated from rats were shown in vitro to also respond to testosterone stimulation by rapidly activating the MAP-kinase signaling pathway (within 1 minute) and CREB transcription factor, in an AR-dependent signaling cascade [7], providing evidence that testosterone can also act in Sertoli cells in a fast, so called non-classical manner. Despite an increasing body of circumstantial evidence amassed over the past decade (reviewed in [8]), it has remained unknown whether testosterone does act via this non-classical pathway in vivo, and if so, what impact it has on spermatogenesis. An unanticipated issue arising from the design of the in vivo cell-specific ablation models of AR is that these knockout models simultaneously destroy signaling via both classical and non-classical pathways, making it impossible to dissect out and assign specific functions to each pathway. In a ground-breaking paper in a recent issue of Biology of Reproduction, Toocheck and colleagues [9] have instead used specific inhibitors and activators of either classical or non-classical testosterone signaling to show that these pathways perform overlapping yet distinct roles, and that both pathways are essential for the support of spermatogenesis. The authors first show that in Sertoli cells within the rat testis phosphorylated ERK, but not total ERK is down-regulated if testosterone levels are reduced, and then rapidly up-regulated following acute stimulation with testosterone, confirming that the non-classical pathway is active in Sertoli cells in vivo. The authors then validated a dominant-negative form of AR, which suppresses expression of a PSA-luciferase construct but does not impact T-mediated increases in ERK phosphorylation (a specific inhibitor of the classical pathway). To complement this, the authors also validated a peptide corresponding to the proline rich domain in AR, and a peptidomimetic of the FXXLF motif of AR, both of which specifically inhibit the non-classical pathway, but do not prevent AR-mediated transcription from a PSA-luciferase reporter construct. (specific inhibitors of the non-classical pathway).Having demonstrated the efficacy of these inhibitors to specifically block each pathway in isolation in vitro, these factors were utilized in a testis explant model. These studies demonstrated that both pathways are active in Sertoli cells with transcription of Rhox5, probably the archetypal AR regulated gene in Sertoli cells [10], being controlled by both classical and non-classical pathways. Wider impacts were also uncovered, revealing that germ cell gene expression can be modulated differentially by the actions of each pathway in Sertoli cells, with some genes activated by both classical and non-classical signaling (cKit), whilst others are more effectively induced by the classical pathway (Plzf). In a further study, delivery of selective pathway inhibitors to Sertoli cells in vivo (via efferent duct injection), showed that function of the blood-testis barrier, and retention of germ cell attachment to Sertoli cells, both testosterone-dependent processes, are impacted by independent loss of the non-classical pathway. However, most importantly, these studies reveal for the first time, that spermatogenesis is blocked during meiosis in vivo if the non-classical pathway is specifically inhibited, even if the classical testosterone signaling pathway is retained and functionally active.In summary, this unique study opens up a new paradigm in testis biology that will provoke a rethink of our understanding of how testosterone controls spermatogenesis. In a wider context, this study has further significant implications for our understanding of androgen signaling throughout all systems of the body, both under normal physiological conditions and also in the context of androgen-related pathologies.