Spermatogenesis requires nuclear androgen receptors in somatic cells and testosterone binding to membrane receptors on germ cells in rat testis

Leydig cell ablation using ethylene dimethane sulfonate (EDS) triggers germ cell apoptosis by depleting testosterone (T) and subsequent loss of spermatogenesis. Male Sprague Dawley rats were injected with EDS at 75 mg/kg body weight to selectively eliminate mature Leydig cells and thus loss of testosterone. Two specific Leydig cell transcriptional markers (3βHSD2 and Insl3), were quantified using qrtPCR and were depleted by >98% with Leydig cell loss that also resulted in complete testosterone loss in serum and testicular tissue and significantly increased germ cell apoptosis and significantly reduced testicular weight. Gene expression levels of pro-apoptotic genes and anti-apoptotic genes all in the Bcl2 protein family in testes were significantly increased 10 days post-EDS as measured by qrtPCR. Immunostaining for the nuclear androgen receptors (nARs) were found in Sertoli cell nuclei, myoid cell nuclei and the stripped cytoplasm of spermatids in seminiferous tubules. Using flow cytometry data from isolated germ cells tagged with Vybrant Dye Cycle (Green DNA dye) confirmed that haploid germ cells were mainly affected by loss of testosterone and this loss was prevented with testosterone replacement. Testosterone
act via both a canonical and a non-canonical pathway, primarily inducing nuclear androgen receptors (nARs) in somatic cells. However, maintenance of spermatogenesis requires testosterone activation of a non-canonical membrane receptor.
We investigated for existence of a functional non-canonical pathway that would be novel for testosterone action in testicular germ cells since testosterone and progesterone have similar affinity for receptor binding with membrane progesterone receptors. Transcription of five mPRs (α, β, γ, δ and ε) were found in male rat testes among which mPRs α and β had a more gene expression level and detection by immunostaining of protein expression. We further investigated whether testosterone binds mPRs in presence of low-level progesterone, mimicking normal testicular environment. We identified testosterone binding on isolated germ cells via IHC. Competitive binding assays were successful in binding testosterone with mPRs even in the presence of low-level progesterone. Thus testosterone may use an alternate pathway for receptor interaction on germ cells that may prevent apoptosis of differentiating germ cells of spermatogenesis.