Effect of Temporal Sertoli Cell Androgen Receptor Expression on Leydig Cell Development and Function in Mice.

Androgens action is vital for the maintenance of quantitatively normal spermatogenesis and male fertility. Androgen receptor (AR) is a ligand-inducible nuclear receptor that regulates gene expression in target cells. Despite this fundamental role, the precise AR-regulated pathways controlling spermatogenic development is poorly defined. Male germ cells do not express AR, highlighting key roles of testicular AR-expressing somatic cells, like Sertoli, Leydig and peritubular cells. Recent studies show genetic loss of Sertoli cell AR impacts on Leydig cell development in mice. To determine the direct role of temporal Sertoli cell AR (SAR) expression in the regulation of Leydig cell development, we established a unique transgenic (Tg) SAR mouse expressing Tg human-AR driven via the Sertoli-specific Abp promoter. Premature TgSAR expression was confirmed by immunofluorescence in postnatal day (PND) 5 testis sections, when wildtype (WT) SAR expression is low and Sertoli-specific expression further verified by TgSAR mRNA detected in enriched Sertoli, but not Leydig and peritubular cell preparations by RT-PCR. To identify SAR-dependent actions on Leydig cell development in vivo, expression levels of Leydig-specific markers were measured by qPCR. We revealed that TgSAR has differential actions upon fetal Leydig cells, with normal testicular Insl3 and Lhr mRNA levels contrasting with elevated Hsd17b3, and reduced Cyp11a1, Cyp17a1 and Star mRNA levels in PND 5 TgSAR relative to WT testes. The developmental upregulation of Leydig cell markers (Cyp11a1, Cyp17a1, Hsd3b6, Hsd17b3, Star, Lhr, and Insl3) was similar during the period of immature Leydig cell differentiation (PND10-30) in TgSAR relative to WT testes. However, intratesticular testosterone (T), dihydrotestosterone (DHT), 3a and 3 -diols levels (measured by LC-MS) were significantly elevated in pubertal (PND 30) TgSAR testes compared to WT [13.2 ± 2.3 vs 3 ± 0.82, 15.3 ± 3.2 vs 5 ± 1.4, 106 ± 21.1 vs 14 ± 3.1, 14 ± 2.1 vs 3.1 ± 0.86 pg/mg respectively], suggesting enhanced steroidogenesis. Adult testes of fertile TgSAR mice were 25% smaller than normal. Despite reductions in total Sertoli (-30%, P < 0.05) and Leydig (-30%, P < 0.05) cell numbers determined by stereology (optical disector technique), normal Leydig:Sertoli cell ratios were maintained in TgSAR mice suggesting SAR control of the ultimate testicular Leydig cell capacity. Serum and intratesticular T, DHT, 3a and 3β-diols levels, and seminal vesicle weights were normal in adult TgSAR vs WT mice. However, total intratesticular T/Leydig cell was increased in adult TgSAR compared to WT testes (3.95 ± 0.352 vs 1.59 ± 0.295 ng/Leydig cell, P < 0.05) respectively. In addition, expression of Hsd3b6, Hsd17b3, Cyp11a1, Cyp17a1, StAR and Insl3 mRNA were elevated (corrected for Leydig cell numbers) in TgSAR versus WT testes, which suggests elevated steroidogenic capacity for Leydig cells in TgSAR mice. In conclusion, our novel gain-of-function TgSAR strategy has revealed that Sertoli cell AR activity has important consequences for Leydig cell function, in particular for the regulation of testicular Leydig cell number and steroidogenic capacity. Our approach provides a unique opportunity to differentiate Sertoli AR regulated pathways involved in Leydig cell development and function.