Abstract
Fetal testis steroidogenesis plays an important role in the reproductive development of the male fetus. While regulators of certain aspects of steroidogenesis are known, the initial driver of steroidogenesis in the human and rodent fetal testis is unclear. Through comparative analysis of rodent fetal testis microarray datasets, 54 candidate fetal Leydig cell-specific genes were identified. Fetal mouse testis interstitial expression of a subset of these genes with unknown expression (Crhr1, Gramd1b, Itih5, Vgll3, and Vsnl1) was verified by whole-mount in situ hybridization. Among the candidate fetal Leydig cell-specific factors, three receptors (CRHR1, PRLR, and PROKR2) were tested for a steroidogenic function using ex vivo fetal testes treated with receptor agonists (CRH, PRL, and PROK2). While PRL and PROK2 had no effect, CRH, at low (approximately 1 to 10) nM concentration, increased expression of the steroidogenic genes Cyp11a1, Cyp17a1, Scarb1, and Star in GD15 mouse and GD17 rat testes, and in conjunction, testosterone production was increased. Exposure of GD15 fetal mouse testis to a specific CRHR1 antagonist blunted the CRH-induced steroidogenic gene expression and testosterone responses. Similar to ex vivo rodent fetal testes, ≥10 nM CRH exposure of MA-10 Leydig cells increased steroidogenic pathway mRNA and progesterone levels, showing CRH can enhance steroidogenesis by directly targeting Leydig cells. Crh mRNA expression was observed in rodent fetal hypothalamus, and CRH peptide was detected in rodent amniotic fluid. Together, these data provide a resource for discovering factors controlling fetal Leydig cell biology and suggest that CRHR1 activation by CRH stimulates rat and mouse fetal Leydig cell steroidogenesis in vivo.
Highlights
Identifying mechanisms regulating fetal Leydig cell differentiation and function is important for understanding hormonedependent male reproductive development
The characteristics of a Leydig cell-specific candidate gene would be: 1) higher testis expression at GD13 compared to GD11; 2) expression in Mafb+ cells but not Sox9+ or Pou5f1+ cells; 3) reduced expression in fetal rat testis but not fetal mouse testis following dibutyl phthalate (DBP) exposure; and 4) increased expression in GD13 fetal testis compared to GD13 fetal ovary
When the expression of five Leydig cell-specific candidate genes with unknown gonadal expression patterns were localized via in situ hybridization (ISH) in GD13 mouse gonads, all showed higher expression in testis compared to ovary
Summary
Identifying mechanisms regulating fetal Leydig cell differentiation and function is important for understanding hormonedependent male reproductive development. Not until the production of LH after GD17 in the rat does LHCGR play a necessary role in rat Leydig cell steroidogenesis [12] It remains unknown what drives Leydig cell steroidogenesis at the beginning of the male programming window in humans and what factor(s) is required to activate Leydig cell steroidogenesis during the masculinization programming window in rodents. To begin closing this knowledge gap, we used a fetal testis comparative genomics approach to identify candidate genes with expression enriched in fetal Leydig cells. We performed in situ hybridization (ISH) to localize a subset of candidate mRNAs in fetal mouse testis and functional tests of candidate receptors in ex vivo fetal rodent testes and murine MA-10 Leydig cells to determine potential modulatory activity on steroidogenesis
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