A transcriptome-wide screen for mRNAs enriched in fetal Leydig cells: CRHR1 agonism stimulates rat and mouse fetal testis steroidogenesis.

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.

Human fetal testis xenografts are resistant to phthalate-induced endocrine disruption.

BACKGROUND: In utero exposure to endocrine-disrupting chemicals may contribute to testicular dysgenesis syndrome (TDS), a proposed constellation of increasingly common male reproductive tract abnormalities (including hypospadias, cryptorchidism, hypospermatogenesis, and testicular cancer). Male rats exposed in utero to certain phthalate plasticizers exhibit multinucleated germ cell (MNG) induction and suppressed steroidogenic gene expression and testosterone production in the fetal testis, causing TDS-consistent effects of hypospadias and cryptorchidism. Mice exposed to phthalates in utero exhibit MNG induction only. This disparity in response demonstrates a species-specific sensitivity to phthalate-induced suppression of fetal Leydig cell steroidogenesis. Importantly, ex vivo phthalate exposure of the fetal testis does not recapitulate the species-specific endocrine disruption, demonstrating the need for a new bioassay to assess the human response to phthalates. OBJECTIVES: In this study, we aimed to develop and validate a rat and mouse testis xenograft bioassay of phthalate exposure and examine the human fetal testis response. METHODS: Fetal rat, mouse, and human testes were xenografted into immunodeficient rodent hosts, and hosts were gavaged with a range of phthalate doses over multiple days. Xenografts were harvested and assessed for histopathology and steroidogenic end points. RESULTS: Consistent with the in utero response, phthalate exposure induced MNG formation in rat and mouse xenografts, but only rats exhibited suppressed steroidogenesis. Across a range of doses, human fetal testis xenografts exhibited MNG induction but were resistant to suppression of steroidogenic gene expression. CONCLUSIONS: Phthalate exposure of grafted human fetal testis altered fetal germ cells but did not reduce expression of genes that regulate fetal testosterone biosynthesis.

Species-specific dibutyl phthalate fetal testis endocrine disruption correlates with inhibition of SREBP2-dependent gene expression pathways.

Fetal rat phthalate exposure produces a spectrum of male reproductive tract malformations downstream of reduced Leydig cell testosterone production, but the molecular mechanism of phthalate perturbation of Leydig cell function is not well understood. By bioinformatically examining fetal testis expression microarray data sets from susceptible (rat) and resistant (mouse) species after dibutyl phthalate (DBP) exposure, we identified decreased expression of several metabolic pathways in both species. However, lipid metabolism pathways transcriptionally regulated by sterol regulatory element-binding protein (SREBP) were inhibited in the rat but induced in the mouse, and this differential species response corresponded with repression of the steroidogenic pathway. In rats exposed to 100 or 500 mg/kg DBP from gestational days (GD) 16 to 20, a correlation was observed between GD20 testis steroidogenic inhibition and reductions of testis cholesterol synthesis endpoints including testis total cholesterol levels, Srebf2 gene expression, and cholesterol synthesis pathway gene expression. SREBP2 expression was detected in all fetal rat testis cells but was highest in Leydig cells. Quantification of SREBP2 immunostaining showed that 500 mg/kg DBP exposure significantly reduced SREBP2 expression in rat fetal Leydig cells but not in seminiferous cords. By Western analysis, total rat testis SREBP2 levels were not altered by DBP exposure. Together, these data suggest that phthalate-induced inhibition of fetal testis steroidogenesis is closely associated with reduced activity of several lipid metabolism pathways and SREBP2-dependent cholesterologenesis in Leydig cells.