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

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.

Figure 1. Flowchart showing the steps used to generate the fetal Leydig cell-specific candidate gene list.

A detailed explanation of this multistep, sequential process is found in the first worksheet of File S1. In step 1, genes were retained that displayed a signal >110 in GD13 testis Mafb-positive cells and an increased expression along with a FDR-corrected p-value <0.25 when comparing GD13 testis Mafb-positive cells to GD11 testis Mafb-positive cells. In step 2, genes were discarded showing an expression signal >200 in GD13 testis Pou5f1-positive cells. In step 3, genes were culled that had higher expression (expression ratio >1) in GD13 testis Sox9-positive cells compared to GD13 testis Mafb-positive cells. In step 4, genes were retained that showed an increased expression (expression ratio >1) and a FDR-corrected p-value <0.25 when comparing GD11 or GD12 to GD14 whole mouse testis. In step 5, genes were retained showing a decrease in gene expression (expression ratio <1) and a FDR-corrected p-value <0.25 after rat DBP exposure. In step 6, genes were culled that displayed reduced expression (expression ratio <1) and a FDR-corrected p-value <0.25 after mouse DBP exposure. In step 7, genes were retained that showed an increase in expression (expression ratio >1) and a FDR-corrected p-value <0.25 in GD13 testis Mafb-positive cells compared to GD13 ovary Mafb-positive cells.

Figure 2. Fetal mouse gonad whole-mount ISH of select candidate Leydig cell-specific genes.

Shown are ISH patterns of GD13 mouse gonads with attached mesonephros below the gonad. The distribution of fetal testis Leydig cells is shown in the male Cyp11a1 image.

Figure 3. GD17 rat testis steroidogenic gene expression after treatment with CRH, PRL, or PROK2.

Testes were exposed for 24 hours in vitro to varying concentrations of CRH, PRL, or PROK2. Taqman-based qRT-PCR was used for determination of mRNA levels. Three to four samples per group. Mean ±SD are shown for all data. C: control. Asterisk indicates significance for p-value of <0.05 when compared with controls.

Figure 4. Effects of CRH on steroidogenesis in GD17 rat testis.

A) Treatment with CRH increased steroidogenesis in GD17 rat testis. Seven to nineteen testes per group were exposed for 24 hours ex vivo. mRNA levels were determined using Taqman-based qRT-PCR. Mean ±SD are shown for all data. Asterisk indicates significance for p-value of <0.05 when compared with controls. B) Testosterone secretion increased in GD17 rat testis treated with CRH. Media were collected from eight to twenty samples per group for testosterone radioimmunoassay analysis. Mean ±SD is shown for all data. An asterisk indicates significance of a p-value <0.05.

Figure 5. Effects of CRH and UCN1 on steroidogenic gene expression in GD19 rat testis.

A) Steroidogenic gene expression in GD19 rat testis after exposure to 1 nM CRH, 1 nM UCN1, and/or 0.1 IU/ml hCG. Testes were exposed for 24 hours, and mRNA levels were determined using Taqman-based qRT-PCR. Mean ±SD are shown for all data. Four to five samples per group. Asterisk indicates significance for p-value of <0.05 when compared with controls. B) Testosterone levels in GD19 rat testis after exposure in vitro to 1 nM CRH, 1 nM UCN1, and/or 0.1 IU/ml hCG. Testes were exposed for 24 hours and media collected from four to five samples/group for testosterone measurement. Mean ±SD shown for all data. Asterisk indicates significance for p-value of <0.05 when compared with vehicle control.

Figure 6. Effects of CRH on steroidogenic gene expression in GD15 mouse testis.

A) Steroidogenic mRNA expression increased after treatment with CRH and/or antagonist in GD15 mouse testis. Eight to twenty-one samples/group were exposed for 24 hours in vitro to varying concentrations of CRH and/or 10 µM of antagonist. mRNA levels were determined by Taqman-based qRT-PCR. Mean ±SD shown for all data. a indicates no significant change between treated samples and vehicle samples. b indicates a significant increase (p-value <0.05) between treated and vehicle samples. c indicates a significant decrease (p-value <0.05) between samples exposed only to 10 nM CRH and samples exposed to 10 nM CRH and 10 µM CRH antagonist. (Ant. = antagonist) B) CRH treatment increased testosterone secretion from GD15 mouse testis. Testes were exposed to varying concentrations of CRH and/or 10 µM of antagonist. Media were collected from seven to fifteen samples/group for testosterone radioimmunoassay analysis. Mean ±SD shown for all data. a, b, and c are described above.

Figure 7. Effect of CRH on MA-10 cell steroidogenesis.

A) CRH exposure of MA-10 cells increased steroidogenic gene expression at different time points. MA-10 cells were exposed to 10 nM CRH for 1, 3, 6 or 24 hours. Three to six replicates were analyzed at each time point. Taqman-based qRT-PCR was used to determine mRNA levels. Mean ±SD are shown for all data. Asterisk indicates significance for p-value of <0.05 when compared with controls. B) CRH concentration-response of MA-10 cell steroidogenic mRNA expression. MA-10 cells, five replicates/group, were exposed to varying concentrations of CRH for 6 hours. Taqman-based qRT-PCR was used to determine mRNA levels. Mean ±SD shown for all data. Asterisk indicates significance for p-value of <0.05 when compared with controls. C) CRH increased MA-10 cell progesterone secretion. Eight replicates/group were treated for 6 hours, and progesterone levels in media were quantified by radioimmunoassay. Mean ±SD shown for all data. C: Control. Asterisk indicates significance of a p-value <0.05.

Figure 8. CRH protein levels are detectable in amniotic fluid from GD17 rat and GD15 mouse testis.

CRH protein levels in amniotic fluid were measured using a fluorescent enzyme immunoassay. Mean ±SD are shown for all data.