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The role of steroidogenic factor 1 (SF-1) in steroidogenic cell function of the testes and ovaries of mature mice
in ReproductionIn brief
The nuclear receptor steroidogenic factor 1 (SF-1) is essential for mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and depletion of SF-1 in steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility.
Abstract
Steroidogenic factor 1 (SF-1 or NR5A1) plays an essential role in the development of fetal gonads and regulates genes involved in steroid biosynthesis. Since SF-1 is expressed in multiple cell types in mouse gonads, we developed three novel conditional knockout (cKO) mouse models employing Cre-recombinase and floxed alleles of SF-1 (Nr5a1f/f) to identify its role in testes and ovaries of mature mice: Cytochrome P450 17α-hydroxylase (Cyp17Cre/+;Nr5a1f/f, Leydig and theca cell-specific), aromatase (Cyp19Cre/+;Nr5a1f/f, Sertoli and granulosa cell-specific), as well as a combination of both (Cyp17+Cyp19-Cre;Nr5a1f/f). Compared to control animals, Cyp19-Cre;Nr5a1f/f cKO males showed normal fertility and testicular function. The Cyp17Cre/+;Nr5a1f/f cKO males had smaller testis, with drastically reduced Leydig cell volumes and impaired steroidogenesis, though their reproductive performance remained comparable to controls. Some 50% of Cyp17Cre/++Cyp19Cre/+;Nr5a1f/f double-cKO (dKO) males were infertile, while the remaining 50% showed significantly reduced fertility. These dKO males also had smaller testis with degenerative seminiferous tubules, abnormal Leydig cell morphology and lower levels of intra-testicular testosterone. Abnormal Sertoli cell localization was noted in dKO testes, with increased Sox9, p27 and inhibin subunit ßb and decreased androgen receptor expression. Female mice from all genotypes showed normal reproductive capacity, though steroidogenic gene expression levels were significantly decreased in both Cyp17Cre/+;Nr5a1f/f cKO and dKO females. These results show the essential role of SF-1 in mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and that depletion SF-1 in all steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility.
Supplementary Materials
- Supplemental figure 1. (A) Examples of morphology of cell in the interstitial space of CON, Cyp17 cKO and dKO testes. Scale bar, 50 µM. (B) Quantitative analysis of Leydig (GATA-4 positive) cells in interstitial space of CON, Cyp19 cKO, Cyp17 cKO and dKO testes. (C) Protein expression of GATA-4 in the interstitial space of CON, Cyp19 cKO, Cyp17 cKO and dKO mature testes. Merged images show both GATA-4 (red) and DAPI (blue) staining. Scale bars 50µm.
- Supplemental figure 2. Protein expression of cytoplasmic 3βHSD in CON, Cyp19 cKO, Cyp17 cKO and dKO mature testes. 3βHSD positive cells (red) are present in the interstitial space. Merged images show both 3βHSD (red) and DAPI (blue) staining. Scale bars 50µm.
- Supplemental figure 3. (A) Protein expression of nuclear specific cleaved caspase-3 in CON, Cyp19 cKO, Cyp17 cKO and dKO mature testes. Cleaved caspase-3 positive cells (red) are present in the testicular cords (white arrow). Merged images show both cleaved caspase-3 (red) and DAPI (blue) staining. Scale bars 50µm. (B) Relative abundance of anti-apoptotic marker Mcl1 in the testes of CON, Cyp19 cKO, Cyp17 cKO and dKO mature males (n= 6 per genotype). Letters indicate significant statistical difference (P < 0.05).
- Supplemental figure 4. (A) mRNA abundance of gap junction marker CX43 (Gja1) mRNA in the testes of CON, Cyp19 cKO, Cyp17 cKO and dKO mature males (n= 6 per genotype). Letters indicate significant statistical difference (P < 0.05). (B) Protein expression of CX43 in CON, Cyp19 cKO, Cyp17 cKO and dKO mature testes. CX43-positive cells are present in interstitial space (arrowheads) and in the testicular cords (arrows). Merged images show CX43 (red) and DAPI (blue) staining. Scale bars 50µm. (C) mRNA abundance of Gja1 normalized to Cyp19a1 in the testes of CON, Cyp19 cKO, Cyp17 cKO and dKO mature males (n= 6 per genotype).
- Supplemental figure 5. (A) Protein expression of AR in CON, Cyp19 cKO, Cyp17 cKO and dKO mature testes. Merged images show AR (red) and DAPI (blue) staining. Scale bars 50µm. (B) Quantitative analysis of mislocated Sertoli (WT-1 positive) cells was calculated as the ratio of mislocated WT-1 positive cells per seminiferous tubule. Data are represented as mean ± SEM. Letters indicate significant statistical difference (P < 0.05). (D) mRNA abundance of Sertoli-specific cell markers normalized to housekeeping genes in the testes of CON, Cyp19 cKO, Cyp17 cKO and dKO mature males (n= 6 per genotype). Letters indicate significant statistical difference (P < 0.05).
- Supplemental figure 6. Effect of steroidogenic cell-specific SF-1 depletion on ovarian steroidogenesis. (A) Protein expression of cytoplasmic StAR in corpora lutea of CON and Cyp17 cKO superstimulated ovaries. Merged images show StAR (red) and DAPI (blue) staining in luteal cells. Scale bars 50µm. (B) Relative mRNA abundance of factors involved in steroidogenesis in CON (n=5) and dKO (n=3) whole ovaries. Asterisk indicates significant statistical difference (P < 0.05).
- Supplemental table 1: List of primer sequences used in this study. All primers are 5’ to 3’.
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