Estrogen suppresses SOX9 and activates markers of female development in a human testis-derived cell line

Abstract

Background: The increasing incidence of reproductive disorders in humans has been attributed to in utero exposure to estrogenic endocrine disruptors. In particular, exposure of the developing testis to exogenous estrogen can negatively impact male reproductive health. To determine how estrogens impact human gonad function, we treated the human testis-derived cell line NT2/D1 with estrogen and examined its impact on SOX9 and the expression of key markers of granulosa (ovarian) and Sertoli (testicular) cell development.

Results: Estrogen successfully activated its cognate receptor (estrogen receptor alpha; ESR1) in NT2/D1 cells. We observed a significant increase in cytoplasmic SOX9 following estrogen treatment. After 48 h of estrogen exposure, mRNA levels of the key Sertoli cell genes SOX9, SRY, AMH, FGF9 and PTGDS were significantly reduced. This was followed by a significant increase in mRNA levels for the key granulosa cell genes FOXL2 and WNT4 after 96 h of estrogen exposure.

Conclusions: These results are consistent with estrogen's effects on marsupial gonads and show that estrogen has a highly conserved impact on gonadal cell fate decisions that has existed in mammals for over 160 million years. This effect of estrogen presents as a potential mechanism contributing to the significant decrease in male fertility and reproductive health reported over recent decades. Given our widespread exposure to estrogenic endocrine disruptors, their effects on SOX9 and Sertoli cell determination could have considerable impact on the adult testis.

Keywords: Estrogen; Sertoli cells; Sex determination; Testis.

Fig. 1

ERα immunofluorescence staining in NT2/D1 cells after 48 h of culture. ERα (green) was primarily cytoplasmic in control cells. EE2 treatment at both 10 nM and 100 nM led to a significant increase in nuclear ERα. Top panel shows ERα, middle panel shows DAPI, bottom panel shows a merge. Nuclei stained with DAPI; magenta. Scale bar 5 μm

Fig. 2

SOX9 immunofluorescence staining in NT2/D1 cells after 48 h of culture. SOX9 (green) was primarily nuclear (nuclei marked by DAPI; magenta) in the control cells. EE2 treatment at both 10 nM and 100 nM led to a greater amount of SOX9 in the cytoplasm. Top panel shows SOX9, middle panel shows DAPI, bottom panel shows a merge. Scale bar 5 μm

Fig. 3

SOX9 immunofluorescence staining in NT2/D1 cells after 96 h of culture. SOX9 (green) was primarily nuclear (nuclei marked by DAPI; magenta) in the control cells. EE2 treatment at both 10 nM and 100 nM led to a greater amount of SOX9 in the cytoplasm. Top panel shows SOX9, middle panel shows DAPI, bottom panel shows a merge. Scale bar 5 μm

Fig. 4

Raincloud plot of the percentage of cytoplasmic SOX9 in NT2/D1 cells. Each filled circle indicates a data point. The error bars delineate the 95% confidence interval of the mean (black square). The density distribution is indicated by the filled area. The percentage of cytoplasmic SOX9 is significantly increased for all treatments (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 compared to the control)

Fig. 5

Gene expression of key gonad markers in NT2/D1 cells. Quantitative mRNA levels of SOX9, SRY, AMH, FGF9 and PTGDS after 48 h (A; n = 10) and SOX9, SRY, AMH, FOXL2 and WNT4 after 96 h (B; n = 5) with either 10 nM or 100 nM of EE2. Expression levels in control cells were designated as 1 and illustrated by the dotted line. Error bars show the 95% confidence interval of the mean. Expression of each gene in the corresponding estrogen treated culture was expressed relative to control levels. All testis markers had significantly reduced mRNA levels following 48 h of EE2 treatment (a). Testis markers did not show reduced mRNA levels after 96 h, however the ovarian markers FOXL2 and WNT4 had significant increases in expression (b). (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 compared to the control)

Fig. 6

Model for estrogen regulation of SOX9 in Sertoli cells. a In a normal Sertoli cell SOX9 increases expression of itself and its downstream targets AMH, FGF9, PTGDS by translocating from the cytoplasm to the nucleus. PGD2 facilitates the nuclear entry of SOX9, while FGF9 inhibits WNT4 and there is no expression of FOXL2. b Exogenous estrogen (E) blocks SOX9 nuclear entry, increasing the amount of SOX9 in the cytoplasm and preventing activation of downstream SOX9 targets. Activated estrogen receptors (ERs) repress SOX9 transcription and promote expression of WNT4 and FOXL2. WNT4 subsequently inhibits FGF9