Oestrogen blocks the nuclear entry of SOX9 in the developing gonad of a marsupial mammal

Abstract

Background: Hormones are critical for early gonadal development in nonmammalian vertebrates, and oestrogen is required for normal ovarian development. In contrast, mammals determine sex by the presence or absence of the SRY gene, and hormones are not thought to play a role in early gonadal development. Despite an XY sex-determining system in marsupial mammals, exposure to oestrogen can override SRY and induce ovarian development of XY gonads if administered early enough. Here we assess the effect of exogenous oestrogen on the molecular pathways of mammalian gonadal development.

Results: We examined the expression of key testicular (SRY, SOX9, AMH and FGF9) and ovarian (WNT4, RSPO1, FOXL2 and FST) markers during gonadal development in the marsupial tammar wallaby (Macropus eugenii) and used these data to determine the effect of oestrogen exposure on gonadal fate. During normal development, we observed male specific upregulation of AMH and SOX9 as in the mouse and human testis, but this upregulation was initiated before the peak in SRY expression and 4 days before testicular cord formation. Similarly, key genes for ovarian development in mouse and human were also upregulated during ovarian differentiation in the tammar. In particular, there was early sexually dimorphic expression of FOXL2 and WNT4, suggesting that these genes are key regulators of ovarian development in all therian mammals. We next examined the effect of exogenous oestrogen on the development of the mammalian XY gonad. Despite the presence of SRY, exogenous oestrogen blocked the key male transcription factor SOX9 from entering the nuclei of male somatic cells, preventing activation of the testicular pathway and permitting upregulation of key female genes, resulting in ovarian development of the XY gonad.

Conclusions: We have uncovered a mechanism by which oestrogen can regulate gonadal development through the nucleocytoplasmic shuttling of SOX9. This may represent an underlying ancestral mechanism by which oestrogen promotes ovarian development in the gonads of nonmammalian vertebrates. Furthermore, oestrogen may retain this function in adult female mammals to maintain granulosa cell fate in the differentiated ovary by suppressing nuclear translocation of the SOX9 protein. See commentary: http://www.biomedcentral.com/1741-7007/8/110.

Figure 1

Quantitative mRNA expression for SRY, SOX9, AMH, FGF9, FOXL2, FST, RSPO1 and WNT4 during normal male (solid circles) and female (open circles) gonadal differentiation. Dotted line indicates day of birth, grey shading in a-d indicates the period of testis patterning and grey shading in e-h indicates the window of ovarian patterning. The critical period of testicular differentiation occurs between day 25 of gestation and day 2 postpartum, and that of ovarian differentiation between days 6-10 postpartum. P values from t-tests conducted to examine whether expression levels of the various genes were significantly different between males and females at each data point are shown in Additional file 1. Raw data for the mean ΔCt and standard deviations for each data point are shown in Additional file 5.

Figure 2

Structure of control and oestrogen-cultured gonads. Oestrogen-cultured XY gonads developed ovarian-like cortical and medullary structures with germ cells distributed at the periphery of the cortex (arrowhead), while control gonads developed normal testicular architecture with germ cells confined to testis cords. Gonad plus mesonephros (meso)complexes were sectioned at 8 μm and stained with Mallory's triple stain. Top: Gonads cultured in control media. Bottom: Gonads cultured in the presence of oestrogen. Control XY gonads have normal male development with the formation of seminiferous cords (SC; outlined by dashed line) delineated by a basal lamina (stained dense blue) with the germ cells contained within these structures. Oestrogen-treated XY gonads formed a cortex (C) which contained germ cells and a central medulla (M), resembling the normal ovarian architecture of equivalent stage XX gonads.

Figure 3

Gene expression in XY oestrogen-treated gonads cultured from day 25 of gestation for 5 days. Quantitative PCR for key male markers SRY, SOX9, AMH and FGF9 (a), and key female markers FOXL2, RSPO1, WNT4 and FST (b) during gonadal culture in control (open bars) and oestrogen-treated (black bars) gonads. Both SRY and AMH mRNA levels were significantly suppressed (P < 0.001) in the sex-reversed gonads. However, SOX9 mRNA levels were unchanged (P > 0.5). There was significant upregulation of the ovarian genes FOXL2 (P < 0.004) and WNT4 (P < 0.02) in sex-reversed gonads, while FST was unchanged and RSPO1 was significantly downregulated (P < 0.001) in the presence of oestrogen.

Figure 4

The distribution of SOX9 protein in oestrogen-treated and control cultures. Immunohistochemistry showing the subcellular distribution of SOX9 in control and oestrogen cultured XY gonads, using AEC+ chromogen red/brown staining (Dako) and counterstained with haematoxylin (blue). In the culture controls (a; magnified in b) localization was nuclear (black arrowhead) and confined to the Sertoli cells of the developing seminiferous cords. In contrast, in the oestrogen-cultured gonads (c; magnified in d), SOX9 was largely cytoplasmic (red arrowheads) and no or very few nuclei were stained. Scale bars are 100 μm in (a) and (b) and 25 μm in (c) and (d).

Figure 5

Model for the role of oestrogen and the gonadal determination pathway. In normal XY gonads, the somatic cells upregulate SRY, which in turn upregulates SOX9. SOX9 can then translocate to the nucleus and activate the expression of AMH to ensure normal male urogenital development. In the presence of oestrogen, the bipotential somatic cell is instead directed into the granulosa cell pathway. SRY fails to upregulate, and any SOX9 protein produced fails to enter the nucleus. In the absence of nuclear SOX9, AMH fails to upregulate and ovarian genes (FOXL2 and WNT4) are activated. Exogenous oestrogen therefore has the ability to direct XY bipotential somatic cells to a granulosa cell fate. Oestrogen may act in a similar manner to maintain granulosa cell fate in mature eutherian ovaries, preventing basal levels of SOX9 protein from translocating to the nucleus and propagating its own upregulation.