Differential susceptibility of male and female germ cells to glucocorticoid-mediated signaling.

While physiologic stress has long been known to impair mammalian reproductive capacity through hormonal dysregulation, mounting evidence now suggests that stress experienced prior to or during gestation may also negatively impact the health of future offspring. Rodent models of gestational physiologic stress can induce neurologic and behavioral changes that persist for up to three generations, suggesting that stress signals can induce lasting epigenetic changes in the germline. Treatment with glucocorticoid stress hormones is sufficient to recapitulate the transgenerational changes seen in physiologic stress models. These hormones are known to bind and activate the glucocorticoid receptor (GR), a ligand-inducible transcription factor, thus implicating GR-mediated signaling as a potential contributor to the transgenerational inheritance of stress-induced phenotypes. Here, we demonstrate dynamic spatiotemporal regulation of GR expression in the mouse germline, showing expression in the fetal oocyte as well as the perinatal and adult spermatogonia. Functionally, we find that fetal oocytes are intrinsically buffered against changes in GR signaling, as neither genetic deletion of GR nor GR agonism with dexamethasone altered the transcriptional landscape or the progression of fetal oocytes through meiosis. In contrast, our studies revealed that the male germline is susceptible to glucocorticoid-mediated signaling, specifically by regulating RNA splicing within the spermatogonia, although this does not abrogate fertility. Together, our work suggests a sexually dimorphic function for GR in the germline, and represents an important step towards understanding the mechanisms by which stress can modulate the transmission of genetic information through the germline.

Differential susceptibility of male and female germ cells to glucocorticoid-mediated signaling.

While physiologic stress has long been known to impair mammalian reproductive capacity through hormonal dysregulation, mounting evidence now suggests that stress experienced prior to or during gestation may also negatively impact the health of future offspring. Rodent models of gestational physiologic stress can induce neurologic and behavioral changes that persist for up to three generations, suggesting that stress signals can induce lasting epigenetic changes in the germline. Treatment with glucocorticoid stress hormones is sufficient to recapitulate the transgenerational changes seen in physiologic stress models. These hormones are known to bind and activate the glucocorticoid receptor (GR), a ligand-inducible transcription factor, thus implicating GR-mediated signaling as a potential contributor to the transgenerational inheritance of stress-induced phenotypes. Here we demonstrate dynamic spatiotemporal regulation of GR expression in the mouse germline, showing expression in the fetal oocyte as well as the perinatal and adult spermatogonia. Functionally, we find that fetal oocytes are intrinsically buffered against changes in GR signaling, as neither genetic deletion of GR nor GR agonism with dexamethasone altered the transcriptional landscape or the progression of fetal oocytes through meiosis. In contrast, our studies revealed that the male germline is susceptible to glucocorticoid-mediated signaling, specifically by regulating RNA splicing within the spermatogonia, although this does not abrogate fertility. Together, our work suggests a sexually dimorphic function for GR in the germline, and represents an important step towards understanding the mechanisms by which stress can modulate the transmission of genetic information through the germline.

Mouse Gonad Development in the Absence of the Pro-Ovary Factor WNT4 and the Pro-Testis Factor SOX9.

The transcription factors SRY and SOX9 and RSPO1/WNT4/ß-Catenin signaling act as antagonistic pathways to drive testis and ovary development respectively, from a common gonadal primordium in mouse embryos. In this work, we took advantage of a double knockout mouse model to study gonadal development when Sox9 and Wnt4 are both mutated. We show that the XX gonad mutant for Wnt4 or for both Wnt4 and Sox9 develop as ovotestes, demonstrating that ectopic SOX9 function is not required for the partial female-to-male sex reversal caused by a Wnt4 mutation. Sox9 deletion in XY gonads leads to ovarian development accompanied by ectopic WNT/ß-catenin signaling. In XY Sox9 mutant gonads, SRY-positive supporting precursors adopt a female-like identity and develop as pre-granulosa-like cells. This phenotype cannot be fully prevented by the deletion of Wnt4 or Rspo1, indicating that SOX9 is required for the early determination of the male supporting cell identity independently of repressing RSPO1/WNT4/ß-Catenin signaling. However, in XY Sox9 Wnt4 double mutant gonads, pre-granulosa cells are not maintained, as they prematurely differentiate as mature granulosa cells and then trans-differentiate into Sertoli-like cells. Together, our results reveal the dynamics of the specific and independent actions of SOX9 and WNT4 during gonadal differentiation: SOX9 is essential in the testis for early specification of male-supporting cells whereas WNT4 functions in the ovary to maintain female-supporting cell identity and inhibit male-specific vascular and steroidogenic cell differentiation.

Sox8 and Sox9 act redundantly for ovarian-to-testicular fate reprogramming in the absence of R-spondin1 in mouse sex reversals.

In mammals, testicular differentiation is initiated by transcription factors SRY and SOX9 in XY gonads, and ovarian differentiation involves R-spondin1 (RSPO1) mediated activation of WNT/ß-catenin signaling in XX gonads. Accordingly, the absence of RSPO1/Rspo1 in XX humans and mice leads to testicular differentiation and female-to-male sex reversal in a manner that does not requireSry or Sox9 in mice. Here we show that an alternate testis-differentiating factor exists and that this factor is Sox8. Specifically, genetic ablation of Sox8 and Sox9 prevents ovarian-to-testicular reprogramming observed in XX Rspo1 loss-of-function mice. Consequently, Rspo1 Sox8 Sox9 triple mutant gonads developed as atrophied ovaries. Thus, SOX8 alone can compensate for the loss of SOX9 for Sertoli cell differentiation during female-to-male sex reversal.

In humans, mice and other mammals, genetic sex is determined by the combination of sex chromosomes that each individual inherits. Individuals with two X chromosomes (XX) are said to be chromosomally female, while individuals with one X and one Y chromosome (XY) are chromosomally males. One of the major differences between XX and XY individuals is that they have different types of gonads (the organs that make egg cells or sperm). In mice, for example, before males are born, a gene called Sox9 triggers a cascade of events that result in the gonads developing into testes. In females, on the other hand, another gene called Rspo1 stimulates the gonads to develop into ovaries. Loss of Sox9 in XY embryos, or Rspo1 in XX embryos, leads to mice developing physical characteristics that do not match their genetic sex, a phenomenon known as sex reversal. For example, in XX female mice lacking Rspo1, cells in the gonads reprogram into testis cells known as Sertoli cells just before birth and form male structures known as testis cords. The gonads of female mice missing both Sox9 and Rspo1 (referred to as "double mutants") also develop Sertoli cells and testis cords, suggesting another gene may compensate for the loss of Sox9. Previous studies suggest that a gene known as Sox8, which is closely related to Sox9, may be able to drive sex reversal in female mice. However, it was not clear whether Sox8 is able to stimulate testis to form in female mice in the absence of Sox9. To address this question, Richardson et al. studied mutant female mice lacking Rspo1, Sox8 and Sox9, known as "triple mutants". Just before birth, the gonads in the triple mutant mice showed some characteristics of sex reversal but lacked the Sertoli cells found in the double mutant mice. After the mice were born, the gonads of the triple mutant mice developed as rudimentary ovaries without testis cords, unlike the more testis-like gonads found in the double mutant mice. The findings of Richardson et al. show that Sox8 is able to trigger sex reversal in female mice in the absence of Rspo1 and Sox9. Differences in sexual development in humans affect the appearance of individuals and often cause infertility. Identifying Sox8 and other similar genes in mice may one day help to diagnose people with such conditions and lead to the development of new therapies.