Sex Maintenance in Mammals

The crucial event in mammalian sexual differentiation occurs at the embryonic stage of sex determination, when the bipotential gonads differentiate as either testes or ovaries, according to the sex chromosome constitution of the embryo, XY or XX, respectively. Once differentiated, testes produce sexual hormones that induce the subsequent differentiation of the male reproductive tract. On the other hand, the lack of masculinizing hormones in XX embryos permits the formation of the female reproductive tract. It was long assumed that once the gonad is differentiated, this developmental decision is irreversible. However, several findings in the last decade have shown that this is not the case and that a continuous sex maintenance is needed. Deletion of Foxl2 in the adult ovary lead to ovary-to-testis transdifferentiation and deletion of either Dmrt1 or Sox9/Sox8 in the adult testis induces the opposite process. In both cases, mutant gonads were genetically reprogrammed, showing that both the male program in ovaries and the female program in testes must be actively repressed throughout the individual’s life. In addition to these transcription factors, other genes and molecular pathways have also been shown to be involved in this antagonism. The aim of this review is to provide an overview of the genetic basis of sex maintenance once the gonad is already differentiated.

Maternal Piwi Regulates Primordial Germ Cell Development to Ensure the Fertility of Female Progeny in Drosophila

In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the Drosophila early embryo. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.

Maternal Piwi Regulates Primordial Germ Cell Development to Ensure the Fertility of Female Progeny in Drosophila

In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the early embryo in Drosophila. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.

TRIM28-dependent SUMOylation protects the adult ovary from the male pathway

Gonadal sexual fate in mammals is determined during embryonic development and must be actively maintained in adulthood. Therefore, gonadal sex-specific transcription factors are required to prevent transdifferentiation of gonadal somatic cells to the other sexual fate. Mouse genetic experiments have shown that oestrogen receptor signalling and the transcription factor FOXL2 protect ovarian granulosa cells from transdifferentiation into Sertoli cells, their testicular counterpart. However, the mechanism underlying this protective mechanism is unknown. Here, we show that one post-translational modification (i.e. SUMOylation catalysed by TRIM28) is sufficient to prevent female-to-male sex reversal of the mouse ovary after birth. We found that upon loss of TRIM28 SUMO-E3 ligase activity, ovarian granulosa cells transdifferentiate to Sertoli cells through an intermediate cell type different from gonadal embryonic progenitors. TRIM28 binds to chromatin close to the critical transcription factor FOXL2 to maintain the female pathway through SUMOylation of specific chromatin regions. Therefore, FOXL2 signalling might maintain the adult ovary cell fate via TRIM28-dependent SUMOylation. Improper SUMOylation of chromatin regions in granulosa cells might lead to female reproductive disorders and infertility, the incidence of which is currently increasing.

Ovarian follicles are resistant to monocyte perturbations—implications for ovarian health with immune disruption

Monocytes and macrophages are the most abundant immune cell populations in the adult ovary, with well-known roles in ovulation and corpus luteum formation and regression. They are activated and proliferate in response to immune challenge and are suppressed by anti-inflammatory treatments. It is also likely they have a functional role in the healthy ovary in supporting the maturing follicle from the primordial through to the later stages, however this role has been unexplored until now. Here we utilised a Cx3cr1-Dtr transgenic Wistar rat model that allows a conditional depletion of circulating monocytes, to investigate their role in ovarian follicle health. Our findings show that circulating monocyte depletion leads to a significant depletion of ovarian monocytes and monocyte-derived macrophages. Depletion of monocytes was associated with a transient reduction in circulating anti-Müllerian hormone (AMH) at 5 days post-depletion. However, the 50–60% ovarian monocyte/macrophage depletion had no effect on ovarian follicle numbers, follicle atresia or apoptosis, within 5 to 21 days post-depletion. These data reveal that the healthy adult ovary is remarkably resistant to perturbations of circulating and ovarian monocytes despite acute changes in AMH. These data suggest that short-term anti-inflammatory therapies that transiently impact on circulating monocytes are unlikely to disrupt ovarian follicle health, findings that have significant implications for fertility planning relative to the experience of an immune challenge or immunosuppression.

84 Characterization of agouti-signalling protein expression within the bovine ovary and early embryo

Factors present in the oocyte and surrounding follicular cells aid in the attainment of oocyte competence. Agouti-signalling protein (ASIP) is a known regulator of melanocyte function through binding to melanocortin receptors including MC1R and MC4R. Additionally, ASIP has been classified as an adipokine due to a link with insulin resistance and obesity in humans. In mice, expression is limited to hair follicles where ASIP regulates hair pigmentation. In cattle, however, ASIP mRNA has been detected in a variety of tissues, including adipose, skin, heart, testis, and the ovary. Despite ovarian expression, the role of ASIP in reproduction remains undetermined. Bovine ASIP is a secreted protein consisting of 133 amino acids. The aim of this experiment was to provide a detailed description of the ASIP expression profile within the bovine ovary and during early embryonic development. Reverse transcription PCR (RT-PCR) was conducted to analyse ASIP, MC1R, and MC4R mRNA expression. Samples examined included fetal ovaries from gestational day 90 to 250, adult ovary, fetal testis, adult testis, and 12 somatic tissues including adrenal, cerebral cortex, gut, heart, intestine, kidney, liver, lung, muscle, pituitary, stomach, and thymus. Amplification of ribosomal protein L19 (RPL19) served as a positive control for all samples. Expression of ASIP was detected in the fetal testis, 9 somatic tissues, and the fetal and adult ovary. In the fetal ovary, ASIP was detected as early as 90 days of gestation and continued throughout gestation. Expression of the ASIP receptors, MC1R and MC4R, were detected exclusively in the fetal ovary. To further characterise ASIP expression, quantitative real-time PCR (RT-qPCR) was utilised to examine samples including germinal vesicle and MII oocytes (pool of 10 oocytes), invitro-produced embryos ranging from the 2-cell to blastocyst stages (pool of 10 embryos), and cumulus and granulosa cells collected from a pool from 5 cumulus–oocyte complexes (COCs) and follicles, respectively. Theca cells from a single follicle were analysed. Samples with cycle threshold values below 35 were considered to express the gene of interest. Of the follicular cells examined, ASIP expression was present in theca, granulosa, and cumulus cells. ASIP expression was detected in both GV and MII oocytes. Early embryonic expression of ASIP was detected in the 2-cell embryo and continued to the blastocyst stage of development. In conclusion, ASIP is present in the bovine adult and fetal ovary, follicular cells including cumulus, granulosa, and theca cells, GV and MII oocytes, and invitro-produced embryos from the 2-cell to blastocyst stages. Future research will focus on identifying the function of ovarian and early embryonic ASIP in cattle.

The Bric-à-Brac BTB/POZ transcription factors are necessary in niche cells for germline stem cells establishment and homeostasis through control of BMP/DPP signaling in the Drosophila melanogaster ovary

Many studies have focused on the mechanisms of stem cell maintenance via their interaction with a particular niche or microenvironment in adult tissues, but how formation of a functional niche is initiated, including how stem cells within a niche are established, is less well understood. Adult Drosophila melanogaster ovary Germline Stem Cell (GSC) niches are comprised of somatic cells forming a stack called a Terminal Filament (TF) and associated Cap and Escort Cells (CCs and ECs, respectively), which are in direct contact with GSCs. In the adult ovary, the transcription factor Engrailed is specifically expressed in niche cells where it directly controls expression of the decapentaplegic (dpp) gene encoding a member of the Bone Morphogenetic Protein (BMP) family of secreted signaling molecules, which are key factors for GSC maintenance. In larval ovaries, in response to BMP signaling from newly formed niches, adjacent primordial germ cells become GSCs. The bric-à-brac paralogs (bab1 and bab2) encode BTB/POZ domain-containing transcription factors that are expressed in developing niches of larval ovaries. We show here that their functions are necessary specifically within precursor cells for TF formation during these stages. We also identify a new function for Bab1 and Bab2 within developing niches for GSC establishment in the larval ovary and for robust GSC maintenance in the adult. Moreover, we show that the presence of Bab proteins in niche cells is necessary for activation of transgenes reporting dpp expression as of larval stages in otherwise correctly specified Cap Cells, independently of Engrailed and its paralog Invected (En/Inv). Moreover, strong reduction of engrailed/invected expression during larval stages does not impair TF formation and only partially reduces GSC numbers. In the adult ovary, Bab proteins are also required for dpp reporter expression in CCs. Finally, when bab2 was overexpressed at this stage in somatic cells outside of the niche, there were no detectable levels of ectopic En/Inv, but ectopic expression of a dpp transgene was found in these cells and BMP signaling activation was induced in adjacent germ cells, which produced GSC-like tumors. Together, these results indicate that Bab transcription factors are positive regulators of BMP signaling in niche cells for establishment and homeostasis of GSCs in the Drosophila ovary.