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REPRODUCTIONFOCUS REVIEW

WOMEN IN REPRODUCTIVE SCIENCE To be or not to be a testis

Blanche Capel Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, USA Correspondence should be addressed to B Capel; Email: [email protected] This paper forms part of a focus section on Women in Reproductive Science. The guest editor for this section was Professor Marilyn Renfree, Ian Potter Chair of Zoology, School of BioSciences, The University of Melbourne, Victoria, Australia

Abstract

Work that established the testis as the driver of male development, and the Y chromosome as the bearer of the male-determining gene, established a working model, and set the stage for the molecular age of mammalian sex determination. The discovery and characterization of Sry/SRY at the top of the hierarchy in mammals launched the field in two major directions. The first was to identify the downstream transcription factors and other molecular players that drive the bifurcation of Sertoli and granulosa cell differentiation. The second major direction was to understand organogenesis of the early bipotential gonad, and how divergence of its two distinct morphogenetic pathways (testis and ovary) is regulated at the cellular level. This review will summarize the early discoveries soon after Sry was identified and focus on my study of the gonad as a model of organogenesis. Reproduction (2019) 158 F101–F111

Setting the stage for the molecular investigation of results together, Anne McLaren and others suggested a sex determination working model in which the testis-determining signal (called Tdy) acted to control the development of the Sex determination is a very old field that has fascinated gonad as a testis or ovary (McLaren 1991) (Fig. 1). scientists and non-scientists alike for thousands of years. Early Greek philosophers suggested that the sex of the child depended on its position in the womb or Identification ofSry on whether the semen came from the right or left testis. The race was on to identify the critical determinant, Tdy. Aristotle, argued against these theories, and instead Candidates came and went, with very little evidence to proposed that the sex of the child depended on the heat support them. For a while, it was believed that a Y-linked of copulation (for review, see Lesky 1951). antigen (H-Y) discovered through male-to-female skin It was Alfred Jost, working in France at the end of grafting experiments, must be the male determinant World War II, who set the stage for the molecular era (Bennett et al. 1977). This idea was followed by the when he established the critical importance of gonad discovery of an evolutionarily conserved Bkm repeat sex specification and differentiation into either a testis sequence on the Y – also proposed to act as the male or ovary (Jost 1947, Ford et al. 1959). Jost surgically determinant for a time (Singh et al. 1984). However, removed the gonads from rabbit embryos in utero in 1989, David Page at MIT put forward the first and showed that this resulted in all female offspring. evidence-based molecular candidate, ZFY, relying on This pivotal experiment established the idea that the the investigation of patients whose sex chromosome ovary was not essential to develop as a female, but the genotype was discordant with their physiological presence of a testis was essential to develop as a male. development as male or female (Page et al. 1987). The By 1959, advances in cytogenetics made it possible to Page lab found that the zinc finger protein,ZFY , was identity the Y chomosome as the mediator of male fate. deleted in an XY female patient, and present in an XX At the time, this result was surprising as the number male. The fact that ZFY was likely to act as a transcription of X chromosomes had been found to mediate sex factor also fit with predictions. determination in Drosophila (Bridges 1921). However, However, very soon after these findings were examination of human aneuploids with XXY and XO published, investigators on the other side of the Atlantic karyotypes showed that it was the presence or absence began to raise doubts that ZFY was the right gene. First, of the Y that controlled male sex (Ford et al. 1959, Jacobs in the absence of germ cells in the mouse, Zfy was not & Strong 1959, Welshons & Russell 1959). Putting these expressed, but testis development occurred normally,

© 2019 Society for Reproduction and Fertility https://doi.org/10.1530/REP -19-0151 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via https://rep.bioscientifica.com Downloaded from Bioscientifica.com at 09/24/2021 06:00:38AM via free access

-19-0151 F102 B Capel

Box 1: An unexpected path into the molecular age of sex determination I grew up in a very conservative Southern family. My father did not believe in educating women and, so, as a compromise, I went to Hollins College in Virginia, famous for producing female writers. I had no concept at all of a career, married a few weeks after graduation, soon had two small children, and was involved in many volunteer organizations that I found frustrating more often than rewarding. I finally decided it was time to stop complaining and do something, so I enrolled in a genetics class at Bryn Mawr as an act of self-defense. I had enjoyed genetics as an elective in my senior year in college, back when genetics meant crossing flies and counting those with red or white eyes. I soon learned that genetics meant something very different 10 years later, which led me to move to Haverford College where a group of strong scientists were teaching Molecular Biology. I will always be grateful to Haverford, a Quaker institution where the focus is on your obligation to share the knowledge you have. It is a powerful view of education that I have carried with me. I was fascinated by this new field and looked forward to every day. When the idea of graduate school came up, it first seemed like a crazy idea, but I eventually decided to pursue a fellowship for the graduate program at UPENN. During a rotation in Will Silvers’s lab, I told him I was interested in developmental biology. He suggested that I might like to work with the renowned mouse embryologist, Beatrice Mintz. Bea had never taken a graduate student, but she was a personal friend of Will’s, and he thought she might consider me because I was older, and (presumably) more certain of my way. I am not at all sure this was true, but I took the confidence Will placed in me very seriously. He talked Bea into interviewing me, and I arrived very scared (her reputation having preceded her). I was wearing a belt that I had made from some soft suede and a hammered frog that caught her eye, and I will always believe this was the reason she took me as a PhD student. It was miraculous being a student of Bea’s. She had an amazing store of knowledge on almost every subject, and her ability to contextualize any new piece of information was very inspiring. Of course, it was not always smooth sailing. Bea had an uncanny ability to turn up behind you just when you dropped your best forceps on the floor. At such moments, she was not the model of patience, but I somehow learned how to diffuse her, and I honestly believe she learned to appreciate my ability to put out fires. It was early days in the effort to establish the existence of hematopoietic stem cells. We used viral integrations to label single donor cells, and showed that a single stem cell could repopulate all hematopoietic lineages in the host. I was very proud of the first manuscript I left on her desk. I knew I was missing some parts of a solid science background, but I was confident that I knew how to write! When she called me in to talk about the paper, her first words were, “Who do you think you are, Edgar Allen Poe? This is a scientific paper, not a mystery novel! You have hidden all the clues in the closet to surprise the reader. No one will ever have the patience to find them.” So I leaned that scientific and literary writing are not the same thing! For a post-doc, I became very interested in the British wing of mouse embryology led by Anne McLaren. Anne and Bea were arch enemies, so I did not consider going to Anne’s lab, but instead I went to the lab of a young trainee of hers, Robin Lovell-Badge. I won an NIH fellowship based on a project to induce homologous recombination in female germ cells during meiotic prophase. However, fortune had a different plan for me. Robin had a long history of searching for the male sex-determining gene. At the time, it appeared that a strong candidate gene, ZFY, had been discovered by the Page lab in the US, which was very disappointing for the London group. I arrived just in time for the experiments that indicated that Zfy was not the male sex determining gene, and the race was on. I was swept into the exciting pursuit of the real Y-linked male-determining gene. We moved quickly to identify and validate a new candidate, Sry, and I took on the project of cloning the Sry transcript. By the time I was ready to leave, most of my colleagues were very excited about identifying the molecular pathways between Sry/SRY expression and testis development. However, I was most interested in using the bipotential gonad as a unique model of organogenesis. I felt this was a direction where I would not have so much competition in setting up my own lab. In fact, I think this was the case, as most people (including granting agencies) didn’t have any idea what I was trying to achieve for several years. I joined the Cell Biology Department in the School of Medicine at Duke, where my colleagues strongly influenced the direction of my work with great ideas about how to study the cell biology of gonadogenesis. I was one of a small handful of developmental biologists and one of the only mouse geneticists at Duke at the time, but this turned out to be a very good thing. I felt that I brought something novel here and I felt valued at Duke because of it. I think this has made all the difference, and I will always feel fortunate for the lucky winds that blew me here.

Key References Capel B, Swain A, Nicolis S, Hacker A, Walter M, Koopman P, Goodfellow P & Lovell-Badge R 1993 Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell 73 1019–1030. (https://doi.org/10.1016/0092-8674(93)90279-y) Kim Y, Kobayashi A, Sekido R, DiNapoli L, Brennan J, Chaboissier MC, Poulat F, Behringer RR, Lovell-Badge R & Capel B 2006 Fgf9 and Wnt4 act as antagonistic signals to regulate mammalian sex determination. PLOS Biology 4 e187. (https://doi.org/10.1371/journal.pbio.0040187) Maatouk DM, DiNapoli L, Alvers A, Parker KL, Taketo MM & Capel B 2008 Stabilization of {beta}-catenin in XY gonads causes male-to-female sex- reversal. Human Molecular Genetics 17 2949–2955. (https://doi.org/10.1093/hmg/ddn193) Munger SC, Aylor DL, Syed HA, Magwene PM, Threadgill DW & Capel B 2009 Elucidation of the transcription network governing mammalian sex determination by exploiting strain-specific susceptibility to sex reversal.Genes and Development 23 2521–2536. (https://doi.org/10.1101/gad.1835809) Defalco T, Potter SJ, Williams AV, Waller B, Kan MJ & Capel B 2015 Macrophages Contribute to the Spermatogonial Niche in the Adult Testis. Cell Reports 12 1107–1109. (https://doi.org/10.1016/j.celrep.2015.07.015) Batchvarov IS, Taylor RW, Bustamante-Marin X, Czerwinski M, Johnson ES, Kornbluth S & Capel B 2016 A grafted ovarian fragment rescues host fertility after chemotherapy. Molecular Human Reproduction 22 842–851. (https://doi.org/10.1093/molehr/gaw064) Ge C, Ye J, Weber C, Sun W, Zhang H, Zhou Y, Cai C, Qian G & Capel B 2018 The histone demethylase KDM6B regulates temperature-dependent sex determination in a turtle species. Science 360 645–648. (https://doi.org/10.1126/science.aap8328) Garcia-Moreno SA, Lin YT, Futtner CR, Salamone IM, Capel B & Maatouk DM 2019 CBX2 is required to stabilize the testis pathway by repressing Wnt signaling. PLoS Genetics 15 e1007895. (https://doi.org/10.1371/journal.pgen.1007895) Ruthig VA, Friedersdorf MB, Garness JA, Munger SC, Bunce C, Keene JD & Capel B 2019 The RNA-binding protein DND1 acts sequentially as a negative regulator of pluripotency and a positive regulator of epigenetic modifiers required for germ cell reprogramming.Development 146 dev175950. (https://doi.org/10.1242/dev.175950)

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Testis 48 h (Hacker et al. 1995). Since it was also expressed male-specific in the adult testis where material was not limiting, genes efforts to clone the cDNA were directed at adult testis libraries. The problem was that screens of testis cDNA Bipotential TDY libraries turned up transcripts with circular permutations Gonad (Fig. 2A). This seemed likely to be an artifact, so it was several years before an RNA protection assay revealed that the Sry transcript in the adult testis is circular – the female specific Ovary first circular transcript documented Capel( et al. 1993b). genes + oocytes In contrast, the transcript in the early gonad is linear, as a result of an alternative start site that eliminates most of Figure 1 Working model of mammalian sex determination (circa the 5′ UTR, including the 5′ end of the inverted repeats 1991). The gonad is initially bipotential. A gene from the Y surrounding the Sry-coding region (Fig. 2B and C) (Capel chromosome (Tdy) is expressed in the early XY gonad and initiates et al. 1993a, Hacker et al. 1995). The regulatory region testis development. Ovary development was known to require oocytes, but no key genes in the ovary pathway were known at that upstream of Sry is complex. Deletion of Y chromosome time (after McLaren 1991). sequences spanning Sx1 repeats located >14 kb upstream of the Sry promoter led to low expression of strongly implying that Zfy could not be the right gene Sry and development of XY females (Capel et al. 1993a). (Koopman et al. 1989). Second, in a group of four XX These findings have not been explained, but may stem patients with Y chromosome translocations and male from chromatin position effects or alterations in the characteristics, the 35 kb region of the Y chromosome epigenetic landscape (Kuroki et al. 2017). that was translocated did not include ZFY, again, inconsistent with the candidacy of ZFY as the male- determining gene (Palmer et al. 1989). Genetic studies identified other key genes in the sex Based on the idea that the mammalian sex-determining determination pathway gene would be conserved among mammals, the The identification of other genes responsible for Goodfellow and Lovell-Badge labs in London used tiling Disorders of Sexual Development in humans by many probes across the 35 kb region of the Y chromosome others helped to build the molecular pathway, upstream present in the four XX patients, to identify a Y-specific and downstream of SRY. A key step was the discovery fragment present on the Y chromosome in a large group of SOX9, identified at the chromosomal breakpoint of mammals, including human, chimp, rabbit, pig, horse, in XY campomelic dysplasia patients who were sex cattle and tiger. This led to the identification of a gene reversed to female (Foster et al. 1994). Experiments encoding an HMG box domain that was named SRY (sex-determining region of the Y) (Sinclair et al. 1990). Validation of SRY as the male sex-determining gene A came from several directions. The orthologous gene was cloned from the mouse genome and used with a 14 kb surrounding region to produce an XX transgenic mouse (Randy) that developed as a male (Koopman et al. 1991). Randy was sterile, but otherwise had all male physiological attributes as well as male mating behavior. B C In addition, mutations within SRY were identified in several XY female patients (Berta et al. 1990). Furthermore, an XY female mouse generated years earlier and predicted to have lost Tdy, was shown to be deleted for the entire Sry locus (Gubbay et al. 1992). The identification ofSry as the Y-linked gene that controls sex determination in mammals launched the molecular age of mammalian sex determination. SRY Figure 2 (A) In the adult testis, Sry is transcribed from a promoter was predicted to act at the top of a pathway to drive testis upstream in the 5′ inverted repeat (TSS). Multiple circular development. It was therefore an immediate priority to permutations of the Sry transcript were recovered from adult testis clone the transcript for Sry, understand its regulation libraries, leading to the hypothesis that the transcript was circular. (B) and begin to identify the downstream genes activated by The Sry transcript in the adult testis was predicted to undergo SRY. This turned out to take some time. circularization based on pairing between the long terminal repeats that bring a splice donor near a splice acceptor. (C) In the fetal testis, By RT-PCR analysis, Sry was found to be expressed in transcription initiates from a promoter internal to the 5′ repeat (GSS), the early mouse gonad, during the bipotential period (as creating a linear transcript. The conserved DNA-binding domain predicted), but at very low levels and for approximately (HMG box) is shown in diagonal stripes. https://rep.bioscientifica.com Reproduction (2019) 158 F101–F111

Downloaded from Bioscientifica.com at 09/24/2021 06:00:38AM via free access F104 B Capel showing that gain or loss of Sox9 had very similar effects Origin of gonadal supporting cells to gain or loss of Sry led to the idea that Sox9 might The gonad forms on the coelomic surface of the be the primary (and perhaps only) target of SRY (Vidal intermediate mesoderm, just above the mesonephric et al. 2001, Chaboissier et al. 2004). Much attention in ducts that are forming in the interior of the tissue (Karl the field turned to addressing the question of how Sox9 & Capel 1995). There are three major lineages in the is regulated. Twenty-five years later, this line of work early gonad, all of which are bipotential: supporting culminated in the discovery of an enhancer element cells (which can become Sertoli or granulosa cells), more than 500 kb upstream of Sox9 based on analysis of steroidogenic cells (which can become Leydig or theca the chromatin landscape (Maatouk et al. 2017, Garcia- cells) and germ cells (which migrate from the posterior of Moreno et al. 2019). Deletion/mutation of this enhancer the embryo and arrive coincident with gonad formation). leads to male-to-female sex reversal in mice and humans Labeling of single cells in the coelomic epithelium (CE) (Gonen et al. 2018). with a vital dye (Karl & Capel 1998), or lineage tracing of Several other seminal discoveries in human patients, dividing cells (Schmahl et al. 2000), showed that Sertoli goats and mice framed the field of ovary development. cells arise from this surface epithelium prior to embryonic FOXL2 was identified as the gene responsible for XX day (E)11.5. After this timepoint, other gonadal cell types blepharophimosis/ptosis/epicanthus inversus syndrome arise from the CE, but it is no longer competent to give (BPES), which is associated with premature ovarian rise to Sertoli cells. Cells in the CE report active Notch failure in humans (Crisponi et al. 2001, Schmidt et al. signaling, but as cells leave the CE, NUMB accumulates 2004). Loss of Foxl2 in goats led to female-to-male sex asymmetrically at their basal surface, and governs the reversal (Pailhoux et al. 2001). However, in mice, loss of competence to differentiate. In gonads lacking Numb Foxl2 led to arrested ovarian somatic cell differentiation, and Numbl (Numb-like), large groups of cells in both XX atresia and infertility, but did not lead to sex reversal and XY gonads fail to differentiate, reducing populations of females to male (Schmidt et al. 2004). However, of supporting and steroidogenic cells in both sexes (Lin Camerino and coworkers discovered another key gene et al. 2017). regulating ovary development in a consanguineous Patterns of proliferation differ between the developing family with XX males, RSPO1 (Parma et al. 2006). Loss testis and ovary. Whereas supporting cells in the ovary of Rspo1 in XX mice led to variable sex reversal with remain quiescent (until after birth) (Mork et al. 2012a), evidence of some well-formed testis structures, strong cells in the testis reenter the cell cycle and expand the SOX9 expression after birth and ambiguous genitalia population of Sertoli progenitors (Schmahl et al. 2000, (Chassot et al. 2008, Tomizuka et al. 2008). RSPO1 Schmahl & Capel 2003). Changes in proliferation of is a secreted activator of Wnt signaling, another gene Sertoli cell progenitors in the CE are contingent on the involved in ovary development in mice and humans expression of Fgf9 and Fgfr2 which are upregulated for (Vainio et al. 1999, Mandel et al. 2008, Biason- a brief period after Sry is expressed (Colvin et al. 2001, Lauber 2012). Schmahl et al. 2004). Loss of Fgf9 leads to disruption of testis development and sex reversal, even though expression of Sry and Sox9 initially occur normally (Kim The gonad as a model of organogenesis et al. 2006). This may be because the reduced Sertoli Apart from being an interesting model for identification population cannot stabilize testis development, but of the transcription factors that act in a cascade to other explanations are possible, including a failure to control sex determination, the gonad is an outstanding propagate the activation of SOX9 across the gonad field model of organogenesis. It is unique in that (unlike a (Hiramatsu et al. 2009). kidney or a lung) the gonad arises as a bipotential organ At least some of the somatic cells in the ovary also with the ability to develop as either a testis or an ovary. arise from the CE (Karl & Capel 1998, Mork et al. Development of the gonad hinges on whether or not 2012a). However, the window of competence to give Sry or Sox9 is expressed to trigger the testis pathway. In rise to granulosa cells extends over a longer period of the absence of either of those genes, and barring other development. DiI labeling of the CE in the XX gonad mutations, the gonad develops as an ovary. These two showed that granulosa cells (which express FOXL2) pathways diverge rapidly after Sry is expressed and show continue to arise until E14.5. FOXL2+ cells in the remarkably different morphogenesis strategies. It was interior of the gonad surround clusters of germ cells therefore very interesting to understand the cell biology of (germ cell cysts) and enter cell cycle arrest. They remain this branchpoint in gonad development. Developments in arrest until birth, when germ cell cysts break apart in confocal imaging made this a particularly good time and granulosa cells surround some individual oogonia, to tackle this problem. In addition to summarizing the reenter active cell cycle and proliferate to form the first early discoveries after Sry was identified, this review wave of growing follicles. When the fetal population of also focuses on my study of the gonad as a model of granulosa cells was lineage traced, they were shown to organogenesis (Box 1). contribute to growing follicles in the medulla, but not to

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Downloaded from Bioscientifica.com at 09/24/2021 06:00:38AM via free access To be or not to be a testis F105 the cortical population. Instead, between birth and P7, gonad. Experiments that block migration disrupted testis a new group of FOXL2+ follicle cells move in from the morphogenesis, suggesting that the vasculature plays a CE and surround individual oogonia in the cortex of the critical role in the structural reorganization of gonadal ovary. This population constitutes the primordial follicle cells into testis tubules. Further experiments showed that pool, the so-called ‘ovarian reserve’ (Mork et al. 2012a). endothelial cells act through Vegf and Pdgf signaling to Progress has been made on the breakdown of germ cell trigger the expansion of interstitial tissue around vessels, cysts, but the hand-off to the new population of granulosa which serves to sub-divide the gonad and reorganize cells arising from the CE is still not clear (Lei & Spradling domains into approximately 12 cord-forming units 2013, 2016). In many ways, this system is similar to (Cool et al. 2011). The vascular niche is also important Drosophila, where the follicle cells that surround germ for the regulation of the steroidogenic progenitors that cells as they first arise are called ‘escort cells’, whose job give rise to Leydig cells (Tang et al. 2008, Defalco et al. is to chaperone the germ cells and transfer them to the 2013). These progenitors arise both from the CE and definitive follicle cells (Sahai-Hernandez et al. 2012). from specialized cells along the gonad-mesonephros These findings required a reassessment of the standard border (Defalco et al. 2011, Kumar & DeFalco 2018). model of sex determination in which the Sertoli and Other signals downstream of Sry are important for granulosa cells of the adult testis and ovary directly stem inducing testis-specific cell types in the XY gonad. For from the supporting cell precursors of the bipotential example, both Dhh (Yao et al. 2002, Yao & Capel 2002) gonad. Although the CE of the gonad, which expresses and Pdgfra (Brennan et al. 2003) are required to induce LGR5 in fetal, neonatal and adult life, appears to be a Leydig cell development. In part, the requirement major, if not the only, source of both Sertoli cells and for Pdgfra may reflect the fact that fetal Leydig cell granulosa cells, fetal and adult granulosa cells are born progenitors are regulated by Notch signaling within a at different stages of development. Whereas the original vascular niche (Tang et al. 2008, Defalco et al. 2013). Sry-expressing cells and their progeny are believed to Yolk sac-derived macrophages also play a role in account for all of the Sertoli cells present in the adult the structural organization of the testis, by engulfing (Sekido et al. 2004), the number of FOXL2-positive Sertoli cells that are not enclosed in cords, eliminating cells increases in the absence of intrinsic proliferation wayward germ cells, and cleaning up other debris by recruitment from the CE after the bipotential period during morphogenesis of the testis (DeFalco et al. 2014). has concluded. The first cells to emerge from the CE Whether they also produce important cytokines is not (prior to E11.5) include stromal cells and the bipotential yet clear. supporting cell precursor population (Karl & Capel Recently, neuronal development was found to differ 1998), competent to differentiation as granulosa cells between testis and ovary development. While neurons or to activate the Sry promoter and differentiate as derived from the neural crest invade the ovary during Sertoli cells (Mork et al. 2012a). In some sense, the fetal the last quarter of fetal development, they are restricted granulosa cells act as ‘place keepers’ for the ovarian to the tunica albuginea of the testis, likely by repulsive pathway until the definitive adult population arises. cues downstream of Sry (McKey et al. 2019). Neither the function of neurons in the ovary, nor the reason they are absent from the testis is yet understood. Whether Recruitment of other cell types in the gonad recruitment of theca cell progenitors from the adjacent Cell types other than supporting cells are present in mesonephros (Liu et al. 2016) is related to recruitment of the early gonad. Some of the cells that make up the neurons in the ovary is not yet been determined. interstitial or stromal populations can also be lineage traced to the CE, but others are recruited from sources Cell fate determination extrinsic to the gonad. Previous work from Anne McLaren’s lab suggested that cells from the adjacent The gonad is also an outstanding model of cell fate tissue, the mesonephros, migrated into the mouse gonad determination. Gonadal sex determination can be (Buehr et al. 1993), but methods at the time did not lend reduced to a question of whether the bipotential themselves to a definitive analysis. The development supporting cells that enter the gonad from the CE of the ROSA-βgal (Soriano 1999), and subsequently, initiate differentiation as Sertoli or granulosa cells. the ROSA-GFP (Giel-Moloney et al. 2007) mouse lines Transcriptome analysis of early gonadal populations facilitated tissue recombination experiments to measure (Munger et al. 2009, Jameson et al. 2012b) and more cell migration between tissues. These experiments recently from single cell analysis (Stevant et al. 2019) showed that vascular endothelial cells migrate into the indicates that the cells in XX and XY bipotential gonads XY but not the XX gonad. Migration was contingent on are initially nearly identical. The only differences at expression of Sry (Martineau et al. 1997, Capel et al. early stages arise from genes that are specific to the sex 1999, Tilmann & Capel 1999, Coveney et al. 2008), and chromosomes such as Xist, Utx and Eif2s3x (only present cells could be induced to migrate into the XX gonad when in XX) and Ddx3y, Eif2s3y and Jarid1d (only present in it was sandwiched between the mesonephros and an XY XY) (Munger et al. 2009). https://rep.bioscientifica.com Reproduction (2019) 158 F101–F111

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Downstream of Sry, the fate of XY gonadal cells depends on the expression of Sox9 and the ability to repress the Wnt/β-catenin pathway that drives the ovary fate (Fig. 3) (Kim et al. 2006, Kim & Capel 2006, Lavery et al. 2012, Nicol & Yao 2015). Wnt4 and Rspo1 may be involved in the establishment of cells in the gonad field. Together, these genes regulate proliferation of CE precursors that give rise to Sertoli cells (Chassot et al. 2012), and loss of Wnt4 was previously shown to affect the SOX9 population (Jeays-Ward et al. 2004). However, once this population is established, repression of Wnt4 signaling is required to stabilize testis fate (Maatouk et al. 2008). Loss of Fgf9 leads to the reversion to ovary fate after SOX9 expression is established, but if Wnt4 is also lost, SOX9 expression and the testis pathway are rescued (Jameson et al. 2012a). These results strongly suggest that there is a second stabilization step controlling testis fate Figure 3 Opposing signals control the fate of the gonad. When SRY governed by repression of Wnt4 signaling. This occurs triggers SOX9 upregulation, FGF9 is expressed and represses Wnt4 and the female pathway. In the absence of SOX9 upregulation, at multiple levels including through the Wnt antagonist, ovarian development ensues, based on Wnt and downstream ZNRF3 (Harris et al. 2018). signaling.

2019), combined with histone data (Garcia-Moreno Epigenetic regulation et al. submitted) and ChIP-seq approaches for important Recent analysis of histone methylation patterns confirm transcription factors, may reveal how male or female that genes associated with both testis and ovary fate is stabilized by repression of the alternative fate. pathways in supporting cells are bivalent in the E10.5 Questions remain about how the male or female gonad, marked with both H3K27me3 (repressing) pathway is activated in mammals. Interestingly, and H3K4me3 (activating) histones marks (Garcia- experiments in the red-eared slider turtle, T. scripta, Moreno et al. 2019). These genes are initially expressed may provide some insight. T. scripta has a temperature- at similarly low levels in XX and XY gonads. At E13.5, dependent sex-determining system, in which the after sex determination has occurred, genes associated temperature of incubation of the egg controls whether with the testis pathway in XY gonads are stripped of the gonad differentiates as a testis or ovary. Evidence their H3K27me3 repressive marks, but genes associated from a transcriptome time course of gonadal expression with the ovary pathway retain their bivalent status. at male- and female-producing temperatures revealed Symmetrical changes occur in the XX gonad, where that Kdm6b is male specific at the earliest stages genes associated with the ovary pathway are stripped of of turtle gonad formation (Czerwinski et al. 2016). their H3K27me3-repressive marks, but genes associated KDM6B is a histone demethylase that specifically with the testis pathway retain their bivalent status. These removes H3K27me3-repressive marks from target findings may explain the ability of Sertoli and granulosa genes. Depletion of Kdm6b in the turtle, using a cells to reverse fate under some circumstances in adult virally transduced shRNA, led to female development life (Uhlenhaut et al. 2009, Matson et al. 2011). at the male-producing temperature (Ge et al. 2018). Several lines of evidence suggest that the polycomb Furthermore, KDM6B was shown to bind the promoter repressive complex 1 (PRC1) is required for testis of Dmrt1, a gene that was previously shown to drive development. Loss of Cbx2, the component of the male sex determination in T. scripta (Ge et al. 2017) PRC1 complex that recognizes the H3K27me3 mark, (and many other species of fish, reptiles, amphibians led to ovary development in XY mice and humans and birds). Loss of Kdm6b was associated with a failure (Katoh-Fukui et al. 1998, Biason-Lauber et al. 2009, to remove H3K27me3 marks from the Dmrt1 locus, Katoh-Fukui et al. 2012). Although this was originally and a failure to activate the gene at the male-producing believed to be due to a failure of Sry expression, it is temperature (Ge et al. 2018). These findings suggest that more likely due to a failure to repress ovary fate and removal of repressive histone marks may be the key to stabilize SOX9 expression. Interestingly, Lef1, which activation of the male pathway and point toward the encodes a protein downstream of Wnt signaling, is exploration of the orthologous H3K27me3 enzymes a direct target of CBX2, as is another key gene in the in mammals. ovary pathway, Foxl2 (Garcia-Moreno et al. 2019). The These experiments have taught us a lot about how identification of regulatory elements across the genome cell fate is established in the gonad (which controls in XX and XY supporting cells using DHS and ATAC- the sexual development of the organism), and the seq methods (Maatouk et al. 2017, Garcia-Moreno et al. findings are likely to be widely applicable to other less

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Downloaded from Bioscientifica.com at 09/24/2021 06:00:38AM via free access To be or not to be a testis F107 accessible and less dramatic cell fate decisions. Many important questions remain. It is known from a group of genetic and gain-of-function experiments that there is a narrow window in development when Sertoli fate can be initiated in gonadal supporting cells (Hiramatsu et al. 2009). If Sry is expressed too late, or at a reduced level, Sertoli cell commitment fails. The molecular explanation for this narrow window of competence to initiate the testis pathway has not been discovered. However, one possibility is that the ovary pathway is on a steady upward trajectory that must be intersected before a female factor accumulates to an insurmountable level (Fig. 4). Antagonism could play out as a competition between the relative levels of a testis and an ovary protein. Even if this is the case, it is unclear which factors are involved in this competition. Another remaining puzzle is the explanation for why in many mutants that impair the male pathway, the central region of the XY gonad shows a stronger tendency to stabilize as testicular while the peripheral regions develop ovarian tissue (Eicher et al. 1982). While this was originally believed to be the result of earlier expression of Sry in the center of the gonad (Bullejos & Koopman 2001), this pattern does not hold up to close scrutiny, thus is unlikely to be the explanation Figure 4 Model to explain the narrow developmental window when Sertoli fate can be initiated. The bipotential gonad is initially (Bunce et al. unpublished). advancing on an ovarian trajectory (solid red line) based on the accumulation of a female regulator. If the male regulator is expressed at the right stage and level (solid blue line), it can intersect the Germ cell development in the fetal gonad trajectory of the female regulator and divert the gonad to testis fate. Germ cells are another major constituent of the fetal However, if the ovarian factor is advanced (broken red line), or the male regulator is delayed (broken blue line), the initiation of Sertoli gonad. They arise at the base of the allantois at ~E6.5 fate fails. and migrate through the gut to the site where the gonad is forming, arriving ~E10.5. Initially, gonadal germ cells in response to RA produced in the mesonephros (Bowles are found in clusters formed by aggregation (based et al. 2006, Koubova et al. 2006). They initiate meiosis on E-cadherin) as well as clonal divisions (Mork et al. in a wave that proceeds from anterior to posterior (Yao 2012b). Although germ cells are not required for the et al. 2003). Although retinoic acid is produced in the structural development of the testis, their absence may mesonephroi of both the ovary and the testis, its meiosis- delay testis cord formation (Merchant 1975, McLaren inducing effect is blocked in the testis by expression of 1985). After birth, germ cells are required in the ovary the RA catabolic enzyme, CYP26B1 (Bowles et al. 2006, for follicle formation and maintenance. In the absence of Koubova et al. 2006), and perhaps other factors produced germ cells, or when they are lost in adult life, the ovary by Sertoli cells. Instead of entering meiosis, male germ undergoes degeneration (Guigon et al. 2005, Guigon cells undergo a period of mitotic arrest extending & Magre 2006). However, fetal ovary development in from ~E15.5 until the end of fetal life (McLaren 1984). the absence of germ cells proceeds normally until birth Experiments suggest that meiotic germ cells antagonize (Maatouk et al. 2012). testis cord formation (Yao et al. 2003). Upon arrival in the gonad, germ cells proliferate Fgf9 is required for germ cell survival and development rapidly in both XX and XY gonads, but by E12.5, when the in the testis but not the ovary environment (DiNapoli somatic cells of the gonad initiate sex-specific behavior, et al. 2006). In Fgf9 mutants, germ cell numbers were the fate of germ cells diverges between the testis and significantly reduced in the testis and could not be ovary (Schmahl et al. 2000). Experiments indicate that rescued unless exogenous FGF9 was added by E11.5, the chromosome constitution of germ cells (XX or XY) suggesting that transition to dependence on FGF9 can be dominated by their gonadal environment: XX occurs between E10.5 and E11.5. It is still unclear what germ cells that arrive in a testis environment enter a entrains germ cells to the presence of FGF9 in the testis. male differentiation pathway, whereas XY germ cells Both suppression of meiosis, and expression of Nanos2, that arrive in an ovary environment enter a female an RNA-binding protein required for male germ pathway (Adams & McLaren 2002). In the ovary, germ cell development, are downstream of FGF9 (Bowles cells upregulate Stra8 (stimulated by retinoic acid (RA)) et al. 2010). https://rep.bioscientifica.com Reproduction (2019) 158 F101–F111

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A large number of RNA-binding proteins are required severely deplete the Sertoli cell population in the testis. for male germ cell development. One of these is Four days after treatment, Sertoli cells were depleted, but DND1. In 2003, the classic Ter mutation was mapped the basal lamina of testis cords and other cell types in the to Dnd1, which was shown to be expressed in germ testis were intact. This created a scaffold for engraftment cells (Youngren et al. 2005). The Ter mutation in Dnd1 of a new population of Sertoli cells that rescued (Dnd1Ter/Ter) leads to severe germ cell loss in both sexes, spermatogenesis from remaining host spermatogonial owing to BAX-mediated cell death pathways (Cook et al. stem cells. This approach might be used to rescue 2009). However, on some genetic backgrounds, germ infertility by the replacement of a defective Sertoli cell cells in male Dnd1Ter/Ter mutants fail to undergo mitotic population in a host. Alternatively, a delay of 7 days arrest and give rise to a very high incidence of testicular between treatment with the drug and injection of donor teratomas that arise between E16.5 and birth (Cook et al. cells from a neonatal mouse, led to the engraftment by 2011). The higher incidence of teratomas in the left testis many cell types in the testis, including spermatogonial compared to the right is correlated with differences in stem cells, peritubular myoid cells and Leydig cells vascular architecture, oxygen availability and metabolic (Yokonishi et al. submitted). Using this method, it might profile Bustamante-Marin( et al. 2015). Transcriptional be possible to establish xenogeneic spermatogenesis profiling comparing WT and Dnd1Ter/Ter germ cells prior in the mouse testis by matching the species origin of to the formation of teratomas, as well as DND1-RNA- somatic and germ cells. Whether a testis depleted for immunoprecipitation experiments, showed that DND1 Sertoli cells has the capacity for repair when donor cells regulates genes associated with pluripotency, the cell are not provided is currently under investigation. cycle and chromatin regulation (Ruthig et al. 2019). It would be especially rewarding if the many years of These findings have led to the idea that reprogramming basic science research in the mouse and turtle one day of pluripotency in germ cells and upregulation of genes paid off in the clinic. essential for spermatogonial stem cell differentiation require cell cycle arrest. Experiments are in progress to investigate the mechanisms involved in this transition Declaration of interest from fetal gonocyte to spermatogonial stem cell, The author declares that there is no conflict of interest that could responsible for the lifetime fertility of the male. be perceived as prejudicing the impartiality of this review.

A new venture: can the adult testis and ovary Funding be rescued? Ongoing funding from NICHD, NIGMS, NSF-IOS, and the Infertility is often the outcome of chemotherapy, which Duke School of Medicine has supported my work reported in is frequently used for treatment of cancers and immune this review. disorders. Can a deeper understanding of the origin of cell types and mechanisms of organogenesis during fetal life help us to devise a scheme to rescue the adult testis Acknowledgements and ovary after damage? This question led us to establish Thanks to members of the lab over the years for the work models of ovarian and testicular damage in the hope of described here; to current members of the lab for their devising a means of rescue for one or both organs. comments on the manuscript. In female FVB mice, three consecutive IP injections with a cytotoxic cocktail of busulfan and cyclophosphamide led to complete infertility (Batchvarov References et al. 2016). However, when an isogenic ovary fragment Adams IR & McLaren A 2002 Sexually dimorphic development of mouse from a healthy female homozygous for a GFP transgene primordial germ cells: switching from oogenesis to spermatogenesis. Development 129 1155–1164. was grafted to the left ovary of CTx-treated hosts, follicle Batchvarov IS, Taylor RW, Bustamante-Marin X, Czerwinski M, Johnson ES, development in the left host ovary was rescued. In Kornbluth S & Capel B 2016 A grafted ovarian fragment rescues host contrast, the ungrafted right ovary underwent complete fertility after chemotherapy. Molecular Human Reproduction 22 842– degeneration. Some host (non-GFP) pups were born 851. (https://doi.org/10.1093/molehr/gaw064) Bennett D, Mathieson BJ, Scheid M, Yanagisawa K, Boyse EA, Wachtel S as late as the sixth litter after grafting, suggesting long- & Cattanach BM 1977 Serological evidence for H-Y antigen in Sxr, term rescue of host fertility. Investigation of the ovary XX sex-reversal phenotypic males. Nature 265 255–257. (https://doi. during and soon after the CTx treatments indicates that org/10.1038/265255a0) Berta P, Hawkins JR, Sinclair AH, Taylor A, Griffiths BL, Goodfellow PN granulosa cells in growing follicles are the primary & Fellous M 1990 Genetic evidence equating SRY and the male sex target of the cytotoxic drugs. Experiments are ongoing determining gene. Nature 348 248–251. to determine the mechanism through which follicles are Biason-Lauber A 2012 WNT4, RSPO1, and FOXL2 in sex development. rescued by the graft. Seminars in Reproductive Medicine 30 387–395. (https://doi. org/10.1055/s-0032-1324722) While (unsuccessfully) trying to block neuronal Biason-Lauber A, Konrad D, Meyer M, DeBeaufort C & Schoenle EJ 2009 development in the ovary, we discovered a drug that can Ovaries and female phenotype in a girl with 46,XY karyotype and

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