158 3 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 of Sry 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.