Gonadal development is a complex process that involves sex determination followed by divergent maturation into ovaries or testes. Historically, limited tissue accessibility and lack of reliable in vitro models have impeded our understanding of human gonadogenesis, despite its importance in gonadal pathologies and infertility. Here, we generated a comprehensive map of first- and second-trimester gonadal development using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and imaging. Using this approach, we identified novel transcription factors and cell states in human germ and supporting cell lineages. We compared them with other mammalian species and found primate-specific regulatory programmes. Our data identified cell context–specific interactions shaping sex specification and development of human germ cells. We defined a novel bipotent progenitor cell (LGR5+, TSPAN8+) in late embryos that can differentiate into early Sertoli in males or pre-granulosa cells in females. In fetal ovaries, we defined two subsets of pre-granulosa cells supporting germ-cell differentiation and distributed across the cortico-medullary axis. We also found a subset of developing granulosa cells appearing during the second trimester of pregnancy that is involved in follicular assembly. In fetal testes, we defined a novel supporting population (sPAX8 cells) located at the poles of the developing testis cords. We also found two tissue-resident myeloid populations that we named microglia-like and SIGLEC15+ fetal testicular macrophages. This study provides an unprecedented spatiotemporal map of human gonadal differentiation that can be utilised as a blueprint for in vitro gametogenesis.

Roser Vento-Tormo, Luz Garcia-Alonso, Valentina Lorenzi, Cecilia Mazzeo, Carmen Sancho-Serra, Kenny Roberts, Justin Engelbert, João Alves-Lopes, Magda Marečková, Rachel Botting, Tong Li, Berta Crespo, Stijn van Dongen, Vladimir Kiselev, Elena Prigmore, Ashley Moffett, Mary Herbert, Omer Ali Bayraktar, Azim Surani, Muzlifah Haniffa
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