It Takes a Cyst: Visualizing the Molecular Dynamics Underlying Mammalian Oocyte Specification and Enrichment During Germline Cyst Breakdown

Abstract

The formation of gametes is a critical event in sexually reproducing organisms, yet little is known about the mechanisms by which mammalian oocytes–arising from structures known as germline cysts–become specified and enriched in size and organelle content. In Drosophila melanogaster, germline cysts, composed of germ cells connected by intercellular bridges, enable oocyte specification through asymmetric distribution of cellular components and promote oocyte enrichment via cytoplasmic transfer through these bridges. Given the germline cyst architecture is conserved in mammals, similar specification and enrichment mechanisms have long been assumed, though direct evidence remains lacking. To investigate this, I constructed a photoconvertible fluorescent reporter to visualize cytoplasmic transfer within germline cysts and developed endogenous photoconvertible reporters for oocyte specification factors to characterize their dynamics during cyst breakdown. I found that GDF9 and FIGLA–key oocyte specification factors–are both expressed during cyst breakdown, with expression increasing during follicle formation and by E18.5, respectively. Additionally, GDF9-Dendra2 expression differed significantly between connected germ cell pairs, suggesting GDF9-Dendra2 heterogeneity within germline cysts may contribute to or result from oocyte specification. Successful photoconversion of FIGLA-Dendra2 was achieved, enabling future spatiotemporal tracking of FIGLA-Dendra2 transfer among germline cysts. The novel imaging tools developed in this study will facilitate the reconstruction of a model of oocyte specification and cytoplasmic transfer in germline cysts, illuminating how mammalian oocytes arise. This knowledge will be critical for advancing infertility treatments and driving innovation in assisted reproductive technologies.