Mechanisms of female germline cyst formation and oocyte selection in mice

Mechanisms of female germline cyst formation and oocyte selection in mice Abstract Oocytes are generated within a cluster of interconnected cells termed the germline cyst, which arise in the ovary as germ cells undergo divisions with incomplete cytokinesis. The germline cyst is a widely conserved structure from insects to mammals, necessary for the oocyte to gain cytoplasmic content, including mitochondria, from sister cells. Cytoplasmic enrichment of the oocyte will in turn be needed for this specialized cell to support the earliest stages of subsequent embryonic development. These processes are best understood from studies in Drosophila melanogaster, which have guided inquiry into cyst formation and selection of the oocyte by cytoplasmic enrichment in mammalian systems, revealing several conserved features of the oocyte program. However, striking differences were also found. Most notably, in contrast to the invariant cyst topology of the fly, germline cysts are highly variable in mammals, both in terms of size and structure. Moreover, as enrichment of the fly oocyte is tied to the stereotypic formation of the cyst, these rules cannot be readily applied to mammalian oocyte selection. As previous studies exclusively relied on the analysis of fixed samples to reconstruct events of cyst formation and oocyte selection in mice, they have offered limited insight into the mechanisms underlying these dynamic and variable processes. Here we develop novel genetically encoded fluorescent reporter mouse lines for key cellular and subcellular features of germline cysts and use our recently established live imaging setup that allows us to directly visualize the dynamics of cyst formation and oocyte selection within the embryonic ovary, with unprecedented spatial and temporal resolution. Using these powerful tools, we will first investigate the mechanisms responsible for generating cysts with variable sizes (Aim 1). We recently uncovered that germ cells are unexpectedly motile, even as they divide to form cysts, and showed that motility can lead to cyst fracture and thus regulates cyst size. We will investigate the mechanisms that promote, as well as eventually down- regulate cell motility, resulting in the stabilization of cysts. Next, we will determine how variable cyst connectivity patterns arise (Aim 2) by visualizing cell division orientation in relation to intercellular connections and probe the mechanisms that regulate mitotic spindle orientation and intercellular connection positioning. We will then directly assay the consequence of these variable cyst features for establishing oocytes (Aim 3). Finally, using a mouse line with a photoconvertible mitochondria reporter, we will investigate organelle transfer between cysts cells and determine how variable cyst topologies influence oocyte selection (Aim 4). This work will reveal fundamental insights into the unique mechanisms of mammalian oocyte formation. Project Number: 1R01HD116856-01A1 | Fiscal Year: 2025 | NIH Institute/Center: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) | Principal Investigator: Eszter Posfai | Institution: PRINCETON UNIVERSITY, Princeton, NJ | Award Amount: $458,236 | Activity Code: R01 | Study Section: Cellular, Molecular and Integrative Reproduction Study Section[CMIR] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01HD11685601A1