Abstract
Within developing ovarian follicles, oocyte maturation requires coordinated interplay between multiple specialized cells: the oocyte, granulosa cells (GCs), and enveloping stromal cells (mainly the theca externa and interna that provide essential structural and steroidogenic support, respectively). Although numerous studies have focused on explaining the interactions that occur within the oocyte and its surrounding GCs throughout maturation, the mechanisms governing theca interna (TI) specification remain elusive. In this study, we aim to identify the molecular pathways that drive the differentiation of stromal cells into TI, and to characterize and localize intermediate cell types that exist along this trajectory. Stromal (CD55+) and TI (ANPEP+) cells were prospectively sorted from different size antral follicles (n=4, 2-4mm) derived either from fresh ovarian tissue or human ovarian xenografts. Samples were submitted for single-cell RNA sequencing (scRNASeq) and analyzed using the Seurat package in R. Immunolabeling studies were done on cryosections of fresh ovarian tissue. Computational analyses allowed us to delineate a gradual progression from an initial stromal progenitor identity toward a steroidogenic TI cell fate. We identified the transcription factor NR5A1/SF1 as a potential driver of commitment toward steroidogenic cell fate, as its graduated increase correlates with upregulated transcription of steroidogenic genes canonically associated with TI Identity, such as LHCGR, STAR, INSL3, and CYP17A1. This trajectory is also supported by the expression of effectors of Hedgehog (Hh) signaling, which animal studies have identified as an essential mediator of theca differentiation. To attribute temporal information to the inferred differentiation trajectory of TI cells, we used immunolabeling on cryosections of developing follicles and observed TI precursors in the stroma adjacent to transitioning primordial follicles, while definitive TI cells first emerged next to the basement membrane of secondary follicles. At the antral stage, these populations display a heterogeneous distribution within the theca layer, suggesting crosstalk as the follicle develops. Using scRNASeq of stromal populations adjacent to antral follicles, we were able to construct a high-resolution trajectory of differentiation, starting from stromal progenitor cells to steroidogenic TI cells. In part, this trajectory seems to be driven by the transcription factor SF1 and the Hh signaling pathway. We were able to localize discrete populations described along this trajectory to different stages of follicle development, suggesting that TI differentiation is dynamic and intricately related to follicle development.
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