The theca cell layers of the ovarian follicle have an essential role in follicle development and ovulation. Current evidence indicates that the theca cell progenitors (TCPs) are present in the ovarian stroma. During folliculogenesis, the mature theca cells are first detected in secondary follicles and form the outermost layers of the follicle. However, the mechanisms governing the transition from TCPs to differentiated theca cells have yet to be fully described. Herein, we used a multi-omic approach integrating single-cell RNAseq (scRNAseq) and spatial transcriptomics to describe the TCP-to-theca cell transition.

Prepubertal (two-week old) mouse ovaries were collected for scRNAseq (n = 3) or spatial transcriptomics (n = 4). Ovaries for scRNAseq were dissociated, and samples with high cell viability (75±2%) were processed using the Chromium Fixed RNA Kit, Mouse Transcriptome (10X Genomics), targeting 10,000 cells per reaction. Libraries were sequenced at a minimum coverage of ~60,000 reads per cell. For spatial transcriptomics, RNA quality was assessed and tissue blocks with RIN >7 were systematically sectioned and ovarian morphology evaluated. Selected sections were mounted onto the Stereo-seq Chip T Slide (STOmics). Slides (n = 2) were imaged by epifluorescent microscopy, and resultant libraries were sequenced on a DNBSEQ-G400 sequencer (MGI).

Differential gene expression and pseudotime trajectory analysis of the scRNAseq data identified two putative TCP clusters within the ovarian stroma subset. Mapping of the respective gene markers onto spatial transcriptomic datasets demonstrated localisation patterns consistent with TCP identity and indicated potential intermediate stages during differentiation to the mature theca cell layers. Our ongoing in vitro studies aim to validate TCP identity and function. These findings demonstrate both the utility of integrating sc- and spatial transcriptomic approaches and provide key datasets for understanding TCPs and the mechanisms underlying theca cell dysfunction.