Potential effect of human chorionic gonadotropin‐induced extracellular vesicle RNAs on mouse ovarian granulosa cells

Gonadotropins regulate granulosa-cell function during ovarian follicular development, and extracellular vesicles (EVs) are potential mediators of intercellular communication within the follicle. However, the regulation of EV RNA cargo following gonadotropin stimulation remains incompletely understood. Here, we investigated human chorionic gonadotropin (hCG)-responsive EV RNA profiles using three experimental models: two Lhcgr-expressing clones of a mouse granulosa-cell tumor-derived cell line and mouse ovaries. The cell models were treated with hCG, and immature mice were primed with serotropin, followed by hCG. EVs were isolated from these cells and ovaries and characterized by transmission electron microscopy and western blot analysis. Total RNAs from these EVs were analyzed by electrophoresis, suggesting global changes in their profiles with hCG. RNA-sequencing identified thousands of potentially differentially expressed genes, including 111 commonly upregulated and 122 commonly downregulated transcripts among all three experimental models. The 111 upregulated transcripts were further narrowed down to 27 candidates based on the abundance, and five protein-coding transcripts (Ccdc200, Gm10337, Gm614, Adad2, and Spata45) were selected for further functional validation. Interestingly, EVs carrying the overexpressed Ccdc200, Gm10337, and Gm614 mRNA significantly increased the number of ovarian cells during 3-day culture. These findings indicate that hCG modulates EV RNA cargo and suggest that EV-mediated RNAs cooperatively function as a regulatory layer of hCG signaling for intercellular communication, fine-tuning granulosa-cell proliferation or survival during follicular development.