mRNA isoform switching plays a crucial role in mural cumulus differentiation

Abstract

During gonadotropin-induced ovarian follicle development, an antral cavity forms within the layers of granulosa cells (GCs). Gonadotropin stimulation also differentiates the GCs into two distinct lineages: mural GCs (mGCs), which surround the antral cavity, and cumulus GCs (cGCs), which stay in contact with the oocyte. We examined the transcriptomes of mouse mural and cumulus cells to understand the mechanism of differentiation. In addition to analyzing a single transcript expression per gene, we also considered multiple isoform expressions to explore differential transcriptomics. GC-specific core transcription factors Foxl2, Nr5a1, Nr5a2, Runx1, and Runx2 were expressed at high levels in mGCs but downregulated in cGCs, indicating that cGCs acquire a more differentiated state. Both single-transcript and multiple-isoform analyses revealed differential expression of about 70% of transcripts between mGCs and cGCs. Although the counts were similar, the differentially expressed genes (DEGs) at the single transcript level did not correlate well with the respective differentially expressed transcript isoforms (DETI). We identified DETIs originating from key epigenetic and transcriptional regulator genes, such as Chd1, Ezh2, Kdm5a, Kdm5b, Gata4, Esr2, Fos, Myc, and Ybx1, that were not differentially expressed at the single-transcript analysis. Further analysis revealed a transcript switch in one-third of the DETIs. Most of the transcript isoforms were protein-coding, followed by non-coding regulatory RNAs. A total of 1,302 transcript isoforms were silenced in cGCs, including those of Adar, Cebpa, Dnmt3a, Foxo4, Pgr, Rest, Runx1, Satb2, Sirt1, Sirt2, and Tead1. Conversely, 529 transcript isoforms were activated in cGCs, including transcripts for Brd7, Crem, Chd1, Med21, Med27, Nfkbia, Rbm39, Rbmx, Suv39h2, Tcf12, Xist, and Ybx3. Additionally, 57 genes exhibited DETIs, with at least one isoform turned off and another turned on in cGCs, including Csde1, Dab2, Ezh2, Gata4, Gnas, Gtf2i, Macf1, Klf10, Setdb1, and Sp3. Finally, we explored the mechanisms underlying transcript switching during the differentiation of mGCs and cGCs. Our findings suggest that gonadotropin-induced transcript switching in GCs is crucial for mural and cumulus granulosa differentiation, a key insight that would be missed without mRNA isoform analysis.