Gene expression by equine oocytes and cumulus cells, a window into the unknown

Abstract

In vitro maturation (IVM) of oocytes (OC) is a critical step for the in vitro production of equine embryos. However, the regulatory mechanisms, biological processes, cellular components, and molecular functions involved in IVM are not entirely understood. We previously analyzed the gene expression dynamics of gonadotropin receptors in cumulus cells (CC) during IVM, demonstrating an upregulation of the follicle-stimulating hormone receptor gene (FSHR) and downregulation of the luteinizing hormone choriogonadotropin receptor gene (LHCGR) in mature compared to immature CC (de la Fuente.et.al. Journal of Equine Veterinary Science 2022; 113). Moreover, we found that CC from in vivo maturing cumulus-oocyte complexes (COC) obtained from dominant gonadotropin-stimulated follicles (DF-CC) had a lower expression of FSHR and a higher expression of LHCGR compared to their in vitro counterparts. However, the effect of IVM medium composition on the expression of gonadotropin receptors has not been investigated. Thus, the aim of this study was to determine differences in the gene expression of OC and CC following IVM of three reported IVM media: A) EQ-IVM (IVF-Bioscience) a commercially available medium, B) DMEM/F12-based medium (Meyers.et.al. Reproduction, Fertility and Development. 2019; 31:1874-1884.) and C) M199-based medium (Choi.et.al. Theriogenology. 2002; 58:771-774) and their difference with DF-CC samples. Extracted total RNA from OC and CC was sequenced and raw reads were trimmed (trimmgalore) and mapped (STAR) to the current equine referencegenome. Mapped reads were quantified (featurecount) and compared between groups, using the DESeq2 package (FDR cutoff 0.1; Min (log2) fold change 1.5). Overall, there was a mapping rate of >85%, and an average of 14,000 expressed genes in OC and CC samples with ∼68% categorized as protein-coding genes. Based on gene expression dis-similarities, OC and CC samples were partially clustered according to their respective maturation media, but all samples showed a different gene expression profile compared to DF-CC samples. Comparing the gene expression of CC between the A and B media, we found 607 differentially expressed genes (DEGs), 1905 DEGs between A and C, and 957 DEGs between B and C. All three media showed a similar DEGs number when compared to DF-CC samples (∼10K DEGs). FSHR showed a higher expression in all IVM samples compared to DF-CC. However, LHCGR presented a lower expression only in two of the IVM media used (B & C) compared to DF-CC, showing no significant expression difference in the commercial IVM media tested. These data highlight the dynamics of gonadotropin receptors during IVM, which may contribute to the understanding of the physiology of oocyte maturation and allow optimization of IVM conditions in horses. Funding: The Theriogenology Foundation, the Population Health and Reproduction Department Seed Grant, and the Center for Equine Health.