Transcriptome Signature of Immature and In Vitro-Matured Equine Cumulus-Oocytes Complex

Abstract

Maturation is a critical step in the development of an oocyte, and it is during this time that the oocyte advances to metaphase II (MII) of the meiotic cycle and acquires developmental competence to be fertilized and become an embryo. However, in vitro maturation (IVM) remains one of the limiting steps in the in vitro production of embryos (IVP), with a variable percentage of oocytes reaching the MII stage and unpredictable levels of developmental competence. Understanding the dynamics of oocyte maturation is essential for the optimization of IVM culture conditions and subsequent IVP outcomes. Thus, the aim of this study was to elucidate the transcriptome dynamics of oocyte maturation by comparing transcriptomic changes during in vitro maturation in both oocytes and their surrounding cumulus cells. Cumulus-oocyte complexes were obtained from antral follicles and divided into two groups: immature and in vitro-matured (MII). RNA was extracted separately from oocytes (OC) and cumulus cells (CC), followed by library preparation and RNA sequencing. A total of 13,918 gene transcripts were identified in OC, with 538 differentially expressed genes (DEG) between immature OC and in vitro-matured OC. In CC, 13,104 genes were expressed with 871 DEG. Gene ontology (GO) analysis showed an association between the DEGs and pathways relating to nuclear maturation in OC and GTPase activity, extracellular matrix organization, and collagen trimers in CC. Additionally, the follicle-stimulating hormone receptor gene (FSHR) and luteinizing hormone/choriogonadotropin receptor gene (LHCGR) showed differential expressions between CC-MII and immature CC samples. Overall, these results serve as a foundation to further investigate the biological pathways relevant to oocyte maturation in horses and pave the road to improve the IVP outcomes and the overall clinical management of equine assisted reproductive technologies (ART).

Keywords: cumulus cell; equine; in vitro embryo production; in vitro oocyte maturation; oocyte; transcriptome.

Figure 1

Gene expression patterns of mature and immature oocytes and cumulus cells. Heat map graphs comparing the immature group to the in vitro-matured group (MII) in OC (A) and CC (B) samples. Samples are in columns, and the 1500 most variable genes are in rows. Expression is indicated by the z-score in a colorimetric scale (blue: −4 to red +4). The principal component analysis (PCA) plots of OC samples (C) (immature samples E, F, G, H; MII samples A, B, C, D) and CC samples (D) (immature samples M, N, O); MII samples I, J, K) show the samples distributed in the graph based on their gene expression similarities. Samples appear to show separation between PC1 and PC2 by maturity status in both types of samples.

Figure 2

Differentially expressed genes (DEG) represented in a volcano plot for oocyte samples (A) and CC samples (B) denoting the downregulated genes (blue) on the left compared to upregulated genes (red) on the right using a fold change cutoff of 1.5. The dotted green line indicates the FDR cutoff of 0.1. GO analysis for enriched pathways of genes highly expressed in OC-MII (C) and lowly expressed in OC-MII (E) relative to immature OC, and those highly expressed within in vitro matured CC (D) and those lowly expressed within in vitro CC (F) relative to immature CC.

Figure 3

Ligand–receptor pairs between oocytes and cumulus cells. Schematic representation of the suggested crosstalk between oocyte and cumulus cells based on DEG between immature and in vitro-matured samples. Genes are colored based on their expression: red represents high expression, and blue represents a low expression of genes in MII samples relative to immature samples. Genes in black correspond to those expressed but not differentially expressed. Illustration made in BioRender.com.