PSII-13 Effect of Clomiphene citrate on the transcriptome of porcine oocytes in vitro.

Abstract In vitro reproductive technologies offer significant potential to accelerate genetic improvement and enhance reproductive efficiency in livestock systems. By reducing generation intervals and increasing selection intensity, these technologies can accelerate genetic improvement. However, current in vitro maturation (IVM) and embryo development rates remain suboptimal, limiting the overall effectiveness of these technologies. One contributor to inefficiencies remains suboptimal oocyte maturation, in vitro, in response to precocious meiotic resumption when oocytes are removed from the follicle and placed in culture. Therefore, our objective was to investigate the effects of Clomiphene citrate (CC), a selective estrogen receptor modulator (SERM), on porcine oocyte maturation and gene expression. Traditionally used in oral form to induce ovulation in anovulatory women, an aqueous formulation of CC was used in this study to investigate the effects on porcine oocyte in vitro maturation and transcriptional activity. To address this, cumulus-oocyte complexes (COCs) were treated for 22 hours in vitro with 10-6M CC, followed by 22 hours in a medium without CC. For the control (untreated) samples, COCs were cultured in the maturation medium without CC for 44 hours. Following 44 hours of maturation, total RNA was extracted from both treated and untreated oocytes (groups of 25 oocytes, n = 6 biological replicates per group) and subjected to library preparation and RNA sequencing using the SMART-seq ultra-low input system. The read counts were obtained after data filtering and quality control using FastQC v0.12.1 and MultiQC v1.21 and alignment to the Ensemble’s Sscrofa11.1 Sus scrofa genome reference using STAR aligner v2.7.11 for the samples. The filtered data was subjected to differential expression analysis using DESeq2. We identified 510 differentially expressed genes (DEGs), including 391 upregulated and 119 downregulated genes in the CC-treated group compared to control (p ≤ 0.05, absolute log2 fold change ≥ 0.5). Functional analysis using KEGG pathway analysis on ClueGO indicated pathways relevant to oocyte development and cellular signaling. Notably, the estrogen, phosphatidylinositol, phospholipase D, cAMP, and ErbB signaling pathways were among the most enriched. These pathways are critical for meiotic progression, follicular development, and cell survival. Several upregulated genes in the CC-treated oocytes—including MAPK3 (mitogen-activated protein kinase 3), AKT1 (AKT serine/threonine kinase 1), TOX3 (TOX high mobility group family member 3), SOCS1 (suppressor of cytokine signaling 1), and NCOA3 (nuclear receptor coactivator 3)—are associated with oocyte competence, estrogen signaling, and embryo development, suggesting that CC could potentially alter the molecular environment of maturing oocytes. However, further studies investigating the effect of CC on oocyte developmental competence are warranted.