Clomiphene Citrate Supplementation During In Vitro Maturation of Porcine Oocytes Reduces ROS Accumulation and Promotes Oxidative Stress Resilience Pathways

Abstract

The research in this thesis addresses key challenges in reproductive biology particularly concerning oocyte maturation and early embryonic development. The efficiency of in vitro reproductive technologies such as oocyte maturation and development, in porcine lags behind other livestock species such as cattle. We aim to explore the potential effect of the selective estrogen receptor modulator (SERM) clomiphene citrate (CC) on porcine oocyte maturation and subsequent in vitro embryo development. Through exploring the effects of CC on porcine oocyte maturation and development we seek to provide new insights to advance the use of in vitro reproductive technologies in pigs. The first study in this thesis investigated the effects of (CC) on porcine oocyte maturation. We investigated nuclear stage of maturation and ROS at 22 h and 44 h in CC treated and control oocytes, neither parameter was affected by CC at 22 h. At 44 h, there was also no significant different in nuclear maturation, but treated oocytes demonstrated significantly reduced levels of reactive oxygen species (ROS) at 44h, suggesting improved cellular conditions during in vitro maturation (IVM) at this time point. To further investigate the biological mechanisms behind this effect, the mRNA expression of genes related to oxidative stress (SOD1, SOD2, GPx) and apoptosis (BAX, CASP3) was analyzed using RT-PCR, with GAPDH and RNA18S as reference genes. Despite the observed reduction in ROS, gene expression analysis did not show any statistically significant differences between treated and control groups. To further investigate the molecular mechanisms underlying improved oocyte quality following clomiphene citrate (CC) treatment, a second study was conducted to achieve deeper molecular characterization through transcriptomic analysis. We sequenced RNA from 44 h in vitro 3 matured porcine oocytes treated with CC for the first 22 h and their untreated counterparts. After filtering out low-expression transcripts, 15,921 genes were retained from a total of 20,391, and 510 differentially expressed genes (DEGs) were identified using a p-value threshold of ≤ 0.05 and |log2 fold change| > 0.5. Among these, 391 genes were upregulated and 119 were downregulated in CC-treated oocytes. ClueGo was used to run a pathway enrichment analysis and among those significantly enriched, two were most notable: the phosphatidylinositol signaling system, and inositol phosphate metabolism—each essential to oxidative damage mitigation, mitochondrial regulation, cell cycle progression, and intracellular signaling fidelity. The activation of key signaling mediators such as AKT1, MAPK3, and INPPL1 reinforces the notion that CC modulates critical pathways associated with oocyte survival and maturation. These findings demonstrate the potential of CC as a functional additive in IVM systems aimed at improving reproductive outcomes in livestock. In our third study, we investigated the effects of Clomiphene citrate (CC) on early embryonic development. Consistent with the previous studies, CC was applied during IVM of oocytes, and we subsequently assessed whether this exposure influenced the ability to undergo successful activation and early embryonic development using parthenogenic activation. Nuclear assessment revealed that oocytes that had been treated during maturation had significantly higher cleavage rates at 30 hours post-activation (38.43%±9.28) compared to controls (26.86%±8.08%, p = 0.022), suggesting enhanced developmental readiness and possibly healthier embryos. There were trending significant differences observed between groups in embryo classification at 60 hours, with more treated oocytes cleaving, and reaching past the 8-cell stage (p<0.1) indicating that CC treatment has promise to benefit cleavage timing and increase the number of transferable stage embryos in vitro. To further explore underlying mechanisms, we assessed the expression of genes related to oxidative stress (SOD1, SOD2), apoptosis (BAX, CASP3), and pluripotency (POU5F1) using RT-PCR, with GAPDH and RNA18S as reference genes. No significant differences were detected in the tested genetic markers in embryos formed from treated and control oocytes, suggesting that CC’s effects are not mediated by changes in expression of these target genes. These results support CC’s potential to enhance early embryo development without inducing cellular stress or compromising embryo development, in vitro.