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Simple Summary  Reduced fertility of modern-day dairy cattle across the world is implicated by the global warming phenomenon. Heat stress (HS) is well-known for compromising the normal physiological functions of granulosa cells (GCs) by promoting reactive oxygen species (ROS), which subsequently induce apoptosis, impair the biosynthesis of estrogen and progesterone, and disrupt the mitochondrial membrane potential. The catalase (CAT) enzyme serves a key antioxidant role in catalyzing the HS-induced ROS, thereby preventing the adverse effects of oxidative stress on cells. Therefore, the regulation of the CAT gene under HS has been the subject of increasing interest among researchers. However, no researches till date performed a functional validation of the CAT gene in bovine GCs under HS. For instance, we silenced the CAT gene using  si RNA in GCs and found that the silencing of CAT aggravated the HS-induced damages to these cells, depicting a protective role of the CAT gene under HS. Thus, the regulation of CAT under HS could be used as an effective molecular marker to enhance the fertility in heat-stressed dairy cows.    Abstract  Heat stress in dairy cattle is recognized to compromise fertility by altering the functions of ovarian follicle-enclosed cells, e.g., oocyte and granulosa cells (GCs). Catalase is an antioxidant enzyme that plays a significant role in cellular protection against oxidative damage by the degradation of hydrogen peroxide to oxygen and water. In this study, the role and mechanism of CAT on the heat stress (HS)-induced apoptosis and altered proliferation of bovine GCs were studied. The catalase gene was knocked-down successfully in bovine GCs at both the transcriptional and translational levels. After a successful knockdown using  si RNA, GCs were divided into HS (40 °C + NC and 40 °C + CAT  si RNA) and 38 °C + NC (NC) groups. The GCs were then examined for ROS, viability, mitochondrial membrane potential (MMP), cell cycle, and biosynthesis of progesterone (P4) and estrogen (E2) hormones. The results indicated that CAT silencing promoted ROS production and apoptosis by up-regulating the Bcl-2-associated X protein (BAX) and Caspase-3 genes both at the transcriptional and translational levels. Furthermore, the knockdown of CAT markedly disrupted the MMP, impaired the production of P4 and E2, altered the progression of the G1 phase of the cell cycle, and decreased the number of cells in the S phase. This was further verified by the down-regulation of proliferating cell nuclear antigen (PCNA), CyclinB1, steroidogenic acute regulatory protein (STAR), and cytochrome P450 family 11 subfamily A member 1 (Cyp11A1) genes. Our study presented a novel strategy to characterize how CAT can regulate cell proliferation and apoptosis in GCs under HS. We concluded that CAT is a broad regulatory marker in GCs by regulating apoptosis, cellular progression, and simultaneously by vital fluctuations in hormonal signaling. Our findings infer a crucial evidence of how to boost the fertility of heat-stressed cows.

RNAi-Mediated Silencing of Catalase Gene Promotes Apoptosis and Impairs Proliferation of Bovine Granulosa Cells under Heat Stress