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

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 siRNA, GCs were divided into HS (40 °C + NC and 40 °C + CAT siRNA) 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.

Joint Transcriptome and Metabolome Analysis Prevails the Biological Mechanisms Underlying the Pro-Survival Fight in In Vitro Heat-Stressed Granulosa Cells

“…The cell collection, culture, and treatment methods followed our previous study [ 21 , 22 , 23 ]. In brief, follicular fluid was collected from small follicles (3–8 mm) of cyclic bovine ovaries followed by filtration and centrifugal washings.…”

“…Intracellular reactive oxygen species (ROS), apoptosis, and hormone measurement methods followed our previous studies [ 21 , 22 , 23 ]. Cells were cultured at the rate of 2 × 10 4 for a colorimetric CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) based on cell viability in each well (of 96-well plates).…”

“…The following supporting information can be downloaded at: , Figure S1: Identification of granulosa cells purity; the figure was taken from a previous study [ 23 ]. PI was used for staining the cellular nuclei (A,D).…”

Joint Transcriptome and Metabolome Analysis Prevails Biological Mechanisms Underlying the Prosurvival Fight in Heat Stressed Granulosa Cells

“…Cell collection, culture and treatment methods followed our previous study [21,22,72]. In brief, follicular fluid was collected from small follicles (3-8 mm) of cyclic bovine ovaries followed by filtration, centrifugal washings.…”

“…Reactive oxygen species, apoptosis and hormone measurement methods followed our previous studies [21,22,72]. Cells were cultured at the rate of 2 × 10 4 for colorimetric CCK-8 and 1 × 10 4 in each well of 96-well plates for fluorocolorimetric ROS assay (DCFDA kit, Abcam, Cambridge, MA, USA).…”