HO-1 reduces heat stress-induced apoptosis in bovine granulosa cells by suppressing oxidative stress

Abstract

Heat stress negatively affects reproduction in cattle by disrupting the normal function of ovarian granulosa cells (GCs), ultimately leading to oxidative damage and cell death via apoptosis. Heme oxygenase-1(HO-1) is a member of the heat shock protein family, which are associated with cellular antioxidant defenses and anti-apoptotic functions. Recent studies demonstrated that HO-1 is upregulated in heat-stressed cells. In the present study, we investigated the expression of HO-1 in bovine GCs transiently exposed to heat stress and characterized the expression and activity of key oxidative stress enzymes and molecules. We show that heat stress induced oxidative stress and apoptosis, and enhanced Nrf2 and HO-1 expression in primary GC cultures. Knocking down HO-1 expression using siRNA exacerbated both oxidative stress and apoptosis, whereas pre-treating GCs with hemin, which induces HO-1 expression, partially prevented these effects. These findings demonstrate that HO-1 attenuates heat stress-induced apoptosis in bovine GCs by decreasing production of reactive oxygen species and activating the antioxidant response.

Keywords: HO-1; apoptosis; granulosa cells; heat stress; reactive oxygen species (ROS).

Figure 1

Heat stress induces ROS generation and apoptosis in bovine ovarian GCs. (A, B) Intracellular accumulation of ROS at different temperatures, quantified by DCF fluorescence. Scale bars, 50 μm. (C–E) Immunoblot analysis of Bax/Bcl-2 and cleaved caspase-3in GCs. (F) Apoptosis induction by heat stress in GCs, analyzed by FACS assay. Data represent mean ± SEM; n = 3 in each group. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Heat stress leads to dysfunction of the antioxidant defense system and oxidative stress in GCs. (A–D) Western blotting showing the expression of Nrf2, HO-1, and SOD2. (E) Nuclear translocation of Nrf2 evaluated by DCF fluorescence. Scale bars, 50 μm. (F) Estimation of SOD activity. (G) Estimation of GSH-Px activity. (H) Nuclear translocation of Nrf2 determined by western blotting. (I) MDA content. Data represent mean ± SEM; n = 3 in each group. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Figure 3

HO-1 gene knockdown enhances ROS generation and induces apoptosis in GCs under heat stress. (A) Western blot analysis of HO-1 expression after siRNA mediated knockdown of HO-1. (B, C) Annexin V/PI FACS analysis of apoptosis. (D–F) Expression of Bax/Bcl-2 and cleaved caspase-3 by western blot. (G, H) Intracellular ROS accumulation detected through DCF fluorescence. Scale bars, 50 μm. Data represent mean ± SEM; n = 3 in each group. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

HO-1 gene knockdown impairs antioxidant defenses in GCs exposed to heat stress. GCs were transfected with NC or siHO-1 and exposed to heat stress for 2 h. (A–C) Western blot expression of HO-1 and SOD2. (D) SOD activity. (E) MDA content. (F) GSH-Px activity. Data represent mean ± SEM; n = 3 in each group. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

Hemin-mediated HO-1 induction decreases oxidative stress and attenuates ROS generation and apoptosis in GCs exposed to heat stress. (A) qRT-PCR analysis of HO-1 gene expression in GCs pre-treated with hemin (10 μmol/L for 48h). (B, C) Effect of HO-1 overexpression on ROS accumulation in GCs under heat stress. (D, E, G, H) HO-1, SOD2, and Bax/Bcl-2 expression determined by western blotting. (F) Hemin pre-treatment reduced apoptosis of heat-stressed GCs, as determined by FACS assay. (I) Enhanced SOD activity in hemin-treated GCs. Data represent mean ± SEM; n = 3 in each group. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Figure 6

Schematic model of HO-1 regulation of oxidative stress and apoptosis in GCs exposed to heat stress.