Expression of STAR and CYP19A1 Genes and Physiological Responses in Buffalo Granulosa Cells Under in vitro Heat Stress and Follicular Cysts Conditions

Abstract

Global warming and heat stress are major challenges affecting livestock productivity and reproductive performance. In Egypt, buffalo milk production has declined markedly in recent years due to climate change and the limited ability of buffaloes to tolerate high environmental temperatures. Heat stress impairs ovarian function by disrupting granulosa cell steroidogenesis, leading to reduced fertility. Therefore, this study aimed to evaluate the effect of heat stress on steroidogenic activity in normal buffalo granulosa cells and to compare it with granulosa cells isolated from cystic ovaries. Granulosa cells were aspirated from normal and cystic buffalo ovaries and cultured under standard conditions until reaching 80–90% confluence. Heat stress was induced in normal granulosa cells, by incubation at 42°C for 2 h, followed by a recovery period under normal culture conditions. Total RNA was extracted, quantified using a NanoDrop spectrophotometer, reverse transcribed into cDNA, and analyzed by quantitative real-time PCR (qRT-PCR). Relative gene expression was calculated using the 2−ΔΔCt method with GAPDH as the reference gene. Cell viability was assessed using a hemocytometer. Heat stress significantly reduced granulosa cell viability during both heat exposure and the recovery period. Moreover, the expression of STAR and CYP19A1 was significantly downregulated, accompanied by a significant decrease in steroid hormone concentrations. In contrast, granulosa cells isolated from cystic follicles showed significantly higher expression of steroidogenic genes and increased hormone concentrations compared with normal granulosa cells. These findings demonstrate that heat stress impairs steroidogenic activity in buffalo granulosa cells, whereas cystic follicles exhibit enhanced steroidogenic activity, highlighting the distinct effects of heat stress and cystic ovarian conditions on buffalo reproductive function.