Molecular Characterization and Gene Expression Profiling of Cultured Theca Cells from Iraqi Buffalo in Relation to Steroidogenic Pathway Genes

Abstract

The present study investigates the molecular characteristics, survivability, and steroidogenic function of cultured theca cells (TCs) isolated from Iraqi buffalo ovaries under heat-stress conditions. Since global warming has been on the rise, affecting livestock fertility, there is a need to comprehend the regulations of thermal tolerance in buffalo reproductive cells to enhance the in vitro reproductive technologies. Primary TCs in both optimal conditions (37°C) and heat stress (40.5°C) conditions were subjected to 24, 48, and 72 hours of culture. Estradiol (E2), progesterone (P4), insulin-like growth factor-1 (IGF-1), and total antioxidant capacity (TAC) of conditioned media and TC pellets. mRNA expression of metabolic (CPT2, ATP5F1A, SREBP1) and stress-responsive genes (SOD2, NFE2L2, TNF-alpha) were analyzed. Buffalo TCs were found to have good thermal resilience with a viability of more than 87 percent in all treatments. Exposure to heat brought about time-dependent hormonal changes such as high secretion of E2 at 72 hours, temporary reduction of P4 at 48 hours, and progressive decrease in the levels of IGF-1. TAC is reduced at 48 hours but recovers in 72 hours, which suggests the activation of antioxidant compensation measures. Analysis of gene expressions revealed that CPT2 and NFE2L2 were downregulated, SREBP1 and TNF-alpha were upregulated in the middle of the exposure, and both SOD2 and ATP5F1A were expressed steadily, indicating selective metabolic and oxidative changes. These data indicate that there is a coordinated response in the genes to allow buffalo TCs to endure moderate heat stress and to regulate steroidogenesis, metabolism, and antioxidant signaling. The findings are helpful to improve buffalo reproduction under hot weather conditions and present possible goals to increase the heat resistance of in vitro culture systems.

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