FSH mediated endocrine disruption potential of Dibutyl phthalate in cultured ovine ovarian granulosa cells: Cytotoxicity, oxidative stress, steroidogenesis, cell senescence, and expression of related key developmental genes

Di-n-butyl phthalate (DBP) is a widely used phthalate ester recognized for its endocrine-disrupting properties and potential reproductive toxicity. The present study aimed to evaluate the concentration-dependent effects of DBP on morphology, cytotoxicity, oxidative stress, cell senescence, steroidogenesis and expression of related key developmental genes in cultured ovine granulosa cells in the presence of FSH. Granulosa cells were isolated from slaughterhouse-derived ovine ovaries and cultured in vitro with exposure to DBP at concentrations of 0, 1, 10, 25, 50, and 100 µM. Cytotoxic responses were assessed by evaluating nuclear damage, reactive oxygen species (ROS) generation, apoptosis, and cellular senescence. In addition, expression levels of key steroidogenic genes (CYP11A1, CYP17A1, CYP19A1, STAR, HSD3B1, HSD17B1), hormonereceptors (ESR1, ESR2, PGR, FSHR), and pro-apoptotic and anti-apoptotic markers (BAX, CASP3, BCL2) were measured using quantitative PCR. Estradiol and progesterone levels were quantified in culture media to evaluate functional steroidogenic output. The results revealed that DBP exposure caused a concentration-dependent increase in ROS levels, apoptosis-related gene expression, and signs of cellular senescence, particularly at concentrations ≥ 25 µM. Interestingly, estradiol production increased significantly with increasing DBP concentration, despite the presence of cellular stress. Gene expression analysis showed a marked upregulation of ESR1, FSHR, and CYP19A1, while ESR2 and the other key steroidogenic genes remained unchanged. These findings suggested that DBP might altered estrogenic activity through selective modulation of estrogen and FSH receptor signaling rather than through global transcriptional activation of steroidogenic enzymes. Moreover, alterations in mitochondrial membrane potential and nuclear morphology suggested the subcellular stress responses to DBP. In conclusion, DBP exerted dual effects on ovine granulosa cells by inducing oxidative stress and cellular dysfunction while simultaneously enhancing estradiol synthesis through selective receptor pathways. FSH stimulated steroidogenesis in GCs culture in the presence of DBP, yet it was unable to mitigate DBP-induced cell toxicity.