Arsenic-Induced Progesterone Production In A Caspase-3 Dependent Manner And Changed Redox Status In Preovulatory Granulosa Cells

Arsenic contamination is a principal environmental health threat throughout the world. Epidemical study demonstrated that arsenite can cause skin diseases, diabetes, high blood pressure, basal cell carcinoma, squamous cell carcinoma. Studies have reported arsenite can interfere with gene expression mediated by hormone receptor. Recent studies demonstrated that sodium arsenite treatment diminished the circulating levels of progesterone, estradiol, FSH and LH level of adult female rat. However, little is known about the effect of arsenic on steroidogenesis of female before sexual maturity.21-day-old female Sprague-Dawley rats were received treatment with arsenite at a dose of 10 mg/L/rat/day. After 7 days, animals were anesthetized. Blood was collected and serum was isolated for steroid radioimmunoassay (RIA). The result showed that arsenite can enhance the level of progesterone in the blood serum, however there was no significant change on the levels of estradiol and testosterone. The granulosa cells were isolated and treated with 0-10μM arsenite for 48 h. The conditioned media were collected and the levels of progesterone were measured by RIA. The result showed that arsenite stimulates progesterone production of cultured preovulatory GCs.Luteinizing hormone (LH) and follicle stimulating hormone (FSH) regulating the progesterone production through cAMP/PKA and ERK1/2 pathways had been reported many times. The gonadotropic hormone enhanced the level of cellular cAMP and actived protein kinase A, phosphorylated ERK1/2, further phosphorylated transcription factors and enhanced the transcription of proteins related with steroidogenesis. Whether the cAMP/PKA and ERK1/2 pathways are involved in progesterone production induced by arsenite? We examine the cellular cAMP levels with cAMP chemiluminescence assay. Our result showed that the cellular cAMP and p-ERK1/2 levels weren't changed in response to arsenite. We also examined the StAR and P450scc downstream of cAMP/PKA and ERK1/2 signaling pathways. The result showed that arsenite didn't alter the levels of StAR and P450scc. However, progesterone production was significantly decreased when cAMP-dependent protein kinase (PKA) or ERK1/2 pathway was inhibited. The result indicated that the production of progesterone may not dependent on cAMP/PKA and ERK1/2 pathways but require them.Progesterone is the marker of granulosa cell differentiation. Arenic can active caspase-3 and induced apoptosis or differentiation. Whether arsenite stimulated progesterone production due to the differentiation by activation of caspase-3? Further study demonstrated that arsenite treatment induced caspase-3 activation, although no apoptosis was observed. Inhibition of caspase-3 activity with broad-spectrum and specific inhibitors can significantly inhibit the basic progesterone production and the progesterone production stimulated by arsenite. Transfection with siRNA targeting caspase-3 in GCs also inhibited progesterone production stimulated by arsenite.Arsenite stimulated the appearance of active/cleaved caspase-3 but without cellular apoptosis.We found that arsenite decreased intracellular reactive oxygen species (ROS) but increased the antioxidant glutathione (GSH) levels and mitochondrial membrane potential (ΔΨM) in parallel to the changes in progesterone production. Progesterone antagonist blocked the arsenic-stimulated increase of GSH. GSH depletion with buthionine sulfoximine led to cell apoptosis in response to arsenite treatment.Collectively, this study demonstrated for the first time that arsenite stimulates progesterone production through cleaved/active caspase-3-dependent pathway, and the increase of GSH level promoted by progesterone production may protect GCs against apoptosis and maintain the steroidogenesis of GCs in response to arsenite treatment.