Effects Of Phoxim And Fenvalerate On Steroidogenesis In Primary Cultured Rat Granulosa Cells In Vitro

The last fifty decades have witnessed growing scientific concerns and public debate over the potential adverse effects that may result from exposure to a group of chemicals that have the potential to alter the normal functions of the endocrine system in wildlife and humans. These chemicals are known as endocrine-disrupting chemicals(EDCs) or environmental endocrine disruptors(EEDs). Among the classes of EDCs, pesticides have been drawing increasing attention since they were commonly used for the control of agricultural and indoor pests. In China, pyrethroid and organophosphorus pesticides are the most popular pesticides used in agriculture. Because they are rapidly degraded in the environment, so they have largely replacedorganochlorine pesticides which can bioaccumulate and biomagnify. Unfortunately, a few studies have shown that these pesticides can cause dysfunction in reproductive system. Therefore, the present study was performed to determine the effects of Fenvalerate(Fen, pyrethroid pesticide) and Phoxim(Pho, organophosphorus pesticide) on steroid hormone production in primary cultured rat granulosa cells(rGCs) in vitro. In addition, their joint action on Steroidogenesis was also studied.Part I Effects of Phoxim and Fenvalerate on Steroidogenesis in Primary Cultured rGCsIn order to study effects of Fen and Pho on steroid hormone production and steroidogenic enzyme mRNA and protein levels, Primary serum-free cultured rat granulosa cells(rGCs) were used as model. rGCs were collected from immature female Sprague-Dawley rats(20-25 days old) and the modified method was described previously. Cells were incubated in different culture plates with Fen and Pho of various concentrations (0, 1, 5, 25, 125 and 625umol/L) for 24h, respectively. In some studies, optimal concentrations of Follicle-Stimulating Hormone (FSH, 500ug/L), forskolin (10umol/L), 22(R)-hydroxycholesterol (22R-HC, 25umol/L), protein kinase inhitor(PKI, 2umol/L), or 8-Bromo-cAMP(1mmol/L) were provided. At the end of all experiments, medium samples were taken and stored at -20C for the tests of17B-estradiol(E2) and progesterone(P4). And cAMP of cells was abstracted by ethanol. Total RNA and protein were extracted for RT-PCR(or real time PCR) and Western' blot, respectively. These experimental results read as follows:1. Fen could significantly decrease the basal production of P4 in rGCs with a dose-dependent manner(especially in 125fimol/L and 625umol/L), especially inhibited FSH-stimulated P4 production. Also Fen could slightly increased E2 levels in medium with or without FSH stimulation.2. Pho could increase the basal production of P4 in rGCs with a dose-dependent manner. But this stimulation to P4 converted to inhibition when rGCs was incubated with 500ug/L FSH. Pho did not affect on basal E2 production and could decrease the level of E2 with 500ug/L FSH.3. When rGCs were incubated with PKI, 8-Bromo-cAMP or forskolin, Fen could also decrease the P4 production. But its stimulation to E2 production existed only with forskolin and disappeared with both PKI and 8-Bromo-cAMR4. Pho did not affect on the P4 production with 8-Bromo-cAMP or forskolin and only increased P4 production at dose of 125umol PKI. Pho also had no effect on E2 production with 8-Bromo-cAMP and could stimulate the steroidogenesis with PKI. This stimulation, however, converted to inhibition when incubated with forskolin.5. When 22R-HC was added to medium with Fen, the inhibition to P4 production was even more significant than basal P4 production.When 22R-HC was added to medium incubated Pho, its stimulation to P4 production was partially reversed.6. Both Fen and Pho had no effects on basal cAMP level of rGCs and could rapidly decrease the FSH-stimulating cAMP level.7. On levels of mRNA, both Fen and Pho could increase the expression of steroidogenic acute regulatory protein (StAR) gene but decrease the expression of cholesterol side chain cleavage enzyme (P450scc) gene. Fen had no effect on the aromatase...