Thymidine incorporation into granulosa cells Granulosa cells were

Thymidine incorporation into granulosa cells Granulosa cells were cultured in 24 well dishes in McCoys 5A medium containing 10% FBS for 48 h and were then serum starved for 24 h in DMEM medium without MG132 CAS glucose then 1 Ci l of thymidine was added in the presence or absence of various con centrations Inhibitors,Modulators,Libraries of glucose and or FSH and IGF 1. Cultures were maintained at 37 C under 5% CO2 in air. After 24 h of culture, excess of thymidine was removed by washing twice with phosphate buffered saline, and the samples were fixed with cold trichloroacetic acid 50% for 15 min and lysed by addition of 0. 5 N NaOH. The radioactivity was determined by addition of scintilla tion fluid and counting in a pho tomultiplier.

Progesterone and oestradiol radioimmunoassay The concentration of progesterone and oestradiol in the culture medium of granulosa cells was measured Inhibitors,Modulators,Libraries after 48 Inhibitors,Modulators,Libraries h of culture by a radioimmunoassay protocol as previously described and adapted to measure ster oids in cell culture media. The limit of detection of P was 12 pg tube and the intra and interassay coef ficients of variation were less than 10% and 11%, respec tively. The limit of detection of E2 was 1. 5 pg tube and the intra and interassay coefficients of varia tion were less than 7% and 9%, respectively. Results are expressed as the amount of steroids secreted per 48 h per 100 g of protein, and values reported are means SE of three cultures of granulosa cells. For each culture, about 30 to 40 rats were used and each condition was ana lyzed four times independently.

After extraction of steroids from serum, the concentration of progesterone was measured by the RIA method as pre viously described. The oestradiol concentration in the serum was measured with a RIA KIT. Glucose, adiponectin, resistin and insulin plasma levels Plasma glucose was assayed by the glucose oxidase method. Plasma adiponectin and resistin were assayed Inhibitors,Modulators,Libraries with a rat adiponectin ELISA kit and a rat resistin ELISA kit, respectively. Serum insulin levels were determined using a rat insulin ELISA kit. Statistical analysis All experimental data are presented as the mean SE. One t test or one way analysis of variance were used to test differences. if ANOVA revealed significant effects, the means were compared by Newmans test, with P 0. 05 considered significant.

Results Effect of glucose Inhibitors,Modulators,Libraries on basal and FSH or IGF 1 stimulated oestradiol and progesterone production in rat granulosa cells The effects of glucose treatment on steroidogenesis in rat granulosa cells were first examined in incubations con read this taining various concentrations of glucose for 48 h. Con centrations of glucose greater than 5 g l were found to inhibit the syntheses of progesterone and oestradiol. We also determined whether glucose affected the production of progesterone and oestradiol in response to FSH or IGF 1.

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