Gonadotropin-Independent Regulation of Steroidogenesis: MATERIALS AND METHODS(5)

6 Nov

Testosterone and cAMP Measurements

Testosterone was measured from diethyl ether extracts of testicular homogenates and incubation media by RIA as described before . The testicular homogenates were composed of pools of two pairs of testes. The assay sensitivity was 2 fmol/tube. The intraassay coefficient of variation (CV) was below 6%, and the interassay CV was below 12%. Extracellular с AMP concentrations in the culture media were assayed by using an RIA as described earlier. Succinyl-cAMP, radioiodinated with Na[125I]iodide (IMS 300; Amersham, Aylesbury, UK) in our laboratory, served as a tracer. buy ortho tri-cyclen

LH Measurements

Plasma and pituitary LH concentrations were measured using the immunofluorometric assay principle (Delfia; Wal-lac Ltd., Turku, Finland), as described by us before . The pituitary homogenates were obtained from the first step of the RNA isolation process (see below), and originated from individual samples of fetal pituitary glands. Individual plasma samples were used from E20.5-21.5 fetuses. Samples of E17.5-19.5 fetuses were pooled from 2-4 animals. The plasma volume used for the LH assay was 50 ц1 or 75 |xl. The sensitivity of the assay was 0.5 pg/tube (of the NIDDK RP-2 standard for rat LH), and at a concentration of 0.2 (Jig/L, the intra- and interassay CVs were 8% and 12.5%, respectively.