Archive for the ‘Gonadotropin’ Category

Southern Hybridization Analysis The cDNA fragments generated by RT-PCR were resolved on 1.2% agarose gel and transferred onto nylon membranes (Hybond-N; Amersham). The membranes were prehybridized for at least 1 h at 65°C in a total volume of 25 ml containing 5-strength SSPE (single-strength SSPE = 180 mM NaCl, 10 mM sodium phosphate, and 1 […]

RNA Extraction Total RNA was isolated from the fetal pituitaries of animals aged E16.5 and older by the CsCl/guanidium isothio-cyanate method as described previously . Because of difficulties in identifying the fetal pituitary at E15.5, total RNA was isolated from the whole fetal head at this age. buy levaquin online

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 […]

Culture of BLT-1 Cells BLT-1 cells are an immortalized Leydig cell line derived from a testicular tumor of a transgenic mouse expressing the SV40 T-antigen under the murine inhibin a-subunit promoter . One day before stimulation, the cells were plated into 24-well culture dishes (Greiner, Frickenhausen, Germany) at a density of 105 cells/well. The next […]

Isolation, Purification, and Culture of Adult Leydig Cells For isolation and purification of adult Leydig cells we used a previously described method . In brief, the testes were decapsulated carefully and incubated in 10 ml of culture medium for 10 min at 34°C in an atmosphere of 5% C02 in air in the presence of […]

Cultures of Dispersed Leydig Cells The medium containing fetal testes was replaced by 15 ml of culture medium containing 0.4% collagenase type II (Sigma) and DNAse I (105 Kunitz U/L; Sigma), and the testes were incubated for 30 min at 37°C in a shaking water bath. Thereafter, the testes were dispersed mechanically by aspirating the […]

Animals and Experimental Design Adult (2- to 3-mo-old) rats of the Sprague-Dawley strain (produced in our own vivarium) were housed under a controlled photoperiod (14L:10D) and fed a commercial diet with water ad libitum. Females were caged with males overnight and checked the following morning for sperm in the vaginal smear. The day after the […]