Gonadotropin-Independent Regulation of Steroidogenesis: MATERIALS AND METHODS(1)

2 Nov
2012

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 night of mating was designated Day 0.5 of gestation. Mothers were killed by decapitation under light C02 anesthesia between 0800 and 1200 h at daily intervals between El5.5 and E21.5. The fetuses were excised and pinned on a silicon rubber mat, and blood was taken by axillary puncture into heparinized syringes kept on ice, and centrifuged; plasma was stored at -20°C until analyzed. The pituitaries were excised, snap-frozen in liquid nitrogen, and stored at -70°C until analyzed. The abdominal wall was opened, and the testes were removed, snap-frozen in liquid nitrogen, and stored at —70°C until analyzed for testosterone content. Alternatively, the testes were excised under sterile conditions and placed into ice-cold medium (Dulbecco’s Modified Eagle’s medium/F12 [1:1], with 0.365 g/L L-glutamine; Life Technologies, GIBCO BRL, Glasgow, Scotland) plus 0.1% BSA (Sigma Chemical Co., St. Louis, MO), 4.5 g/L 20 mM HE-PES, and 0.1 g/L gentamicin (Biological Industries, Bet HaEmek, Israel), which was used for all incubations and cultures (see below). All animal experiments were approved by the Turku University Committee on ethics of animal experimentation and were carried out according to the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction. Buy Asthma Inhalers Online

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