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Characterization of Inhibin/Activin Subunit: MATERIALS AND METHODS(2)

Mouse Oocytes and Embryos

Female B6C3F1 mice (22-43 days of age) were superovulated by administration of eCG (5 IU; Professional Compounding Centers of America, Houston, TX), followed by hCG (5 IU; Serono Laboratories, Norwell, MA) 48 h later. Mature (M2) oocytes were collected from the oviducts 16 h after hCG and denuded as described above. Fertilized eggs were obtained from females that were caged with males immediately after hCG induction. Fertilized eggs were cultured in IVF50 medium until the time of embryo hatching (3-5 days; preimplantation embryos). This protocol was approved by the Massachusetts General Hospital Subcommittee on Research Animal Care.

Total RNA Extraction

Individual denuded human oocytes or the detached cumulus mass was extracted with 500 ^l of Trizol (Gibco-BRL, Grand Island, NY) according to the manufacturer’s protocol in the presence of 5 ^g Escherichia coli tRNA as a carrier. Mouse oocytes or embryos were similarly treated except that they were pooled into groups of 1-5 before extraction. buy birth control online

RT-PCR

The entire RNA sample extracted from oocytes, or 25100% of the extracted cumulus RNA, was treated with DNase I (1 U; GibcoBRL) to remove DNA contamination; this was followed by first strand cDNA synthesis in a 20-^l reaction containing 5 mM oligo(dT), 0.5 mM dNTP mix, and 100 U of SuperScript II (GibcoBRL).