FIG. 8.

SH2-B is essential for developing cumulus oophori. A COC expansion assay was performed with wild-type (A, B, and C) and SH2-B-deficient (D, E, and F) COCs. Forty-eight hours after administration of 5 IU of PMSG, Graafian follicles were punctured with needles and the COCs were removed into control Waymouth's medium. COCs were cultured in Waymouth's medium containing 3 mg of BSA/ml without FCS overnight at 37°C (A and D). Wild-type COCs were expanded with 1 μg of FSH/ml (B and E) in Waymouth's medium. COCs were also cultured in Waymouth's medium containing 10 ng of IGF-I/ml for 4 h, followed by 1 μg of FSH/ml (C and F). Similar results were obtained with up to 100 ng of IGF-I/ml. Magnification, ×100. Representative results of three independent experiments are shown. (G) Expression of FSH-R. Total RNA was extracted from ovaries of wild-type (+/+) and SH2-B-null (−/−) mice and hybridized with FSH-R (upper panel) and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (lower panel). (H) Tyrosine phosphorylation of cellular proteins and ERK2 phosphorylation in COCs in response to IGF-I. COCs were collected from two female mice and stimulated with 100 ng of IGF-I/ml for 20 min. Cell extracts were immunoblotted with antiphosphotyrosine (αPY), anti-phosphorylated ERK (αP-ERK), or anti-ERK2 antibodies.