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Insulin Signaling in Mouse Oocytes¹

Departments of Molecular and Integrative Physiology,⁴ Urology,⁵ and Obstetrics and Gynecology,⁶ and the Reproductive Sciences Program,⁷ University of Michigan, Ann Arbor, Michigan 48109

ABSTRACT

Continuous exposure of follicles/oocytes to elevated levels of insulin compromises embryonic developmental competence, although the underlying cellular mechanisms are unknown. The objectives of the present study were to determine whether mouse oocytes have insulin receptors and a functional insulin signaling cascade, and whether insulin exposure during oocyte growth or maturation influences meiotic progression and chromatin remodeling. Immunoblot and immunocytochemical analyses of germinal vesicle-intact (GVI) oocytes demonstrated the presence of insulin receptor-ß. Insulin receptor expression in oocytes was increased by gonadotropin stimulation, and remained elevated throughout meiotic maturation. Fully grown GVI oocytes contained 3-phosphoinositide-dependent protein kinase-1 (PDPK1), thymoma viral proto-oncogene 1 (AKT1), and glycogen synthase kinase 3 (GSK3). In vitro maturation of GVI oocytes in 5 µg/ml insulin had no influence on meiotic progression or the incidence of normal metaphase II (MII) chromosome condensation. Treatment of oocytes during maturation had no effect on GSK3A/B protein expression or phosphorylation of S21/9. However, the culturing of preantral follicles for 10 days with 5 µg/ml insulin increased the phosphorylation of oocyte GSK3B, indicating GSK3 inactivation. The rates of development to metaphase I (MI) were similar for oocytes obtained from insulin-treated follicles and controls, whereas the incidence of abnormal MI chromatin condensation was significantly higher in oocytes obtained from follicles cultured with insulin compared to those cultured without insulin. These results demonstrate that oocytes contain a functional insulin signaling pathway, and that insulin exposure during oocyte growth results in chromatin remodeling aberrations. These findings begin to elucidate the mechanisms by which chronic elevated insulin influences oocyte meiosis, chromatin remodeling, and embryonic developmental competence.

chromatin remodeling, gamete biology, glycogen synthase kinase-3, insulin, kinases, meiosis, oocyte, oocyte development

³These authors contributed equally to this work.

¹Supported by NIH grant HD046768 (to G.D.S.). Support for N.A. was provided by NIH Training Grant in Reproductive Sciences T32-HD07048 (to D. Foster). This work utilized the Morphology and Image Analysis Core of the Michigan Diabetes Research and Training Center, which is funded by NIH5P60 DK20572 from the National Institute of Diabetes and Digestive and Kidney Diseases.

Correspondence: ²Gary D. Smith, 6428 Medical Sciences I, 1301 E. Catherine St, Ann Arbor, MI 48109-0617. FAX: 734 936 8617; e-mail: smithgd{at}umich.edu