Role of glycogen synthase kinase-3 in regulation of mammalian oocyte developmental competence.

Mammalian oocyte or follicle growth in prolonged exposure to elevated insulin has been shown to impair oocyte developmental competence, yet the mechanism is unknown. The overall hypothesis of this dissertation is that insulin regulates oocyte developmental competence via its terminal kinase, glycogen synthase-kinase-3alpha/beta (GSK-3alpha/beta), and that elevated insulin negatively impacts fertility by dysregulation of oocyte GSK-3, leading to abnormal chromatin segregation during early embryonic cleavage events. We have determined a functional insulin signaling cascade in the mammalian oocyte, through protein expression of IR-beta, PDK-1, Akt/PKB, and GSK-3alpha/beta, as well as endogenous GSK-3 activity in fully-grown oocytes. Prolonged insulin exposure during in vitro oocyte growth resulted in increased phosphorylation (inactivation) of GSK-3beta (S9) in GVI oocytes and impaired chromatin remodeling and condensation during meiosis. Pharmacological inhibition of oocyte GSK-3 during in vitro maturation caused a delay in meiotic spindle formation and polar body extrusion, and resulted in severe chromatin condensation errors similar to those evidenced following insulin exposure during oocyte growth. We characterized GSK-3alpha/beta protein expression/phosphorylation in mouse preimplantation embryos and assessed the effect of GSK-3 inhibition on early mitotic events. Immunocytochemical analysis demonstrated co-localization of GSK-3 with the first mitotic spindle. Both immunoneutralization and pharmacological inhibition of GSK-3 in pronuclear stage embryos significantly compromised embryo development past the 2-cell stage, and arrested embryos showed abnormal chromatin segregation. Time-lapse imaging of GSK-3 inhibited embryos showed a delay in both karyokinesis and cytokinesis. To address a maternal insulin effect on oocyte developmental competence, we employed leprs1138 homozygous (s/s) females that exhibit obesity, hyperinsulinemia, hyperleptinemia, but not hyperglycemia. In vitro development at 2-cell and blastocyst stage was significantly compromised in embryos derived from hyperinsulinemic females. Evaluation of oocyte meiotic competence showed no effect of maternal insulin levels on rate of in vitro oocyte maturation to MII. However, analysis of MII oocytes derived from hyperinsulinemic demonstrated a significant impairment in normal chromatin condensation. In conclusion, we have confirmed through in vitro and in vivo studies that elevated insulin during oocyte growth can negatively impact oocyte developmental competence through impaired chromosomal integrity and this regulation is likely mediated via actions of the terminal enzyme, GSK-3.PhDAnimal PhysiologyBiological SciencesHealth and Environmental SciencesMolecular biologyObstetricsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126349/2/3253206.pd