title: Expression of focal adhesion kinase in mouse cumulus-oocyte complexes, and effect of phosphorylation at Tyr397 on cumulus expansion.
abstract: We investigated the expression of focal adhesion kinase (FAK) in mouse cumulus-oocyte complexes (COCs), as well as the role of FAK phosphorylation at Tyr397 during oocyte maturation. The effect of inhibiting FAK phosphorylation at Tyr397 during in vitro maturation (IVM) on subsequent fertilization and preimplantation embryo development was also examined. Western blotting analyses revealed that total and Tyr397-phosphorylated FAK were expressed in vivo in both cumulus cells and oocytes. Immunocytochemical studies localized this kinase throughout the cytoplasm of cumulus cells and oocytes; in particular, Tyr397-phosphorylated FAK tended to accumulate in regions where cumulus cells contact each other. Interestingly, the in vivo level of Tyr397 phosphorylation in cumulus cells was significantly lower after compared to before cumulus expansion. Addition of FAK inhibitor 14, which specifically blocks phosphorylation at Tyr397, stimulated oocyte meiotic maturation and cumulus expansion during IVM in the absence of follicle-stimulating hormone (FSH). Reverse-transcriptase PCR showed that the mRNA expression of hyaluronan synthase 2 (Has2), a marker of cumulus expansion, was significantly induced in cumulus cells. Subsequent in vitro fertilization and culture showed that more oocytes developed to the blastocyst stage when they were treated with FAK inhibitor 14 during IVM, although the blastocyst total cell number was lower than in oocytes stimulated with FSH. These results indicate that FAK is involved in the maturation of COCs; specifically, phosphorylation at Tyr397 may regulate cumulus expansion via the expression of Has2 mRNA in cumulus cells, which could affect the developmental competence of oocytes.
authors.affiliation: Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ohtake
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sakurai
authors.forename: Yumi
authors.initials: Y
authors.lastname: Hoshino
authors.forename: Kentaro
authors.initials: K
authors.lastname: Tanemura
authors.forename: Eimei
authors.initials: E
authors.lastname: Sato
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation / Q000502:physiology*; D054885:Cumulus Cells / Q000201:enzymology* / Q000502:physiology; D017931:DNA Primers / Q000235:genetics; D047108:Embryonic Development / Q000502:physiology; D005306:Fertilization / Q000502:physiology; D005307:Fertilization in Vitro / Q000379:methods; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism; D000076002:Hyaluronan Synthases; D007150:Immunohistochemistry; D059471:In Vitro Oocyte Maturation Techniques / Q000379:methods*; D051379:Mice; D009865:Oocytes / Q000201:enzymology*; D010766:Phosphorylation; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D014453:Glucuronosyltransferase; C491099:Has2 protein, mouse; D000076002:Hyaluronan Synthases; D051416:Focal Adhesion Protein-Tyrosine Kinases