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Epidermal Growth Factor-Receptor Tyrosine Kinase Activity Regulates Expansion of Porcine Oocyte-Cumulus Cell Complexes In Vitro¹

^a Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech Republic ^b Department of Molecular and Cellular Biology, University of California at Berkeley, Berkeley, California 94720

We have recently shown that epidermal growth factor (EGF) strongly stimulates expansion of porcine oocyte-cumulus complexes (OCCs) isolated from large follicles (>6 mm) and does not promote expansion of OCCs from small (3–4-mm) follicles. In order to elucidate the role of EGF in OCCs expansion, in the present study, we first examined the presence of EGF receptors (EGFRs) in cumulus cells isolated from follicles of different sizes. Surprisingly, immunoblotting showed that cumulus cells obtained from all follicular size categories contained similar amounts of EGFR protein. On the other hand, we found a dramatic difference in the pattern of protein tyrosine phosphorylation in a comparison of cumulus cells isolated from small and large follicles treated by EGF. Furthermore, tyrosine-phosphorylated EGFR was specifically immunoprecipitated with antiphosphotyrosine antibodies from EGF-treated cumulus cells isolated from the large follicles. This result strongly indicates that only OCCs from the large follicles contain mature EGFRs that are capable of becoming activated by EGF. Remarkably, preincubation of cumulus cells from small follicles (3–4 mm) with FSH strongly increased EGF-stimulated tyrosine phosphorylation to levels comparable with OCCs from large follicles. The FSH-dependent activation of EGFRs was beneficial for expansion of OCCs isolated from the small follicles since OCCs treated sequentially by FSH (3 h) and EGF (1 h) underwent expansion significantly better then OCCs cultured in FSH or EGF alone. We conclude that a FSH-dependent pathway has an important role in the maturation of the EGFR in cumulus cells and that activation of EGFR-dependent signaling is sufficient to induce expansion.

¹ This work was supported by grants 524/01/0903 from Grant Agency of the Czech Republic, A5045102 from Grant Agency of the Academy of Sciences of the Czech Republic, and LN 00A065 from Czech Ministry of Education.

² Correspondence: Radek Prochazka, Academy of Sciences of the Czech Republic, Institute of Animal Physiology and Genetics, Rumburska 89, 277 21 Libechov, Czech Republic. FAX: 420 315 69 71 86; e-mail: prochazka{at}iapg.cas.cz