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Covalent Transfer of Heavy Chains of Inter--Trypsin Inhibitor Family Proteins to Hyaluronan in In Vivo and In Vitro Expanded Porcine Oocyte-Cumulus Complexes¹

Institute of Animal Physiology and Genetics,³ Libechov, Czech Republic Department of Public Health and Cell Biology,⁴ Faculty of Medicine, University of Rome, Rome, Italy

Previous studies have shown that the heavy chains (HCs) of serum-derived inter-alpha-trypsin inhibitor (II) molecules become covalently linked to hyaluronan (HA) during in vivo mouse cumulus expansion and significantly contribute to cumulus matrix organization. Experiments with mice suggest that the incorporation of such proteins in cumulus matrix appears to be rather complex, involving LH/hCG-induced changes in blood-follicle barrier and functional cooperation between cumulus cells, granulosa cells, and oocyte within the follicle. We demonstrate here that HC-HA covalent complexes are formed during in vivo porcine cumulus expansion as well. Western blot analysis with II antibody revealed that follicular fluids from medium-sized follicles and those from large follicles unstimulated with hCG contain high levels of all forms of II family members present in pig serum. The same amount of HCs were covalently transferred from II molecules to HA when pig oocyte-cumulus complexes (OCCs) were stimulated in vitro with FSH in the presence of pig serum or follicular fluid from unstimulated or hCG-stimulated follicles. In addition, HC-HA coupling activity was stimulated in cumulus cells by FSH treatment also in the absence of oocyte. Collectively, these results indicate that II molecules can freely cross the blood follicle barrier and that follicular fluid collected at any stage of folliculogenesis can be successfully used instead of serum for improving OCC maturation. Finally, pig cumulus cells show an autonomous ability to promote the incorporation of II HCs in the cumulus matrix.

¹ Supported as follows: Collaboration via a fellowship (E.N.) under the OECD Cooperative Research Programme: Biological Resource Management for Sustainable Agricultural Systems, grant A 504 5102/01 from Grant Agency of the Academy of Sciences of the Czech Republic (E.N.), grant 523/04/0574 from Grant Agency of the Czech Republic (R.P.), and grant 2003 from Ministero Italiano dell'Istruzione, dell'Università e della Ricerca Scientifica (A.S.).

² Correspondence: Nagyova Eva, Academy of Sciences of the Czech Republic, Institute of Animal Physiology and Genetics, Rumburska 89, 277 21 Libechov, Czech Republic. FAX: +420 315 63 95 10; nagyova{at}iapg.cas.cz