Covalent Transfer of Heavy Chains of Inter-{alpha}-Trypsin Inhibitor Family Proteins to Hyaluronan in In Vivo and In Vitro Expanded Porcine Oocyte-Cumulus Complexes
Nagyova E, et al 2004 .
Previous studies have shown that the heavy chains (HCs) of serum-derived inter-alpha-trypsin inhibitor (IalphaI) 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 IalphaI antibody revealed that follicular fluids from medium size follicles and those from large follicles unstimulated with hCG contain high levels of all forms of IalphaI family members present in pig serum. The same amount of HCs were covalently transferred from IalphaI 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 IalphaI molecules can freely cross the blood follicle barrier and that follicular fluid collected at any stage of folliculogenesis can be successfully utilized instead of serum for improving OCC maturation. Finally, pig cumulus cells show an autonomous ability to promote the incorporation of IalphaI HCs in the cumulus matrix.
Formation of the Ovarian Follicular Antrum and Follicular Fluid. Rodgers RJ et al. The formation of the follicular antrum and fluid has received scant attention from researchers and yet both are important processes in follicular development. The central hypothesis on follicular fluid formation suggests that production by granulosa cells of hyaluronan and the chondroitin sulfate proteoglycan versican generates an osmotic gradient. This gradient draws in fluid derived from the thecal vasculature. Inter-alpha-trypsin inhibitor is also present in follicular fluid of species with large follicles, and inter-alpha-trypsin inhibitor and versican could additionally bind or cross-link with hyaluronan, resulting in the retention of these molecules within the follicular antrum. In mice inter-alpha-trypsin inhibitor, derived from serum, is not present in the follicular antrum until after the LH surge showing that there are clearly species differences. Barriers to the movement of fluid across the membrana granulosa are apparently minimal as even relatively large serum proteins are present in follicular fluid. Despite the relative permeability of the follicular wall aquaporins are present in granulosa cells and could be actively involved in the transport of water into the follicle. The formation of an antrum also requires movement of granulosa cells relative to each other to allow the fluid to accumulate. This presumably involves remodeling of cell-cell junctions and in species with small follicles may involve death of centrally located granulosa cells. Remodeling of the stroma and thecal layers also accompanies growth and expansion of the antrum, and presumably involves similar processes that accompany growth of other glands.