Abstract
The formation of the hyaluronan-rich cumulus extracellular matrix is crucial for female fertility and accompanied by a transesterification reaction in which the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI)-related proteins are covalently transferred to hyaluronan. Tumor necrosis factor-induced protein-6 (TNFIP6) is essential for this transfer reaction. Female mice deficient in TNFIP6 are infertile due to the lack of a correctly formed cumulus matrix. In this report, we characterize the specificity of TNFIP6-mediated HC transfer from IalphaI to hyaluronan. Hyaluronan oligosaccharides with eight or more monosaccharide units are potent acceptors in the HC transfer, with longer oligosaccharides being somewhat more efficient. Epimerization of the N-acetyl-glucosamine residues to N-acetyl-galactosamines (i.e. in chondroitin) still allows the HC transfer although at a significantly lower efficiency. Sulfation of the N-acetyl-galactosamines in dermatan-4-sulfate or chondroitin-6-sulfate prevents the HC transfer. Hyaluronan oligosaccharides disperse cumulus cells from expanding cumulus cell-oocyte complexes with the same size specificity as their HC acceptor specificity. This process is accompanied by the loss of hyaluronan-linked HCs from the cumulus matrix and the appearance of oligosaccharide-linked HCs in the culture medium. Chondroitin interferes with the expansion of cumulus cell-oocyte complexes only when added with exogenous TNFIP6 before endogenous hyaluronan synthesis starts, supporting that chondroitin is a weaker HC acceptor than hyaluronan. Our data indicate that TNFIP6-mediated HC transfer to hyaluronan is a prerequisite for the correct cumulus matrix assembly and hyaluronan oligosaccharides and chondroitin interfere with this assembly by capturing the HCs of the IalphaI-related proteins.
Highlights
The formation of the hyaluronan-rich cumulus extracellular matrix is crucial for female fertility and accompanied by a transesterification reaction in which the heavy chains (HCs) of inter-␣-trypsin inhibitor (I␣I)-related proteins are covalently transferred to hyaluronan
Our data indicate that Tumor necrosis factor-induced protein-6 (TNFIP6)-mediated HC transfer to hyaluronan is a prerequisite for the correct cumulus matrix assembly and hyaluronan oligosaccharides and chondroitin interfere with this assembly by capturing the HCs of the I␣I-related proteins
In order to investigate the size specificity of the TNFIP6-mediated HC transfer to HA, small oligosaccharides of defined sizes were incubated with mouse serum in the presence of recombinant TNFIP6 at 37 °C for 24 h
Summary
The formation of the hyaluronan-rich cumulus extracellular matrix is crucial for female fertility and accompanied by a transesterification reaction in which the heavy chains (HCs) of inter-␣-trypsin inhibitor (I␣I)-related proteins are covalently transferred to hyaluronan. I␣I and P␣I are covalent complexes of bikunin (the light chain with trypsin inhibitor activity), and either one or two of three heavy chains (HCs) linked through a chondroitin sulfate chain [24] In mice, these serum proteins diffuse into the preovulatory follicle from the capillaries after the LH surge [25], and their HCs incorporate into the cumulus matrix through covalent linkages to HA [26]. We and others have demonstrated that the presence of TNFIP6 is crucial for the transfer of HCs from I␣I to HA [21, 32] This transesterification reaction appears to be necessary for the formation of the COC extracellular matrix. HA oligosaccharides inhibit COC expansion with the same size specificity as the HC transfer, strongly suggesting a direct functional link between the transesterification reaction and the assembly of the cumulus matrix
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