Inhibitors of zinc-dependent metalloproteases hinder sperm passage through the cumulus oophorus during porcine fertilization in vitro.
In this study, we report for the first time on a possible contribution of metalloproteases in sperm passage through the cumulus matrix in pigs. The presence of 20 μM 1,10-phenanthroline (1,10-PHEN), inhibitor of zinc-dependent metalloproteases, strongly inhibited the degree of sperm penetration in cumulus-intact (CI), but not in cumulus-free (CF), porcine oocytes during IVF. The inhibitory effect of 1,10-PHEN was due to the chelation of metal ions as a non-chelating analog (1,7-PHEN) did not affect IVF rates. Furthermore, incubation with 1,10-PHEN did not affect sperm binding to the zona pellucida nor sperm motility, membrane integrity, or acrosomal status. These findings led to the assumption that 1,10-PHEN interacts with a sperm- or cumulus-derived metalloprotease. Metalloproteases are key players in physiological processes involving degradation or remodeling of extracellular matrix. In vivo, their proteolytic activity is regulated by tissue inhibitors of metalloproteases (TIMP1-TIMP4). We tested the effect of TIMP3 on fertilization parameters after porcine IVF. Similar to 1,10-PHEN, TIMP3 inhibited total fertilization rate of CI but not CF oocytes and did not influence sperm quality parameters. Although the inhibitory effect was stronger in CI oocytes, TIMP3 also reduced the degree of sperm penetration in CF oocytes, suggesting the involvement of a metalloprotease in a subsequent step during fertilization. In conclusion, our results indicate the involvement of TIMP3-sensitive, zinc-dependent metalloprotease activity in sperm passage through the cumulus oophorus in pigs. The results should provide the basis for further biochemical research toward the localization and identification of the metalloprotease involved.
Department Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium. josine.beek@ugent.be
J
J
Beek
H
H
Nauwynck
D
D
Maes
A
A
Van Soom
D000177:Acrosome / Q000502:physiology; D000818:Animals; D054885:Cumulus Cells / Q000502:physiology; D005260:Female; D005307:Fertilization in Vitro; D008297:Male; D045726:Metalloproteases / Q000378:metabolism*; D009865:Oocytes / Q000502:physiology*; D010618:Phenanthrolines; D013081:Sperm Motility; D013084:Sperm-Ovum Interactions; D013552:Swine / Q000378:metabolism*; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D010618:Phenanthrolines; D019717:Tissue Inhibitor of Metalloproteinase-3; D045726:Metalloproteases; D015032:Zinc; C025205:1,10-phenanthroline