198 FUNCTIONAL EVIDENCE FOR THE EXISTENCE OF AN OVIDUCTAL FACTOR THAT INDUCES ZONA PELLUCIDA HARDENING AND REGULATES POLYSPERMY IN THE PIG AND COW

Abstract

Many differences between in vivo and in vitro fertilization (IVF) efficiency in mammals are related to the differences between IVF media and oviductal fluid. One of the best known examples is the frequency of polyspermy observed under in vitro conditions in cattle (Roh S et al. 2002 J. Vet. Med. Sci. 64, 667–671) and, in particular, in pigs (Coy P and Romar R 2002 Reprod. Fertil. Dev. 14, 275–286). Zona pellucida (ZP) resistance to pronase digestion (ZP hardening) has been considered as a postfertilization event contributing to the block of polyspermy in mammals (Green D 1997 Rev. Reprod. 2, 147–156). However, pig and cow unfertilized ovulated oocytes show a ZP hardening of hours or days (Katska L et al. 1999 Reprod. Dom. Anim. 34, 255–259; Kolbe T and Holtz W 2005 Theriogenology 63, 1695–1705) compared with the minutes or seconds observed in the in vitro-matured oocytes, even after fertilization (Coy P et al. 2002 Reproduction 124, 279–288; Coy P et al. 2005 Reproduction 129, 19–26). Consequently, we propose the existence of an oviductal factor that induces ZP hardening before any contact of the oocyte with the sperm, thus regulating polyspermy. Porcine and bovine oviductal fluid was obtained by aspiration of oviducts collected at the slaughterhouse and stored frozen. In vitro-matured porcine and bovine oocytes were incubated for 30 min in the oviductal fluid, washed thoroughly in fresh medium, and either assessed for ZP digestion time or in vitro fertilized. The results, analyzed by ANOVA, showed a very strong ZP hardening in oviductal-treated oocytes (2866.83 ± 94.4 s in the pig and 4301.1 ± 441.7 s in the cow) compared with control oocytes (63.5 ± 2.9 s in the pig and 124.2 ± 5.9 s in the cow). Moreover, the percentage of monospermy for the oviductal-treated oocytes was significantly higher in both species (50.0 ± 10.0% in the pig and 91.7 ± 3.0% in the cow) compared with the control groups (5.56 ± 3.8% in the pig and 80.8 ± 3.5% in the cow). Percentage penetration did not change in porcine oocytes but decreased in bovine oocytes (58.1 ± 3.3 v. 38.4 ± 3.3, P ≤ 0.001), whereas the mean number of sperm per oocyte decreased for the porcine-treated oocytes (2.7 ± 0.2 v. 8.2 ± 0.4, P ≤ 0.001) and did not change for the bovine oocytes. These results support the hypothesis that an oviductal factor induces ZP hardening, contributing to the control of polyspermy in the pig and cow, and that it could be used to improve the output of IVF.

Supported by MEC and FEDER (AGL2006-03495).