The human is an exception to the evolutionarily-conserved phenomenon of pre-fertilization zona pellucida resistance to proteolysis induced by oviductal fluid

Abstract

Is zona pellucida (ZP) resistance to proteolysis, induced by oviductal fluid (OF), a mechanism common to species other than the pig and cow? ZP resistance to proteolysis induced by OF was observed in the mouse, rat, hamster, rabbit, sheep, goat, pig and cow, but not in humans. Oviductal ZP resistance to proteolysis occurs in the pig and cow where it influences the incidence of fertilization and polyspermy. The effect is observed after incubation of ZP in OFs from pig (pOF), cow (cOF), rabbit (rOF) and sheep (sOF). Oocytes from nine different species, including ungulates, rodents, lagomorphs and primates were incubated in rOF, sOF, gOF, cOF, pOF and human oviductal fluid (hOF). ZP digestion times for the matured oocytes of these nine species, without any treatment or incubated in 5 (mouse, rat, hamster, rabbit, cow, ewe and goat) or 6 (pig and humans) of the OFs collected were compared using three replicates per treatment and at least three oocytes per replicate. In vivo matured oocytes from rat, hamster, mouse, rabbit and humans, in vitro matured oocytes from cow, goat, ewe and pig and rOF, cOF, gOF, sOF, pOF and human (hOF) were collected and processed for the study. Oocytes from each species were incubated in the different OFs for 30 min. The resistance of the ZP of the oocytes to enzymatic digestion in a pronase solution (0.5% in PBS) was measured and registered as ZP digestion time. rOF increased ZP resistance to proteolytic digestion in the range of between 96 and 720 h for any of the species tested, whereas the corresponding increase in human ZP was only 1 min. OFs from the remaining species also had a significant effect, with variations among the cross-species experiments (P < 0.05). hOF, which was only tested on human and porcine oocytes, had no effect on ZP chemical hardening. Measurements of ZP digestion times are not of extreme accuracy and errors of a few seconds can be assumed in the experimental data. However, when differences are in the range of hours among treatments, variations measured in seconds do not alter the robustness of the findings. Human oocytes and OF were of limited access, compared with oocytes from species collected in slaughterhouses. OFs from mouse, rat and hamster were not tested due to the small size of the genital tract in these species and the small volume of fluid available. Since oviductal modification of ZP resistance to proteolytic digestion has been demonstrated to influence fertilization and this pre-fertilization mechanism is considered to contribute to the control of polyspermy, the apparent absence of this mechanism in humans suggests that the regulation of polyspermy depends mainly on other mechanisms, most probably of cortical granule origin. Investigation into a possible relationship between the lack of oviductal ZP hardening in human oocytes and the existence of tubal ectopic pregnancies in this species is proposed. This work was supported by the Spanish Ministry of Science and Innovation and FEDER, Grant AGL2009-12512-C02-01-02. The authors declare no competing interest.