Polyspermy is an important anomaly of fertilization in placental mammals, causing premature death of the embryo. It is especially frequent under in vitro conditions, complicating the successful generation of viable embryos. A block to polyspermy develops as a result of changes after sperm entry (i.e., cortical granule exocytosis). However, additional factors may play an important role in regulating polyspermy by acting on gametes before sperm-oocyte interaction. Most studies have used rodents as models, but ungulates may differ in mechanisms preventing polyspermy. We hypothesize that zona pellucida (ZP) changes during transit of the oocyte along the oviductal ampulla modulate the interaction with spermatozoa, contributing to the regulation of polyspermy. We report here that periovulatory oviductal fluid (OF) from sows and heifers increases (both, con-and heterospecifically) ZP resistance to digestion with pronase (a parameter commonly used to measure the block to polyspermy), changing from digestion times of Ϸ1 min (pig) or 2 min (cattle) to 45 min (pig) or several hours (cattle). Exposure of oocytes to OF increases monospermy after in vitro fertilization in both species, and in pigs, sperm-ZP binding decreases. The resistance of OF-exposed oocytes to pronase was abolished by exposure to heparin-depleted medium; in a medium with heparin it was not altered. Proteomic analysis of the content released in the heparin-depleted medium after removal of OFexposed oocytes allowed the isolation and identification of oviduct-specific glycoprotein. Thus, an oviduct-specific glycoproteinheparin protein complex seems to be responsible for ZP changes in the oviduct before fertilization, affecting sperm binding and contributing to the regulation of polyspermy.sperm-oocyte interaction ͉ oviductal fluid ͉ ZP hardening P olyspermy (the penetration of the egg cytoplasm by more than one spermatozoa) is a pathologic condition in placental mammals, usually causing early death of the embryo (1). Although the prevalence of polyspermy under natural conditions is moderate, in in vitro fertilization (IVF) systems polyspermy remains a major obstacle to successful development of viable embryos in different species, including humans (2). Mechanisms underlying the block to polyspermy in mammals have been partially uncovered and characterized, mainly with use of rodents as animal models and usually related to events occurring after sperm entry into the oocyte.The entrance of the spermatozoon into the oocyte's cytoplasm induces the release of cortical granule contents, which modify the vitelline membrane, the zona pellucida (ZP), or both, rendering the oocyte refractory to additional sperm binding and penetration (3) and ending in changes in the mechanical properties and resistance to protease throughout the ZP (4). Yet assuming strong similarities in fertilization mechanisms among rodents and ungulates, observations in ovulated unfertilized porcine and bovine oocytes, showing that ZP resistance to pronase lasts from hours to days (5-8), contras...

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Oviduct fluid increases the time required for digestion of the zona pellucida (ZP) by proteolytic enzymes (ZP hardening). This effect has been associated with levels of monospermy after in vitro fertilization (IVF) in the pig and cow, but the possible existence of a directly proportional relationship between hardening and monospermy remains unknown. To investigate whether variations in hardening of different oviductal fluids (OFs) are correlated with variations in levels of monospermy after IVF, porcine oocytes were incubated with three batches of OFs known to produce different ZP hardening effects (3, 7, and 25 min); after IVF, monospermy levels were 0%, 14.58% ± 5.14%, and 35.14% ± 7.95%, respectively. These results could partially explain the lack of polyspermy found during in vivo fertilization in pigs (with a hardened oviductal ZP) compared with levels found during IVF (with no hardened ZP). Using the bovine model, OF was fractionated by heparin affinity chromatography, and the hardening effect on the ZP was tested for each fraction obtained from a linear gradient of sodium chloride concentration. The highest effect was obtained with the fraction eluted with 0.4 M sodium chloride. Fractions with high-level or low-level effects were processed by on-chip electrophoresis and high-performance liquid chromatography-tandem mass spectrometry. A list of potential proteins responsible for this effect includes OVGP1 and members of the HSP and PDI families.

The mammalian oviduct is an anatomical part of the female reproductive tract, which plays several important roles in the events related to fertilization and embryo development. This review examines and compares several studies related to the proteomic and transcriptomic profile of the oviduct in different domestic animals. This information could be important for clarifying the role of oviductal factors in different events regulating fertilization and early embryo development, as well as for improving synthetic media for in vitro maturation/in vitro fertilization/embryo culture techniques (IVM/IVF/EC).

Our results describe a novel mechanism for the success or failure of fertilization in mammals, by which molecules present in the oviductal environment are activated by molecules originating within the gametes. We anticipate that therapeutic up- or down-regulation of this physiological mechanism may be used to help in conception or as a contraceptive tool. Since components of the PLG-PLA system are already available as drugs for heart attacks or cancer therapies, basic research on this novel function would be rapidly transferable for clinical application.

Plasminogen is present in the oviduct, on the zona pellucida (ZP) and on oolemma, and reduces the number of sperm penetrating the oocyte during in vitro fertilization in pig and cow. It is unknown how this reduction occurs. We tested whether plasminogen (1) changed the ZP resistance to enzymatic digestion thus making the passage of the spermatozoa across it difficult; (2) reduced the sperm functionality, assessed by sperm viability, motility, spontaneous acrosome reaction and membrane lipid disorder; or (3) affected the sperm-ZP binding before or after sperm-ZP interaction. The mechanism by which plasminogen/plasmin system contributes to regulate sperm entry into the oocyte is not inducing a ZP hardening or a decrease in sperm functionality but detaching more than 50% of sperm bound to the ZP. It is suggested that the fertilizing spermatozoon activates plasminogen into plasmin at the oocyte surface and that plasmin removes additional spermatozoa attached to the ZP.

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.

This study was designed to determine whether calreticulin (CRT), a chaperone protein, is present in in vitro-matured (IVM) pig oocytes and to study its potential role in the block to polyspermy. Western blot analysis, using an anti-CRT antibody, of oocyte lysate showed an immunoreactive band of w60 kDa. Simultaneous labeling of IVM oocytes with anti-CRT antibody and peanut agglutinin lectin (PNA lectin, a porcine cortical granules (CG)-specific binding lectin) revealed localization of CRT in the subplasmalemmal region with a 27.7% colocalization with PNA staining. After IVF, PNA labeling was not observed and anti-CRT labeling decreased significantly in zygotes and disappeared in two-cell embryos. Western blot analysis of oocyte exudate obtained from zona pellucida (ZP)-free oocytes activated with calcium ionophore confirmed the presence of a band that reacted with an anti-CRT antibody. Anti-CRT antibody and PNA labeling were not observed in activated oocytes despite being detectable in non-activated oocytes. The presence of CRT in vesicles located under the oolemma was demonstrated using immunogold cytochemistry at the ultrastructural level. To study the role of CRT in fertilization, ZP-enclosed and ZP-free oocytes were incubated with exogenous CRT and then inseminated. Whereas ZP-free oocytes showed fewer penetrating sperm and lower polyspermy rates than untreated oocytes, the opposite effect was observed in ZP-enclosed oocytes. In conclusion, CRT is confined to subplasmalemmal vesicles partially overlapping with CG contents. Its exocytosis after the oocyte activation seems to participate in the membrane block to polyspermy in pigs but is not involved in the ZP block.