Oviduct-specific glycoprotein and heparin modulate sperm–zona pellucida interaction during fertilization and contribute to the control of polyspermy

Abstract

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...

Rat recombinant β-defensin 22 is a heparin-binding protein with antimicrobial activity

“…15 Here, we report that a soluble recombinant protein of b-defensin 22 has antimicrobial and heparin-binding activities, which matches its possession of the defensin-like and lectin-like motifs. 15 Because heparin contributes to sperm chemo-attraction, 16,17 capacitation, 18 acrosome reaction, 19 regulation of sperm-zona pellucida interactions and polyspermy, 20 our findings suggest that b-defensin 22 might function not only in the innate immunity but also in sperm fertilisation abilities via its heparin-binding property.…”

Considerations of viscosity in the preliminaries to mammalian fertilisation

“…However, this does not happen in cow eggs. On the contrary, the resistance of the zona increases due to addition of oviduct fluid molecules [47].…”

Sperm surface changes and physiological consequences induced by sperm handling and storage

“…In some species, additional factors are included, such as negatively charged polysaccharides (e.g. heparin with bull spermatozoa (Parrish et al 1988)), to further enhance stripping of extracellular sperm coating material, possibly mimicking the surface effects elicited by glycosaminoglycans that are present in oviductal fluids (Parrish et al 1989, Coy et al 2008, Killian 2011.…”