Hardening of the zona pellucida of unfertilized eggs can reduce polyspermic fertilization in the pig and cow

Abstract

One of the proposed mechanisms of polyspermy block is an increased resistance of the zona pellucida (ZP) to proteolytic digestion (ZP hardening) as a consequence of cortical granule exocytosis that occurs soon after fertilization. However, evidence is available that the zonae pellucidae of freshly ovulated pig and cow oocytes harden considerably before fertilization. It was thought that such pre-fertilization ZP hardening could be involved in the control of polyspermy, and its lack in the oocytes matured in vitro could be one of the reasons for the extremely high incidence of polyspermy in pig in vitro fertilization (IVF). To test this hypothesis, two different types of cross-linking reagents were employed and their effects on ZP hardening and IVF efficiency were examined. The sulfhydryl-reactive cross-linkers produced a slight hardening of ZP (P!0.001) of treated oocytes compared with control oocytes, and totally inhibited sperm penetration into pig oocytes after IVF. In the cow, sperm penetration into eggs was reduced to 10%. It is proposed that formation of disulfide bonds in ZPor blocking of SH groups in the oocyte plasma membrane proteins prevents sperm penetration. An amine-reactive cross-linker was then assayed and produced strong ZP hardening, increasing the incidence of monospermy in both pig and cow oocytes after fertilization. When the cross-linker concentration was optimized, a 45% improvement for pig IVF efficiency was reached. It is proposed that the observed physiological ZP hardening is a mechanism to control polyspermy, differentially affecting various mammalian species and can be imitated by the use of amine-reactive cross-linkers during IVF.

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

“…Hence, the oviductal-acquired resistance of the ZP seems to be a relatively common event, not species specific, as shown in sheep, rabbit, cow, and pig oviducts (8,31). In mice or hamster, however, the ZP resistance to proteinases is not acquired in the oviduct (10,32), but the postfertilization increased resistance of the ZP to pronase arises from the cortical reaction (3,33,34).…”

“…Unless otherwise indicated, all chemicals and reagents were purchased from Sigma-Aldrich Química. The medium used for oocyte maturation in pig was NCSU-37 supplemented as described previously (8). The basic medium used for IVF in the pig, designated as TALPp medium, consists of 114.06 mM NaCl, 3.2 mM KCl, 8 mM Ca-lactate.5H2O, 0.5 mM MgCl2.6H2O, 0.35 mM NaH2PO4, 25.07 mM NaHCO3, 10 mM Na-lactate, 1.1 mM Na-pyruvate, 5 mM glucose, 2 mM caffeine, 3 mg/ml BSA-FAF(A-6003), 1 mg/ml PVA, and 0.17 mM kanamycin sulfate.…”

“…The basic medium used for IVF in the pig, designated as TALPp medium, consists of 114.06 mM NaCl, 3.2 mM KCl, 8 mM Ca-lactate.5H2O, 0.5 mM MgCl2.6H2O, 0.35 mM NaH2PO4, 25.07 mM NaHCO3, 10 mM Na-lactate, 1.1 mM Na-pyruvate, 5 mM glucose, 2 mM caffeine, 3 mg/ml BSA-FAF(A-6003), 1 mg/ml PVA, and 0.17 mM kanamycin sulfate. The in vitro maturation medium for cow was TCM-199 with Earle's salts, supplemented as described previously (8). The IVF medium for this species was designated as TALPb and consists of 114 mM NaCl, 3.2 mM KCl, 10 mM Na lactate, 0.5 mM MgCl2 6H2O, 0.3 mM NaH2PO4 H2O, 25 mM NaHCO3, 0.2 mM Na-pyruvate, 2 mM CaCl2.2H2O, 6 mg/ml BSA-FAF(A-6003), 1.75 units/ml heparin, 50 units/ml penicillin, and 50 g/ml streptomycin.…”

“…Methods for in vitro maturation in pig and cow were those described previously (8). Ovaries from Landrace ϫ Large White gilts and Charolais, Limousine, and Simmental heifers were collected at the slaughterhouse.…”

“…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)(6)(7)(8), contrast with the much shorter resistance (seconds to minutes) exhibited by ZP of mouse or hamster oviductal oocytes before fertilization (9,10). Similarly, ZP from oocytes of follicular, preovulatory origin and from in vitro-matured (IVM) porcine and bovine oocytes before and even after fertilization (8,(11)(12)(13) show shorter digestion times (i.e., resistance to pronase). These observations prompted us to hypothesize whether the ZP in ungulates could be undergoing modifications during transit in the oviduct (before fertilization) that affect its resistance to pronase digestion and consequently its interaction with the sperm, and whether this may represent an additional mechanism to control polyspermy, different from the changes brought about by the cortical reaction.…”