Pre-fertilization zona pellucida hardening by different cross-linkers affects IVF in pigs and cattle and improves embryo production in pigs

Abstract

Zona pellucida (ZP) hardening (resistance to proteolysis) has been classically identified as a post-fertilization event that contributes to the block to polyspermy. Di-(N-succinimidyl)-3,3 0 -dithiodipropionate (DSP), a permeable amine-reactive cross-linker, was recently shown to induce pre-fertilization ZP hardening and to improve porcine IVF productivity. The objectives of this study were to investigate i) how DSP affects pre-fertilization ZP hardening and IVF in cattle, ii) if a non-permeable amine-reactive cross-linker such as bis(sulfosuccinimidyl) suberate (BS3) affects ZP hardening and IVF in cattle and pigs, and iii) whether DSP or BS3, if improvement in IVF productivity was demonstrated in either species, affects in vitro embryo development. Bovine and porcine in vitro matured oocytes were incubated with the cross-linkers (0.06, 0.3, and 0.6 mg/ml) for 30 min. Then they were subjected to ZP digestion or IVF. In cattle, both DSP and BS3 induced ZP hardening and decreased the penetration rate, although monospermy, penetration, or male pronuclear formation was not affected. In pigs, BS3 treatment induced ZP hardening, decreased penetration and male pronuclear formation, and increased monospermy. IVF productivity only improved when porcine oocytes were exposed to DSP. When porcine zygotes derived from this treatment were further cultured in vitro, the cleavage and blastocyst formation rates increased. These results support the idea that mechanisms involved in the prevention of polyspermic fertilization in cattle and pigs have different efficiencies, and ZP hardening induced by DSP cross-linker may be useful for improving porcine embryo production.

Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer (SMGT) technique

“…In vitro oocyte maturation and IVF were made as we have previously described (Canovas et al, 2009), with minor modifications. Groups of 30–40 mature bovine oocytes were transferred to wells containing IVF media and 25 µl of penicillamine–hypotaurine–epinephrine mix was added to each well ∼30 min before insemination.…”

“…For the IVF experiment, the semen samples were processed by a Percoll gradient and incubated with or without exogenous DNA before being used in our standard IVF system (Canovas et al, 2009). In 3 replicates, 237 oocytes in the control group and 215 oocytes in the DNA group were inseminated.…”

What controls polyspermy in mammals, the oviduct or the oocyte?

“…Additionally, intramolecular disulphide bonds in ZP2 and intra‐ and intermolecular disulphide bonds in ZP4 are formed during fertilisation in the cow and pig (Iwamoto et al , 1999; Topfer‐Petersen et al , 2008), probably making the ZP much less porous or accessible to proteolytic enzymes. It also has been observed that treatment of oocytes with a crosslinking reagent [Di‐ (N‐succinimidyl)‐3,3′‐dithiodipropionate (DSP)] which forms bonds between the NH 2 groups of proteins increases ZP resistance to pronase digestion and to sperm penetration in the pig and cow (Coy et al , 2008 b ; Cánovas et al , 2009). For these reasons, the block to polyspermy and the hardening of the ZP have been associated with the formation of new bonds among proteins (Iwamoto et al , 1999; Topfer‐Petersen, Ekhlasi‐Hundrieser & Tsolova, 2008) and with changes in the secondary structure of the zona proteins (Nara et al , 2006) that take place at fertilisation and could modify the accessibility of the ZP receptors for spermatozoa and proteolytic enzymes.…”

Heat stress during in vitro fertilization decreases fertilization success by disrupting anti‐polyspermy systems of the oocytes

“…Following fertilization of the first sperm, the ZP is normally modified by cortical‐granule contents in a process called “hardening,” which prevents the binding and penetration of additional sperm (Vincent et al, ). In some species, artificial induction of ZP hardening can reduce the rate of polyspermy and improve embryonic development (Coy et al, ; Canovas et al, ). According to Edwards et al, (), heat stress during in vitro maturation induced the rate of dispersed cortical granules.…”