Abstract
The egg is a spherical cell encapsulated by the zona pellucida (ZP) which forms a filamentous matrix composed of several glycoproteins that mediate gamete recognition at fertilization. Studies on molecular mechanisms of sperm-egg binding are limited in many mammalian species by the scarcity of eggs, by ethical concerns in harvesting eggs, and by the high cost of producing genetically modified animals. To address these limitations, we have reproduced a three-dimensional (3D) model mimicking the oocyte’s shape, by means of magnetic sepharose beads coated with recombinant ZP glycoproteins (BZP) and cumulus cells. Three preparations composed of either ZP2 (C and N-termini; BZP2), ZP3 (BZP3) or ZP4 (BZP4) were obtained and characterized by protein SDS-PAGE, immunoblot and imaging with confocal and electron microscopy. The functionality of the model was validated by adhesion of cumulus cells, the ability of the glycoprotein-beads to support spermatozoa binding and induce acrosome exocytosis. Thus, our findings document that ZP-beads provide a novel 3D tool to investigate the role of specific proteins on egg-sperm interactions becoming a relevant tool as a diagnostic predictor of mammalian sperm function once transferred to the industry.
Highlights
The egg is a spherical cell encapsulated by the zona pellucida (ZP) which forms a filamentous matrix composed of several glycoproteins that mediate gamete recognition at fertilization
ZP2C and ZP2N glycoproteins showed a molecular weight of 100 kDa, ZP3 reached 55 kDa, and ZP4 was 65 kDa on immunoblots probed with anti-Flag (ZP2C and ZP2N), anti-ZP3 (ZP3) and anti-V5 (ZP4) antibodies (Fig. 1b, Supplementary Material Fig. S1)
The proteins were not detected in the medium confirming that all the glycoprotein secreted in the Chinese Hamster Ovary (CHO) medium was successfully bound to beads
Summary
The egg is a spherical cell encapsulated by the zona pellucida (ZP) which forms a filamentous matrix composed of several glycoproteins that mediate gamete recognition at fertilization. In mouse and human, sperm bind to the N-terminus of ZP220,21 whereas in pigs and cows, ZP3 and/or ZP4 has been implicated in sperm-egg interaction[18,19] This suggests that the role of individual ZP glycoproteins during fertilization may differ among mammals and needs to be investigated independently rather than extrapolating the findings of one species to another. With individual zona glycoproteins were studied: 1) for their ability to support adhesion of in vitro matured porcine cumulus cells; 2) their potential to bind spermatozoa; 3) their ability to induce acrosome exocytosis; and 4) determine if these interactions were affected by the protocol used for sperm capacitation This system recreates a 3D environment of ovulated eggs that is scalable and will offer insights into molecular mechanisms of gamete recognition in mammals
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