Mammalian spermatozoa and cumulus cells bind to a 3D model generated by recombinant zona pellucida protein-coated beads

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.

Sperm‐Binding Assay Using an In Vitro 3D Model of the Mammalian Cumulus‐Oocyte Complex

“…Once the protein is incubated with the magnetic Sepharose beads (Basic Protocol 1), both correct protein expression and complete conjugation to the beads are confirmed with a quality-control step (Basic Protocol 2). If the FLAG peptide tag has been correctly incorporated into the sequence, the protein should be detected in SDS-PAGE electrophoresis and western blot, as shown in 'figure 2A' of Hamze et al (2019). In this case, three lanes can be observed (as the ones described in Basic Protocol 3): (1) the medium containing the secreted recombinant protein before its conjugation; (2) the beads once the conjugation process is completed; and (3) the medium where the beads are conjugated after the overnight co-incubation is finished.…”

“…If the protein was successfully conjugated to the beads, a positive signal in the western blot is expected for samples 'a'and 'b', but not for sample 'c', thus indicating that all available protein has been bound to the beads during the conjugation process (Hamze et al, 2019).…”

“…The model has one internal layer of recombinant protein over a 3D spherical shape, and, potentially, a second layer of cumulus oophorus cells, thus mimicking the native cumulusoocyte complex. The potential applications of this model are wide, since it can be used to study gamete interaction, aiding in the characterization of how the sperm interact with a protein over a three-dimensional surface (Hamze et al, 2019), to improve in vitro fertilization efficiency rates by adding the model when native gametes are being co-incubated (Hamze et al, 2020a), and as a tool to predict sperm fertilizing capacity (Hamze et al, 2020a).…”

“…The choice of protein expression system (Strategic Planning) is essential to ensure the functionality of the designed recombinant protein. If the protein is a mammalian glycoprotein, it is advisable to use a mammalian cell line such as CHO to ensure proper glycosylation, as described in this work and in our previous studies with porcine ZP recombinant proteins (Hamze et al, 2019(Hamze et al, , 2020b and bovine IZUMO1 receptor (JUNO protein; Hamze et al, 2020a). Moreover, when designing the protein construct, it is recommended to add the histidine tail (6×His) at the N-or C-terminus, to try to avoid altering protein domains.…”

“…Thanks to the ability of this model to decoy sperm, once the beads are coated with proteins (and cumulus cells, if desired), they can be subjected to sperm-binding assays, with the objective of gaining knowledge of the molecular basis of gamete interaction, selecting sperm with the highest fertilizing ability, or even predicting sperm fertility in the future (Hamze et al, 2019(Hamze et al, , 2020a(Hamze et al, , 2020b.…”