Department of Cell Biology and Histology, School of Medicine, University of Murcia, Campus Mare Nostrum and IMIB-Arrixaca, Murcia, Spain.
Department of Physiology, Faculty of Veterinary, University of Murcia, Campus Mare Nostrum and IMIB-Arrixaca, Murcia, Spain.
Reproduction. 2020 Nov;160(5):725-735. doi: 10.1530/REP-20-0123.
The role of specific zona pellucida (ZP) glycoproteins in gamete interaction has not yet been elucidated in many species. A recently developed 3D model based on magnetic sepharose beads (B) conjugated to recombinant ZP glycoproteins (BZP) and cumulus cells (CBZP) allows the study of isolated ZP proteins in gamete recognition studies. The objective of this work was to study the role of porcine ZP2, ZP3 and ZP4 proteins in sperm binding, cumulus cell adhesion and acrosome reaction triggering. ZP protein-bound beads were incubated with fresh ejaculated boar spermatozoa and isolated cumulus cells for 24 h. The number of sperm bound to the beads, the acrosomal shrouds (presence of acrosomal content) on the bead's surface, and the acrosome integrity (by means of PNA-FITC lectin) in bound and unbound sperm were studied. Finally, in vitro matured porcine oocytes mixed with BZP2 were inseminated in vitro using fresh sperm and fertilisation results evaluated. Over 60% of beads had at least one sperm bound after 2 h of coincubation. ZP2-beads (BZP2) and cumulus-ZP2-bead complexes (CBZP2) reached the highest number of sperm per bead, whereas BZP3 and BZP4 models showed the highest number of unbound reacted sperm cells and acrosomal shrouds. Fertilisation efficiency and monospermy rate increased when oocytes were fertilised in the presence of BZP2. We, therefore, conclude that in pigs, it is mainly ZP2 that is involved in sperm-ZP binding whereas ZP3 and ZP4 induce acrosome reaction. Using magnetic sepharose ZP2-bound beads might be a valuable tool to improve the fertilisation rate in pigs.
许多物种的精卵相互作用中，特定透明带（ZP）糖蛋白的作用尚未阐明。最近开发的一种基于磁琼脂糖珠（B）与重组 ZP 糖蛋白（BZP）和卵丘细胞（CBZP）偶联的 3D 模型，允许在配子识别研究中研究分离的 ZP 蛋白。本研究的目的是研究猪 ZP2、ZP3 和 ZP4 蛋白在精子结合、卵丘细胞黏附和顶体反应触发中的作用。将 ZP 蛋白结合珠与新鲜射出的公猪精子和分离的卵丘细胞孵育 24 小时。研究了珠表面结合的精子数量、珠表面的顶体被（存在顶体内容物）和结合和未结合精子的顶体完整性（通过 PNA-FITC 凝集素）。最后，将与 BZP2 混合的体外成熟的猪卵母细胞与新鲜精子混合，在体外受精，并评估受精结果。在 2 小时共孵育后，超过 60%的珠上至少有一个精子结合。ZP2 珠（BZP2）和卵丘-ZP2 珠复合物（CBZP2）达到了每个珠上精子数量最多，而 BZP3 和 BZP4 模型显示了最高数量的未结合反应精子和顶体被。当卵母细胞在 BZP2 存在下受精时，受精效率和单精子受精率增加。因此，我们得出结论，在猪中，主要是 ZP2 参与精子-ZP 结合，而 ZP3 和 ZP4 诱导顶体反应。使用磁琼脂糖 ZP2 结合珠可能是提高猪受精率的有价值工具。
