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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.
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Department of Cell Biology and Histology, Universidad de Murcia. International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), Murcia, Spain
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In brief
Bovine embryos lacking SMC2 (a core component of condensins I and II) are unable to survive maternal recognition of pregnancy. SMC2 KO embryos are able to form blastocysts, exhibiting a reduced cell proliferation ability, and arrest their development shortly after hatching.
Abstract
Condensins are large protein complexes required for chromosome assembly and segregation during mitosis and meiosis. Mouse or bovine embryos lacking SMC2 (a core component of condensins I and II) do not complete development to term, but it is unknown when they arrest their development. Herein, we have assessed the developmental ability of bovine embryos lacking SMC2 due to a naturally occurring mutation termed HH3 (Holstein Haplotype 3) or by CRISPR-mediated gene ablation. To determine if embryos homozygous for the HH3 allele survive to maternal recognition of pregnancy, embryonic day (E)14 embryos were flushed from superovulated carrier cows inseminated with a carrier bull. Mendelian inheritance of the HH3 allele was observed at E14 conceptuses but conceptuses homozygous for HH3 failed to achieve elongation and lacked an embryonic disc. To assess the consequence of the ablation of condensins I and II at earlier developmental stages, SMC2 KO bovine embryos were generated in vitro using CRISPR technology. SMC2 KO embryos were able to form blastocysts but exhibited reduced cell proliferation as evidenced by a significantly lower number of total, trophectoderm (CDX2+), and inner cell mass (SOX2+) cells at Day (D) 8 post-fertilization compared to their WT counterparts and were unable to survive to D12 in vitro. SMC2 ablation did not alter relative telomere length at D8, D12, or E14. In conclusion, condensins I and II are required for blastomere mitosis during early development, and embryos lacking those complexes arrest their development shortly after blastocyst hatching.
