SMC2 ablation impairs bovine embryo development shortly after blastocyst hatching

Abstract

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.

Figure 1

Conceptus elongation is impaired in HH3 double-carrier (DC) conceptuses. A) Superovulation protocol to obtain in vivo elongated conceptuses. AI, Artificial Insemination; E, Embryonic day. B) Immunohistochemistry to detect SOX2 (epiblast) and SOX17 (hypoblast); cell nuclei counterstained with DAPI. Representative pictures of tubular NC and HH3 SC conceptuses, and the two HH3 DC embryos obtained at E14. Scale bar: 100 µm for conceptuses; 50 µm for embryonic disc magnifications. C) Representative Sanger sequencing chromatograms from PCR products of conceptuses recovered from a cross of SC individuals. The image on the left corresponds to an NC conceptus (displaying TTC codon), the middle image depicts an SC conceptus (carrying both TTC and TCC –HH3- alleles), and the image on the right shows a DC conceptus (harboring the TCC codon –HH3 allele- in both chromosomes).

Figure 2

Development of cell lineages in D8 blastocysts. A) Representative pictures of WT, edited in-frame (IF), and SCM2 KO blastocysts after immunohistochemistry for SOX2 (ICM) and CDX2 (TE); nuclei counterstained with DAPI. Scale bar: 100 µm. B) Scatter plots of DAPI+, SOX2+, and CDX2+ cell numbers in WT, IF and SCM2 KO blastocysts. Different letters indicate statistically significant differences between groups (mean ± s.e.m.; Kruskal–Wallis test, P < 0.05). C) Representative pictures of D12 structures in C and C+G groups. Dead structures (KO embryos) showing a collapsed blastocoel are indicated with arrows. Scale bar: 500 µm.

Figure 3

Relative telomere length in bovine embryos lacking condensins I and II. A) Relative telomere length in WT, IF, or SMC2 KO D8 blastocysts. B) Relative telomere length in WT, IF, or SMC2 KO D12 structures. C) Relative telomere length in NC, SC, or DC E14 conceptuses for HH3 allele. No significant differences were observed between groups (mean ± s.e.m.; Kruskal–Wallis test, P > 0.05).