3 ID2 is not required for bovine blastocyst formation

The first lineage differentiation in the mammalian embryo precedes blastocyst formation and gives rise to the inner cell mass (ICM) and the trophectoderm (TE), the first extra-embryonic lineage. This differentiation event is regulated by specific transcription factors (TFs) that have been thoroughly studied in the mouse model, but the roles of key regulators such as OCT4, CDX2, and TEAD4 are not conserved in other mammals, including ungulates. Inhibitor of DNA binding 2 (ID2) is one of the earliest markers of inner and outer cells in the mouse blastocyst, and it is also expressed exclusively in the ICM of bovine embryos according to transcriptional data. Although Id2 ablation in mice does not prevent first lineage differentiation and blastocyst formation, it is possible that—given the dispensable role of other TFs—ID2 may play a relevant role in bovine ICM commitment. To test that hypothesis, we analyzed the developmental ability of bovine embryos lacking ID2 (ID2 KO) generated by CRISPR technology. Bovine IVM oocytes were microinjected with Cas9-encoding mRNA and an sgRNA against ID2 (C+G group, containing KO embryos) or with Cas9-encoding mRNA alone (C group, formed by wild-type [WT] embryos) as a microinjection control. Microinjected oocytes were fertilized in vitro and allowed to develop to Day 8 (D8) blastocysts in conventional culture, at which point they were fixed. Fixed specimens and subjected to immunohistochemistry to detect ICM (SOX2⁺) and TE (CDX2⁺) cells and finally genotyped by miSeq to identify KO embryos in the C+G group. Cleavage and blastocyst rates out of microinjected oocytes were similar between microinjection groups (for C and C+G groups, respectively: cleavage, 72.7 ± 4.9% vs. 69.4 ± 2%; blastocyst 23.3 ± 3.4% vs. 18.9 ± 4.5%; t-test P > 0.05). In C+G group ~55% (21/38) of the blastocysts genotyped were edited and ~29% (11/38) were KO (i.e. contained only KO alleles generated by frame-disrupting indels). ID2 KO blastocysts were morphologically normal and displayed a similar diameter to their edited non-KO or KO counterparts (WT and edited non-KO and KO, respectively: 240 ± 12 µm vs. 215 ± 13 µm vs. 204 ± 21 µm; ANOVA P > 0.05). Cell numbers were numerically reduced in KO embryos, but no significant differences were observed (total cells, 126 ± 10 vs. 100 ± 10 vs. 88 ± 13; ICM, 37 ± 4 vs. 31 ± 4 vs. 22 ± 5; TE, 88 ± 7 vs. 78 ± 7 vs. 66 ± 12 for WT and edited non-KO and KO; ANOVA P > 0.05). In conclusion, ID2 is not essential for first lineage differentiation and blastocyst formation in cattle embryos.