Disruption of CSF2RA in the bovine preimplantation embryo reduces development and affects embryonic gene expression in utero

The hypothesis that colony-stimulating factor 2 (CSF2) plays a role in the preimplantation development of the bovine embryo was tested by evaluating consequences of inactivation of CSF2RA (the functional receptor in the embryo) for the development of embryos in utero. CRISPR/Cas9 was used to alter sequences on exon 5 and intron 5 of CSF2RA, Control embryos were injected with Cas9 mRNA only. Embryos > 16 cells at day 5 after insemination were transferred to synchronized recipient females in groups of 7–24. Embryos were flushed from the uterus 2 days later. The proportion of recovered embryos that developed to the blastocyst stage was lower for knockout embryos (39%) than for control embryos (63%). RNA sequencing of individual morulae and blastocysts indicated a total of 27 (morula) or 15 (blastocyst) differentially expressed genes (false discovery rate <0.05). Gene set enrichment analysis indicated that the knockout affected genes playing roles in several functions including cell signaling and glycosylation. It was concluded that signaling through CSF2RA is not obligatory for the development of the bovine preimplantation embryo to the blastocyst stage but that CSF2 signaling does enhance the likelihood that the embryo can become a blastocyst and result in specific changes in gene expression. LAY SUMMARY: Development of the early embryo depends upon regulation by chemical signals produced by the uterus. One of these signals is a protein called colony-stimulating factor 2 (CSF2) that can affect the development of embryos in culture. To test whether CSF2 also regulates the embryo in the uterus, where development ordinarily occurs, we evaluated development in the uterus of embryos in which the receptor for CSF2 was disrupted. Embryos without the receptor gene were less likely to develop to the typical stage of development than control embryos and experienced some differences in the expression of specific genes. We conclude that CSF2 regulates embryonic development in the uterus.