62 Investigating differences in gene expression between in vitro-produced bovine embryos and parthenotes

Abstract

Parthenogenic activation allows for the development of an oocyte without male gamete contribution and may serve as a suitable model for understanding maternal and paternal contributions during development. In the bovine, parthenotes lack the ability to survive to term once transferred into females. The goal of this study was to investigate the gene expression profile at the blastocyst stage in parthenotes and embryos by characterising expression of developmentally important genes, such as markers for pluripotency (OCT4, NANOG), hypoblast (GATA6), epiblast (SOX2), trophectoderm (CDX2), and maternal-embryo communication (IFNT2). To test this, IVM oocytes were either fertilized to a bull with known fertility in vitro or activated. To activate, oocytes were denuded, and placed in ionomycin calcium salt for 5 min. They were incubated for 3 h in 6-(dimethylamino)purine (6-DMAP) and placed in synthetic oviductal fluid (SOF). Putative zygotes and activated oocytes were cultured in SOF for 8 days. Cleavage (at least one cellular division) was recorded on Day 3 and development to the blastocyst stage was recorded on Day 8 after insemination or activation. Cleavage rate was 85.9% and 83.2% for parthenotes and embryos, respectively. Both groups produced a similar blastocyst rate, 32.3% for parthenotes and 33.7% for embryos. On Day 8, blastocyst stage parthenotes and embryos were collected. Data were analysed by ANOVA (Tukey HSD post hoc test) using the GLM procedure of SAS version 9.4. Pools of 5 embryos or parthenotes (3 replicates) were flash frozen and stored until RNA isolation (PicoPure™ RNA Isolation Kit). Real-time PCR was used for quantification of gene expression. Genes were analysed relative to a housekeeping gene, GAPDH. There was no difference in gene expression detected for OCT4 (P = 0.25), NANOG (P = 0.11), GATA6 (P = 0.32), SOX2 (P = 0.25), or IFNT2 (P = 0.52). Expression of CDX2 was lower in the parthenotes than the embryos (P = 0.05). In a second experiment, the proteins GATA6, NANOG, and CDX2 were immunolocalized in 17 parthenotes and 15 embryos. Fixed embryos were permeabilized, blocked, and placed in primary antibodies overnight. After, they were placed into the secondary antibody for 1 h, followed by nuclear stain. There was a decreased mean intensity of CDX2 in the parthenotes compared to embryos (P = 0.005). No difference (P > 0.05) in GATA6 or NANOG was observed between the 2 groups. The ratio of inner cell mass to trophectoderm was higher (P = 0.04) in the parthenotes (2.5 ± 0.23) than in the embryos (1.7 ± 0.25). Here we analysed and confirmed the expression of developmentally important genes at the blastocyst stage in embryos and parthenotes. CDX2, a marker of the trophectoderm that will later give rise to the placenta, was downregulated in parthenotes. This highlights the importance of the contribution of the paternal genome to development. Further research is necessary to elucidate the ability of the parthenotes to establish and maintain pregnancy.

This research was supported by the National Needs Fellowship funded by USDA NIFA Grant 2019-38420-28972.