54 Evidence of sexual dimorphism in transcriptome of in vitro- versus in vivo-derived bovine embryos

This study compared the gene expression of extraembryonic membranes (EEM) from in vitro-produced (IVP) and in vivo-derived embryos. A total of 172 Nelore (Bos indicus) cows were used; from which 40 cows were selected as oocyte donors. The remaining 132 cows were synchronised. On the day of AI, cows were inseminated (AI: n = 50) or assigned to receive a single IVP embryo 6.5 days later if a corpus luteum was present (IVP: n = 82). The embryo transferred was produced from the donors, and the same bull was used for AI. Females were slaughtered and uteri collected on Days 18 or 32 of pregnancy. Pregnancy was confirmed by the presence of an elongated conceptus (Day 18 uteri), or by ultrasonography on Day 31 before slaughter (Day 32 uteri). A total of 57 conceptuses were collected (26 AI and 31 IVP). The EEM samples were a piece of the trophectoderm (Day 18) or chorioallantois (Day 32). It was sectioned from the conceptuses and used for DNA and RNA isolation, and embryos were sexed by amplification of the Y-linked region of the DNA. The male and female ratios were analysed by Chi-squared of SAS (SAS Institute Inc.). A subset of three female and three male EEM samples from each group (AI and IVP, Days 18 and 32) were used for transcriptome analysis. Samples were sequenced using 75 bp paired-end sequencing carried out on the Illumina NovaSeq (Illumina Inc.). Differentially expressed genes (DEGs) between groups, sex, and days were determined using edgeR-robust. A false discovery rate (FDR) < 0.05 was used for statistical significance, and the Toppgene platform (https://toppgene.cchmc.org/) was used for biological process analysis. The Day 32 IVP group had a greater number of females than males (75 vs. 25%, P = 0.004). On Day 18 female embryos, out of 71 DEGs identified, 50 were up-regulated in IVP and 21 in AI. A trophectoderm marker (TKDP2) was up-regulated in the AI group, whereas IGF2, which is involved in placenta development, and APOA2, APOB, and APOE, which are important for lipid metabolism, were up-regulated in the IVP group. Male embryos on Day 18 had 22 DEGs, 15 of which were up-regulated in AI and seven in IVP. Female embryos on Day 32 had 74 DEGs, with 21 up-regulated in AI and 53 in IVP embryos. Some of the genes with increased expression in the IVP group were PTGIS, APOB, and APOH, which are related to lipid synthesis and metabolism. Male embryos on Day 32 presented 899 DEGs, 564 up-regulated in AI and 335 in IVP. Embryos from IVP had increased expression of genes related to embryo development, morphogenesis, neurogenesis, and developmental growth. In addition, male IVP embryos had decreased expression of genes related to lipid and carbohydrate metabolism. Interestingly, PLAC8B, BRB, and pregnancy-associated glycoproteins (PAG) 7, 9, 10, and 19, which are markers of placental function and have been previously associated with pregnancy loss, were also down-regulated in IVP male EEM. In conclusion, IVP-derived male embryos were more susceptible to alterations in gene expression, and these effects extend to the peri-implantation period, including genes associated with placental development.

This research was supported by FAPESP Project 2018/03798-7 and USDA-NIFA AFRI Grant No. 2019-67015-28998.