102 A delay in maternal zygotic transition may lead to early embryonic loss in poor-quality bovine blastocysts

Abstract

Increased genetic potential and performance of dairy cows has coincided with a decline in fertility. Early embryonic mortality accounts for 75-80% of this decline in fertility, costing the industry over $1.28 trillion worldwide. Despite advancements in assisted reproductive technologies and embryo transfer, many transferred embryos do not survive past Day 24 of gestation, suggesting flaws in embryo selection for transfer. It was hypothesised that visually lower-quality IVF Day 7 blastocysts were developmentally delayed as a result of altered mitotic signalling and were at higher risk of embryo mortality. To identify potential causes for early embryo mortality in IVF embryos, RNA-Seq was performed on 6 categories of Day 7 blastocysts: stages (S) 5 (early), 6 (full), and 7 (expanded), with quality scores (Q) of 1 or 2. Oocytes were matured, fertilized by routine procedure, and cultured for 7 days. Blastocysts were classified and graded, separated into the six categories, and subjected to Pronase digestion of the zona pellucida. From three biological replicates of each blastocyst group, RNA was extracted and submitted for RNA-sequencing. Secondary bioinformatics and analyses were performed using R to determine differentially expressed genes. When S7.Q1 blastocysts were compared to other categories, 55 genes were consistently differentially expressed (P < 0.05) in S5.Q1 or 2 and S6.Q2. Of these 55 genes, 15 were significantly upregulated (>1.5 fold change), and 40 were downregulated (<−1.5 fold change). The nine most common upregulated genes in S5.Q1 or 2 and S6.Q2, compared with S7.Q1, were BTG4, ARGFX, GPC4, BOC, CNTNAP2, NR3C2, CCDC7, and PHYHIPL. The five most common downregulated genes included MUC1, HSD3B1, ADAM19, EVPL, and TGM1. The EVPL and TGM1 proteins are associated with cell barrier permeability, and a lack of TGM1 has been shown to cause neonatal death in mice. Therefore, early embryo mortality may begin with decreased EVPL and TGM1, limiting cell permeability and communication between blastomeres. This limited communication might delay gene expression in the embryo at the 4- to 8-cell stage, delaying the maternal zygotic transition (MZT), in spite of continued cell division. This explanation is supported by the observed increase in ARGFX and BTG4 mRNA. Normally, stored maternal BTG4 mRNA becomes translated during the MZT and degrades maternal mRNA. The increase of BTG4 mRNA in poor-quality embryos may reflect delayed translation of BTG4 and delayed MZT. The mRNA transcripts increased in poor-quality blastocysts may be excess maternal mRNA not yet degraded, like BTG4. The decreased mRNA transcripts observed may be indicative of zygotic genes which have not yet been transcribed. For instance, MUC1 is necessary for proper embryo implantation, and HSD3B1 converts placental pregnenolone to progesterone and produces a precursor to oestradiol. The delayed transcription of both MUC1 and HSD3B1 may impair maternal recognition of pregnancy, implantation, and communication to the maternal endometrium via oestradiol, thereby causing embryo mortality.

This research was supported by USDA NNF 2016-38420-25289 and Zoetis Inc.