129 KNOCKDOWN OF WBP1 DECREASES TROPHECTODERM FORMATION IN THE PRE-IMPLANTATION BOVINE EMBRYO

Abstract

A single nucleotide polymorphism (SNP) in WBP1 has been previously associated with embryonic development to the blastocyst stage. WBP1 interacts with WW domain containing proteins including YAP1 from the hippo signalling pathway that is involved in trophectoderm (TE) formation. Here we tested whether reduction in mRNA abundance for WBP1 would reduce development to the blastocyst stage and formation of cells in the inner cell mass (ICM) and TE. Knockdown was performed using a GapmeR LNA^TM antisense oligonucleotide designed to target WBP1. A scrambled version of the same sequence was used as a control. Embryos were produced in vitro from slaughterhouse oocytes and bulls from Bos taurus and Bos indicus breeds. At 20 to 22 h after insemination (hpi), embryos were treated with 5 µM anti-WBP1 GapmeR (KD), 5 µM scrambled GapmeR (SC), or vehicle (CTL). At 72 to 75 hpi (the time of maximal WBP1 expression), groups of 18 to 20 embryos were collected from each treatment to evaluate WBP1 expression. Other cultured embryos (minimum of 50/treatment for each replicate) were cultured until Day 8 after insemination. Cleavage was assessed at Day 3 and blastocyst formation at Day 7 and 8. Embryos were collected at Day 8 to determine ICM and TE cell number by determining nuclear immunoreactive CDX2. All experiments were replicated 5 times. Fold change was calculated relative to the CTL group. Data were analysed by analysis of variance for gene expression and cell number, and through logistic regression for embryonic development. WBP1 expression was reduced (P = 0.04) in KD embryos compared to CTL (least squares means ± SEM: 1 ± 0.19 v. 0.64 ± 0.19 fold change) or SC (1.05 ± 0.19). There was no difference in expression between CTL and SC. Percent of embryos that cleaved was not affected by treatment (P > 0.05); however, percent of inseminated oocytes that became blastocysts tended to be lower in KD compared to CTL and SC at Day 7 (P = 0.09) [10.8 ± 2.8, 20 ± 3.0, and 16.3 ± 3.1% for KD, CTL, and SC, respectively] and 8 after insemination (P = 0.06) [13.7 ± 3.3, 24.2 ± 3.3, and 22.9 ± 3.6%]. Knockdown of WBP1 caused a reduction in number of total (P = 0.0004) and TE (P < 0.0001) cells with no effect on ICM cell number (P = 0.83). Total cell numbers for KD, SC, and CTL were 124.2 ± 6.4, 157.75 ± 7.4, and 124.28 ± 6.4 and numbers of TE cells were 59.7 ± 3.8, 90.0 ± 4.47, and 90.0 ± 4.4. Results show that reduction in mRNA for WBP1 decreases TE formation and tends to reduce competence of embryos to become blastocysts.

This study was supported by USDA AFRI 2013–68004–20365.