47 Regulation of bovine embryonic development by WNT5A is modified by the source of albumin and is independent of RAC1 signalling

WNT5A is the most abundant embryokine gene expressed in the bovine endometrium at Day 5 after oestrus. Earlier, we showed that WNT5A improved embryo competence to become a blastocyst and increased the number of inner cell mass (ICM) cells when embryos were cultured in medium containing bovine serum albumin (BSA). Given possible contaminants in BSA that could alter effects of WNT5A, the objective of experiment (Exp) 1 was to evaluate whether actions of WNT5A are different when BSA is replaced with recombinant human serum albumin (rHSA). One signalling pathway for WNT5A involves activation of RAC1 signalling. The objective of Exp 2 was to determine whether inhibition of RAC1 blocks actions of WNT5A and the objective of Exp 3 was to determine whether JNK, a downstream mediator of RAC1, is phosphorylated after WNT5A treatment. Oocytes from abattoir ovaries were fertilised using conventional semen. Presumptive zygotes were cultured in synthetic oviducal fluid (SOF) containing BSA for 5 days. In Exp 1 (n = 1847 zygotes; n = 10 replicates), embryos were placed in new drops of medium on Day 5 in a 2 × 2 factorial arrangement of treatments with SOF containing BSA or rHSA and with 0 or 200 ng mL⁻¹ recombinant human WNT5A. Percent cleaved embryos becoming a blastocyst at Day 7.5 was affected by the interaction between treatments (P < 0.01). Percent blastocysts was increased by WNT5A in rHSA-containing medium (31.1 ± 2.4% for 0 ng mL⁻¹ vs. 42.9 ± 2.6% for 200 ng mL⁻¹) but not in medium with BSA (37.1 ± 2.6% vs. 35.6 ± 2.6%). A subset of blastocysts (n = 162; n = 5 replicates) was labelled with Hoechst 33342 (total cells) and antibodies to SOX2 (i.e. ICM) and NANOG. The number of trophectoderm (TE) cells was calculated from total cells minus ICM. Blastocysts treated with WNT5A had increased number of ICM (P < 0.01) and a decreased number of TE (P < 0.01) regardless of albumin source. WNT5A did not affect number of NANOG-positive cells but numbers were lower (P < 0.01) for rHSA than for BSA. For Exp 2, embryos (n = 1003 zygotes; n = 6 replicates) were placed in new medium with rHSA at Day 5 and with treatments in a 2 × 2 factorial arrangement with WNT5A (0 or 200 ng mL⁻¹) and NSC23766 RAC1 inhibitor (0 or 50 µM). There was no interaction between WNT5A and inhibitor. WNT5A increased development to the blastocyst stage (P = 0.01) regardless of presence of the RAC1 inhibitor. For Exp 3, Day 5 morulae were treated with 0 or 200 ng mL⁻¹ WNT5A in BSA containing medium (n = 400; n = 2 replicates) and collected 30 min later for evaluation of pJNK by western blotting. Immunoreactive pJNK was detected but was very low for both groups. Our results demonstrate that BSA can obscure some actions of WNT5A on the bovine embryo. Moreover, actions of WNT5A to increase development is independent of the RAC1-JNK pathway.

Research was supported by USDA-AFRI 2017-67015-26452 and NIH R01 HD088352.