109 Agouti-signalling protein impacts blastocyst development in cattle

In 2016, it was reported that a greater number of bovine embryos were generated via in vitro fertilisation (IVF) and transferred worldwide rather than in vivo. Despite advancements in oocyte and embryo culture systems, only 20–40% of presumptive zygotes will reach the blastocyst stage in vitro. Factors limiting the further advancement of bovine IVF include the current lack of knowledge of mRNAs and proteins imperative to oocyte quality and early embryonic development. Therefore, the identification and characterisation of factors present in the oocyte are crucial for further optimisation of in vitro bovine embryo production. Analysis of RNA-Seq data of bovine oocytes obtained by our laboratory revealed agouti-signalling protein (ASIP), a gene shown to be involved in lipid metabolism in cattle, was more than 1,000-fold more abundant in the bovine oocyte than in other somatic tissues. Further, ASIP is expressed in the early embryo, with high expression at the blastocyst stage. Presently, the role of ASIP in the bovine oocyte and early embryo is unknown. In order to determine the impact of exogenous ASIP on oocyte maturation, recombinant ASIP (rASIP) protein was supplemented to cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) at the following concentrations: 0, 1, 10, or 100 ng/mL (n = 10–30 COCs/well; n = 4 replicates/treatment). Oocytes were aspirated from abattoir-derived bovine ovaries, washed, and placed in BO-IVM (IVF Bioscience) containing the rASIP for 22–24 h. Following IVM, oocytes were fertilised and, 12 h later, placed in BO-IVC until Day 8, when blastocyst rates were obtained. Percentage data were transformed using arcsine and analysed for statistical differences using one-way ANOVA. Data are presented as the mean ± standard error of the mean. The control (0 ng/mL rASIP) blastocyst rate was 29% ± 1.8. Addition of rASIP increased Day 8 blastocyst rates to 32% ± 10 (1 ng/mL), 45% ± 4.2 (10 ng/mL), and 45% ± 5.5 (100 ng/mL). Overall, the addition of 100 ng/mL of rASIP led to a significant increase in blastocyst development (P = 0.032). Additional COCs were matured in IVM without rASIP and fertilised to produce presumptive zygotes for microinjection of small interfering RNA (siRNA) targeting ASIP to determine the effect of ASIP knockdown on early embryonic development. Presumptive zygotes (n = 30–37 zygotes/treatment) were collected 12–16 h post-fertilisation and injected with ∼20 pl of either ASIP siRNA (25 µM), negative siRNA (25 µM), or remained as uninjected controls. The experiment was repeated four times. On Day 8, blastocyst development was examined. Both uninjected and negative siRNA blastocyst rates were 45% ± 2.98 and ± 3.8, respectively, while the ASIP siRNA rate was 29% ± 2.98. Statistical analysis revealed there was a tendency for ASIP knockdown to result in a decreased blastocyst rate (P = 0.06). Overall, these data show that the addition of ASIP during IVM increases blastocyst development and decreasing ASIP levels may impair blastocyst development.

Funding for this research was provided by the National Institute of Food and Agriculture, USA Department of Agriculture, award number 2020-38640-31520 through the North-east SARE program under subaward number GNE21-252-35383, and USDA National Institute of Food and Agriculture, Multistate Research Project 1014002.