175 EFFECT OF ACETYL-CoA CARBOXYLASE (ACC) INHIBITOR ON THE LIPID CONTENT AND NUCLEAR MATURATION OF CANINE OOCYTES

Abstract

Compared with other domestic species, embryo technologies are least developed for the dog. This is mainly due to difficulties in producing mature oocytes in vitro. Canine oocytes contain exceptionally high amounts of lipid. High lipid content increases the chilling sensitivity of oocytes and embryos. Mechanical and chemical reductions of the lipid content have been used to improve the cryotolerance of oocytes. Additionally, chemical stimulation of lipid catabolism improved oocyte in vitro maturation (IVM) rates in other species (You et al. 2012 Theriogenology 78, 235–543). Acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme in de novo lipogenesis and its expression has been reported in oocytes and embryos. In somatic cells, inhibition of ACC reduces lipogenesis and enhances β-oxidation. Our hypothesis is that treatment of oocytes with an inhibitor of ACC (CP640186, Pfizer Animal Health, New York, NY, USA) reduces lipid content and improves IVM rate of oocytes. Ovaries were collected from a spay clinic and sliced in HEPES-buffered TCM-199 to recover oocytes. In vitro maturation was conducted at 38.5°C, 5% CO2, and high humidity in TCM-199 supplemented with 1% fetal bovine serum, glutamine, sodium pyruvate, β-mercaptoethanol, oestradiol, epidermal growth factor, and antimicrobial agents (Songsasen et al. Mol. Reprod. Dev. 79, 186–196). During the first 19 to 21 h, the IVM media contained 4 concentrations of the inhibitor (0+DMSO, 0.02, 0.1, and 0.5 μM, designated as treatments 1, 2, 3, and 4, respectively) and then oocytes were transferred to a medium without the inhibitor and cultured for an additional 27 to 29 h. At the end of culture (total of 48 h), oocytes were denuded of cumulus layers, washed, fixed, and stained with Nile red (lipid) and Hoechst-33342 (chromatin), and then mounted on a microscope slide. Lipid content and chromatin status were evaluated using fluorescent microscopy (TRITC and DAPI filters, respectively). The relative lipid content was measured by the corrected total cell fluorescence (CTCF) using ImageJ software (http://rsbweb.nih.gov/ij/). Data on CTCF and proportions of chromatin status of oocytes were analysed using one-way ANOVA (SigmaPlot 11.0). The mean CTCF for each treatment was 5.5 × 109 (n = 51, 5.2 × 109 (n = 44), 4.5 × 109 (n = 31), and 4.8 × 109 (n = 34), respectively (P = 0.3; 4 replicates). At the highest dose, the agent induced relatively more cumulus cell layer expansion but inhibited their attachment to the dish; the latter effect was reversible because cumulus cells attached and proliferated after washing the oocytes of the agent. Metaphase II was rare (≤3.1%); however, the proportion of oocytes developing to ≥GVBD stage (Trt 1 14%, n = 37; Trt 2 41%, n = 56; Trt 3 5%, n = 22; Trt 4 11%, n = 43) was affected by treatments. Our preliminary data indicate that a low concentration of ACC inhibitor has a positive effect on the nuclear maturation of canine oocytes but the effect on lipid content as estimated by using Nile red fluorescence intensity appears to be minimal.