164 Evaluation of the lipid content of in vitro-matured bovine cumulus - oocyte complexes in the presence of natriuretic peptides A, B, and C types

Abstract

One of the difficulties still observed in in vitro production (IVP) of bovine embryos is the lower cryotolerance of such embryos, which has been related to their increased lipid accumulation during culture in the presence of fetal bovine serum (FBS). Previous studies have indicated that the cyclic guanosine monophosphate (cGMP) pathway may be involved in the lipid metabolism of bovine cumulus-oocyte complexes (COC). Synthesis of cGMP can be caused by activation of membrane guanylate cyclase (mGC), also called natriuretic peptide receptors (NPR1 and NPR2), which are activated by natriuretic peptides (NP) A, B, and C types (NPPA, NPPB, and NPPC). The objective of this study was to investigate the influence of supplementation with NP during in vitro maturation (IVM) on lipid content and nuclear maturation of bovine COC. Pools of 25 COC were submitted to IVM in TCM-199 with 0.2mM sodium pyruvate, 10μg mL−1 gentamicide, 0.5μg mL−1 FSH, 10% FBS, and NPPA (10−5 M), NPPB (10−7 M), or NPPC (10−5 M). The control group was matured without NP. After 24h, cumulus cells (CC) were removed and oocytes (OO) were fixed and permeabilized in 4% paraformaldehyde+0.5% Triton for 20min, stained with 10μg mL−1 Hoechst 33342 for 15min and 1μg mL−1 Nile Red for 30min. Then, the OO were placed in 13μL of ProLong (Thermo Fisher Scientific, Waltham, MA, USA) on glass slides, covered with a coverslip, and submitted to epifluorescence microscopy to evaluate nuclear maturation (emission 445-450nm and excitation 475-490nm) and lipid content (emission 590nm and excitation 516-560nm). Data for 4 replicates/group were tested for normality of results and homogeneity of variance, and then submitted to ANOVA, followed by Tukey test using a GraphPad Prism software (GraphPad Inc., San Diego, CA, USA), with a significance level of 5%. Nuclear maturation was influenced only by one of the NP added to IVM medium, NPPC, which reduced maturation rate (68.3% metaphase II, MII) compared with the control (82.7% MII; P<0.05). Maturation rates of NPPA (79.5% MII) and NPPB (85.1% MII) did not differ from the control (P>0.05). Activation of mGC by NPPB generated oocytes with lower lipid content (34.02±1.2 IF/μm2) compared with control (36.98±0.7 IF/μm2; P<0.05). Neither NPPA (36.5±1.4 IF/μm2) nor NPPC (39.3±1.4 IF/μm2) was able to reduce the lipid content in oocytes matured in vitro relative to control (P>0.05). Different NP may have different effects on maturation and lipid content in in vitro matured bovine oocytes; NPPB may be favourable for reducing the lipid content of matured bovine oocytes in vitro.