187 Effect of cytokines during invitro maturation of bovine oocytes on the development potential of parthenogenetic embryos

Abstract

The efficiency of assisted reproductive technologies is critically dependent on the quality of the oocytes used to produce the embryos. The aim of the present research was to study dose-dependent effects of three cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) individually on IVM in bovine oocytes and their consecutive development to blastocysts after artificial activation. Slaughterhouse-derived cumulus-oocyte complexes (COC) (n=2052 COC) were cultured for 22h in either standard maturation medium [TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2mM sodium pyruvate, 10μgmL−1porcine FSH, and 10μgmL−1 ovine LH; control] or maturation medium supplemented with different concentrations (5-160ngmL−1) of FGF2, LIF, and IGF1. After IVM, matured oocytes were activated by culturing in 5μM ionomycin solution for 5min followed by 4h in 2mM 4-dimethylaminopyridine (DMAP) and 10mgmL−1 cyclohexamide. Activated oocytes were cultured in CR1aa medium until Day 5 and then transferred to the same medium supplemented with 5% FCS and cultured until Day 7. All cultures were performed at 38.5°C and 5% CO2 in humidified air. At Days 2 and 7 after activation, cleavage and blastocyst rates were determined. In addition, obtained blastocysts were fixed with 4% paraformaldehyde, and the total cell number was determined by 4′,6-diamidino-2-phenylindole (DAPI) staining. The data from 4 to 6 replicates (77-140 oocytes per treatment) were analysed by ANOVA. After 22h of culture, the rate of MII oocytes, assessed by the presence of the first polar body, did not differ between the groups and ranged from 74.4 to 90.7%. No statistical differences were found in the cleavage rate between oocytes matured in cytokine-treated groups compared with the control. Cleavage rates for the LIF experimental groups was 63.5 to 77.2%, for the IGF1 experimental groups was 68.1 to 80.7%, and for the FGF experimental groups 63.7 to 77.0%. Optimal concentrations of LIF (5 and 20ngmL−1), and FGF2 (20 and 40ngmL−1) increased (P<0.05) the blastocyst rate from 21.7±1.5 (control for LIF-treated groups) to 32.7±7.1 and 27.1±3.4 and from 19.6±1.8% (control for FGF-treated groups) to 29.8±1.9 and 31.1±2.1%, respectively. Furthermore, the addition of FGF2 in IVM medium (except at 5ngmL−1) led to an increase in the total cell number in embryos that developed to the blastocyst stage, whereas LIF did not have this effect. The maturation of COC in the presence of IGF1 had no effect on the yield of parthenogenetic blastocysts or on the total cell number in blastocysts compared with the control medium. However, the blastocyst rate was lower in groups with IGF1 at 40 to 80ngmL−1 compared with those with IGF1 at 5 to 20ngmL−1 (P<0.05). Thus, both LIF and FGF2 (each individually) (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro, and FGF2 additionally can improve parthenogenetic blastocyst quality. This research was supported by RFBR (Project no. 18-29-07089).