187 Effect of cytokines during in vitro 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 22 h in either standard maturation medium [TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg mL⁻¹porcine FSH, and 10 μg mL⁻¹ ovine LH; control] or maturation medium supplemented with different concentrations (5-160 ng mL⁻¹) of FGF2, LIF, and IGF1. After IVM, matured oocytes were activated by culturing in 5 μM ionomycin solution for 5 min followed by 4 h in 2 mM 4-dimethylaminopyridine (DMAP) and 10 mg mL⁻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 22 h 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 20 ng mL⁻¹), and FGF2 (20 and 40 ng mL⁻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 5 ng mL⁻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 80 ng mL⁻¹ compared with those with IGF1 at 5 to 20 ng mL⁻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 in vitro, and FGF2 additionally can improve parthenogenetic blastocyst quality.

This research was supported by RFBR (Project no. 18-29-07089).