In vitro maturation (IVM) is an important process in the in vitro production of embryos. It has been recently shown that 3 cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) have increased the efficiency of IVM, blastocyst production, and in vivo development in pig (Yuan et al. 2017 Proc. Natl. Acad. Sci. USA 114, E5796-E5804). In vitro maturation in medium supplemented with cytokines doubled the blastocyst rate and quadrupled the litter size when transferred. It was observed that the addition of cytokines to IVM medium had an effect on the regulation of pMAPK1/3, cumulus cell expansion, and transzonal projections in cumulus-oocyte complexes (COC). This study was designed to assess the effect of these 3 cytokines on IVM in bovine oocytes and their consecutive development to blastocyst. Intracellular glutathione level (GSH), frequently used as an indicator of metaphase II (MII) oocyte quality, was also evaluated. The COC were retrieved from abattoir-derived ovaries and matured for 21h in either our standard maturation medium [TCM-199 (Gibco/Life Technologies, Grand Island, NY, USA), containing 10% fetal bovine serum, 0.5µg mL−1 FSH, 5µg mL−1 LH, and 100U mL−1 penicillin/streptomycin] or maturation medium supplemented with 20ng mL−1 human LIF, 20ng mL−1 human IGF1, and 40ng mL−1 human FGF2. After IVM, COC were placed in fertilization medium and incubated with frozen-thawed sperm for 20h. Cumulus cells were removed from fertilized COC and cultured in SOF culture medium at 38.5°C in 5% CO2/humidified air. Cleavage and blastocyst rates were assessed at 48h and Day 8 post-IVF, respectively. To assess GSH level, MII oocytes were incubated in 20 µM CellTracker Blue CMF2HC (Thermo Fisher Scientific, Waltham, MA, USA) and observed under blue fluorescent light. All statistical analysis was performed using one-way ANOVA and data are presented as mean±s.e.m. The MII rate, assessed by the presence of the first polar body, was significantly higher in the maturation medium supplemented with cytokines compared with the control medium (167/202; 82.4±2.02% v. 136/198; 68.8±1.1%; P<0.05, 4 replicates). For IVF, no statistical difference was found in the cleavage rate between oocytes matured in the medium supplemented with cytokines compared with control medium (351/473; 74.3±4.86% v. 358/573; 63.9±4.03%; P>0.05, 5 replicates), respectively. However, a significant increase in blastocyst rate was observed in the cytokine-containing medium (64/351; 17.7±2.06%) compared with the control group (42/358; 11.0±1.96%; P<0.05, 5 replicates). Furthermore, our preliminary data indicate an increase in GSH in MII oocytes matured in the cytokine-containing medium. In conclusion, the addition of FGF2, LIF, and IGF1 to maturation media improves bovine IVM efficiency and quality of the MII oocytes, leading to a greater blastocyst development rate.
Supported by RFBR (18-29-07089) and UAES (1343).
Pengaruh urea dalam media maturasi in vitro terhadap tingkat maturasi oosit sapi
