162 EFFECTS OF PROLACTIN AND DITHIOTHREITOL ON THE QUALITY AND DEVELOPMENTAL CAPACITY OF IN VITRO MATURED BOVINE OOCYTES

Abstract

In vitro maturation (IVM) and IVF of domestic animal oocytes is widely used for commercial and research purposes. The oocyte quality and capacity for further development acquired during in vitro maturation and reduced during the subsequent aging are the main limitative factors affecting the embryo production (Miao et al. 2009 Hum. Reprod. Update 15, 573–585). Our objective was to evaluate effects of prolactin (PRL) and dithiothreitol (DTT) on apoptosis and the embryo development of bovine oocytes matured in vitro using 2 different systems. A total of 1437 slaughterhouse-derived cumulus-oocyte complexes (COC) were matured for 24 h in TCM-199 supplemented with 10% FCS, 0.2 mM sodium pyruvate, 10 μg mL–1 porcine FSH, and 5 μg mL–1 ovine LH. In system 1, 251 COC from a total of IVM oocytes were transferred to the aging medium (TCM-199 supplemented with 10% FCS) and cultured for 24 h in the absence (control) and the presence of either PRL (20 and 50 ng mL–1) or DTT (2.5, 5, and 10 μM). At the end of culture, oocyte apoptosis was detected using the TUNEL method. In system 2, another part of IVM oocytes (1186 COC) was co-incubated for 18 h with sperm in Fert-TALP medium modified by addition of 10 μg mL–1 heparin, PHE (20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine), and 0.1% modified Eagle’s medium (MEM) nonessential amino acids. In this case, PRL and DTT (at the above listed concentrations) were added directly to the fertilization medium. After IVF, oocytes were cultured in CR1aa medium for assessment of the cleavage and blastocyst rates on Days 2 and 8, respectively. The nuclear status of blastocysts was evaluated by the cytogenetic method. The data from 3–7 replicates were analysed by ANOVA. Culture of matured COC in the aging medium (system 1) increased the rate of apoptotic oocytes from 8.1 ± 4.7% (0 h) to 48.6 ± 5.8% (24 h) (P < 0.01). This rate was reduced (P < 0.05) up to 22.5 ± 3.1% and 17.8 ± 5.1% in the presence of PRL (20 and 50 ng mL–1) and up to 15.0 ± 6.9% and 19.5 ± 3.7% in the presence of DTT (2.5 and 5 μM). The direct addition of PRL at a concentration of 20 ng mL–1 to the IVF medium raised the blastocyst rate from 21.6 ± 2.2% to 29.8 ± 2.4% (P < 0.05) but did not affect the cleavage rate (72.1 ± 2.2% v. 74.3 ± 2.1%). By contrast, 50 ng mL–1 PRL did not increase the yield of blastocysts and decreased the cleavage rate (from 74.3 ± 2.1% to 62.9 ± 2.4%, P < 0.05). When added to the IVF medium, DTT raised the blastocyst rate only at a concentration of 2.5 μM (P < 0.05). No effects of PRL and DTT on the number of cells in embryos at the blastocyst stage were found. Our findings indicated that PRL and DTT supplements during in vitro fertilization of bovine oocytes may improve their capacity for the subsequent embryo development. This effect was probably due to the inhibitory influence of PRL and DTT on apoptosis of matured oocytes. The study was supported by the Federal Agency for Scientific Organizations and RFBR (project No. 14–48–03681).