185 Serum-dependent and serum-independent effects of prolactin and progesterone during the completion of invitro maturation on metaphase II chromosomes in bovine oocytes

Abstract

The final stages of IVM are of great importance for the developmental competence of mammalian oocytes. The goal of the present work was to study effects of prolactin (PRL) and progesterone (PG) during the completion of IVM in serum-containing and serum-free media on destructive and apoptotic changes of MII chromosomes in bovine oocytes. Cumulus-oocyte complexes (COCs) were matured for 16h in TCM-199 containing 10% fetal calf serum (FCS), 10μgmL−1 porcine FSH, and 10μgmL−1 ovine LH at 38.5°C and 5% CO2. Thereafter, the COCs were transferred to and cultured for either 8 or 26h in the following systems: TCM-199 containing 10% FCS (Control-1) and TCM-199 containing 3mgmL−1 bovine serum albumin (BSA; Control-2). In both systems, the medium of experimental groups was supplemented with 50ngmL−1 bovine PRL or 50ngmL−1 PG. All treatments were repeated 4 to 5 times using 80 to 108 oocytes per group. The state of the oocyte nuclear material was evaluated by Tarkowski's method. Apoptosis in MII oocytes was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Arcsine-transformed data were analysed by ANOVA. At the end of culture, the rate of MII oocytes was similar in all the groups (83.4-93.9%). Following 8h of IVM, the frequency of MII chromosome abnormalities (decondensation, adherence, clumping) was reduced (P<0.01) in the PRL-1 and PG-1 groups compared with Control-1 (Table 1). This effect was still observed in the PG-1 group (P<0.001) but disappeared in the PRL-1 group after 26h of prolonged oocyte culture. In contrast, a decrease in the rate of MII oocytes with abnormal chromosomes occurred only in the PG-2 group by the end of 8h of culture (P<0.01), whereas the decrease was revealed in both the PG-2 and PRL-2 groups after 26h of culture (P<0.001). Furthermore, the rate of MII oocytes with apoptotic signs following 8h was lower in the PRL-1 and PG-1 groups than in Control-1 (P<0.05). During the 26-hculture, this rate increased in all groups, being higher in Control-1 than in the PG-1 group (P<0.05). The rate of apoptotic oocytes was 2.8 times (8h) and 1.8 times (26h) lower in Control-2 than in Control-1 (P<0.01). Neither PRL nor PG affected oocyte apoptosis in system 2. Thus, during the completion of IVM, PRL can exert a short-term and serum-dependent inhibitory effect on destructive changes of MII chromosomes in bovine oocytes, whereas the similar effect of PG is long-term and serum-independent. Anti-apoptotic effects of both PRL and P4 on the oocytes are determined by serum. Table 1.Effects of prolactin (PRL) and progesterone (PG) on MII chromosomes in bovine oocytes Group % of MII oocytes With abnormal chromosomes With apoptotic signs 8 h 26 h 8 h 26 h Control-1 36.5±3.1a 68.0±2.2a 19.4±1.1a 65.5±2.9a PRL-1 23.6±3.2bc 68.3±2.6a 9.8±3.3b 51.2±7.8abc PG-1 23.4±2.4bc 49.4±0.6b 8.0±3.1b 53.6±0.7b Control-2 31.7±2.7ac 62.7±1.0a 6.9±1.3b 37.3±3.3cd PRL-2 32.6±2.5ac 45.9±0.9b 7.4±2.9b 28.1±2.1d PG-2 20.0±2.5b 47.2±1.6b 6.8±0.9b 29.9±3.9cd a-dValues within a column differ (at least P<0.05). This study was supported by RFBR (No. 17-29-08035).