172 Expression of proliferation and apoptosis markers in cumulus cells surrounding matured and aged oocytes exposed to luteotropic factors during the second phase of in vitro maturation

Abstract

The quality and developmental capacity of mammalian oocytes depends on cooperation with surrounding cumulus cells. The functional state and activity of cumulus cells changes with oocyte maturation, especially during the oocyte transition from metaphase I (MI) to metaphase II (MII) stage. In the present work, effects of 3 luteotropic factors, progesterone (P4), prolactin (PRL), and LH, during the second phase of in vitro maturation (IVM) on the subsequent expression of proliferation and apoptosis markers in bovine cumulus cells surrounding matured and aged oocytes were studied. A total of 1532 cumulus-oocyte complexes (COC) were cultured for 12h in TCM-199 containing 10% fetal calf serum (FCS), 10μg mL−1 porcine FSH, and 10μg mL−1 ovine LH at 38.5°C and 5% CO2. Thereafter, COC were transferred to the following IVM systems: (1) TCM-199 containing 10% FCS (Control 1) and (2) a monolayer of granulosa cells (GC) precultured for 12h in TCM-199 containing 10% FCS (Control 2). In both systems, the medium of experimental groups was supplemented with either P4 (50 ng mL−1) or bovine PRL (50ng mL−1) or ovine LH (5μg mL−1); then, the COC were matured for next 12h. Half of the COC matured for 12h in both systems were cultured for an additional 24h in fresh TCM-199 containing 10% FCS to test long-term hormonal effects during oocyte aging. After culture, the cumulus expression of the proliferation marker proliferating cell nuclear antigen (PCNA) and the pro-apoptotic markers caspase-3 and Bax was assessed by the immunocytochemical method. The data from 4 to 5 replicates using 84 to 106 COC per treatment were analysed by ANOVA. After IVM in System 1, the rate of PCNA-positive cumulus cells was higher (P<0.05) in the PRL-treated group (41.3±1.6%) than in the control (34.6±2.3%) or LH-treated group (29.9±2.9%), but did not differ from that in the P4-treated group (38.2±4.8%). In the presence of GC (System 2), the respective rates did not change but were more variable. Aging of COC matured in both systems led to a 1.4- to 1.9-fold reduction in the proportion of the cells containing the proliferation marker PCNA (P<0.05). Meanwhile, none of the hormones tested had any long-term effect on the proliferative activity of senescent cumulus cells. The rate of cumulus cells expressing caspase-3 in different groups varied from 48.5±4.9 to 53.8±5.8% and did not depend on the hormones, IVM system, or oocyte aging. The proportion of the Bax-positive cells was also unaffected by luteotropic factors but increased 1.4 to 1.6 times (P<0.01) following 24h of COC aging. Our findings indicate that PRL can exert a short-term stimulatory action on the proliferative activity of bovine cumulus cells in the course of the second phase of IVM. Meanwhile, the cumulus expression of pro-apoptotic markers caspase-3 and Bax is not responsive to P4, PRL, or LH during the second step of IVM. The study was supported by the Russian Science Foundation (project 16-16-10069).