Prolactin supports the developmental competence and apoptosis resistance of aging bovine oocytes through the same signaling pathway

The time-dependent senescence of mammalian oocytes attained the M-II stage results in a decline of their quality in vivo and in vitro. We have previously shown that the decelerating effect of prolactin (PRL) on age-associated alterations in M-II chromosomes in bovine cumulus-enclosed oocytes is related to activation of Src-family tyrosine kinases, Akt, and protein kinase C (Lebedeva et al., Front Genet, 6:274, 2015). The aim of the present research was to study mechanisms of PRL actions on the developmental competence and apoptosis resistance of bovine oocytes aging in vitro. Bovine cumulus-oocyte complexes (COCs) were cultured for 20 h in TCM 199 containing 10% fetal calf serum, 10 μg/ml porcine FSH, and 10 μg/ml ovine LH at 38.5°C and 5% CO2. Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). After IVM, COCs were transferred to the aging medium (TCM 199 supplemented with 10% fetal calf serum) and cultured for 12 or 24 h in the absence (Control) or in the presence of 50 ng/ml bovine PRL (Research Center for Endocrinology, Moscow, Russia) and/or protein kinase inhibitors. The following inhibitors were applied: (1) PP2 (an inhibitor of Src-family tyrosine kinases), (2) triciribine (an inhibitor of Akt kinase), and (3) calphostin C (a protein kinase C inhibitor; Calbiochem, Darmstadt, Germany). After the prolonged culture for 12 h, oocytes underwent IVF and IVC as described previously (Singina et al., Reprod Fert Dev, 26:154, 2014). The cleavage and blastocyst rates were assessed at Days 2 and 8, respectively. Apoptosis was detected in oocytes following 24 h aging using the TUNEL kit (Roche, Indianapolis, USA). The data for apoptosis (52-57 oocytes per treatment) and IVF/IVC (177-212 oocytes per treatment) were analyzed by ANOVA. For oocytes fertilized just after IVM, the cleavage and blastocyst rates were 67.9 ± 4.2% and 22.1 ± 1.6%, respectively. After 12 h aging, the blastocyst yield declined to 7.7 ± 1.2% (Control), whereas PRL raised the yield to 14.9 ± 2.6% (P < 0.01). Calphostin C (0.5 µM) eliminated (P < 0.01) this effect of PRL on aging oocytes, although it did not affect the blastocyst rate in the control medium. Triciribine (25 µM) reduced the yield of blastocysts both in the control and PRL-treated groups (to 3.3 ± 1.2 and 7.9 ± 2.8%, respectively, P < 0.05), whereas PP2 (10 µM) did not. Furthermore, PRL decreased the apoptosis frequency in aging oocytes from 23.3 ± 3.4% (Control) to 9.0 ± 2.4% (P < 0.01), while this frequency was 4.1 ± 2.3% before aging. The hormonal action on apoptosis was abolished by calphostin C (1 µM) but not by triciribine (50 µM) or PP2 (20 µM). Our findings indicate that PRL can maintain the developmental competence and apoptosis resistance of bovine cumulus-enclosed oocytes aging in vitro by activating protein kinase C. Thus, the supporting action of PRL on the oocyte developmental capacity is likely to be related to its pro-survival action.