Progesterone is involved in anti-aging effects of prolactin on bovine cumulus-enclosed oocytes matured in vitro

In matured oocytes, aging processes are accelerated that reduces the ovum fitness. However, little is known about physiological factors responsible for the oocyte protection from precocious senescence. We have found previously that prolactin (PRL) can maintain the developmental capacity and apoptosis resistance of in vitro matured bovine oocytes aging in vitro (Singina et al., Reprod Fert Dev, 27:204, 2015; Singina et al., Reprod Domest Anim, 50(S3):77, 2015). The goal of this study was to test a hypothesis that these anti-aging effects of PRL might be achieved through stimulation of progesterone production by cumulus cells (CCs). Bovine cumulus-enclosed oocytes (CEOs) were cultured for 20 h in the IVM medium (TCM 199 supplemented with 10% fetal calf serum, 10 μg/ml FSH, and 10 μg/ml LH). Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). A part of in vitro matured oocytes was set free from their CCs. Then CEOs and denuded oocytes (DOs) were cultured for additional 12 or 24 h in the aging medium (TCM 199 containing 10% fetal calf serum) without (Control group) or with bovine PRL (50 ng/ml; Research Center for Endocrinology, Moscow, Russia) and/or trilostane (an inhibitor of progesterone synthesis). Apoptosis was detected in oocytes aged for 24 h using the In Situ Cell Death Detection Kit (Fluorescein, Roche, Indianapolis, USA). Oocytes aged for 12 h were subjected to the IVF procedure described previously (Singina et al., Reprod Fert Dev, 26:154, 2014). The embryo development was evaluated at Days 2 and 8 for cleavage and blastocyst formation. The data for apoptosis (n = 4, 80-90 oocytes per treatment) and IVF/IVC (n = 5-6, 135-175 oocytes per treatment) were analyzed by ANOVA. After 24 h aging, the rate of apoptotic CEOs in the PRL-treated group was reduced as compared to the Control group (8.2 ± 3.3 vs. 24.5 ± 3.3%, P u003c 0.001), but did not differ from that prior to aging (3.5 ± 1.8%). Meanwhile, PRL did not affect this rate in the medium containing 1 or 10 μM trilostane (1 μM: 23.8 ± 3.4 vs. 29.0 ± 1.6%; 10 μM: 30.7 ± 3.4 vs. 36.2 ± 2.0%). Furthermore, the effect of PRL on oocyte apoptosis disappeared when removing CCs. After IVM for 20 h, the blastocyst rate in our IVF/IVP system was 25.9 ± 3.0%. Following the prolonged culture of CEOs for 12 h, the blastocyst yield in the Control group decreased to 9.9 ± 0.9% and was lower than in the PRL group (18.5 ± 2.8, P u003c 0.01). The addition of 1 μM trilostane to the aging medium containing PRL (but not to the Control medium) caused the blastocyst yield to decline up to 11.7 ± 2.4% (P u003c 0.05). At the same time the developmental capacity of cultured DOs was unaffected by PRL. Thus, the supporting effects of PRL on the developmental capacity and apoptosis resistance of aging oocytes are mediated through CCs and related to stimulation of progesterone synthesis. Therefore, the anti-aging effects of PRL may be achieved, at least in part, due to a pro-survival action of progesterone on mature oocytes.