168 THE DEVELOPMENTAL POTENTIAL OF PARTHENOGENETIC EMBRYOS IS AFFECTED BY PROLACTIN DURING THE PROLONGED CULTURE OF BOVINE CUMULUS-ENCLOSED OOCYTES

Abstract

The technique of somatic cell nuclear transfer includes a delayed activation of reconstituted oocytes (4–6 h after IVM). The prolonged culture of mammalian oocytes is known to be associated with aging of the ova leading to a decline in their quality and developmental capacity. The aim of the present research was to study effects of 2 closely related hormones, prolactin (PRL) and growth hormone (GH), during the prolonged culture of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-enclosed oocytes (CEO) were cultured for 16 h in TCM 199 containing 10% FCS, 10 μg mL−1 of porcine FSH, and 10 μg mL−1 of ovine LH. After 16 h of maturation, CEO or denuded oocytes were transferred to the fresh medium consisting of TCM 199 supplemented with 10% FCS and cultured for 12 h in the absence (Control) or in presence of 50 ng mL−1 of bovine PRL or 10 ng mL−1 of recombinant bovine GH or protein kinase inhibitors. The following inhibitors were used: (1) PP2 (an inhibitor of Src-family tyrosine kinases, 10 mM), (2) triciribine (an inhibitor of Akt kinase, 25 μM), and (3) calphostin C (a protein kinase C inhibitor, 0.5 μM). After the prolonged culture for 12 h, oocytes were activated by sequential treatment with ionomycin (5 μM for 5 min) immediately followed by 6-dimethylaminopurine (2 mM for 4 h). Activated oocytes were cultured in CR1aa medium until Day 5 postactivation and then transferred to the same medium supplemented with 5% FCS and cultured up to Day 7. The cleavage and blastocyst rates were assessed at Day 2 and 7, respectively. The data from 4 to 6 replicates (106–169 oocytes per treatment) were analysed by ANOVA. After the prolonged culture of CEO, the cleavage rate did not differ between the control and the hormone-treated groups, varying from 64.3 to 70.1%. Meanwhile, the blastocyst yield in the control group (12.5 ± 1.6%) was lower than in the PRL group (21.5 ± 2.4%, P < 0.05), but was similar to that in the GH group (13.9 ± 1.8%). Furthermore, the developmental capacity of cultured denuded oocytes was unaffected by both GH and PRL. The enhancing effect of PRL on the yield of parthenogenetic blastocysts derived from CEO was also observed in the presence of PP2 (26.8 ± 2.4 v. 15.7 ± 2.0%; P < 0.01). By contrast, calphostin C abolished this effect of PRL, although it did not affect the developmental potential of CEO in the control medium. At the same time triciribine reduced the blastocyst rate both in the control and PRL-treated groups (from 16.5 ± 1.8 to 8.1 ± 0.8% and from 24.3 ± 1.6 to 16.3 ± 2.4%, respectively; P < 0.05). Our data indicate that PRL can support the developmental potential of parthenogenetic embryos by affecting bovine oocytes during the prolonged culture, with the effect being mediated by cumulus cells and achieved via activation of protein kinase C. This research was supported by the Federal Agency for Scientific Organizations and RFBR (project No. 15-08-99473).