284 INTRACELLULAR PATHWAYS MEDIATING DIRECT EFFECTS OF PROLACTIN AND GROWTH HORMONE ON BOVINE METAPHASE-II OOCYTES AGING IN VITRO

Abstract

Maturation of mammalian oocytes coincides with various aging processes, which negatively affect the oocyte quality. Cellular and molecular changes in matured oocytes aging in vivo and in vitro are very similar, suggesting similarities in the underlying mechanisms. The goal of the present research was to study direct effects of prolactin (PRL) and growth hormone (GH) on bovine metaphase-II (M-II) oocytes aging in vitro and intracellular pathways mediating these effects. Cumulus-enclosed oocytes were matured for 20 h in TCM 199 containing 10% fetal calf serum (FCS), 10 µg mL–1 of porcine FSH, and 10 µg mL–1 of ovine LH. After IVM, oocytes were set free from cumulus and denuded oocytes (DO) were cultured for 44 h in the aging medium consisting of TCM 199 supplemented with 10% FCS in the absence of PRL and GH (Control), then in the presence of either 50 ng mL–1 of bovine PRL or 10 ng mL–1 of bovine GH or protein kinase inhibitors. The following inhibitors were added: (1) PP2 (an inhibitor of Src-family tyrosine kinases, 20 µM), (2) U0126 (a MEK inhibitor, 20 µM), and (3) triciribine (an inhibitor of Akt kinase, 50 µM). At the end of culture, the state of the nuclear material of oocytes and embryos was evaluated by the method of Tarkowski. The number of oocytes undergone spontaneous parthenogenetic activation was determined by summarising the numbers of embryos cleaved and oocytes reached anaphase-II to pronucleus stages. The data from 4 replicates were analysed by ANOVA. During aging in the control medium, the rate of M-II oocytes with destructive changes of chromosomes (decondensation, clumping, fragmentation) increased from 20.3 ± 1.7 to 65.1 ± 3.5% (P < 0.001) and was unaffected by PRL and GH. At the same time, the frequency of oocyte parthenogenetic activation (18.7 ± 4.9%) was reduced (P < 0.001) by both PRL and GH (up to 6.0 ± 2.9 and 5.9 ± 2.4%, respectively). The inhibitor of Src-family tyrosine kinases PP2 eliminated (at least P < 0.05) the supporting effects of PRL and GH on the meiotic arrest at M-II. The MEK inhibitor U0126 also abolished the effect of PRL (but not that of GH), increasing the frequency of the oocyte activation from 5.9 ± 2.3 to 21.6 ± 1.9% (P < 0.01). Concurrently, both inhibitors did not affect the meiotic arrest in the control group. The inhibitor of Akt kinase triciribine did not influence the frequency of the oocyte activation in the PRL- and GH-treated groups. Meanwhile, triciribine decreased the rate of M-II oocytes with chromosome abnormalities in the control medium (from 71.2 ± 1.7 to 47.0 ± 2.7%; P < 0.001). When added to the aging medium, PRL enhanced the triciribine effect, while GH suppressed it (P < 0.01). Thus, PRL and GH can directly support the meiotic arrest at M-II in aging oocytes by activating MAP kinases or Src-family tyrosine kinases. Besides, GH may enhance the chromosome destruction in DOs through activation of Akt kinase.The research was supported by RFBR (No. 13-04-01888).