Reproductive biotechnology such as in vitro fertilization, the creation of transgenic animals or cloning by nuclear transfer depends on the use of fully grown, meiotically competent oocytes capable of completing meiotic maturation by reaching the stage of metaphase II. However, there exists only a limited quantity of these oocytes in the ovaries of females. In view of their limited number, growing oocytes without meiotic competence represent a possible source. The mechanisms controlling the acquisition of meiotic competence, however, are still not completely clear. A gas with a short half-life, nitric oxide (NO), produced by NO-synthase (NOS) enzyme can fulfill a regulatory role in this period. The objective of this study was to ascertain the role of NO in the growth phase of pig oocytes and its influence on the acquisition of meiotic competence with the help of NOS inhibitors, NO donors and their combinations. We demonstrated that the selective competitive iNOS inhibitor aminoguanidine and also the non-selective NOS inhibitor L-NAME block meiotic maturation of oocytes with partial or even full meiotic competence at the very beginning. NOS inhibitors influence even competent oocytes in the first stage of meiotic metaphase. However, blockage is less effective than at the beginning of meiotic maturation. The number of parthenogenetically activated competent oocytes greatly increased in a pure medium after inhibitor reversion. A large quantity of NO externally added to the in vitro cultivation environment disrupts the viability of oocytes. The effectiveness of the inhibitor can be reversed in oocytes by an NO donor in a very low concentration. However, the donor is not capable of pushing the oocytes farther than beyond the first stage of meiotic metaphase. The experiments confirmed the connection of NO with the growth period and the acquisition of meiotic competence. However, it is evident from the experiments that NO is not the only stimulus controlling the growth period.

Porcine oocytes matured in vitro were activated for parthenogenetic development using either calcium ionophore (50µM for 10 min) or nitric oxide donor SNAP (2mM for 23.5 hours). Protein kinase C (PKC) inhibitors, bisindolylmaleimide I or rottlerin, are able to inhibit parthenogenetic activation induced by calcium ionophore. The rate of activated oocytes decreased from 69% to 2% (P < 0.05) under the effect of bisindolylmaleimide I at a concentration of 0 or 20nM, respectively. The activation rate decreased from 68% to 0% (P < 0.05) under the influence of 0 or 20µM rottlerin, respectively. PKC inhibitors Go6976 or hispidin had no effect on the oocyte activation using calcium ionophore or on oocytes activated by a nitric oxide donor. The activation of oocytes by a nitric oxide donor is not significantly influenced even under the effects of bisindolylmaleimide I or rottlerin. Based on these data we can conclude that the oocyte activation induced by calcium ionophore depends on PKC, especially on PKC-δ. On the other hand, the oocyte activation induced by nitric oxide is independent of the tested isotypes of PKC.

ABSTRACT:Oocyte ageing is a complex of processes that occur when matured in vitro oocytes are, after reaching the metaphase II stage, exposed to further in vitro culture. Aged oocytes remaining at the metaphase II stage undergo spontaneous parthenogenetic activation, or cellular death, through apoptosis (fragmentation) or lysis. The key factor in apoptotic pathway regulation is c-Jun-N-terminal kinase (JNK), stress kinase from the mitogene-activated protein kinase (MAPK) family. To investigate the effect of JNK inhibition on porcine oocytes ageing, cleavage rate, and embryonic development after parthenogenetic activation, DNA fragmentation, and pro-apoptotic factor Bax expression, we cultured in vitro matured oocytes for another 1-4 days in the presence of a JNK inhibitor. The inhibition of JNK significantly protected the oocytes from fragmentation (0% of fragmented oocytes under JNK inhibition vs. 13.4% of fragmented oocytes in the control group, 2 nd day of ageing) and increased the percentage of parthenogenetically activated oocytes (82 vs 57.7%, 2 nd day of ageing). The embryonic development of oocytes parthenogenetically activated after 24 h of ageing was influenced by JNK inhibition as well. The percentage of oocytes at the morula stage, after seven days of cultivation, was significantly increased when oocytes aged in the presence of a JNK inhibitor (42.5%) by comparison to the percentage of oocytes exposed to ageing in an inhibitor-free medium (23.3%). DNA fragmentation was significantly suppressed by JNK inhibition from the 1 st day of ageing, but the expression of pro-apoptotic factor Bax in the oocytes was not influenced. On the basis of our experiments, we can conclude that JNK inhibition suppresses apoptosis and DNA fragmentation of aged oocytes and improves their embryonic development following the parthenogenetic activation. However, to completely eliminate all ageing related processes is insufficient.

The objective of this study was to assess the effect of vitamin E and selenium (Se) supplementation on the plasma levels of F 2 -isoprostanes as a marker of oxidative stress in horses in their training period. Twelve healthy 3-year-old English thoroughbred horses were divided into two groups: control (n = 6) and experimental (n = 6). Feeding rations were adapted to a moderate workload. The horses of the experimental group received supplements of DL--tocopheryl acetate E (2 250 mg/day/horse) and of sodium selenite (0.5 mg/day/horse). The plasma concentrations of both antioxidants and F 2 -isoprostanes were monitored on days 0, 44 and 70. After 70 days of supplementation, the concentrations of selenium in the experimental group were significantly higher (P  0.05) compared to the beginning of the experiment (mean ± SE: 135. ). In the horses of the experimental group, plasma -tocopherol levels significantly increased from the 44 th day of supplementation compared to the beginning of the study as well to the control group (5.23 ± 0.52 mg·l -1 vs. 2.45 ± 0.25 mg·l -1 or 3.46 ± 0.34 mg·l -1 , respectively). The plasma concentration of F 2 -isoprostanes tended to be lower in the experimental group at the end than at the beginning of monitoring (156.8 ± 12.89 pg·ml -1 vs. 170.3 ± 60.8 pg·ml -1 ), although the control group showed the opposite trend (181.2 ± 15.67 pg·ml -1 vs. 137.0 ± 47.05 pg·ml -1 ). Nevertheless, none of these differences were significant because of the large variability of the individual values. It can be stated that supplementation of the diet used with selenium and vitamin E caused a non-significant decrease of F 2 -isoprostane concentration in the blood plasma only, and a significant increase of plasma concentrations of these antioxidants. The variation of isoprostane levels probably reflected rather the individual responses of the horses' organisms to the training workload.