Nitric oxide acts through different signaling pathways in maturation of cumulus cell-enclosed mouse oocytes

Nitric oxide [NO] have a dual action in mouse oocyte meiotic maturation which depends on its concentration, but the mechanisms by which it influences oocyte maturation has not been exactly clarified. In this study different signaling mechanisms which exist for in vitro maturation of meiosis was examined in cumulus cell-enclosed oocytes [CEOs] after injection of pregnant mare's serum gonadotropin [PMSG] to immature female mice. The CEOs were cultured in spontaneous maturation and hypoxanthine [HX] arrested model. Sodium nitroprusside [SNP, an NO donor, 10mM] delayed germinal vesicle breakdown [GVBD] significantly during the first 5 hrs of incubation and inhibited the formation of first polar body [PB1] at the end of 24 hrs of incubation. SNP [10-5M] stimulated the meiotic maturation of oocytes significantly by overcoming the inhibition of HX. Sildenafil [a cGMP stimulator, 100 nM], had a significant inhibitory effects on both spontaneous meiotic maturation and HX-arrested meiotic maturation. Forskolin [an adenylate cyclase stimulator, 6 micro M] and SNP [10mM] had the same effects on GVBD. Forskolin reversed the SNP [10-5M] stimulated meiotic maturation. These results suggest that differences in pathways are present between SNP-inhibited spontaneous meiotic maturation and SNP-stimulated meiotic maturation in mouse oocytes

Presence of antioxidant in vitro maturation medium and its effects on glutathione level, spindle area and rate of in vitro fertilization

Effect of different doses of cysteamine on rate of in vitro maturation [IVM], in vitro fertilization [IVF] and glutathione [GSH] level was studied. Metaphase II [MII] spindle area was analyzed for quantification of shape and size of oocytes. Female mice were primed with 5 IU of pregnant mare's stimulating gonadotrophin. Germinal vesicle [GV] oocytes were retrieved 48 hrs later. IVM medium was supplemented with 0, 50, 100, 200 and 500 mM of cysteamine. For IVM and IVF assessment in each group, 150 GV oocytes were used. Experiments also included a group of ovulated oocytes [matured in vivo] after priming with pregnant mare's stimulating gonadotrophin and human chorionic gonadotropin. GSH level was measured by 5, 5-Dithio-bis [2nitrobenzoic acid] DTNB-GR recycling protocol in GV and MII oocytes. For IVF, MII oocytes were inseminated with mature mouse sperm and rate of two-cell embryo was measured. For immunocytochemistry of microtubule and chromosomes, MII oocytes were fixed by methanol and immunostained with alpha-and beta-microtubule antibody and Hoechst. The spindle area was then analyzed. A dose-dependent improvement was observed in IVM and IVF rate. MII development and two-cell embryo formation were increased significantly in group which received 200 micro m cysteamine compare to the control group. GSH level was increased in presence of cysteamine in group which received 200 micro m cysteamine. Spindle area was increased in all groups in vitro except for the group which received 500 micro m cysteamine. The difference between spindle area in 200 micro m cysteamine and in vivo group was not significant [P>0.05]. Administration of cysteamine improves IVM and IVF rate in a dose-dependant manner. Also cysteamine induces glutathione synthesis in MII oocyte and improves microtubule when administered at a dose of 200 micro m. Therefore, addition of cysteamine as an antioxidant can improve IVM and IVF rate by increasing of oocytes quality

[ effects of hyaluronan on fertilization capability of mouse sperm before cryopreservation and after thawing]

Freezing and thawing induce a number of insults to the sperm cells, such as low motility and low fertilization capability. For evaluation of hyaluronan [HA] supplementation on sperm characteristics, we investigated the effect of hyaluronan [HA] on mouse sperm before freezing and after thawing. For this purpose we removed cauda epididimes from 24 male mice with aseptic method and freezed the semen in 1.8ml cryotubes with%18 raffinose and%3 skim milk cryoprotectant solution.We had 4 groups: group 1[fresh control] group 2 [freeze control] group 3[supplemented 750 micro g/ml HA to sperm before freezing] and group 4[supplemented 750 micro g/ml HA to sperm after thawing]. Fertility rate evaluated after routine IVF by counting two-cell stage embryos. HA supplementation [750 micro g /ml] after thawing improved fertilization capability parameters but supplementation before freezing had no effect on mentioned characteristic. Acording to data of present study the hyaluronan supplemen- tation [750 micro g /ml] after thawing has the greatest effect on the fertility rate of sperms

[Ischemic and reperfusion injury of adult rat sciatic nerve]

Ischemia plays a major role in development of pathological changes in various neuropathies. Reperfusion amplifies physiological and pathological abnormalities in ischemic nerves. In this research, we studied ischemic-reperfusion [IR] injury of sciatic nerve up to 14 days of reperfusion. IR was produced by ligation and release of nooses around supplying vessels to the sciatic nerve. 30 rats were assigned into 5 groups of 6. Group 1 [control] did not undergo IR while the 4 remaining groups after three hours of complete hind leg ischemia underwent reperfusion within 0hr, 3hrs, 7 days and 14 days. Pathologically, two phases were identifiable. During phase 1 [0-3 hrs] fiber degeneration and endoneurial edema were observed. During phase 2 [7 days and, 14 days] prominent fiber degeneration and prominent endoneurial edema were observed. Loss of function occurred in more than 75% of the rats with ischemia alone, in comparison with the control group the maximum reduction in activities was observed amongst the group of rats reperfused within 3 hours. IR injury depends on duration of reperfusion. Microvascular events during reperfusion may enhance the nerve fiber damage following the ischemia period