Egg activation induced by osmotic pressure change and the effects of amiloride on the cryopreservation of mouse oocytes.
Activation of oocytes is caused by osmotic pressure change in some species. However, cryopreservation of oocytes occurs in the presence of osmotic pressure change induced by cryoprotectants. We investigated the effect of 5-(N,N,-dimethyl)-amiloride (NNDMA), a selective inhibitor of Na+/H+ exchange, on the cryopreservation and osmotic activation of mouse oocytes. The percentage (23.2%) of degenerate oocytes after cryopreservation in the presence of NNDMA was found to be lower than that (39.5%) of untreated oocytes. After thawing, the percentage (23.6%) of oocytes which could be fertilized following cryopreservation in the presence of NNDMA was significantly higher than that of untreated (18.0%) oocytes. These results suggest that amiloride increased the survival rate after thawing following cryopreservation. To investigate the effect of NNDMA on oocyte activation caused by the cryoprotectant, dimethyl sulphoxide (DMSO) was used to induce osmotic pressure change. NNDMA was found to inhibit cortical granule exocytosis, the second polar body emission and pronuclear formation which occurs upon activation due to osmotic pressure change. It also inhibited the increase in phosphorylation of many proteins including 33 and 45 kDa proteins, which occurs, during fertilization and chemical oocyte activation. In contrast, protein phosphorylation was not inhibited by W7, a calmodulin inhibitor. The actions of these inhibitors suggest that oocyte activation induced by osmotic pressure change involves a pathway mediated by Na+/H+ exchange which may be distinct from the Ca-calmodulin pathway. Amiloride may be a useful drug for increasing the rate of survival of cryopreserved oocytes.
Inagaki
N
N
Department of Obstetrics and Gynecology, School of Medicine, Keio University, Tokyo, Japan.
Suzuki
S
S
Kuji
N
N
Kitai
H
H
Nakatogawa
N
N
Nozawa
S
S
D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002869:Chromosome Aberrations; D015925:Cryopreservation / Q000379:methods*; D004121:Dimethyl Sulfoxide; D005089:Exocytosis / Q000187:drug effects; D005260:Female; D005307:Fertilization in Vitro; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D017136:Ion Transport / Q000187:drug effects; D051379:Mice; D008813:Mice, Inbred ICR; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D009997:Osmotic Pressure; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism
D011506:Proteins; D017923:Sodium-Hydrogen Exchangers; C039465:5-dimethylamiloride; D000584:Amiloride; D004121:Dimethyl Sulfoxide