Inducible nitric oxide synthase-derived nitric oxide regulates germinal vesicle breakdown and first polar body emission in the mouse oocyte.
The present study investigated the subcellular localization of inducible nitric oxide synthase (iNOS) during mouse oocyte meiotic maturation and fertilization using confocal microscopy, and further studied the roles of iNOS-derived NO in oocyte maturation by using an iNOS-specific inhibitor aminoguanidine (AG) and iNOS antibody microinjection. In germinal vesicle-stage oocytes, iNOS immunoreactivity was mainly localized in the germinal vesicle. Shortly after germinal vesicle breakdown, the iNOS immunoreactivity accumulated around the condensed chromosomes. At metaphase I and metaphase II, with the organization of chromosomes to the equatorial plate, iNOS immunoreactivity was concentrated around the aligned chromosomes, putatively the position of the metaphase spindle. The accumulation of iNOS immunoreactivity could not be detected at anaphase I and anaphase II. However, at telophase I and telophase II, the staining of iNOS was concentrated in the region between the separating chromosomes/chromatids. Furthermore, the staining of iNOS also accumulated in the male and female pronuclei in fertilized eggs. Germinal vesicle breakdown and the first polar body emission of the oocytes were significantly blocked by the iNOS-specific inhibitor AG in a dose-dependent manner. The germinal vesicle breakdown in oocytes injected with iNOS antibody was also inhibited. We found that the phosphorylation of mitogen-activated protein kinase in oocytes after germinal vesicle breakdown was inhibited by AG treatment. The control oocytes extruded a normal first polar body, while the AG-treated oocytes exhibited an elongated protrusion or no elongated protrusion. The results of confocal microscopy showed that the AG-treated oocytes were arrested at anaphase I-telophase I. Our results suggest that the iNOS-derived NO pathway plays important roles in mouse oocyte meiotic maturation, especially in germinal vesicle breakdown and the anaphase-telophase transition.
Huo
Li-Jun
LJ
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
Liang
Cheng-Guang
CG
Yu
Ling-Zhu
LZ
Zhong
Zhi-Sheng
ZS
Yang
Zeng-Ming
ZM
Fan
Heng-Yu
HY
Chen
Da-Yuan
DY
Sun
Qing-Yuan
QY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage; D015153:Blotting, Western / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006146:Guanidines / Q000494:pharmacology; D008297:Male; D051379:Mice; D008845:Microinjections; D018613:Microscopy, Confocal; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009865:Oocytes / Q000201:enzymology*; D009866:Oogenesis / Q000187:drug effects*; D013084:Sperm-Ovum Interactions
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D006146:Guanidines; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C004479:pimagedine