Nitric oxide influences the maturation of cumulus cell-enclosed mouse oocytes cultured in spontaneous maturation medium and hypoxanthine-supplemented medium through different signaling pathways.
Nitric oxide (NO) has been recently shown to act with a dual action in mouse oocyte meiotic maturation depending on its concentration, but the mechanism(s) through which it influences oocyte maturation has not been fully clarified to date. The purpose of this study was to test the hypothesis that different signaling mechanisms exist for NO-stimulated and NO-inhibited in vitro maturation of meiosis in cumulus cell-enclosed oocytes (CEOs) from PMSG-primed immature female mice. CEOs were cultured in both spontaneous maturation model and hypoxanthine (HX) arrested model to investigate the mechanism(s). Sodium nitroprusside (SNP, an NO donor) at a concentration of 1mM delayed significantly germinal vesical breakdown (GVBD) during the first 5 h of incubation period and further inhibited the formation of first polar body (PB1) at the end of 24 h of incubation. While SNP, at a concentration of 10 microM, stimulated significantly the meiotic maturation of oocytes by overcoming the inhibition of HX. Methinine blue (MB, 10 microM) or 1-H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 microM)), two soluble guanylate cyclase (sGC) inhibitors, could reverse SNP-inhibited spontaneous oocyte maturation, but had no effect on SNP-stimulated meiotic maturation in the presence of HX. 8-Br-cGMP (1mM), a cell-permeating cGMP analogue, demonstrated a significant inhibitory effect on both spontaneous meiotic maturation and HX-arrested meiotic maturation. The delay effect of SNP on GVBD occurrence was similar to that of forskolin (6 microM, an adenylate cyclase stimulator) and rolipram (250 microM, a phosphodiesterase 4 inhibitor), two cAMP elevating reagents. Both forskolin and rolipram reversed significantly the SNP-stimulated meiotic maturation, but did not reverse the SNP-inhibited spontaneous meiotic maturation. Cilostamide (1 microM), the selective inhibitor of phosphodiestrase 3 (PDE3), could mimic the inhibitory effect of HX on the spontaneous meiotic maturation in CEOs and this inhibitory effect could also be reversed by SNP (10 microM). Moreover, sphingosine (3 microM), a protein kinase C (PKC) inhibitor, blocked the SNP-inhibited spontaneous meiotic maturation, but did not block the SNP-stimulated meiotic maturation. Clearly, these results suggest that pathway differences are present between SNP-inhibited spontaneous meiotic maturation and SNP-stimulated meiotic maturation of mouse oocytes.
College of Biological Sciences, China Agricultural University, Beijing 100094, PR China.
Shumin
S
Bu
Huirong
H
Xie
Yong
Y
Tao
Jianhong
J
Wang
Guoliang
G
Xia
D000818:Animals; D002478:Cells, Cultured; D003470:Culture Media; D000242:Cyclic AMP / Q000502:physiology; D006152:Cyclic GMP / Q000502:physiology; D005260:Female; D006162:Guanylate Cyclase; D019271:Hypoxanthine / Q000494:pharmacology*; D008540:Meiosis / Q000187:drug effects / Q000502:physiology; D051379:Mice; D009569:Nitric Oxide / Q000502:physiology*; D009599:Nitroprusside / Q000494:pharmacology; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000254:growth & development*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000502:physiology; D015398:Signal Transduction; D000071756:Soluble Guanylyl Cyclase
D003470:Culture Media; D018160:Receptors, Cytoplasmic and Nuclear; D009599:Nitroprusside; D019271:Hypoxanthine; D009569:Nitric Oxide; D000242:Cyclic AMP; D011493:Protein Kinase C; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP