Obox4 critically regulates cAMP-dependent meiotic arrest and MI-MII transition in oocytes
ABSTRACT Extra follicular oocytes spontaneously resume meiosis in vitro, but the intact germinal vesicle (GV) is retained if the oocytes are cultured in medium containing phosphodiesterase (PDE) inhibitors or cAMP analogues. On the basis of our finding that Obox4 is prominently expressed in oocytes, the present study was conducted to determine the functional role of the homeodomain-containing factor Obox4 during in vitro oocyte maturation. After microinjection of Obox4 dsRNA into the cytoplasm of GV oocytes cultured in M16 medium, oocytes were arrested at metaphase I (MI, 77.7%) and metaphase II (MII, 22.3%). Surprisingly, however, 89% of Obox4 RNAi-treated oocytes resumed meiosis and developed to MI and MII when cultured in medium containing 0.2 mM 3-isobutyl-1-methyl-xanthine (IBMX), in which untreated oocytes maintain intact GVs. Spindles were aberrant, and chromosomes were severely aggregated with decreased MPF and MAP kinase activities in arrested MI oocytes after exposure to Obox4 RNAi. Oocytes overexpressing Obox4 retained intact GVs when cultured in M16 medium. Taken together, for the first time to our knowledge, these findings indicate that Obox4 plays a key role in the cAMP-dependent signaling cascades that maintain GV arrest. Oocytes not expressing Obox4 failed to maintain intact GVs in IBMX-supplemented medium, while GVs remained intact when oocytes were kept in plain medium and overexpressing Obox4, suggesting that Obox4 plays a critical role in cAMP-dependent cascade for maintaining intact GVs.
- [Show abstract] [Hide abstract]
ABSTRACT: 1. In mice, acetylcholine (ACh) plays an important role in oocyte activation and embryonic development. However, the role of ACh in mouse oocyte maturation has not been investigated. 2. In the present study, the effects of 100 μmol/L and 1 mmol/L ACh on maturation processes of murine germinal vesicle (GV) intact oocytes (GV oocytes) exposed to 10 and 100 μmol/L 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, were evaluated morphologically and immunologically. It has been shown that IBMX inhibits the resumption of meiosis by preventing cAMP breakdown. 3. In the present study, at the start of in vitro culture 100% of oocytes were at the GV stage. After 18 h culture, 95 ± 3, 0 and 85.8 ± 10.2% of oocytes had passed the GV stage in the control, IBMX and IBMX + ACh groups, respectively. The IBMX-induced inhibition of the maturation process was significantly attenuated by approximately 90% by ACh in groups treated with 10 μmol/L IBMX + 100 μmol/L ACh and 100 μmol/L IBMX + 1 mmol/L ACh. Although cAMP levels were high in oocytes treated with 100 μmol/L IBMX, levels were reduced in groups treated simultaneously with 100 μmol/L ACh. Furthermore, compared with mature oocytes, ACh-treated GV oocytes exhibited significantly lower (by approximately 2.3-fold) or absent Ca(2+) peaks. 4. The results of the present study indicate that maturation of GV oocytes, arrested by IBMX treatment, is resumed following ACh treatment and that this effect is due to downregulation of cAMP rather than changes in intracellular Ca(2+) levels.Clinical and Experimental Pharmacology and Physiology 05/2011; 38(7):435-7. DOI:10.1111/j.1440-1681.2011.05533.x · 2.41 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Previously, we found that oocyte specific homeobox (Obox) 4 plays significant role in completion of meiosis specifically at meiosis I-meiosis II (MI-MII) transition. The purpose of this study was to determine the mechanism of action of Obox4 in oocyte maturation by evaluating downstream signal networking. The Obox4 dsRNA was prepared by in vitro transcription and microinjected into the cytoplasm of germinal vesicle oocytes followed by in vitro maturation in the presence or absence of 0.2 mM 3-isobutyl-1-metyl-xanthine. Total RNA was extracted from 200 oocytes of each group using a PicoPure RNA isolation kit then amplified two-rounds. The probe hybridization and data analysis were used by Affymetrix GeneChip® Mouse Genome 430 2.0 array and GenPlex 3.0 (ISTECH, Korea) software, respectively. Total 424 genes were up (n=80) and down (n=344) regulated after Obox4 RNA interference (RNAi). Genes mainly related to metabolic pathways and mitogen-activated protein kinase (MAPK) signaling pathway was changed. Among the protein kinase C (PKC) isoforms, PKC-alpha, beta, gamma were down-regulated and especially the MAPK signaling pathway PKC-gamma was dramatically decreased by Obox4 RNAi. In the cell cycle pathway, we evaluated the expression of genes involved in regulation of chromosome separation, and found that these genes were down-regulated. It may cause the aberrant chromosome segregation during MI-MII transition. From the results of this study, it is concluded that Obox4 is important upstream regulator of the PKC and anaphase-promoting complex action for maintaining intact germinal vesicle.06/2011; 38(2):68-74. DOI:10.5653/cerm.2011.38.2.68
- [Show abstract] [Hide abstract]
ABSTRACT: Previously, we found that the growth arrest-specific gene 6 (Gas6) is more highly expressed in germinal vesicle (GV) oocytes than in metaphase II (MII) oocytes using annealing control primer (ACP)-PCR technology. The current study was undertaken to investigate the role of Gas6 in oocyte maturation and fertilization using RNA interference (RNAi). Interestingly, despite the specific and marked decrease in Gas6 mRNA and protein expression in GVs after Gas6 RNAi, nuclear maturation including spindle structures and chromosome segregation was not affected. The only discernible effect induced by Gas6 RNAi was a change in maturation promoting factor (MPF) activity. After parthenogenetic activation, Gas6 RNAi-treated oocytes at the MII stage had not developed further and arrested at MII (90.0%). After stimulation with Sr(2+), Gas6-silenced MII oocytes had markedly reduced Ca(2+) oscillation and exhibited no exocytosis of cortical granules. In these oocytes, sperm penetration occurred during fertilization but not pronucleus (PN) formation. By roscovitine and colcemid treatment, we found that the Gas6 knockdown affected cytoplasmic maturation directly, independent to the changed MPF activity. These results strongly suggest that 1) the Gas6 signaling itself is important to the cytoplasmic maturation, but not nuclear maturation, and 2) the decreased Gas6 expression and decreased MPF activity separately or mutually influence sperm head decondensation and PN formation.PLoS ONE 08/2011; 6(8):e23304. DOI:10.1371/journal.pone.0023304 · 3.53 Impact Factor