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Obox4-silencing activated STAT3 and MPF/MAPK signaling accelerate GVBD in mouse oocytes. Lee HS et al. (2016) Mouse oocytes begin to mature in vitro once liberated from ovarian follicles. Previously, we showed that oocyte-specific homeobox (Obox) 4 is critical for maintaining the intact nuclear membrane of the germinal vesicle (GV) in oocytes and for completing meiosis at the metaphase I-metaphase II (MI-MII) transition. This study further examines the molecular mechanisms of Obox4 in regulating GV nuclear membrane breakdown. Maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are normally inactive in GV stage oocytes but were activated prematurely in arrested GV stage oocytes by 3-isobutyl-1-metyl-xanthine (IBMX) in vitro after Obox4 RNA interference (RNAi). Furthermore, signal transducer and activator of transcription 3 (STAT3) was significantly activated by Obox4 RNAi. We confirmed that this Obox4 RNAi-induced premature STAT3 and MPF/MAPK activation at the GV stage provoked subsequent germinal vesicle breakdown (GVBD) despite the opposing force of high cAMP in the IBMX-supplemented medium to maintain intact GV. When cumulus-oocyte complexes were exposed to interferon α (IFN-α), a STAT3 activator, oocytes matured and cumulus cells expanded to resume nuclear maturation in IBMX-supplemented medium, suggesting that STAT3 activation is sufficient for stimulating the continuation of meiosis. Using Stattic, a specific STAT3 inhibitor, we confirmed that GVBD involves STAT3 activation in Obox4-silenced oocytes. Based on these findings, we concluded that 1) Obox4 is an important upstream regulator of MPF/MAPK and STAT3 signaling and 2) Obox4 is a key regulator of the GV arrest mechanism in oocytes.//////////////////
Changes in gene expression associated with oocyte meiosis after Obox4 RNAi. Lee HS et al. 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. Obox4 critically regulates cAMP-dependent meiotic arrest and MI-MII transition in oocytes. Lee HS et al. 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-metyl-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.-Lee, H.-S., Kim, E.-Y., Kim, K.-H., Moon, J., Park, K.-S., Kim, K.-S., Lee, K.-A. Obox4 critically regulates cAMP-dependent meiotic arrest and MI-MII transition in oocytes.
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Obox4 regulates the expression of histone family genes and promotes differentiation of mouse embryonic stem cells. Kim HM et al. Obox genes are preferentially expressed in the ovary, testis and oocyte, and play important roles in many developmental processes. In this study, we report that Obox4 and Obox6 are expressed in mouse embryonic stem cells (mESCs) and that Obox4 regulates histone family gene expression in mESCs. Obox4 protein expressing mESCs formed colonies with a flattened and irregular morphology, and exhibited decreased expression levels of self-renewal related proteins, such as Oct4 and Sox2, as well as reduced alkaline phosphatase activity. The results of microarray analysis and siRNA mediated knockdown experiments suggest that Obox4 is an upstream regulator of the histone gene family.
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