Kyeoung-Hwa Kim

Role of Bcl2-like 10 (Bcl2l10) in Regulating Mouse Oocyte Maturation

Previously, we have shown that Bcl2l10 is highly expressed in metaphase II (MII)-stage oocytes. The objective of this study was to characterize Bcl2l10 expression in ovaries and to examine the function of Bcl2l10 in oocyte maturation using RNA interference. Bcl2l10 transcript expression was ovary and oocyte specific. Bcl2l10 was highly expressed in oocytes and pronuclear-stage embryos; however, its expression decreased at the two-cell stage and dramatically disappeared thereafter. Microinjection of Bcl2l10 double-stranded RNA into the cytoplasm of germinal vesicle oocytes resulted in a marked decrease in Bcl2l10 mRNA and protein and metaphase I (MI) arrest (78.9%). Most MI-arrested oocytes exhibited abnormalities in their spindles and chromosome configurations. Bcl2l10 RNA interference had an obvious effect on the activity of maturation-promoting factor but not on that of mitogen-activated protein kinase. We concluded that the role of Bcl2l10 is strongly associated with oocyte maturation, especially at the MI–MII transition.

SEBOX Is Essential for Early Embryogenesis at the Two-Cell Stage in the Mouse

Abstract Previously, we found high levels of skin-embryo-brain-oocyte homeobox (Sebox) gene expression in germinal vesicle (GV)-stage oocytes. The objective of the present study was to determine the role played by SEBOX in oocyte maturation and early embryogenesis using RNA interference (RNAi). Microinjection of Sebox double-stranded RNA into GV oocytes resulted in a marked decrease in Sebox mRNA and protein expression. However, Sebox RNAi affects neither oocyte maturation rate nor morphological characteristics, including spindle and chromosomal organization of metaphase II oocytes. In addition, Sebox RNAi had no discernible effect on the activities of M-phase promoting factor or mitogen-activated protein kinase. In contrast, microinjection of Sebox double-stranded RNA into pronuclear-stage embryos resulted in holding embryo development at the two-cell (84.9%) and the four- and eight-cell (15.1%) stages. We concluded that Sebox is a new addition to maternal effect genes that produced and stored in oocytes and function in preimplantation embryo development.

Maternal effect genes: Findings and effects on mouse embryo development

Stored maternal factors in oocytes regulate oocyte differentiation into embryos during early embryonic development. Before zygotic gene activation (ZGA), these early embryos are mainly dependent on maternal factors for survival, such as macromolecules and subcellular organelles in oocytes. The genes encoding these essential maternal products are referred to as maternal effect genes (MEGs). MEGs accumulate maternal factors during oogenesis and enable ZGA, progression of early embryo development, and the initial establishment of embryonic cell lineages. Disruption of MEGs results in defective embryogenesis. Despite their important functions, only a few mammalian MEGs have been identified. In this review we summarize the roles of known MEGs in mouse fertility, with a particular emphasis on oocytes and early embryonic development. An increased knowledge of the working mechanism of MEGs could ultimately provide a means to regulate oocyte maturation and subsequent early embryonic development.

Obox4 critically regulates cAMP-dependent meiotic arrest and MI-MII transition in oocytes.

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 RNAitreated 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. FASEB J. 24, 2314–2324 (2010). www.fasebj.org

Gas6 Downregulation Impaired Cytoplasmic Maturation and Pronuclear Formation Independent to the MPF Activity

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 Sr2+, Gas6-silenced MII oocytes had markedly reduced Ca2+ 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.

Associations among Sebox and Other MEGs and Its Effects on Early Embryogenesis

In a previous report, we identified Sebox as a new candidate maternal effect gene that is essential for embryonic development and primarily impacts the two-cell (2C) stage. The present study was conducted to determine the mechanism of action for Sebox in this capacity, as shown by changes in the expression levels of other known MEG mRNAs after Sebox RNA interference (RNAi) in oocytes. Sebox-knockdown metaphase II (Mll) oocytes displayed normal morphology, but among the 23 MEGs monitored, 8 genes were upregulated, and 15 genes were unchanged. We hypothesized that the perturbed gene expression of these MEGs may cause the arrest of embryo development at the 2C stage and examined the expression of several marker genes for the degradation of maternal factors and zygotic genome activation. We found that some maternal mRNAs, c-mos, Gbx2, and Gdf9, were not fully degraded in Sebox-knockdown 2C embryos, and that several zygotic genome activation markers, Mt1a, Rpl23, Ube2a and Wee1, were not fully expressed in conjunction with diminished embryonic transcriptional activity. In addition, Sebox may be involved in the formation of the subcortical maternal complex through its regulation of the upstream regulator, Figla. Therefore, we concluded that Sebox is important in preparing oocytes for embryonic development by orchestrating the expression of other important MEGs.

Obox4-silencing-activated STAT3 and MPF/MAPK signaling accelerate nuclear membrane breakdown in mouse oocytes

Mouse oocytes begin to mature in vitro once liberated from ovarian follicles. Previously, we showed that oocyte-specific homeobox 4 (Obox4) is critical for maintaining the intact nuclear membrane of the germinal vesicle (GV) in oocytes and for completing meiosis at the metaphase I-II (MI-MII) transition. This study further examines the molecular mechanisms of OBOX4 in regulating GV nuclear membrane breakdown. Maturation-promoting factor (MPF) and 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 GV 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 (IFNA), 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 i) Obox4 is an important upstream regulator of MPF/MAPK and STAT3 signaling, and ii) Obox4 is a key regulator of the GV arrest mechanism in oocytes.

RASD1 Knockdown Results in Failure of Oocyte Maturation

Background: Ras dexamethasone-induced protein (RASD1) is a member of Ras superfamily of small GTPases. RASD1 regulates various signaling pathways involved in iron homeostasis, growth hormone secretion, and circadian rhythm. However, RASD1 function in oocyte remains unknown. Methods: Using immunohistochemistry, immunofluorescence, and quantitative real-time RT-PCR, RASD1 expression in mouse ovary and RASD1 role in oocyte maturation-related gene expression, spindle formation, and chromosome alignment were analyzed. RNAi microinjection and time-lapse video microscopy were used to examine the effect of Rasd1 knockdown on oocyte maturation. Results: RASD1 was highly detected in oocytes transitioning from primordial to secondary follicles. Rasd1 was highly expressed in germinal vesicle (GV), during GV breakdown, and in metaphase I (MI) stage as oocytes mature, and its expression was significantly downregulated in MII stage. With knockdown of Rasd1, maturation in GV oocytes was arrested at MI stage, showing disrupted meiotic spindling and chromosomal misalignment. In addition, Obox4 and Arp2/3, engaged in MI-MII transition and cytokinesis, respectively, were misregulated in GV oocytes by Rasd1 knockdown. Conclusion: These findings suggest that RASD1 is a novel factor in MI-MII oocyte transition and may be involved in regulating the progression of cytokinesis and spindle formation, controlling related signaling pathways during oocyte maturation.

The role of Rad51 in safeguarding mitochondrial activity during the meiotic cell cycle in mammalian oocytes

Rad51 is a conserved eukaryotic protein that mediates the homologous recombination repair of DNA double-strand breaks that occur during mitosis and meiosis. In addition, Rad51 promotes mitochondrial DNA synthesis when replication stress is increased. Rad51 also regulates cell cycle progression by preserving the G2/M transition in embryonic stem cells. In this study, we report a novel function of Rad51 in regulating mitochondrial activity during in vitro maturation of mouse oocytes. Suppression of Rad51 by injection of Rad51 dsRNA into germinal vesicle-stage oocytes resulted in arrest of meiosis in metaphase I. Rad51-depleted oocytes showed chromosome misalignment and failures in spindle aggregation, affecting the completion of cytokinesis. We found that Rad51 depletion was accompanied by decreased ATP production and mitochondrial membrane potential and increased DNA degradation. We further demonstrated that the mitochondrial defect activated autophagy in Rad51-depleted oocytes. Taken together, we concluded that Rad51 functions to safeguard mitochondrial integrity during the meiotic maturation of oocytes.

Function of COP9 signalosome in regulation of mouse oocytes meiosis by regulating MPF activity and securing degradation.

The COP9 (constitutive photomorphogenic) signalosome (CSN), composed of eight subunits, is a highly conserved protein complex that regulates processes such as cell cycle progression and kinase signalling. Previously, we found the expression of the COP9 constitutive photomorphogenic homolog subunit 3 (CSN3) and subunit 5 (CSN5) changes as oocytes mature for the first time, and there is no report regarding roles of COP9 in the mammalian oocytes. Therefore, in the present study, we examined the effects of RNA interference (RNAi)-mediated transient knockdown of each subunit on the meiotic cell cycle in mice oocytes. Following knockdown of either CSN3 or CSN5, oocytes failed to complete meiosis I. These arrested oocytes exhibited a disrupted meiotic spindle and misarranged chromosomes. Moreover, down-regulation of each subunit disrupted the activity of maturation-promoting factor (MPF) and concurrently reduced degradation of the anaphase-promoting complex/cyclosome (APC/C) substrates Cyclin B1 and Securin. Our data suggest that the CSN3 and CSN5 are involved in oocyte meiosis by regulating degradation of Cyclin B1 and Securin via APC/C.