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Immature Oocyte-Specific Zap70 and Its Functional Analysis in Regulating Oocyte Maturation
Kim Yun-Na,Kim Eun-Ju,Kim Eun-Young,Lee Hyun-Seo,Kim Kyeoung-Hwa,Lee Kyung-Ah 한국발생생물학회 2009 발생과 생식 Vol.13 No.3
Previously, we obtained the list of genes differentially expressed between GV and MII oocytes. Out of the list, we focused on functional analysis of Zap70 in the present study, because it has been known to be expressed only in immune cells. This is the first report about the expression and its function of Zap70 in the oocytes. Synthetic 475 bp Zap70 dsRNA was microinjected into the GV oocytes, and the oocytes were cultured in vitro. In addition to maturation rates, meiotic spindle and chromosome rearrangements, and changes in expression levels of transcripts of three kinases, Erk1/2, JNK, and p38, were determined. Zap70 is highly expressed in immature GV oocytes, and gradually decreased as oocyte matured. When dsRNA of Zap70 was injected into the GV oocytes, Zap70 mRNA specifically and completely decreased by 2 hr and its protein expression also decreased significantly. Absence of Zap70 resulted in maturation inhibition at meiosis I (57%) with abnormalities in meiotic spindle formation and chromosome rearrangement. Concurrently, mRNA expression of Erk2, JNK, and p38, were affected by Zap70 RNAi. Therefore, we concluded that Zap70 is involved in MI-MII transition by affecting expression of MAP kinases.
Maternal effect genes: Findings and effects on mouse embryo development
Kim, Kyeoung-Hwa,Lee, Kyung-Ah The Korean Society for Reproductive Medicine 2014 Clinical and Experimental Reproductive Medicine Vol.41 No.2
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.
The role of maternal effect genes, Gas6 and Sebox
Kyeoung-Hwa Kim,Kyung-Ah Lee 한국발생생물학회 2017 한국발생생물학회 학술발표대회 Vol.2017 No.8
The conserved The oocytes acquire competence to undergo complex processes, oocyte growth and oocyte maturation, and the capacity for fertilization and preimplantational embryo development, by accumulating RNAs and proteins in the ooplasm. Therefore, the identification of the genes expressed in the oocyte and its functional analysis will provide valuable resources to study molecular regulatory mechanism of oocyte maturation, fertilization and early embryogenesis. To better understand these mechanisms, a decade ago, we identified a list of differentially expressed genes between GV and MII oocytes using annealing control primer (ACP)-PCR technology. Among these genes, we selected two genes, Gas6 (growth arrest-specific 6), and Sebox (skin-embryobrain- oocyte homeobox) that expressed significantly higher levels in GV than MII and analyzed its functions by using RNA interference (RNAi). Unexpectedly, and fortunately, it turned out that both of genes are new candidate of maternal effect genes (MEGs) that is important for fertilization and/or early embryogenesis but not crucial for oocyte meiotic maturation. In particular, Gas6 is essential in maintaining the proper mitochondrial function, and biosynthesis of heparan sulfate and glutathione, which are required for normal sperm chromatin decondensation, pronuclear formation, and mainly for the sufficient cytoplasmic maturation of oocytes. We suggest that the correction in the Gas6 signaling network in oocytes may improve the embryonic developmental capacity caused by deterioration of the mitochondrial functions and/or contents during oocyte maturation. Meanwhile, Sebox is crucial for zygotic genome activation (ZGA) required for subsequent embryonic development beyond the 2-cell stage by coordinating the expression of other maternal factors, such as c-mos, Gdf9, Ube2a and Wee1. In conclusion, the observed failure of fertilization after Gas6 RNAi and the embryonic development at the 2-cell stage after Sebox RNAi was similar to the loss-of-function of the previously well-known MEGs. Based on these findings, we added Gas6 and Sebox as new mammalian MEGs. Findings of our research would broaden our knowledge regarding MEGs and a field of maternal programming in oocytes.
Kyeoung-Hwa Kim,이상은,이경아 한국발생생물학회 2012 발생과 생식 Vol.16 No.3
Recently, we reported growth arrest-specific gene 6 (Gas6) as a new maternal effect gene (MEG), that expressed in the oocytes but functioned principally during embryogenesis. Gas6 RNAi-treated oocytes developed to metaphase II (MII) stage but they have affected M-phase promoting factor (MPF) activity and incurred abnormal pronuclear (PN) formation during fertilization. Gas6 is a ligand of TAM family members (Tyro3, Axl and Mertk) of receptor tyrosine kinase (RTK). Purpose of the present study was to evaluate the expression of Tyro3, Axl and Mertk transcripts in oocytes and early embryos. Expression of Gas6 and Mertk mRNA was detectable in oocytes and follicular cells, while Tyro3 and Axl mRNA was expressed only in follicular cells. Expression of Mertk mRNA was relatively constant during oocytes maturation and embryogenesis, but the other receptors, Tyro3 and Axl, were not expressed in oocytes and PN stage of embryos at all. Knockdown of Mertk mRNA and protein by using sequence-specific Mertk double strand RNA (dsRNA)did not affect oocytes maturation. In this case, however, contrary to the ligand Gas6 RNA interference (RNAi), MPF activity had not been changed by Mertk RNAi. Therefore, we concluded that the Gas6-Mertk signaling is not directly related to the oocyte maturation. It is still required to study further regarding the function of Mertk as the receptor of Gas6 during preimplantational early embryogenesis.
Kim Kyeoung-Hwa,Lee Sang-Eun,Lee Kyung-Ah 한국발생생물학회 2012 발생과 생식 Vol.16 No.3
Recently, we reported growth arrest-specific gene 6 (Gas6) as a new maternal effect gene (MEG), that expressed in the oocytes but functioned principally during embryogenesis. Gas6 RNAi-treated oocytes developed to metaphase II (MII) stage but they have affected M-phase promoting factor (MPF) activity and incurred abnormal pronuclear (PN) formation during fertilization. Gas6 is a ligand of TAM family members (Tyro3, Axl and Mertk) of receptor tyrosine kinase (RTK). Purpose of the present study was to evaluate the expression of Tyro3, Axl and Mertk transcripts in oocytes and early embryos. Expression of Gas6 and Mertk mRNA was detectable in oocytes and follicular cells, while Tyro3 and Axl mRNA was expressed only in follicular cells. Expression of Mertk mRNA was relatively constant during oocytes maturation and embryogenesis, but the other receptors, Tyro3 and Axl, were not expressed in oocytes and PN stage of embryos at all. Knockdown of Mertk mRNA and protein by using sequence-specific Mertk double strand RNA (dsRNA) did not affect oocytes maturation. In this case, however, contrary to the ligand Gas6 RNA interference (RNAi), MPF activity had not been changed by Mertk RNAi. Therefore, we concluded that the Gas6-Mertk signaling is not directly related to the oocyte maturation. It is still required to study further regarding the function of Mertk as the receptor of Gas6 during preimplantational early embryogenesis.
Kim, Kyeoung-Hwa,Kim, Eun-Young,Lee, Su-Yeon,Ko, Jung-Jae,Lee, Kyung-Ah S. Karger AG 2018 CELLULAR PHYSIOLOGY AND BIOCHEMISTRY Vol.45 No.1
<P><B><I>Background/Aims:</I></B> Previously, we found that silencing of growth arrest-specific gene 6 (<I>Gas6</I>) in oocytes impaired cytoplasmic maturation, resulting in failure of sperm chromatin decondensation (SCD) and pronuclear (PN) formation after fertilization. Thus, we conducted this study to determine the effect of <I>Gas6</I> RNAi on downstream genes and to elucidate the working mechanism of <I>Gas6</I> on oocyte cytoplasmic maturation and SCD. <B><I>Methods:</I></B> Using RT-PCR, Western blot and immunofluorescence, the expression levels of various target genes and the localization of heparan sulfate (HS) were analyzed after <I>Gas6</I> RNAi. The roles of Gas6 in HS biosynthesis, production of ATP and GSH, ROS generation and ΔΨm were also investigated. SCD and micrococcal nuclease (MNase) analyses were used to examine the effects of HS on the open chromatin state in sperm and somatic cell nuclei, respectively. <B><I>Results:</I></B><B><I></I></B> Disruption of <I>Gas6</I> expression led to the inhibition of HS biosynthesis through the reduction of several HS biosynthetic enzymes. The rescue experiment, HS treatment <I>in vitro</I>, significantly recovered SCD and PN formation, confirming that HS had the ability to induce sperm head remodeling during fertilization. Interestingly, excessive mitochondrial activation in <I>Gas6</I>-depleted MII oocytes caused ROS generation and glutathione (GSH) degradation via mitochondrial activation, such as elevated ΔΨm and ATP production. Indeed, HS-treated NIH3T3 cell nuclei showed an open chromatin state, as determined by diffuse DAPI staining and increased sensitivity to MNase. <B><I>Conclusion:</I></B> We propose that the addition of HS to sperm and/or oocyte maturation would improve the efficiency of <I>in vitro</I> fertilization and somatic cell nuclear transfer (SCNT) reprogramming.</P>
Downstream Genes Regulated by Bcl2l10 RNAi in the Mouse Oocytes
Kim Eun-Ah,Kim Kyeoung-Hwa,Lee Hyun-Seo,Lee Su-Yeon,Kim Eun-Young,Seo You-Mi,Bae Jee-Hyeon,Lee Kyung-Ah 한국발생생물학회 2011 발생과 생식 Vol.15 No.1
Previously, we have shown that Bcl2l10 as a member of Bcl-2 family, key regulators of the apoptotic process, is dominantly expressed in oocytes of ovary but several member of the Bcl-2 family are not expressed in oocytes. Recent our studies had been processed about roles and regulatory mechanisms of Bcl2l10 in oocytes. Microinjection of Bcl2l10 RNAi into the cytoplasm of germinal vesicle oocytes resulted in metaphase I (MI) arrest and exhibited abnormalities in their spindles and chromosome configurations (Yoon et al., 2009). The present study was conducted to elucidate the downstream genes regulated by Bcl2l10 and signaling networks in Bcl2l10 RNAi microinjected oocytes by using microarray analysis. Surprisingly, we found that a large proportion of genes regulated by Bcl2l10 RNAi were involved in the cell cycle and actin skeletal system regulation as important upstream genes of Bcl2l10. Among the transcripts with highly significant fold changes more than 2-fold, Tpx2 and Cep192 are 16.1- and 8.2-fold down regulated respectively by Bcl2l10 RNAi. Tpx2 and Cep192 are known as cofactors that control Aurora A kinase activity and localization. Therefore, we concluded that Bcl2l10 may have important roles during oocyte meiosis as functional upstream regulator of Tpx2 and Cep192.