Intact germinal vesicle (GV) arrest and release are essential for maintaining the fertility of mammals inducing human. Intact germinal vesicle release, maturation of oocytes is maintained by very complex procedures along with folliculogenesis and is a critical step for embryonic development. Cyclic guanosine monophosphate (cGMP) has been suggested a key factor for meiotic arrest but so far its mechanisms are controversy. In this study we examine the effects of cGMP on germinal vesicle breakdown in cumulus-enclosed oocytes and denuded oocytes. Spontaneous maturation was inhibited by a cGMP agonist, 8-Br-cGMP with concentration dependent manners both in cumulus-enclosed oocytes and denuded oocytes. The inhibitory effect was more severe in denuded oocytes than cumulus-enclosed oocytes. The Rp-8-Br-cGMP and Rp-pCPT- 8-Br-cGMP did not severely block GVB compared to 8-Br-cGMP. The spontaneous GVB inhibitory effects were different by the existence of cumulus. Based on them it is suggested that the cumulus modulates the role of cGMP in GV arrest.

Primary oocytes that are arrested in first meiotic prophase for years enter maturation process to meet a critical precondition for successful fertilization. During maturation, oocyte finishes meiosis I and progresses to the metaphase II stage, achieving meiotic maturity. Although importance of oocyte maturation for oocyte quality has been recognized, it is not fully understood for molecular mechanisms underlying oocyte maturation. Here, we found that dexamethasone-induced Ras-related protein 1 (RASD1), a member of RAS superfamily of small GTPases, was expressed in the mouse ovary. Immunohistochemical analysis revealed that Rasd1 expression was dominant in oocyte cytoplasm. Real-time PCR and RT-PCR analyses showed that Rasd1 mRNA was steadily expressed in germinal vesicle (GV), germinal vesicle break down (GVBD), metaphase I (MI) oocytes, but decreased in metaphase II(MII) oocytes during oocyte maturation. Konckdown of Rasd1 using RNAi system in the GV oocytes suppressed oocyte maturation through disruption of meiotic spindle and formation of misarranged chromosomes. Taken together, Rasd1 is a critical factor for MI-MII transition of oocyte and is involved in the regulation of spindle formation during oocyte maturation. Further study is needed to examine relationship between Rasd1 and spindle formation in MI-MII transition.

Cryopreservation has become a powerful method of the assisted reproduction technology and supports fertility preservation of cancer and other indication patients. After controlled ovarian hyperstimulation, surplus oocytes and embryos were recommended to store using cryopreservation. Recently, vitrification is replaced with traditional slow freezing protocol, because of improved survival rates and clinical outcomes. Vitrification requires a high concentration of CPAs that may induce significant osmotic and metabolic damage to cells including oocytes even in a short exposure of a few minutes. Generally, MPF plays a crucial role in the cell cycle regulation and maintaining the meiotic arrest of oocytes. In fact, it has been observed to decline in MII ovine oocytes after vitrification and would be suggested that one of the main causes of low fertilization rate and developmental competence derived from cryoinjury during vitrification. Therefore, the aim of this study was to evaluate the effect of caffeine treatment on the activity of MPF, MAPK level in vitrified/warmed mouse mature eggs. Caffeine, Phosphataseinhibitor, may maintain active form of MPF. We evaluated their survival after warming procedure, fertilization, cleavage, and developmental rates. Ovulated MII eggs were retrieved from 6 weeks old B6D2F1 female mouse at 14hr post hCG injection. Collected MII eggs were maintained in HTF medium containing 10% KSR with or without caffeine for 1hr. Eggs were vitrified in 7.5%EG +7.5%DMSO equilibrium solution, 15%EG + 15%DMSO + 0.5M sucrose vitrification solution with or without caffeine. Also warming solution contained sucrose (0.5M, 0.25M, 0.125M, and 0M) with or without caffeine. After warming, eggs were cultured in HTF medium with or without caffeine for 2 hr then fertilized with epididymal sperm in vitro and cultured in KSOM for 5 days to analyze embryonic development. Survival rates were similar in all experimental groups. However, fertilization rate was higher in with caffeine group compare to without caffeine significantly (80% vs. 85%, p<0.05). 2-cell and blastocyst formation were increased in caffeine group (p<0.05). MPF activity and MAP kinase activity were recovered in with caffeine group after vitrification/warming process. In conclusion, Caffeine may maintain MPF and MAPK level in vitrified/warmed MII eggs, and enhance fertilization and further embryonic development.

Dynamic reorganization of actin filaments is essential for various stages of mammalian oocyte maturation, including spindle migration, actin cap formation, polar body extrusion, and cytokinesis. Various actin binding proteins (ABPs) have been known to be involved in the regulation of actin filament remodeling. We elucidate roles of three different actin binding proteins in mouse oocyte maturation. The heterodimeric actin-capping protein (CP) binds to the fast-growing(barbed) ends of actin filaments and plays essential roles in various actin-mediated cellular processes. When CP is knockdowned or inhibitory component was overexpressed, asymmetric division of oocyte have been compromised. It turns out that knockdown or inhibition of CP deplete cytoplasmic actin mesh level, which have been known to be essential for maintain cytoplasmic actin mesh. Another actin binding proteins, tropomodulin 3 (Tmod3), binds to the slow-growing end of actin filaments and knockdown or expression deletion mutant of Tmod3 also decrease actin mesh level in maturing oocyte and it severely ablated asymmetric division of oocyte. Finally, tropomyosin 3, actin filament binding proteins protect actin filament from depolymerization, is also important to maintain cortex integrity in maturing oocyte. Taken together, these finding showed the essential roles of actin binding proteins in remodeling of actin filaments in mammalian oocyte development.

The aim of this study was to analyze the effect of antifreeze proteins (AFPs) on vitrification of mouse mature (MII) oocytes. We studied about 3 types of AFPs from different origins (FfIBP, LeIBPand Type III AFP). The MII oocytes were obtained from 4-week-old BD-F1 mice. Vitrification of oocyte was performed by 2 steps using the Cryotop (equilibration: 7.5% EG + 7.5% PROH for 5 min, vitrification: 15% EG + 15% PROH + 0.5M sucrose for 1 min). The concentrations of AFPs added to these solutions were 0.05 mg/ml for FfIBP and 0.1 mg/ml for LeIBP and Type III AFP. After fertilization, embryo development was assessed up to 5 days. Through immunostaining of vitrified-warmed oocytes, we assessed the normal meiotic spindle. Also, intracellular ROS and mitochondrial activity was analyzed. In the developmental stages, FfIBP and LeIBP groups showed significantly higher survival rates. In the blastocyst and apoptotic blastomere rates were significant differences in AFPs treated groups. AFPs treated groups were significantly higher in blastocyst cell numbers than control group. Among the AFPs treated groups, FfIBP, LeIBP groups were significantly higher rates. And, in cleavage rates, FfIBP group was significantly higher rates than the other groups. In vitrified-warmed MII oocytes, the normal meiotic spindle organization and chromosome alignment rate was significantly higher in FfIBP and LeIBP groups. And in intracellular ROS levels, control group was significantly increased than AFPs treated groups. However, in the mitochondrial activity, LeIBP group was significantly higher than control, FfIBP and LeIBP groups. AFPs treated groups were significant differences in development, meiotic spindle organization and intracellular ROS levels. And in the AFPs treated groups, FfIBP and LeIBP groups were significantly higher rates in normal meiotic spindle and mitochondrial activity than Type III AFP group respectively. In conclusion, FfIBP and LeIBP can be thought to improve oocyte cryopreservation efficiency.

Throughout their meiotic maturation in most mammals, oocytes are arrested twice, prophase I and metaphase II. Being released from these arrests, transient or oscillation of intracellular Ca2+ concentration is observed in the ooplasm, which is not answered in relation to the specific role in the resumption of meiotic arrest. Recently, Ca2+/calmodulin-dependent protein kinase II (CaM KII) has been known as a Ca2+ oscillation decoder from the in vitro experiment. CaM KII is multifunctional serine/threonine kinase observed in most cells. Present studies were performed to investigate the role of CaM KII during resumption of meiotic arrest and activation in vitro of mouse oocytes. It was questioned whether CaM KII might be involved in the meiotic resumption of mouse oocytes. Compared to the control, both of CaM KII inhibitors, KN-93 and KN-62, significantly inhibited germinal vesicle breakdown (GVBD) of mouse oocytes in a dose-dependent manner. As the concentration of KN-93 increased, concomitant decrease of intracellular Ca2+ concentration ([Ca2+]i) was also observed using confocal laser scanning microscope (CLSM) and an intracellular Ca2+ indicator, fluo 3-AM. When GVBD oocytes were treated with 6% ethanol, small [Ca2+]i transient was observed in oocytes bathed with Ca2+-free medium and large increase was observed in oocytes bathed with Ca2+-containing medium, suggesting that [Ca2+]i transient could happen from intracellular Ca2+ store as well as Ca2+ influx through Ca2+-channel on the oolemma. However, KN-93 inhibited the [Ca2+]i transient of GVBD oocytes in both cases. Using monoclonal antibodies against α-subunit of CaM KII, tubulin and microtubule-assocaited proteins (MAPs), CaM KII has been colocalized on the spindle with tubulin and MAPs. The present study also demonstrated the presence of α-subunit of CaM KII in heart, kidney, testes, ovary as well as in brain of the mouse. In ovarian follicles, CaM KII was expressed in granulosa cells and oocytes. Based on overall the above results, followings are suggested. First, CaM KII might be involved in the regulatory mechanism of meiotic resumption. Second, CaM KII might play a regulatory role in the stabilization of microtubule.

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.

본 실험은 생쥐 난자의 성숙과 생존에 미치는 selenium의 영향을 알아보고자 수행하였다. 난자의 성숙은 현미경을 통해 관찰하였으며, 핵막 붕괴(germinal vesicle breakdown, GVBD)와 극체 형성(polar body formation, PB)은 체외 배양 시작 후 각각 2.5, 13시간에 확인하였다. 난자의 생존은 72 시간동안 체외 배양하면서 형태학적 차이로 정상 난자와 비정상 난자를 판별하였다. 또한 각 단계별로 수집된 난자의

세포내 칼슘은 다양한 세포에서 중요한 생리적 반응을 일으키며, ruthenium red와 ryanodine은 중요한 칼슘 조절자로 작용한다. Ruthenium red는 세포내 칼슘 저장고에서의 calcium induced calcium release(CICR)를 저해한다. Ryanodine은 ryanodine 통로를 통한 칼슘 방출을 촉진한다. 본 실험은 두 조절자가 생쥐 난자와 초기배아의 세포내 칼슘이온 농도에 영향을 미치는지 여부와 그 유효농도를 알

대부분의 포유동물에서 수란관내로 배란된 난자는 정자에 의해 수정이 된 후 개체발생을 시작한다. 그러나 수정이 되지 못한 난자들은 난구세포와 함께 수란관내에서 퇴화하여 제거되는데, 그 기작에 대해서는 구체적으로 알려져 있지 않다. 따라서 본 연구는 포유동물의 수란관내 물질이 난자-난구 복합체에 미치는 영향을 알아보고자 사람의 난포액과 소의 수란관 조직 추출액을 생쥐의 난자-난구 복합체에 처리하고 난자의 생존율 및 난구세포의 세포자연사(apoptosis)를

Phospholipase C (PLC)는 다양한 세포주에서 세포내 신호전달에 중요한 역할을 한다고 알려져 있으나, 생쥐 난자성숙 과정과 착성전 배아발생 과정에서 PLC의 역할과 발현은 아직 연구된 바 없다. 본 연구에서는 난자성숙과 착상전 배아발생 과정에서 생쥐의 PLC β1과 γ1의 유전자 발현을 조사하기 위하여 한 개의 난자 혹은 배아에서 추출된 total RNA를 사용하여 경쟁적 RT-PCR 방법으로 mRNA를 정량하였다. PL

Befor fertilization, mammalian oocytes undergo meiotic maturation, which consists of nuclear and cytoplasmic differentiation. In this study, changes of stores in mouse oocytes were examined during meiotic maturation and the role of in the regulation of the maturation was investigated by using monoclonal antibodies against smooth endoplasmic reticulum -ATPase(SERCA-ATPase) and calreticulin. Observations were made under epifluorescence microscope and/or confocal laser scanning microscope. In immature oocytes which did not resume meiotic maturation, SERCA-ATPases were mostly localized in the vicinity of the germinal vesicle and calreticulins were distributed evenly throughout the cytoplasm. In mature oocytes, SERCA-ATPases were observed throughout the cytoplasm, butwere absent from the nuclear region. In contrast, calreticulins were localized mostl in the cortex of the oocyte and were absent from the cytoplasm. However, bright fluoresence stainings were wbserved in the perimeiotic spindle region of mature oocyte when labeled with antibodies against calreticulin. These results indicate that mouse oocytes undergo distinct rearrangement of the localization of -ATPases and calreticulins during meiotic maturation. Thus it can be suggested that redistribution of the stores, as revealed by differential fluorescence stainings, is deeply involved in the regulatory mechanism of mammalian oocyte maturation.