To verify the sex steroids which are involved in oocyte maturation of the blacktip grouper, , we incubated vitellogenic oocytes (0.41 and 0.50 mm in average diameter) in the presence of exogenous steroid precursor (-hydroxyprogesterone). Steroids were extracted, separated and identified by thin layer chromatography. The major metabolites produced were androstenedione, estradiol-, estrone and progestogens. Progestogen metabolites in the oocytes of 0.50 mm were more abundant than those of 0.41 mm. Also, we investigated the effects of human chorionic gonadotropin (HCG; 5, 50 and 500 ), -dihydroxy-4-pregnen-3-one () and -trihydroxy-4-pregnen-3-one (; 5, 50 and 500 , respectively) on oocyte maturation. In the oocytes of 0.41 mm, treatment with 50 IU HCG stimulated GVBD () compared with controls (, <0.05). In the oocytes of 0.50 mm, treatment of (50 and 500 ) stimulated GVBD ( and , respectively) compared with controls (, <0.05). Treatment with 500 IU HCG also stimulated GVBD () compared with controls (<0.05). Taken together, these results suggested that both HCG and were effective on in vitro oocyte maturation and may act as a maturation inducing hormone in blacktip grouper.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants derived from incomplete combustion of carbons and crude oil. In this study, we investigated the effects of benzo[a]pyrene (B[a]P), a representative PAHs on in vitro sex steroid hormone production and germinal vesicle breakdown (GVBD) using isolated oocytes of longchin goby (Chasmichthys dolichognathus) and chameleon goby (Tridentiger trigonocephalus). Oocytes in diameters of 0.8-0.9 (end vitellogenic stage) and 0.9-1.0 mm (germinal vesicle migratory stage) from longchin goby and 0.5 mm (fully vitellogenic stage) from chameleon goby were used. In GVBD assay, B[a]P at 10 nM stimulated GVBD in the oocytes of 0.8-0.9 mm from longchin goby. B[a]P at 1 nM stimulated GVBD in the oocytes with diameter 0.5 mm from chameleon goby. In steroid production from oocytes of longchin goby, B[a]P at 100 nM decreased testosterone (T) production, B[a]P at 1,000 nM increased estraiol-17 (J (E2) production and 10 and 100 nM increased -dihydroxy-4-pregnen-3-one () production in the oocytes with diameter 0.8-0.9 mm. B[a]P at 1,000 nM increased E2 production, 100 and 1,000 nM increased production in the oocytes with diameter 0.9-1.0 mm. In steroid production of oocytes from chameleon goby, B[a]P at 1,000 nM increased production. B[a]P at 10 nM increased production. In the ratio of to T (/T), B[a]P at 100 and 1,000 nM increased /T in the oocytes of longchin goby. B[a]P at 100 nM also increased /T in the oocytes of chameleon goby. Taken together, these results suggest that B[a]P have not only weak estrogenic effects but progestogenic effects on oocyte maturation.

Lhx8 is a member of the LIM-homeobox transcription factor family expressed in the mouse ovary. We discovered that Lhx8 knockout females lose oocytes within 7 days after birth. Lhx8–/–ovaries fail to maintain the primordial follicles and growing follicles. Lhx8–/–ovaries misexpress numerous oocyte-specific genes such as H1foo and Nlrp14. The molecular mechanism of there gulation of Lhx8 in the oocyte has not been described. We examined to characterize Lhx8 DNA binding elements and to identify its direct target genes in the oocyte. CAST was performed using glutathione transferase Lhx8 homeodomain fusion protein (GST-LHX8HD). A 15-bp random sequence flanked by 20-bp of fixed sequences were incubated with purified GST-LHX8HD protein. Unbound DNA was washed with binding reaction buffer. Bound DNA was eluted and re-amplified by PCR for the next round of CAST. Final PCR products were cloned and sequenced to derive consensus binding sequence. EMSA was performed using 32P-labeled oligomers. Binding reactions were conducted by incubating 32P-labeled probes with purified protein. Dual luciferase assays were carried out with extracts of total HEK293 cell which was transfected by the pGL4-promoter vector containing three artificial repeats of LBE(3xLBE-Luc) and overexpression vector carrying the Lhx8 homeodomain as recommended by Promega. We identified several cis-acting sites, TGATTG as Lhx8 DNA binding elements (LBE) using a library of randomly generated oligonucleotides by CAST. EMSA reslut shows that Lhx8 preferentially binds to the oligomer including Lhx8 binding element (TGATTG) with high affinity. In addition, we found that the relative luciferase activity of reporter construct containing three copies of TGATTG was increased by 2.3-fold with Lhx8 overexpression. These results suggest that Lhx8 preferentially binds Lhx8 DNA binding element, TGATTG, and can transactivate reporter genes through the LBE. The transcription of Lhx8 target gene in oocytes directly might be regulated by its during early folliculogenesis.

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 연구에서는 해산어를 이용하여 bisphenol A(BPA)와 nonylphenol(NP)이 난모세포 성숙 과정에 어떤 영향을 미치는지 조사하기 위해 성숙단계에 있는 노래미(Hexagrammos agrammus) 난모세포(난경 약 1.88 ㎜)를 대상으로 in vitro에서 BPA와 NP 처리에 의한 난모세포의 성스테로이드 생성농도를 조사하였다. 난모세포에 BPA와 NP를 농도구별(0.1, 1, 10, 100, 1,000 ng/㎖)로 첨가하고, 50 IU의 human chorionic gonadotropin(HCG)를 농도구별 BPA 또는 NP와 함께 첨가하거나 하지 않고 48시간 동안 배양하였다. 배양 후 배양액 내의 17α,20β-dihydroxy-4-pregnen-3-one(17α20βOHP), estradiol-17β(E2) 그리고 testosterone(T)의 농도를 방사면역측정법(RIA)을 통해 정량하였다. BPA 처리구에서는 100 ng/㎖의 농도구에서 HCG 처리 유무에 상관없이 E2 생성이 촉진되었다. HCG 처리하에서 0.1 ng/㎖의 농도구에서 T 생성은 촉진되었으나, HCG를 처리하지 않은 실험구의 모든 농도구에서 T 생성은 저해되었다. NP 처리구에서는 HCG를 처리하지 않은 실험구의 10 ng/㎖의 농도구에서 17α20βOHP와 T 생성이 촉진되었고, 1 ng/㎖의 농도구에서는 E2 생성이 억제되었다. 이상의 결과를 종합하면, 노래미의 성숙단계의 난모세포에서 BPA는 약한 estrogen-agonistic 효과를, NP는 estrogen- antagonistic 효과를 지니는 것으로 사료된다.

점망둑의 난모세포 성숙과정에서 생성되는 주요 성 스테로이드 호르몬, -스테로이드를 분석하고자 전구물질 ()를 성숙기 난모세포(난경 ) 배양초기에 첨가하여 24시간 배양하였다. 스테로이드 대사물질 분석과 동정은 thin layer chromatography와 gaschromatography-mass spectrometry로 이루어졌다. 로부터 생성된 주요 성 스테로이드 대사물질은 -hydroxy, -dihydroprogesterone ()와 -hydrox

한국 곰소만산 암컷 바지락(Ruditapes philippinarum)의 난모세포의 발달 및 퇴화과정 중 일어나는 미세구조적 변화에 관해 기술하였다. 난소소낭은 영양성분을 저장하는 포상결체조직세포(VCT cell)들의 기질에 의해 둘러싸여 있다. 난 형성과정 초기에 초기난황형성난모세포와 보조세포(follicle cell)들 사이에 desmosome-like gap junction들이 나타났다. 난황형성과정은 골지체, 미토톤드리아, 조면소포체가 결합되어

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

The aims of this study are 1) to test oocytes and embryos collected from in-vivo and in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to the procedures of Funahashi et al. Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at 39, and 10% fetal bovine serum was added to the culture medium thereafter. Embryos were treated with 7.5g/ml cytochalasin-B for 30 min, centrifuged at 13,000 g for 13 min and then exposed sequentially to an ethylene glycol (EG) vitrification solution, aspirated into OPSs, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three donors after Al. Forty-six embryos (18, 9 and 19 embryos, respectively) were washed 3 times in mPBS+10%FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients received surgically 34(control), 188 and 184 embryos (derived from abattoir), respectively. Another three recipients were received nonsurgically 150, 100 and 150 embryos, respectively. All recipient sows exhibited delayed returns to estrus. To our knowledge, these results suggest that required an improved techniques, more vigorous embryos preparation and cleaner uterous condition(use gilt).

The successful development of embryos cloned by nuclear transfer (NT)have been dependent on a wide range of known factors including cell cycle of donor and recipient ooplast, oocyte quality, NT procedure and oocyte activation. The present study compared the development of cloned porcine embryos following different activation treatments. Cumulus-oocyte complexes (COCs) were aspirated from 26 mm follicles of slaughterhouse ovaries and cultured for 22 h in NCSU #23 medium supplemented with 10% porcine follicular fluid, 0.57 mM cysteine, 0.5 g/mL LH, 0.5 g/mL FSH and 10 ng/mL EGF. The COCs were further cultured for an additional 22 h in the same medium at in an atmosphere of 5% in air, without hormonal supplements. Primary cultures of fibroblasts isolated from a female fetus on day 40 of gestation were established in DMEM + 15% FCS. For nuclear donation, cells at the 5th-6th passage were cultured in DMEM +0.5% FCS for 5 days in order to arrest the cells in G0/Gl. After enucleation, oocytes were reconstructed by transfer of donor cells and fusion with three DC pulses (1.4 KV/cm, 30 sec) in 0.28 M mannitol containing 0.01 mM and 0.01 mM . Eggs were then divided into three treatment groups, control (without further treatment, Group 1), eggs cultured in 10 g/ml cycloheximide (CHX) for 5 h (Group 2), and eggs cultured in 1.9 mM 6-dimethylaminopurine (6-DMAP) for 5 h (Group 3). The eggs were then cultured in sets of 30 in 60 I drops of NCSU#23 supplemented with 4mg/ml BSA (essentially fatty acid free) until day 7 at in a humidified atmosphere of 5% . On day 4 the culture were fed by adding 20 I NCSU #23 supplemented with 10% FBS. Development rates into blastocysts were significantly higher (P<0.05) in Group 3 embryos compared to Group 1 controls (, respectively), but rates did not differ in Group 2 compared to control (). Total cell number in Group 3 blastocysts was however significantly higher (P<0.05) than in Groups 1 and 2 (, respectively). These results suggest that 6-DMAP is more efficient than cycloheximide in the activation of electrically fused NT oocytes during in vitro production of cloned porcine embryos.

The birth of the clone animals is influencing the frontier of research of animal biotechnology. It has effects on research of animal biotechnology itself by necessitating setting of new research subjects, modifications of the strategy of ongoing research projects, and challenges to schemes formerly considered impossible. In my talk, such topics including mass production of fertile ova and oocyte maturation will be discussed. (1) Oocytes are needed for the production of a clone by nuclear transplantation. Mitochondrial DNA inherited via the oocyte are involved also in the morphogenesis. Therefore, oocytes from the same animal must be used as recipients to produce genuine clones by nuclear transplantation. Experimenting on the assumption that selective oogenesis can be avoided, and apoptosis of oocytes can be prevented, by using ovarian angiogenic factos will be introduced. (2) It is important to clarify the factors of oocytes involving in reprogramming of somatic cells. Such factors are thought to be expressed in oocytes during oogenesis and oocyte maturation. Therefore, molecular mechanisms of oogenesis and oocyte maturation must be clarified extensively. Topics in this field including our recent advances will be discussed. (중략)

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

포유동물의 난포내 난자의 성숙 시에는 난자를 둘러싸고 있는 난구세포의 확장 현상이 일어나는데 이 현상에는 hyaluronic acid 뿐만 아니라 다른 성분도 관여하는 것으로 알려져 있다. 본 연구는 조직 재구성 과정에서 중요한 역할을 하는 matrix metalloproteinase(MMP)가 사람의 성숙한 난자-난구 복합체의 extracellular matrix(ECM)에 존재하는지의 여부를 알아보고자 하였다. 체외수정 시술 시에 얻어지는 사람의 난

난자와 난포세포는 gap junction channel을 통해 긴밀한 연락을 주고받음으로써 난자의 발달이나 난포의 성장에 영향을 미친다. 본 연구는 같은 크기의 난포에서 나온 난자라고 하여도 cumulus cell(난구세포)들이 붙어있는 상태가 다르다는 것을 관찰, 이렇게 난구세포와의 서로 다른 연결상태를 갖는 난자의 재질에 차이가 있을 것으로 가정하고, 난자의 competence와 이때 난자와 난구세포 사이의 cell-to-cell communicat