• Title/Summary/Keyword: 생식세포

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Effect of TGF-${\beta}$ Supplementation on In Vitro Maturation of Hanwoo COCs (Cumulus Oocytes Complexes) (TGF- ${\beta}$ 첨가가 한우 난포란의 체외성숙에 미치는 영향)

  • Choi, Sun-Ho;Lee, Hye-Hyun;Yeon, Seong-Heum;Han, Man-Hye;Kim, Hyun-Jong;Cho, Sang-Rae;Woo, Jae-Seok;Baek, Kwang-Soo;Ryu, Il-Sun;Son, Dong-Soo
    • Development and Reproduction
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    • v.8 no.2
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    • pp.119-122
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    • 2004
  • It is well known that unidentified factors in sera, hormones and growth factors promote the proliferation of granulosa cells and nuclear maturation of bovine COCs in vitro. Attemps had been developed the simple composition of culture media and similar system to in vivo conditions has been applied. In the present study, we investigated the effect of TGF-${\beta}$ on in vitro maturation and in vitro development of Hanwoo COCs. When the COCs were matured in TCM 199 containing 0.1, 1 or 10 ng/ml TGF-${\beta}$ for 24 hrs, metaphaseⅡ of COCs were obtained 95.8%, 100% of matured COCs, respectively and there were no differences among the concentrations of TGF-${\beta}$. Matured COCs with TGF-${\beta}$ cultured in maturation medium after in vitro fertilization, developmental rate to blastocyst were 0~0.8%. Matured COCs with TGF-${\beta}$ were cultured in TCM 199+10% FBS, 0.8% BSA, 0.1% PVA, blastocyst formation were showed in 12.4%, 12.8%, 8.5% of those and cultured in IVMD or IVD without serum were 38.4%, 34.8%, respectively. There were significant differences among the media (P<0.05). TGF-${\beta}$ is available for i vitro maturation of bovine COCs, but further investigation would be need for finding the synergistic autocrine/paracrine fashion of other growth factors in early bovine development.

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Simultaneous Detection of Seven Phosphoproteins in a Single Lysate Sample during Oocyte Maturation Process (난자성숙 과정의 단일 시료에서 일곱 가지 인산화 단백질의 동시 분석 방법)

  • Yoon, Se-Jin;Kim, Yun-Sun;Kim, Kyeoung-Hwa;Yoon, Tae-Ki;Lee, Woo-Sik;Lee, Kyung-Ah
    • Clinical and Experimental Reproductive Medicine
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    • v.36 no.3
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    • pp.187-197
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    • 2009
  • Objective: Phosphorylation and dephosphorylation of proteins are important in regulating cellular signaling pathways. Bead-based multiplex phosphorylation assay was conducted to detect the phosphorylation of seven proteins to maximize the information obtained from a single lysate of stage-specific mouse oocytes at a time. Methods: Cumulus-oocyte complexes (COCs) were cultured for 2 h, 8 h, and 16 h, respectively to address phosphorylation status of seven target proteins during oocyte maturation process. We analyzed the changes in phosphorylation at germinal vesicle (GV, 0 h), germinal vesicle breakdown (GVBD, 2 h), metaphase I (MI, 8 h), and metaphase II (MII, 16 h in vitro or in vivo) mouse oocytes by using Bio-Plex phosphoprotein assay system. We chose seven target proteins, namely, three mitogen-activated protein kinases (MAPKs), ERK1/2, JNK, and p38 MAPK, and other 4 well known signaling molecules, Akt, GSK-$3{\alpha}/{\beta}$, $I{\kappa}B{\alpha}$, and STAT3 to measure their phosphorylation status. Western blot analysis and kinase inhibitor treatment for ERK1/2, JNK, and Akt during in vitro maturation of oocytes were conducted for the confirmation. Results: Phosphorylation of ERK1/2, JNK, p38 MAPK and STAT3 was increased over 3 folds up to 20 folds, while phosphorylation of the other three signal molecules, Akt, GSK-$3{\alpha}/{\beta}$, and $I{\kapa}B{\alpha}$ was less than 3 folds. All of these results except for Akt were statistically significant (p<0.05). Conclusion: This is the first report on the new and valuable method measuring many phosphoproteins simultaneously in one minute sample such as oocyte lysates. All of the three MAPKs, ERK1/2, JNK, and p38 MAPK are involved in the process of mouse oocyte maturation. In addition, STAT3 might be important regulator of oocyte maturation, while Akt phosphorylation at Serine 473 may not be involved in the regulation of oocyte maturation.

The Effect of Low Concentrated Hypoxanthine and FSH in 10% FBS Supplemented Medium on Immature Oocyte in vitro Maturatio (낮은 농도의 Hypoxanthine과 FSH가 미성숙난자의 체외성숙에 미치는 영향)

  • Han, Hyuck-Dong;Lim, Chang-Kyo;Youm, Hyun-Sik;Hyon, Naomi Na-Hyoung;Lee, Ji-Hyang;Hong, Me
    • Clinical and Experimental Reproductive Medicine
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    • v.36 no.3
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    • pp.175-186
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    • 2009
  • Objective: We examined the effect of different culture media on oocyte maturation. Methods: Four groups of media, (1) 0.3% BSA mBASAL-XI-HTF, (2) 0.3% BSA mBASAL-XI-HTF with FSH, (3) 10% FBS mBASAL-XI-HTF and (4) 10% FBS mBASAL-XI-HTF with FSH were prepared. Mouse cumulus enclosed oocytes (CEOs) were incubated in each group of medium. Hypoxanthine (Hx) was mixed to each group of medium in increasing concentrations of 1 mM, 2 mM and 4 mM. CEOs were incubated and assessed for GVBD and MII development at 3, 6, 18 hours. Results: CEOs maturation to GVBD was seen in all four groups during 3 hours of culture, however MII stage of oocytes was seen after 6 hours. Complete arrest of GV stage in 4 mM Hx media without FSH and partial arrest in 2 mM Hx media without FSH were seen during 18 hours of culture but development was not suppressed in 1 mM Hx media without FSH. More prominent GVBD suppression was noted at early 3, 6 hours culture in 1 mM, 2 mM Hx media with FSH compared to media without FSH. But the suppression was recovered at 18 hours. This result suggests that low concentrated Hx and FSH supplemented media can suppress CEOs maturation during early culture period but recovery is resumed or even stimulated at late period. 1 mM, 2 mM Hx 10% FBS medium with FSH had significantly higher rates of MII development (71.7%, 66.7%) at 18 hours compared to other media. Conclusion: Our results show that low concentrated Hx and FSH supplemented 10% FBS media may stimulate MII development after an initial inhibitory effect.

Discovery of UBE2I as a Novel Binding Protein of a Premature Ovarian Failure-Related Protein, FOXL2 (조기 난소 부전증 유발 관련 단백질인 FOXL2의 새로운 결합 단백질 UBE2I의 발견)

  • Park, Mira;Jung, Hyun Sook;Kim, Hyun-Lee;Pisarska, Margareta D.;Ha, Hye-Jeong;Lee, Kangseok;Bae, Jeehyeon;Ko, Jeong-Jae
    • Development and Reproduction
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    • v.12 no.3
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    • pp.289-296
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    • 2008
  • BPES (Blepharophimosis/Ptosis/Epicanthus inversus Syndrome) is an autosomal dominant disorder caused by mutations in FOXL2. Affected individuals have premature ovarian failure (POF) in addition to small palpebral fissures, drooping eyelids, and broad nasal bridge. FOXL2 is a member of the forkhead family transcription factors. In FOXL2-deficient ovaries, granulosa cell differentiation dose not progress, leading to arrest of folliculogenesis and oocytes atresia. Using yeast two-hybrid screening of rat ovarian cDNA library with FOXL2 as bait, we found that small ubiquitin-related modifier (SUMO)-conjugating E2 enzyme UBE2I protein interacted with FOXL2 protein. UBE2I also known as UBC9 is an essential protein for processing SUMO modification. Sumoylation is a form of post-translational modification involved in diverse signaling pathways including the regulation of transcriptional activities of many transcriptional factors. In the present study, we confirmed the protein-protein interaction between FOXL2 and UBE2I in human cells, 293T, by in vivo immunoprecipitation. In addition, we generated truncated FOXL2 mutants and identified the region of FOXL2 required for its association with UBE2I using yeast-two hybrid system. Therefore, the identification of UBE2I as an interacting protein of FOXL2 further suggests a presence of novel regulatory mechanism of FOXL2 by sumoylation.

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Studies on the Life History of Bacciger harengulae (Bacciger harengulae의 생활사에 관한 연구)

  • KIM Young-Gill;CHUN Seh-Kyu
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.17 no.5
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    • pp.449-470
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    • 1984
  • The cercaria of Bacciger herengulae which is parasitized on the gonad of Solen strictus was investigated in order to reveal its entire life history. The area covered for the study was in the vicinity sea of Naechodo, the estuary of the Kum river in the western coast of Korea during the period of 1980-1983. Morphology and development as well as infection rates of sporocyst and cercaria within Solen strictus were examined. For accomplishing the objectives of this study, an artificial infection experiment and some investigations on the second intermediate host, the final host and the growing stages were also studied in both laboratory and natural habitat of Solen strictus. According to the study, it was revealed that the first intermediate hosts were Meretrix lusoria, Solen strictus, Tapes japonica and Laternula limicola, the second intermediate host was Palaemon (Exopalaemon) carinicauda and the final hosts were Konosirus punctatus and Harengula zunasi. A mature sporocyst which was found in the gonad of Solen strictus was $4.0-4.3{\times}0.2-0.21\;mm$ insize, and the cercaia with 27 pairs of setae, each seta consisting of 6 tufts, was $270{\times}147{\mu}m$ in body size and $550{\times}52{\mu}m$ in tail size. Oral sucker($52{\times}42{\mu}m$), pharynx, vental sucker and two testese were obviously seen within the cercaria. The excretory vesicles of cercaria were in V-shape and the flame cell were formula was expressed as 2[(3+3)+(3+3)]=24. The infection of cercaria in the first intermediate host, Solen strictus, was found throughout the year regardlless of the water temperature, and its mean infection rate was $9.67\%$ during the study period. The infection rate fluctuated with temperature, the highest being $28.0\%\;at\;28.0^{\circ}C$ water temperature in July and the lowest $2.4\%\;at\;19.5^{\circ}C$ in October, and it increased in proportion to the shell length on the host. But cercaria was not detected at below 4.0 cm in size of the host. Mature cercariae were found 6 months from May to October when water temperature was above $19.5^{\circ}C$. On the other hand, when water temperature was below $19.5^{\circ}C$, only immature cercariae and sporocysts were found. The cercariae were active for 35 hours and survived for 71 hours at $20^{\circ}C$, and 29 and 34 hours at $25^{\circ}C$ respectively, whereas the cercariae were inactive at less than $20^{\circ}C$ in water temperature. Cercaria, from Solen strictus, approached shrimp of 1-3 cm in body length as its second host. Then, it began to intrude in to the muscle of shrimp after 2-3 hours. The infected cercaria formed cyst after 7-8 hours, and became mature metacercaria. $420{\times}310{\mu}m$ in size, 15 days afer infection. The infection rate of metaceria to shrimp in the laboratory was highest, at $25^{\circ}C$ being $61\%$ and at $20^{\circ}C\;17%$. The infection rate of metacearia in shrimp was highest in the first abdominal segment, followed by cephalothorax, the second, and fifth abdominal segments, and in that order. Also, the infection rate of metacercaria in wild shrimp was high $9.6-11.1\%$ at $26.5^{\circ}C$ in June, and low $1.56-2.5\%$ at $28-29.5^{\circ}C$ from July to August. The infected shrimp with metacercaria was experimentally fed to Konosirus punctatus in the laboratory in order to know its final host. The metacercaria developed into the adult worm, $440-520{\times}310-360{\mu}m$ in size, within the intestine of Konosirus punctatus 20 days after infection. The adult worm was oval shape and $20-24{\times}11-20{\mu}m$ in size. The infection rate of adult worm to Konosirus punctatus and Harengula zunasi ranged 87.3 to $100\%$, the mean being $95.2\%$, regardless of the body length of their hosts. The infection rate was $100\%$ in June and July, but it decreased in September and October. The size and body structure of the trematode observed during the present study were well agreed with those ievestigated by Yamaguti(1938), thus, it may be concluded that the adult worm it identified as Bacciger harengulae.

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Establishment and Application of Molecular Genetic Techniques for Preimplantation Genetic Diagnosis of Osteogenesis Imperfecta (골형성부전증의 착상전 유전진단을 위한 분자유전학적 방법의 조건 확립과 적용)

  • Kim, Min-Jee;Lee, Hyoung-Song;Choi, Hye-Won;Lim, Chun-Kyu;Cho, Jae-Won;Kim, Jin-Young;Song, In-Ok;Kang, Inn-Soo
    • Clinical and Experimental Reproductive Medicine
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    • v.35 no.2
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    • pp.99-110
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    • 2008
  • Objectives: Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples carrying genetic conditions that may affect their offspring. Osteogenesis imperfecta (OI) is an autosomal dominant disorder of connective tissue characterized by bone fragility and low bone mass. At least 95% of cases are caused by dominant mutations in the COL1A1 or COL1A2. In this study, we report on our experience clinical outcomes with 5 PGD cycles for OI in two couples. Methods: Before clinical PGD, we assessed the amplification rate and allele drop-out (ADO) rate of alkaline lysis and nested PCR protocol using heterozygous patient's single lymphocytes in the pre-clinical diagnostic tests for OI. We performed 5 cycles of PGD for OI by nested PCR for the causative mutation loci, COL1A1 c.2452G>A and c.3226G>A, in case 1 and case 2, respectively. The PCR products were analyzed by agarose gel electrophoresis, restriction fragment length polymorphism (RFLP) analysis with HaeIII restriction enzyme in the case 1 and direct DNA sequencing. Results: We confirmed the causative mutation loci, COL1A1 c.2452G>A in case 1 and c.3226G>A in case 2. In the pre-clinical tests, the amplification rate was 94.2% and ADO rate was 22.5% in case 1, while 98.1% and 1.9% in case 2, respectively. In case 1, a total of 34 embryos were analyzed and 31 embryos (91.2%) were successfully diagnosed in 3 PGD cycles. Eight out of 19 embryos diagnosed as unaffected embryos were transferred in all 3 cycles, and in the third cycle, pregnancy was achieved and a healthy baby was delivered without any complications in July, 2005. In case 2, all 19 embryos (100.0%) were successfully diagnosed and 4 out of 11 unaffected embryos were transferred in 2 cycles. Pregnancy was achieved in the second cycle and the healthy baby was delivered in March, 2008. The causative locus was confirmed as a normal by amniocentesis and postnatal diagnosis. Conclusions: To our knowledge, these two cases are the first successful PGD for OI in Korea. Our experience provides a further demonstration that PGD is a reliable and effective clinical techniques and a useful option for many couples with a high risk of transmitting a genetic disease.

Studies on the Internal Changes and Germinability during the Period of Seed Maturation of Pinus koraiensis Sieb. et Zucc. (잣나무 종자(種字) 성숙과정(成熟過程)에 있어서의 내적변화(內的變化)와 발아력(發芽力)에 대(對)한 연구(硏究))

  • Min, Kyung-Hyun
    • Journal of Korean Society of Forest Science
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    • v.21 no.1
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    • pp.1-34
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    • 1974
  • The author intended to investigate external and internal changes in the cone structure, changes in water content, sugar, fat and protein during the period of seed maturation which bears a proper germinability. The experimental results can be summarized as in the following. 1. Male flowers 1) Pollen-mother cells occur as a mass from late in April to early in May, and form pollen tetrads through meiosis early and middle of May. Pollen with simple nucleus reach maturity late in May. 2) Stamen number of a male flower is almost same as the scale number of cone and is 69-102 stamens. One stamen includes 5800-7300 pollen. 3) The shape is round and elliptical, both of a pollen has air-sac with $80-91{\mu}$ in length, and has cuticlar exine and cellulose intine. 4) Pollen germinate in 68 hours at $25^{\circ}C$ with distilled water of pH 6.0, 2% sugar and 0.8% agar. 2. Female flowers 1) Ovuliferous scales grow rapidly in late April, and differentiation of ovules begins early in May. Embryo-sac-mother cells produce pollen tetrads through meiosis in the middle of May, and flower in late May. 2) The pollinated female flowers show repeated divisions of embryo-sac nucleus, and a great number of free nuclei form a mass for overwintering. Morphogenesis of isolation in the mass structure takes place from the middle of March, and that forms albuminous bodies of aivealus in early May. 3. Formation of pollinators and embryos. 1) Archegonia produce archegonial initial cells in the middle and late April, and pollinators are produced in the late April and late in early May. 2) After pollination, Oespore nuclei are seen to divide in the late May forming a layer of suspensor from the diaphragm in early June and in the middle of June. Thus this happens to show 4 pro-embryos. The organ of embryos begins to differentiate 1 pro-embryo and reachs perfect maturation in late August. 4. The growth of cones 1) In the year of flowering, strobiles grow during the period from the middle of June to the middle of July, and do not grow after the middle of August. Strobiles grow 1.6 times more in length 3.3 times short in diameter and about 22 times more weight than those of female flower in the year of flowering. 2) The cones at the adult stage grow 7 times longer in diameter, 12-15 times shorter diameter than those of strobiles after flowering. 3) Cone has 96-133 scales with the ratio of scale to be 69-80% and the length of cone is 11-13cm. Diameter is 5-8cm with 160-190g weight, and the seed number of it is 90-150 having empty seed ratio of 8-15%. 5. Formation of seed-coats 1) The layers of outer seed-coat become most for the width of $703{\mu}$ in the middle of July. At the adult stage of seed, it becomes $550-580{\mu}$ in size by decreasing moisture content. Then a horny and the cortical tissue of outer coats become differentiated. 2) The outer seed-coat of mature seeds forms epidermal cells of 3-4 layers and the stone cells of 16-21 layers. The interior part of it becomes parenchyma layer of 1 or 2 rows. 3) Inner seed-coat is formed 2 months earlier than the outer seed-coat in the middle of May, having the most width of inner seed-coat $667{\mu}$. At the adult stage it loses to $80-90{\mu}$. 6. Change in moisture content After pollination moisture content becomes gradually increased at the top in the early June and becomes markedly decreased in the middle of August. At the adult stage it shows 43~48% in cone, 23~25% in the outer seed-coat, 32~37% in the inner seed-coat, 23~26% in the inner seed-coat and endosperm and embryo, 21~24% in the embryo and endosperm, 36~40% in the embryos. 7. The content compositions of seed 1) Fat contents become gradually increased after the early May, at the adult stage it occupies 65~85% more fat than walnut and palm. Embryo includes 78.8% fat, and 57.0% fat in endosperm. 2) Sugar content after pollination becomes greatly increased as in the case of reducing sugar, while non-reducing sugar becomes increased in the early June. 3) Crude protein content becomes gradually increased after the early May, and at the adult stage it becomes 48.8%. Endosperm is made up with more protein than embryo. 8. The test of germination The collected optimum period of Pinus koraiensis seeds at an adequate maturity was collected in the early September, and used for the germination test of reduction-method and embryo culture. Seeds were taken at the interval of 7 days from the middle of July to the middle of September for the germination test at germination apparatus.

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