• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.117-122
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• 2019

The objective of this study was to establish an in vitro culture system for ovarian preantral follicles of B6D2F1. First, we optimized the in vitro preantral-follicle culture by culture duration, follicle stimulating hormone (FSH) type, and activin A concentration. Duration of in vitro culture for 9, 11, and 13 days was sufficient for the normal development of preantral follicles to antral follicles. Formation of cumulus cell-oocyte complex (COC) was induced by treatment with human chorionic gonadotropin (hCG; 2.5 IU/mL) and epidermal growth factor (EGF; 5 ng/mL). In addition, metaphase II (MII) oocytes formed during this in vitro culture of preantral follicles. In vitro preantralfollicle culture for 9 days showed higher rates of growth and maturation, thus yielding a greater number of antral follicles, and there were significant differences (p < 0.05) in the number of MII oocytes (that formed from these preantral follicles via differentiation) between the 9-day culture and 11-day or 13-day culture. The follicles cultured for 9 days contained a tightly packed well-defined COC, whereas in follicles cultured for 11 days, the COC was not well defined (spreading was observed in the culture dish); the follicles cultured for 13 days disintegrated and released the oocyte. Second, we compared the growth of the preantral follicles in vitro in the presence of various FSH types. There were no significant differences in the growth and maturation rates and in differentiation into MII oocytes during in vitro culture between preantral follicles supplemented with FSH from Merck and those supplemented with FSH from Sigma. To increase the efficiency of MII oocyte formation, the preantral follicles were cultured at different activin A concentrations (0 to 200 ng/mL). The control follicles, which were not treated with activin A, showed the highest rate of differentiation into antral follicles and into MII oocytes among all the groups (0 to 200 ng/mL). Therefore, activin A (50 to 200 ng/mL) had a negative effect on oocyte maturation. Thus, in this study, we propose an in vitro system of preantral-follicle culture that can serve as a therapeutic strategy for fertility preservation of human oocytes for assisted reproductive medicine, for conservation of endangered species, and for creation of superior breeds.

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.135-141
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• 2006

The present study was conducted to investigate the effects of cumulus cells and porcine follicular fluid (pFF) on plasminogen activator (PA) activity and oocytes maturation in vitro in the pig. The cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were incubated in NCSU-23 medium with or without 10% pFF for 0, 24, or 48 hr. In the presence of cumulus cells, the proportions of oocytes matured to metaphase-II stage were significantly (P<0.05) higher in medium with pFF than without pFF (69.8 vs. 37.7%, respectively). When COCs and DOs were cultured in the presence of pFF, tissue-type PA (tPA), urokinase-type PA (uPA), and tPA-PA inhibitor (tPA-PAI) were observed in COCs, and PA activities were higher at 48 hr than 24 hr. When COCs and DOs were cultured in the absence of pFF, tPA and tPA-PAI were observed in COCs, and PA activities were increased as duration of culture increased. No PA activities were detected in DOs regardless of pFF supplementation. When porcine oocytes were cultured in the presence of pFF for 24 and 48 hrs, the activities of tPA-PAI, tPA, and uPA were observed in both COCs and DOs. In medium of absence of pFF, PA activities were observed in oocytes with cumulus cells only. On the other hand, three plasminogen-dependent lytic bands (tPA-PAI, tPA, and uPA) were observed in pFF cultures. Particularly uPA activity was higher than the other kinds of PA activity. When oocytes and cumulus cells were separated from porcine COCs at 0 hr of culture, tPA-PAI, tPA, and uPA were detected in cumulus cells at 48 hr of culture, but no PA activities were in DOs. The presence of pFF and cumulus cells in maturation medium stimulated not only nuclear and cytoplasmic maturation in porcine COCs, but also PA production by cumulus cells and COCs. It is possible that PAs produced by cumulus cells migrated through the gap junction between oocyte and cumulus cells. These results suggest that porcine oocytes have no ability to produce PA themselves.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.149-157
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• 1993

A new technique was established to maximize the numbers of follicular oocytes recovered from the ovaries obtained at the slaughter house. And their further developmental capacity was demonstrated. There recovery techniques were used; aspiration (ASP, control), slicing (SLC) and slicing combining aspiration (ASP+SLC). Recovered oocytes were cultured in TCM 199+15% FCS+gonadotrophins in an atmosphere of 5% CO$_2$ in air at 39$^{\circ}C$ for 24 h. The nuclear maturation was detemined with chromo-some configuration by rapid staining. And cytoplasmic maturation was examined by the formation of female pronuclei with parthenogenetic activation of the matured oocyte after 18 h of co-culture with granulosa cell monolayer. Total 1,641 bovine follicular oocytes recovered from 245 ovaries. The number of oocytcs per ovary was 1.87 in ASP, 11.05 in SLC and 7.88 in ASP+SLC, respectively. SLC would yield 5.9 folds increase, compared with ASP. The rate of maturation were 92.9% in ASP, 79.1% in SLC and 71.7% in ASP+SLC, respectively. Although the maturation rate in ASP was the highest, metaphase II oocytes per ovary in SLC was 5 times higher than that of ASP. The rates of pronuclei formation upon ethanol activation were 75% in ASP, 67% in SLC and 62.5% in ASP+SLC, respectively. The results demonstrate that it should be possible to maximize the number of the follicular oocyte from the ovary for mass production of bovine embryos. Thus the established technique may provide efficient supply of bovine embryos for biochemical and molecular study of early bovine embryos.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.3
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• pp.159-166
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• 2002

연구목적: 본 연구에서는 생쥐 Dazla 유전자의 난소내 발현 위치를 확인하고, 배아의 발달에 따른 Dazla 유전자의 발현 양상을 관찰하고자 하였다. 연구재료 및 방법: 임신 제 7일, 10일, 11일, 14일의 태자 (각각 n=9)와 생후 27일된 암컷 미성숙 생쥐 (n=32), 8주령의 암컷 성숙 생쥐 (n=9)로 부터 난자, 과립막세포, 난소 조직을 획득하였으며, 9주령의 수컷 성숙 생쥐 (n=3)로부터 고환 조직을 획득하였다. 각각의 획득된 조직과 세포에서 Dazla mRNA의 발현 여부를 RT-PCR, in situ hybridization (ISH) 방법 등으로 확인하였다. 성선자극호르몬을 투여하지 않은 미성숙 및 성숙 생쥐에서 획득한 미성숙 난자 (GV)와 PMSG와 hCG를 투여한 미성숙 및 성숙 생쥐에서 획득한 성숙 난자 (MII)에서 RT-PCR로 Dazla 유전자의 발현 여부를 확인하였다. 미성숙 및 성숙 생쥐의 난소 조직과 성숙 생쥐의 고환 조직에서 RT-PCR 및 ISH 방법으로 Dazla 유전자의 발현 여부를 확인하였다. 결 과: 난소의 과립막세포에서는 미성숙 및 성숙 생쥐, PMSG와 hCG 투여 여부 등과 상관없이 모두 Dazla 유전자의 발현은 음성으로 판정되었다. PMSG 및 hCG를 투여하지 않은 난소에서 획득한 미성숙난자 (germinal vesicle, GV) 또는 PMSG 및 hCG 투여 후 채취한 성숙 난자 (metaphase II, MII) 모두 Dazla 유전자가 발현되었다. Dazla 유전자의 발현은 수정 직후 (2PN) 음성으로 전환되었으며, 착상 직전의 배반포 시기까지 유전자 발현이 음성으로 지속되었다. Dazla 유전자는 임신 제 7일 (PCD 7), 10일 (PCD 10), 11일 (PCD 11)의 태자에서도 유전자 발현이 계속 음성으로 관찰되었으나, 성 분화가 일어나기 시작하는 임신 제 $12{\sim}14$일 (PCD $12{\sim}14$)의 태자에서 유전자 발현이 다시 관찰되었다. 결 론: Dazla 유전자는 난소 내 난자에서만 특이적으로 발현하는 난자 특이 유전자 (oocyte specific factor)로서 Dazla 유전자가 난소 내 난포 생성과 관련성이 있음을 제시하고 있다. 향후 조기 폐경 환자에서의 연관성 등을 확인한다면 임상적으로 유용한 지표가 될 수 있을 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• v.40 no.1
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• pp.7-11
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• 2013

Objective: We evaluated the fertilization potential of immature oocytes obtained from controlled ovarian hyperstimulation cycles of patients undergoing ICSI. Methods: We retrospectively analyzed 463 ICSI cycles containing at least one immature oocyte at oocyte denudation. ICSI was performed on mature oocytes at oocyte denudation (metaphase-II [MII] oocytes) and the oocytes that extruded the first polar body between oocyte denudation and ICSI (MI-MII oocytes). Fertilization and early embryonic development were compared between MII and MI-MII oocytes. To investigate the pregnancy potential of MI-MII oocytes, the pregnancy outcome was analyzed in 24 ICSI cycles containing only immature oocytes at retrieval. Results: The fertilization rate of MI-MII oocytes (37.0%) was significantly lower than that of MII oocytes (72.3%). The rates of delayed embryos and damaged embryos did not significantly differ. Eighty-one immature oocytes were retrieved in 24 cycles that retrieved only immature oocytes and 61 (75.3%) of them were in the MI stage. ICSI was performed on 36 oocytes (59.0%) that extruded the first polar body before ICSI and nine MI-MII oocytes (25.0%) were fertilized. Embryo transfers were performed in five cycles. Pregnancy was observed in one cycle, but it ended in biochemical pregnancy. Conclusion: In ICSI cycles, oocytes that extruded the first polar body between denudation and ICSI can be used as a source of oocytes for sperm injection. However, their fertilization and pregnancy potential are lower than that of mature oocytes. Therefore, ovarian stimulation should be performed carefully for mature oocytes obtained at retrieval, especially in cycles with a small number of retrieved oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.1
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• pp.23-28
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• 2003

The present study was undertaken to investigate the effects of PVP concentration and exposure temperature to vitrification solution on the post-thaw survival, in vitro maturation and development of immature bovine oocytes (germinal vesicle stage). The vitrification solution (VS) consisted of 40% ethylene glycol (EG)+0.5 M sucrose (S)+10% FBS. PVP was added to VS: 0%, 5% or 10%. The cumulus-oocyte complexes (COCs) were diluted in VS as one step, after 2 min the COCs were loaded in straw and vitrified by direct immersion into liquid nitrogen. For thawing, the straws were plunged into $30^{\circ}C$ water bath for 10s. After thawing, the oocytes were diluted in 0.5 M (in DPBS with 10% FBS) sucrose solution for 5 min. The survival rate (FDA-test and trypan blue) of immature bovine oocytes was measured. The survival rate was higher in 5% PVP (91.5%) than in 0% (64.2%) or in 10% PVP (79.7%). The proportion of metaphase II formation was 69.35% in control (no vitrified COCs), 9.3% in 40% EG+0.5 M S+0% PVP and 21.05% in 40% EG+0.5 M S+5% PVP (p<0.05). The effect of room temperature ($25^{\circ}C$ for 10 min) and cold temperature ($4^{\circ}C$ for 10 min) on COCs were determined in this study. After IVF, the cleavage and blastocysts rate of oocytes exposed to room temperature and cold temperature in VS+5% PVP was significantly different (2 cell: 63.20% vs 37.97%, blastocysts: 18.40% vs 2.53%). The cleavage rates of frozen-thawed oocytes were 20.53% with PVP and 22.13% without PVP (p>0.05). Two out of 151 oocytes (1.32%) developed to blastocyst stage after frozen-thawed with 5% PVP (p>0.05). Development of oocytes after frozen-thawing to the 2 cell were not significantly affected with or without PVP following IVF. However, the vitrification of immature bovine oocytes with PVP maintained the ability to develop to the blastocyst stage after IVM-IVF and IVC, while no blastocysts were obtained from oocytes vitrified without PVP. These results suggested that PVP has a protective role for vitrification of immature bovine oocytes as far as survival is concerned, however, the protection was not sufficient enough to support blastocyst formation.

• Clinical and Experimental Reproductive Medicine
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• v.39 no.3
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• pp.107-113
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• 2012

Objective: To determine whether animal age impacts in vitro preantral follicle growth. Effects of hCG, stem cell factor (SCF), and/or insulin-like growth factor (IGF) supplementation in growth medium were also investigated. Methods: Intact preantral follicles were mechanically isolated from fresh ovaries of BDF1 mice and cultured in growth medium for 9 to 11 days. Surviving follicles with antrum formation were transferred to maturation medium for 14 to 18 hours. Follicle survival, antrum formation, and retrieval of metaphase II (MII) oocytes were compared among three age categories (4-5, 7-8, and 10-11 week-old). By using 7- to 8-week-old mice, preantral follicles were cultured in growth medium supplemented with hCG (0, 5, or 10 mIU/mL), SCF (50 ng/mL), IGF-1 (50 ng/mL), and SCF+IGF-1. Results: Seven- to eight-week-old mice showed a higher follicle survival and antrum formation and produced more MII oocytes compared to other groups. In the 7- to 8-week-old mice, supplementation of 5 mIU/mL hCG significantly enhanced the antrum formation but the percentage of MII oocytes was similar to that of the control. Supplementation of SCF+IGF-1 did not enhance follicle survival or antrum formation but the percentage of MII oocytes increased modestly (39.1%) than in the control (28.6%, p>0.05, statistically not significant). Conclusion: Seven- to eight-week-old mice showed better outcomes in growth of preantral follicles in vitro than 4- to 5- or 10- to 11-week-old mice. Supplementation of hCG enhanced antrum formation and supplementation of SCF+IGF-1 yielded more mature oocytes; hence, these should be considered in the growth of preantral follicles in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.47 no.3
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• pp.213-220
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• 2020

Objective: The aim of this study was to explore the potential adverse effect of spontaneously decreasing serum estradiol (SE) levels on in vitro fertilization (IVF) outcomes. Methods: This retrospective single-subject study analyzed IVF cycles conducted at a hospital IVF unit between 2010 and 2017. Overall, 2,417 cycles were analyzed. Only cycles with spontaneously decreasing SE before human chorionic gonadotropin (hCG) triggering were included. Each patient served as her own control, and subsequent cycles were analyzed for recurrent SE decreases. The main outcome was the number of oocytes retrieved. Results: Cycle characteristics were similar between the study (SE decrease) and control groups, with the exception of the median SE on the day of hCG triggering (899.7 pg/mL; interquartile range [IQR], 193-2,116 pg/mL vs. 1,566.8 pg/mL; IQR, 249-2,970 pg/mL; p< 0.001). The study group, relative to the control group, had significantly fewer total oocytes (5 [IQR, 2-9] vs. 7 [IQR, 3-11]; p= 0.002) and significantly fewer metaphase II (MII) oocytes (3 [IQR, 1-6] vs. 4 [IQR, 2-8]; p= 0.001) retrieved. The study group had fewer cleavage-stage embryos than the control cycles (3 [IQR, 1-6] vs. 4 [IQR, 2-7]; p= 0.012). Compared to cycles with a ≤ 20% SE decrease, cycles with a > 20% decrease had significantly fewer total and MII oocytes retrieved. SE decrease recurred in 12% of patients. Conclusion: A spontaneous decrease in SE levels adversely affected IVF outcomes, with a linear correlation between the percentage decrease and the number of oocytes retrieved. SE decrease can repeat in later cycles.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.117-117
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• 2002

The oocytes from most of animal species accumulate genetic information and other necessary materials during oogenesis for the later use in the early development. Over the years oocyte maturation has been studied extensively both in vitro and in vivo. Particularly, maturation of follicular oocyte in vitro becomes one of the important tools for the studies of basic cell biology, the in vitro technology of animal production, and in particular, the somatic cell cloning by nuclear transfer. We examined meiotic maturation and cumulus expansion in the presence of translation or transcription inhibitors for varying periods of in viかo maturation (IVM) of pig oocyte. In Experiment 1, the results revealed that translation and transcription inhibitors inhibited cumulus expansion and meiotic maturation during 35h of IVM. However, 50 to 60% of the oocytes underwent nuclear maturation without cumulus expansion during 75h of IVM. The rest of the oocytes were arrested at metaphase I (40-50%) in the presence of the inhibitors. In Experiment II, the OCCs were exposed to the drugs only for 15h to examine translation and transcription inhibitors on cumulus expansion and meiotic maturation. Transcription inhibitors for 15h did not arrest meiotic maturation when the oocytes were cultured for subsequent, necessary period of IVM, whereas cumulus expansion was completely inhibited, suggesting that initial 15h is critical transcription activity far cumulus expansion. Translation inhibitors for 15h exposure did not alter cumulus expansion and meiotic maturation during subsequent culture in the absence of the drugs. In Experiment III, the OCCs were exposed to the drugs only for later 30h to examine the influence of transcription and translation inhibitors on oocyte maturation. Interestingly, all meiotic maturation underwent normally with full expansion of cumulus. Similar results were obtained from Experiment IV where 5h of exposure from 15 to 20h of IVM culture to the drugs was performed and subsequently cultured for same period in fresh medium. Taken there results together, both transcription and translation are necessary for nuclear maturation and cumulus expansion, and first 15h IVM for cumulus expansion is critical. The arrested oocytes by the drugs were still capable of undergoing nuclear maturation, although cumulus expansion was affected.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.76-76
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• 2002

본 실험은 Piezo-미세조작기(PrimeTech Ltd., Japan)를 사용하여 마우스 핵이식 후 재구축배를 CZB와 KSOM 두가지 배양액을 사용하여 체외배양성적을 비교 검토하였다. MII의 미수정란은 성숙한 4~5주령 B6D2Fl에 hCG 주사 후 14시간째에 과적 방법을 통해 난관의 팽대부로 부터 회수하였고, metaphase II chromosome-spindle complex와 최소량의 세포질을 내경이 10$\mu\textrm{m}$인 피펫으로 흡입하여 탈핵하였다. 핵이식에 사용된 난구세포(8-l0$\mu\textrm{m}$)는 3시간동안 12% PVP 에처리 하여 piezo-미세조작기를 이용하여 세포질에 세포의 핵을 직접 미세주입 하였다. 핵이식 후 생존한 재구축배는 2시간동안 배양한 후 10mM SrC1$_2$와 5$\mu\textrm{g}$/$m\ell$의 cytochalasin B가 첨가된 $Ca^{2＋}$-free CZB에서 6시간 활성화 처리하였다, 활성화 처리 후 위전핵이 관찰된 재구축란을 CZB 와 KSOM 배지에서 배양하면서 발달률을 비교하였고, 상실배 및 배반포배로 발달한 재구축배를 day 3 대리모에 이식하였다. 표 1에서 보는 바와 같이 재구축배의 2-cell로의 발달률에 있어서 KSOM이 CZB에 비하여 유의적으로 높게 나타났으며(P<0.05), 또한 4-cell과 상실배/배반 포배로의 발달률에 있어서도 KSOM이 CZB에 비하여 유의적으로 높은 발달률을 나타내었다(P<0.01). 또한 KSOM 배지에서 배양된 상실배/배반포배를 대리모에 이식한 경우에 11.5 d.p.c에 생존한 태아가 관찰되었다. 이상의 결과로 핵이식 재구축배의 활성화 처리 후의 발생에는 KSOM 배지가 CZB 배지에 비하여 유효함을 확인 할 수 있었다.그와 같은 배양 기술을 이용하여 외래유전자를 도입한 일련의 결과에 관하여 보고 하고자한다., 이것은 세포내 유전자가 transfection되지 않은 세포도 neo selection에서 선발된다는 것을 제시하고 있다. 따라서 체세포를 이용한 형질전환동물 생산을 위해서는 세포내 유전자 도입과 선발 과정에서 나타난 colony에 대하여 보다 엄격한 screen을 하는 것이 필요한 것으로 생각된다.로 우점하였다. 여름철 식물플랑크톤 대발생에 영향은 수온과 직산염이 중요하였으나, 부유물질 크게 기여하지 못하였다.애를 확인하고 지도 관점을 파악하는 것을 포함한다. 그러나 본 논문은 역사발생적 수학 학습-지도 원리의 실제적인 적용에 관하여는 기초적인 연구에 지나지 않기 때문에, 역사발생적 원리를 학교수학에 실제적으로 적용하기 위해서는 각각의 내용에 대한 철저한 역사적 분석을 바탕으로 하는 후속 연구가 필요하다./TEX>구성교육${\lrcorner}$이 조선총독부의 관리하에서 실행되었다는 것을, 당시의 사범학교를 중심으로 한 교육조직을 기술한 문헌에 의해 규명시켰다.nd of letter design which represents -natural objects and was popular at the time of Yukjo Dynasty, and there are some documents of that period left both in Japan and Korea. "Hyojedo" in Korea is supposed to have been influenced by the letter design. Asite- is also considered to have been "Japanese Letter Jobcheso." Therefore, the purpose of this study is to look into the origin of the letter designs in t