• Asian-Australasian Journal of Animal Sciences
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• 제23권1호
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• pp.17-24
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• 2010

In the antral follicle phase, several layers of cumulus cells surround the oocyte and play an important support and regulation role in oocyte development and maturation via intercellular communications and interactions between oocytes and cumulus cells. However, information on stage specific gene expression in swine during the phase is not well understood. To investigate the function of cumulus cells during in vitro maturation of porcine oocytes and gene expression, suppression subtractive hybridization (SSH) was performed to screen genes that were differentially expressed between cumulus-oocyte complexes (COCs) and naked oocytes (NOs). Utilizing mRNAs from in vitro maturation oocytes, a SSH cDNA library from COCs as the tester and NOs as the driver was constructed. The SSH cDNA library was then screened using dot blot analysis. Results showed that a total of 70 clones randomly selected from the library were differentially expressed. Among these, 41 exhibited high homology to known genes and 11 were novel expressed sequences tags (ESTs). Four differentially expressed genes, including bfgf, sprouty 2, egr and btc, were further studied by real time quantitative PCR; results confirmed an increased expression of respective mRNA in COCs compared with NOs, which suggests that these factors may play an important role in oocyte development and maturation.

• 한국수정란이식학회지
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• 제33권3호
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• pp.99-105
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• 2018

To determine the differences in the in-vitro ovum maturation process of bovine, we compared the expression of MMPs in these oocytes and cumulus cell throughout oocytes maturated. In an attempt to investigate the effect of MMP activation and inhibitors in total protein of cumulus cell and, oocytes during oocytes maturation, we examined and monitored the localization and expression of MMPs (MMP-2 and MMP-9), TIMPs (TIMP-2 and TIMP-3), as well as their expression profiles (Real-time PCR, Gelatin Zymography and ELISA). Our results that the bovine oocytes MMP-2 and MMP-9 level was significantly associated with the rate of maturity of oocytes (P<0.05). In cumulus cell, MMP-2 was highly expressed in all stages of the oocyte's maturation. The final oocytes maturation exhibited strong gelatinase activity. There was no significant correlation between cumulus cell MMP-9 and the maturation rate of oocytes. However, for the oocyte cytoplasm MMP-9 expression was significant correlation to the maturation oocytes. There was no significant correlation between cumulonimbus cells MMP-9 and oocyte maturation rates; however, for oocyte cytoplasm, MMP-9 expression was significantly correlated with mature oocyte. However, the TIMP-1 and TIMP-2 protein expression patterns are not correlated with the maturation rate of the oocyte. Our results suggest that MMP different expression pattern may regulate the morphological remodeling of oocyte's in the cumulus cell. Further, the MMP-2 expression has a strong relation with a higher maturation rate of the oocyte.

• 한국가축번식학회지
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• 제17권2호
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• pp.93-101
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• 1993

These experiments were carried out to investigate whether zona hardening affect the efficiency of in vitro fertilization in mouse oocytes. The soluble properties for zona pellucida of oocytes matured in vivo, aged oocytes, and ovarian oocytes matured in vitro have been analyzed with proteolytic enzyme, 3mg/ml of $\alpha$-chymotrypsin. The mean solubility(t50) for the zona of unfertilized oocytes, oocytes not fertilized at the first inseminati and in vitro produced zygotes were 10.1, 20.3 and 32.3min., respectively. The t50 for zona lysis of fertilized oocytes was significantly difference than those observed for unfertilized oocytes and oocytes not fertilized at the first insemination(P<0.01). In addition, the t50 of zona in ovulated oocytes with and without cumulus cells incubated for 0, 3, 6, 9 and 12 hr in vitro, t50 were 13.9, 11.1, 20.7 and 28.0min., and 22.3, 21.0, 30.0 and 33.5min., respectively. In these experiments, the zona pellucida showed a gradual increase in resistance to dissolution by $\alpha$-chyjotrypsin with in vitro aging for more than 6 hrs. This effect was greater in cumulus-free as compared to cumulus-intact oocytes. Finally, in cumulus-intact and cumulus-free ovarian oocytes matured for 0, 5, 10 and 15 hr in vitro the t50 of zona pellucida were 3.0, 10.6, 18.4 and 24.5 min., and 3.0, 14.0, 26.2 and 32.0 min., respectively. Clear differences in solubility between the zona pellucida of oocytes matured in vivo and in vitro. This data were found suggest that under in vitro conditions there is a gradual change in the soluble properties of the zona pellucida, particularly in the absence of the cumulus cells.

• 한국발생생물학회지:발생과생식
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• 제17권1호
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• pp.63-72
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• 2013

A specific inhibitor of RNA polymerase II, ${\alpha}$-amanitin is broadly used to block transcriptional activities in cells. Previous studies showed that ${\alpha}$-amanitin affects in vitro maturation of cumulus-oocyte-complex (COC). In this study, we evaluated the target of ${\alpha}$-amanitin, and whether it affects oocytes or cumulus cells (CCs), or both. We treated ${\alpha}$-amanitin with different time period during in vitro culture of denuded oocytes (DOs) or COCs in comparison, and observed the changes in morphology and maturation status. Although DOs did not show any change in morphology and maturation rates with ${\alpha}$-amanitin treatment, oocytes from COCs were arrested at metaphase I (MI) stage and CCs were more scattered than control groups. To discover causes of meiotic arrest and scattering of CCs, we focused on changes of cumulus expansion, gap junctions, and cellular metabolism which to be the important factors for the successful in vitro maturation of COCs. Expression of genes for cumulus expansion markers (Ptx3, Has2, and Tnfaip6) and gap junctional proteins (Gja1, Gja4, and Gjc1) decreased in ${\alpha}$-amanitin-treated CCs. However, these changes were not observed in oocytes. In addition, expression of genes related to metabolism (Prps1, Rpe, Rpia, Taldo1, and Tkt) decreased in ${\alpha}$-amanitin-treated CCs but not in oocytes. Therefore, we concluded that the transcriptional activities of CCs for supporting suitable transcripts, especially for its metabolic activities and formation of gap junctions among CCs as well as with oocytes, are important for oocytes maturation in COCs.

• 한국가축번식학회지
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• 제17권4호
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• pp.271-278
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• 1994

The aim of this study was to compare the two culture systems 1) co-culture with cumulus cells and 2) chemically defined medium supplemented with amino acids (CR1aa) and fetal calf serum (FCS) of in vitro produced bovine embryos from follicular oocytes in vitro. Bovine follicular oocytes were collected from ovaries of slaughtered cows and matured in TCM199 supplemented with 10% FCS and hormones (1$\mu\textrm{g}$/ml FSH-P and 1$\mu\textrm{g}$/ml oestradiol-17$\beta$)24 hours at 39$^{\circ}C$ under 5% CO2 in air. The capacitation of spermatozoa from ejaculated or frozen bull semen was induced by centrifugation through Percoll density gradient (45%, 90%). Then capacitated spermatozoa (1$\times$106/ml) were inseminated into 50${mu}ell$ droplet containing matured follicular oocytes and incubated for 40~42 hours. Cleaved embryos of 2~4cell stage were transferred to the co-culture with cumulus cells and/or CR1aa medium supplemented with FCS. In semen source, the developmental rates to the blastocyst and the hatched blastocyst stages were higher in ejaculated semen(27.6% and 14.9%) than those of frozen-thawed semen(18.3% and 11.8%), respectively. In two culture systems, the proportions of embryonic development upto the blastocysts and the hatched blastocysts were higher of CR1aa medium (22.1% and 12.1%) than those of cumulus cell co-culture (16.8% and 5.1%), respectively. The number of cells in exapnded blastocysts was slightly higher in cumulus cells co-culture (122.6$\pm$8.5) than that in CR1aa medium (117.9$\pm$5.9). The present results indicated that the early development of in vitro produced bovine embryos can be maintained efficiently in CR1aa medium as well as in co-culture with cumulus cells.

• 한국가축번식학회지
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• 제20권3호
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• pp.353-360
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• 1996

The study was conducted to determine the optimal cumulus cells, glucose and superoxide dimutase(SOD) levels during the in vitro culture of bovine oocytes matured and fertilized in vitro for morulae and blastocyst development. Oocytes were cultured for 0~8 days in TCM-199 medium supplemented with 20% FCS, cumulus cells and with different glucose and SOD levels. The results are summarized as follows; 1. The in vitro developmental rates of bovine oocytes cultured in TCM-199 medium containing cumulus cells and 0.1, 0.5, 1.0, 5.0mM glucose levels 0~3 and 0~8 days after insemination were 21.1, 25.0, 23.3, 17.9 and 26.3, 25.7, 23.1, 19.4% respectively and there was significant differences on the development to the molurae and blastocysts stage among the cumulus cells and glucose levels. 2. The in vitro developmental rates of bovine oocytes cultured in TCM-199 medium containing 0.1, 0.5, 1.0, 5.0mM glucose levels 0~3 and 0~8 days after insemination were 11.3~24.5% and 17.3~25.0%, respectively. 3. The in vitro developmental rates bovine oocytes cultured in TCM-199 medium containing 100, 200, 300, 500 $\mu\textrm{g}$/ml SOD levels 0~3 and 0~8 days after insemination were 12.5~22.9% and 12.9~22.2%, respectively. Hight levels of SOD(500$\mu\textrm{g}$/ml) significantly reduced the rates ofmolurae and blastocysts stage(P<0.05).

• 한국동물학회지
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• 제31권4호
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• pp.273-282
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• 1988

생쥐에 PMSG와 hOG를 주사한 후 난자-난구복합체의 미세구조의 변화를 환찰함으로세 난구세포의 분산현상을 규명하고자 본 실험을 행하였다. 난자는 PMSG 주사후 48시간까지 별 다른 변화가 없었고 다만 표면막에 miGrOVilli와 Coaled pit의 수가 감소하는 경향을 보였다. 그러나 PMSG-hCG주사 12시간 후에 배란된 난자의 표면은 microvilli와 coated pit가 사라져서 평평하게 되었다. 방사관세포는 PMSG주사 48시간 후메 밀착해 있던 투명대와 간격이 생기기 시작하였고, 투명대를 통관하여 난자의 표면막과 desmosome으로 연결되어 있던 세포질돌기도 퇴화의 징후를 보였다. PMSG-hCG주사 후에는 급속히 격리, 분산되고 세포질돌기는 퇴화하였으며 dermo-some도 사라겼다. 난구세포들은 대조군에서 밀집되어 있었고 거의 gap junction으로 연결되어 있었는데, PMSG주사 24시간 후에는 모양이 등글게 되고 더욱 밀집되었으며, 48시간 후에는 거의 loose junction으로 연결되었고 분산되기 시작하였다. 결국 PMSG-hCG주사 If시간 후에는 완전히 분산되었고 거의 모두 핵응축과 괴사현상을 보였다. 난자- 난구 복합체의 분산은 배란전에 PMSG에 의하여 시작되고 hCG에 의하여 촉진 완결된다는 것이 확실하다. The ultrastructural changes of the oocyte-cumulus complexes of mouse alter injection of PMSG and hOG have been investigated in order to elucidate expansion phenomenon of the cumulus cells. The oocytes until 48 hours after PMSC injection showed no change except a tendency of decrease in numbers of microvilli and the coated pelts on surface membrane. However, surface membrane of the ovulated oocytes 12 hours after PMSC-hCC injection changed to be smooth due to disapperance of microvilli and coated pits. Corona radiate cells tightly attaching to zona pe]lucida 48 hours after PMSC injection began to be detached and their cytoplasmic processes connected by desmosome to oocyte surface membrane showed a degeneration symptom. Thereafter the detachment and degeneration were accelerated by hCG injection and followed by disappearence of desmosome. The cumulus cells in control group were compacted and connected by almost 9aP junction each another. Ite cumulus cells 24 hours after PMSG injection were changed to be round form and more tightly compacted. However, the cumulus cells 48 hours after PMSG injection were connected by almost loose junction and showed the beginning of expansion. Eventuallv, the cumulus cells 12 hours a%or PMSG-hCG injection were completely expanded, and became pvknotic and necrotic in most It is clear that the expansion of oocyte-cumulus complex were initiated by PMSC, then accelerated and completed by hCG before ovulation.

• 한국동물학회지
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• 제30권1호
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• pp.1-9
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• 1987

생쥐 난자-난구 복합체를 인공배양하면서 adenylate cyclase의 촉진제인 forskilin과 phosphodiesterase의 저해제인 3-isobutyl-1-methylxanthine(IBMX)을 배양액에 첨가하여 이들이 난구세포의 분산과 난자의 성숙(핵붕괴)에 미치는 효과를 관찰한 결과 다음과 같았다. 1. Forskilin은 0.001$\mu$M에서 부터 난구세포의 분산을 유도하기 시작하여 (36%) 0.1-10$\mu$M 구간에서 최대의 분산율을 나타내었고 (80-90%) 100$\mu$M에서는 그 효과가 줄어들었다(60%). 이때 난자의 핵 붕괴는 10$\mu$M까지 정상으로 일어나다 (75-80%) 100$\mu$M에서 부분적으로 억제되기 시작하였다(40%). 2. IMBX는 0.01$\mu$M에서 부터 난구세포의 분산을 유도하기 시작하여 (30%) 1-1,000$\mu$M의 전 구간에서 최대의 분산율(81-89%)을 나타내었다. 난자들은 10$\mu$M의 농도까지 정상적으로 핵 붕괴를 일으키었으나(90% 이상) 100$\mu$M 이상에서 급격히 억제되었다(14%). 3. 난구세포의 분산을 유도하는데 필요한 최소의 자극기간을 조사해 본 결과 HCG는 2분, FSH와 forskilin은 15-30분, IBMX는 2시간이었다. 위 결과로 부터 생쥐 난구세포에서 cAMP의 농도를 높이는데 adenylate cyclase 와 phosphodiesterase의 두 효소가 모두 중요한 기여를 하며 난구세포는 단 기간에 생긴 cAMP의 peak로서 분산이 유도될 수 있으나 난자의 성숙억제 과정에서는 지속적인 cAMP의 존재가 필요하다는 것을 알았다.

• 농업과학연구
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• 제23권2호
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• pp.206-211
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• 1996

본 실험은 수정배양액 및 난구세포가 돼지난포란의 체외수정에 미치는 영향을 구명하고자 실시하였는 바, 그 결과는 다음과 같다. 1. 체외수정배양액에 따른 수정률은 BO, mTALP 및 TCM-HEPES 배양액에서 각각 14.0~24.3%, 30.8~32.7% 및 21.4~23.9%의 정상수정률을 나타내서 mTALP 배양액이 가장 적합한 수정배지였다. 2. 수정에 이용된 두 종류 정액간의 정상수정률은 정소상체미부정액이 BO, mTALP 및 TCM-HEPES 배양액에서 각각 24.3%, 30.8% 및 23.9%이었고, 사출정액이 14.0%, 32.7% 및 21.4%로 나타나서 mTALP 배양액을 수정배지로 하여 사출정액을 사용한 경우가 가장 높은 정상수정률(32.7%)을 나타냈다 3. 난자-난구세포복합체와 난구세포를 제거한 나화난자의 정자침투률은 각각 54.0% 및 72.0%로 나화난자에서 높게 나타났다 그러나, 정상수정률은 난자-난구세포복합체 및 나화난자에서 각각 11.9% 및 21.5%로 난구세포를 제거하지 않은 난자-난구세포복합체에서 유의적(P<0.05)으로 높은 성적을 나타냈다.

• 한국발생생물학회지:발생과생식
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• 제6권2호
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• pp.97-104
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• 2002

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 조사하였다. 수란관 조직 추출액을 처리한 미성숙난자는 난포액만을 처리한 난자와 비교하여 난자의 성숙률에 차이를 보이지 않았다. 그러나 난구세포에서 난포액만을 처리한 경우 확장을 일으키면서 배양접시 바닥에 붙어서 배양 후 72시간까지 분열 증식을 계속하는 것이 관찰된 반면, 수란관 조직 추출액을 처리한 난구세포는 확장이 억제되며 72시간 배양 후 모두 죽었다. 한편 난포액과 수란관 조직 추출액을 함께 처리한 난구세포는 배양 후 24시간에 화장을 일으켰으나 72시간 후에는 수란관 조직 추출액 만을 처리했을 때 와 마찬가지로 모든 난구세포가 죽었다. 이러한 난구세포의 죽음이 세포자연사에 의한 것인지를 알아보기 위하여 DAPI로 핵을 염색한 후 관찰한 결과 세포자연사의 특징인 응축되고 분절화된 핵을 관찰 할 수 있었고, 특히 수란관 조직 추출액을 처리한 난구세포에서 많이 관찰되었다. 또한 TUNEL 방법으로 세포자연사를 확인한 결과 응축되고 분절화된 핵을 가진 난구세포에서 염색되는 것을 확인하였다. 본 연구의 결과들을 종합해 볼 때 소의 수란관 조직 추출액에 의한 생쥐 난구세포의 죽음은 세포자연사에 의한 것으로 판단되며, 이로 미루어 수란관 조직세포에는 난구세포의 세포자연사를 유도할 수 있는 물질을 포함하고 있는 것으로 사료된다.or teacher educators to re-conceptualize teacher education in Korea. 것이 필요하다.량은 264,000$m^2$, 79,200$m^3$이였으며, 우려기준의 40% 농도 이상의 경우 복원 면적과 물량은 779,000$m^2$, 233,700㎥, 배경치 농도 이상의 복원 목표 설정의 경우에는 각각 969,200$m^2$, 290,760$m^3$ 이였다. 토양오염 우려기준 농도의 40%를 복원 목표로 하는 경우와 배경치 농도를 복원 목표로 하는 경우, 복원 물량은 우려기준 농도를 복원 목표로 하는 경우의 3.0배와 3.7배정도 증가하는 것으로 나타나 복원 목표치 설정시 우려기준 농도의 40%와 배경농도의 차이에 다른 복원 물량의 차이는 적어서, 복원 목표 설정시 배경치 농도로 복원계획을 세우는 것이 가장 바람직한 것으로 판단되었다.amma}$D)안정동위원소값이 광화I시기에는 각각 11∼9.${\textperthansand}$, -92∼-86${\textperthansand}$, 광화 II시기에는 각각 0.3${\textperthansand}$(${\gamma}^{18}O_{H2O}$),-93${\textperthansand}$({\gamma}$D)이며, 리본-호상구조를 보이는 것으로 보아 대봉광상의 광화유체에 대한 기원과 진화과정을 두 가지로 생각할 수 있다. 1) 마그마유체로부터 광화작용이 진행됨에 따라 계속적인 순환수의 혼입이 있었으며 2) 조기 마그마${\pm}$변성유체에서 유체압력의 차에