• The Korean Journal of Zoology
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• v.31 no.4
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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.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.1
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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.

• Clinical and Experimental Reproductive Medicine
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• v.13 no.2
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• pp.201-206
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• 1986

These experiments were conducted to know whether RNA syntheis is involved in the cumulus expansion and oocyte maturation of mouse cumulus-oocyte complexes in vitro. Mouse cumulus-oocyte complexes(COC's) were cultured in the presence of cordycepin, an inhibitior of RNA synthesis and its effect on the cumulus expansion and oocyte maturation were examined. The results were as follows. 1. Continuous presence of cordycepin in the medium(200${\mu}g/ml$) inhibited the HCG-induced cumulus cell expansion of mouse complexes. This inhibition was reversible. 2. When the COC'S were preincubated with different concentration of cordycepin plus HCG for 3 hours and then transferred to the plain medium, the HCG induced cumulus expansion was suppressed at $100{\mu}g/ml$ of cordycepin. 3. When the COC'S were cultured with cordycepin after HCG stimulation(3hrs), the cumulus expansion were not suppressed by cordycepin. 4. Oocyte meiotic maturation did not appear to be affected by cordycepin. The data presented here imply that RNA synthesis is involved in the cumulus expansion process and that mouse oocytes are more resistant to RNA synthesis inhibitor than cumulus cells.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.759-764
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• 2001

This study was carried out to investigate the effect of activation timing, cell cycle and passage on the development of embryos produced by cumulus cell nuclear transfer in Hanwoo (Korean cattle). Nuclear donor cumulus cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at $38.5^{\circ}C$ in a humidified atmosphere of 5% $CO_2$ in air. The 1~6 passages of serum deprived or actively dividing cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One pulse of 180 volts for $15{\mu}s$ was applied to induce the fusion between karyoplast and cytoplast. The activation was done before or after the fusion. To activate, oocytes were treated with $10{\mu}M$ calcium ionophore for 5 min immediately followed by 2 mM 6-dimethylaminopurine for 3 h. The nuclear transfer embryos were cultured in $500{\mu}l$ of modified CRlaa supplemented with 3 mg/ml BSA in four well dish covered with mineral oil. After 3 days culture, culture medium was changed into modified CRlaa medium containing 1.5 mg/ml BSA and 5% FBS for 4 days. The incubation environment was 5% $CO_2$, 5% $O_2$, 90% $N_2$ at $38.5^{\circ}C$. There was no blastocyst formation when the nuclear transfer embryos were activated before the fusion, whereas, 29.9% of blastocyst formation was shown when the nuclear transfer embryos were activated after the fusion. When serum deprived and actively dividing cumulus cells were used as nuclear donor cells, the developmental rates to blastocyst were 38.5% and 40.6%, respectively. There was no significant difference between serum deprived and actively dividing cells in the developmental rates. The developmental rates to blastocyst according to 1~6 passages were 37.5~44.4%. However, there were no significant differences among passages. These results indicate that 1~6 passage cumulus cell irrespective of cell cycle could support development of nuclear transfer embryos activated after the fusion.

• Development and Reproduction
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• v.22 no.4
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• pp.297-307
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• 2018

The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on cumulus expansion, nuclear maturation, fertilization capacity and subsequent development in porcine oocytes. The oocytes were incubated with 0, 25, 50, and $100{\mu}M$ ALA. Cumulus expansion was measured at 22 h, and gene expresison and nuclear maturation were analyzed at 44 h after maturation. Then, mature oocytes with ALA were inseminated, and fertilization parameters and embryo development were evaluated. In results, both of cumulus expansion and nuclear maturation were increased in $50{\mu}M$ ALA groups compared to control groups (p<0.05). However, expression of gap junction protein alpha 1 (GJA1, cumulus expansion-related gene), delta-6 desaturase (FADS1, fatty acid metabolism-related gene), and delta-5 desaturase (FADS2) mRNA in cumulus cells were reduced by $50{\mu}M$ ALA treatment (p<0.05). Cleavage rate was enhanced in 25 and $50{\mu}M$ ALA groups (p<0.05), especially, treatment of $50{\mu}M$ ALA promoted early embryo develop to 4 and 8 cell stages (p<0.05). However, blastocyst formation and number of cells in blastocyst were not differ in 25 and $50{\mu}M$ ALA groups. Our findings show that ALA treatment during maturation could improve nuclear maturation, fertilization, and early embryo development through enhancing of cumulus expansion, however, fatty acid metabolism- and cumulus expansion-related genes were down-regulated. Therefore, addition of ALA during IVM of oocytes could improve fertilization and developmental competence, and further studies regarding with the mechanism of ALA metabolism are needed.

• Journal of Genetic Medicine
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• v.18 no.1
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• pp.1-7
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• 2021

The oocyte quality is of great importance in infertility as it reflects the follicle developmental potential and further affects the embryo development, clinical pregnancy outcomes. The analysis of gene expression in somatic cells is an important study to better clinical in vitro fertilization (IVF) outcomes in embryo selection reflecting the appropriate communication between the oocyte and somatic cells. Specifically, somatic cell transcriptomic technology can help assess biomarkers of oocyte and embryo ability. The present article aims to overview the basic aspect of folliculogenesis and review studies involving changes in candidate gene expression of cumulus or granulosa cell related to clinical outcomes in human IVF.

• Development and Reproduction
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• v.6 no.2
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• pp.97-104
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• 2002

In most mammals, mature oocyte-cumulus complexer(OCCs) ovulate into the oviduct where fertilization by sperm takes place. However, the complex that fail to fertilize eventually undergoes degeneration while they reside in the oviduct. Yet there is no blown mechanism how both oocyte and cumulus cells degenerate. Using human follicular fluid (hFF), bovine oviductal tissue extract (BOX) and mouse OCC, the present study aimed to find how the oviduct influence the viability of the oocyte and cumulus cells in vitro. There was no difference of oocyte maturation rate between the control and BOX-treated groups. However, there was a significant difference in the survival of cumulus cells between two groups. Cumulus cells cultured in the presence of hFF alone underwent initially expansion and then they formed monolayer in the culture dish. Even after 72 hr, they proliferated well and showed fibroblast-like morphology. Cumulus cells cultured in the presence of both hFF and BOX also expanded after 24 hr, however, after 72 hr culture, they eventually detached and degenerated. Cumulus cells cultured in the BOX alone gave a similar drastic result. When the cumulus cells cultured in the presence of BOX were stained with DAPI, their nuclei showed partial condensation and fragmentation. After detailed analysis of these cells by TUNEL assay, many nuclei of them exhibited well stained spots indicating the signs of apoptosis. Based upon these observations, it is suggested that BOX might possess a factor that leads mouse cumulus cells to undergo apoptosis in vitro.

• Korean Journal of Animal Reproduction
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• v.16 no.4
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• pp.311-316
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• 1993

Bovine follicular oocytes were matured, fertilized and cultured in vitro. Oocytes with or without cumulus cells were transferred into TC-I99 medium supplemented with 10% FCS 8 h after insemination, and embryos cleaved to 2- to 8-cell stage 56 h after insemination were cultured further for 5 days. The proportions of embryos developed to morular and blastocyst stages were significantly higher (P<0.0l) in those cultured with(31%) than without(15%) cumulus cells. When the embryos were cultured with cumulus cells in the medium with different protein sources, the highest proportion(59%) of embryos developed to morular or blastocyst stage was obtained in medium with BSA(P<0. O01). Higher proportions of embryos were degenerated during culture with FCS(15%) or CS(14%) compared with BSA(3%). The present results indicate the early development of in-vitro fertilized bovine embryos can be maintained efficiently by BSA when they were co-cultured with cumulus cells.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.99-103
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• 2011

Transducin-like enhancer protein 1(TLE-1) is protein associated with cell proliferation. This study analyzed change of TLE-1 mRNA expression during in vivo and in vitro maturation in porcine oocytes. Oocytes and granulose cells were collected from follicles of <2 mm, 2~6 mm and >6 mm in diameter in slaughtered pig's ovaries. Oocytes collected from follicles of 2~6 mm in diameter were used after in vitro maturation for 0, 10, 20 and 44 h. Cumulus cells and granulose cells were collected after treatment with hyaluronidase. In results, TLE-1 mRNA expression in oocytes collected from follicle >6 mm in diameter is increased, TLE-1 RNA expression in cumulus cells and granulosa cells from follicles <2 mm, 2 mm 6 mm and >6 mm in diameter. However, there is no significant difference. On the other hand, TLE-1 mRNA expression from oocytes cultured for 10 hand 44 h is increased, TLE-1 mRNA in cumulus cells cultured for 10 h is significant increased(p<0.05) than other culture periods. In conclusion, these results show that TLE-1 is expressed in all cell types of oocytes, cumulus cells and granulose cells, and associated with oocyte maturation.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.113-120
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• 1993

To provide the optimal culture conditions for the developm,ent of in vit개 produced embryos, we have been investigated various culture media as well as co-cultrue systems using porcine cumulus cells or mouse fetal fibroblast cells. Porcine ovaries were brought to the laboratory from local slaughter house within 1 hour after slaughtering and cumulus oocytes complexes were recovered from antral follicles(3~5mm) with 23 gauge needle. To maturate follicular oocytes, cumulus oocytes complexes were washed three times with TCM-199 containing 25mM HEPES and incubated(39$^{\circ}C$, 5% CO2 in air) in various maturation media for 42 hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were rpepared for fertilizing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${\mu}\ell$ fo capacitated spermatozoa containing 1~5$\times$105 sperm/ml to droplets. Eighteen to twenty-eight hours after sperm insemination, fertilized eggs were washed three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three different media, m-KRB, BECM and TCM-HEPES were 0~1.0%, showing extremely lower rates. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. The rates of embryos developed to 2-, 4-, 8-, 16-, 32-cell and morula or blastocyst stage in co-culture with porcine cumulus cells and mouse fetal fibroblast cells were 61.1~67.0%, 59.0~58.0%, 42.5~43.1%, 28.4~30.2% and 20.4~21.0%, respectively. These development rates upto morula or blastocyst stages were significantly higher than those of the embryos cultured in the basic culture medium(P<0.01). These findings suggest that co-culture of in vitro fertilized eggs with porcine cumulus cells or mouse fetal fibroblast cells enhance the development of fertilized eggs to morula or blastocyst stage in vitro.