• Development and Reproduction
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• v.5 no.2
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• pp.115-122
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• 2001

Production of a mature oocyte is a complex process that requires the close association between oocyte and follicular cells. The present study was conducted to investigate the difference between oocytes with and without close junctional communication with cumulus cells and the involvement of two connexins(CXs) in the interactions. Follicles at different sizes(small: 200～400 ㎛; large:>450 ㎛) were mechanically isolated from PMSG-primed mouse ovaries, and punctured to get cumulus-oocyte complex(COC). Oocytes were released themselves(denuded), with partially attached(partial), or with tightly attached(intact) cumulus cells. Maturation and fertilization capacity of the COCs were measured. Expression of CX 37 and CX 43 was examined by RT-PCR and in situ hybridization. The ratio between intact COC and denude/partial oocytes was 30％(SI) and 70％(SPD) in small follicles, while 55％(LI) and 45％(LPD) in large follicles, respectively. Maturation and fertilization rates of the released oocytes were similar among SI, LPD, LI groups, but those were significantly lower in SPD oocytes. In oocytes, CX 37 was the major CX and CX 43 was not expressed, whereas in the cumulus cells, CX 43 was the major, and CX 37 was the next. Results of the present study suggest that 1) immature oocytes from small follicles with intact cell-to-cell communication with cumulus have the similar quality to that of the oocytes from larger follicles, 2) gap junction between oocyte and cumulus cells may be the heterotypic channel, and 3) we could not explain the difference in the cell-to-cell communication between intact and partial/denuded COCs through the expression of the two CXs.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.2
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• pp.177-182
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• 1994

ICR female mice aged 3 to 4 weeks, were stimulated with 7.5 IU PMS injection. At 48-52h post-PMS injection, ovaries were dissected out and oocytes-cumulus complexes(OCCs) were divided into three groups, cumulus-free oocytes(O), cumulus-free oocyte cocultured with cumulus cells(O+C) and OCC. The oocyte were cultured in TCM199 containing various protein sources, FCS, BSA or PVP with gonadotropins(Gns) for 24h. Spermatozoa were collected from cauda epididymis and capacitated in T6 + BSA for 2h. After oocyte maturation in vitro(IVM) in different experimental groups, matured oocytes were inseminated with the capacitated spermatozoa in T6 + BSA for 6h. In the groups of IVM in TCM + BSA or PVP, fertilization(IVF) did not occur efficiently. However, increased fertilization was found in TCM+ FCS group. The oocytes groups, with cumulus cells showed decreased polyspermy in FCS group (O; 31.8 %, O + C; 12.2 %, OCC; 16%), the addition of Gns did not prevent polyspermy in all three groups. The rates of fertilization increased in zona-free oocytes in PVP group. This results showed that culture system for IVM and IVF could be improved. Furthermore, PVP can be used for the substitution of protein source during maturation, and its low rate of fertilization has been found due to zona hardening which occurred in FCS-free medium.

• Korean Journal of Animal Reproduction
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• v.14 no.3
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• pp.183-190
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• 1990

These studies were carried out ot investigate the effects of estrous cow serum(ECS), fetal calf serum(FCS), bovine follicular fluid(BFF) and matured cumulus cell(MCC) on in vitro maturation and fertilization of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3-5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, ECS, BFF and MCC for 24~48 hrs. in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 18$^{\circ}C$20 hrs. with motile capacitated sperm in the TCF(Tyrode calcium-free) solution containing 200$\mu\textrm{g}$/ml of heparin. The results obtained in these experiments were summarized as follows : 1. The oocytes classified as "A, B, C, D and Degenerative" depending morphological integrity and those were 61.4%, 12.1%, 19.2%, 4.2% and 3.0% of the total oocytes harvested, respectively. The maturation and fertilization rate of the A, B, C class follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS were 89.1%, 78.0%, 52.6% and 78.1%, 33.3%, respectively. 2. The maturation and fertilization rate of the follicular oocytes cultured in TCM-199 medium supplemented with 5%~20% ECS and FCS were 74.0%~80.6, 26.2%~30.0% and 71.7%~76.9%, 51.9%~58.0%, and those values were higher the supplement of ECS than FCS. 3. The maturation rate(68.0%~64.6%) and fertilization rate(59.6%~60.4%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 20~30% BFF were higher than those of follicular oocytes cultured TCM-199 medium supplemented with 10% FCS and 10% and 50% BFF. 4. The maturation rate(76.5%) and fertilization rate(61.7%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 1$\times$106/ml cumulus cells were higher than those of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 1$\times$104~5/ml and 1$\times$108/ml cumulus cells.lus cells.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.105-109
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• 2017

Autophagy is an intracellular degradation and recycling system. Oocyte maturation is dynamic process, in which various proteins should be synthesized and degraded. In our previous study, we reported the loci of autophagosome and dynamics of autophagic activity in porcine oocytes during in vitro maturation. In this study, we verified loci of autophagosome in porcine follicular cumulus-oocyte complex by detection of microtubule-associated protein 1A/1B-light chain 3 (LC3) which is the reliable marker of autophagosome. Porcine ovary including various sizes of follicles was fixed within 1 hour after collection from slaughterhouse. After fixation, immunohistochemistry was conducted on sliced ovary tissue containing various sizes of follicles by using LC3 antibody. As a result, LC3 signal was clearly detected in both cumulus and oocytes of various sizes of follicles. We also found ring shaped signal which represent autophagosome near oocyte membrane. Most of the signals in oocytes were localized nearby cellular membrane while evenly dispersed in cumulus cells. Therefore, this result suggests that autophagy occurs in porcine COCs (cumulus-oocyte complexes) at follicular stage.

• The Korean Journal of Zoology
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• v.30 no.1
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• pp.1-9
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• 1987

Cyclic AMP (cAMP) was known to play a key role in the regulation of cumulus expansion and oocyte maturation of mammalian cumulus-oocyte complexes (COC's) in vivo and in vitro. The present experiments were conducted to know how intracellular level of cAMP in these cells is controlled. Intracellular cAMP level was modulated by culturing mouse CGC's with an adenylate cyclase stimulator, forskolin, a phosphodiesterase inhibitor, 3-isobutyl-1-methyixanthine (IBMX), human chorionic gonadotrophin (HCG), or follicle stimulating hormone (FSH). The rate of cumulus expansion and germinal vesicle break-down (GVBD) was checked after culture and used as a biological end point. Forskolin in the medium began to stimulate the expansion of the complexes at 1 nM and induced maximum expansion (80~90%) at 0 1~10 $\mu$M. The expansion rate was reduced to 60% when forskolin concentration was increased to 100 $\mu$M. Oocyte GVBD occurred normally (75~82%) in the presence of 10 $\mu$M of forskolin, but partial suppression was appeared at 100 pM of the drug (40%). IBMX also stimulated the expansion from the concentration of 0.01 pM and induced full expansion (81~89%) between the concentration of 1-1000 $\mu$M. Meiotic resumption was occurred normally under 10 $\mu$M of IBMX, but suppressed drastically from the concentration of 100 $\mu$M. The minimum exposing time to hormone or drugs required to trigger cumulus expansion was two minutes with HCG, 15~30 minutes with FSH and fors kolin, and two hours with IBMX. The data presented here seemed to imply that intracellular cAMP level in cumulus cells is regulated by both adenylate cyclase and phosphodiesterase and cumulus expansion is induced by a peak of cAMP while meiotic arrest is maintained by continuous presence of cAMP.

• Journal of Embryo Transfer
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• v.25 no.3
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• pp.171-177
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• 2010

Plasminogen activators (PAs) are serine proteases that convert plasminogen to plasmin. The PA/plasmin system has been associated with a number of physiological processes such as fibrinolysis, ovulation and fertilization. Although correlations have been reported between reactive oxygen species (ROS) and oocyte maturation, the relationship between PA activity and ROS is unknown. The present study was undertaken to determine the effects of cumulus cells on PA activity in matured porcine oocytes under xanthine (X)-xanthine oxidase (XO) system. When oocytes were matured under the X-XO system, the proportion of oocytes remaining GV stage was higher (p<0.05) in oocytes without cumulus cells. The incidence of degenerated oocytes was higher (p<0.05) in the X+XO ($11.1{\pm}6.1$ and $21.6{\pm}3.4%$) than in the control group ($2.9{\pm}1.8$ and $4.0{\pm}1.6%$). The proportion of TUNEL-positive oocytes and activity of caspase-3 were higher (p<0.05) in cumulus-free oocytes and oocytes exposed to ROS. Tissue-type plasminogen activator-plasminogen activator inhibitor (tPA-PAI) and tissue-type plasminogen activator (tPA) activity were detected in oocytes that were separated from cumulus-oocytes complexs (COCs) at 44 h of maturation culture, and only tPA was produced in oocytes that were denuded before the onset of maturation culture. On the other hand, the activities of PA were increased (p<0.05) when oocytes were cultured under the X-XO system. The higher activity of tPA was observed in denuded oocytes (DOs) underwent apoptotic changes by oxidative stress. In COCs, however, tPA-PAI as well as tPA activity was detected and apoptotic changes such as DNA cleavage or caspase-3 activation were not observed. These results suggest that tP A may be relevant to apoptotic cell death in porcine oocytes by oxidative stress.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.8
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• pp.1057-1061
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• 2001

In this study we explored the possibility of performing nuclear transfer in the domestic cat and assessed the ability of different culture media to support in vitro development of reconstructed cat embryos. Donor somatic cells were derived from cultured cumulus cells or explants of oviduct tissue, and recipient cytoplasts from in vitro matured oocytes. A higher percentage of cleavage (84.6% and 86.5%) and development to the morula stage (35.9% and 44.2%) was found when reconstructed embryos receiving cumulus or oviduct cells were cultured in MK1 medium, compared with those cultured in CR1aa (58.7% and 72.5%, 13.8% and 13.6%, respectively). There was no significant difference between MK1 and CR1aa media with respect to the proportion developing to the blastocyst stage (15.4% and 17.3% vs 6.8% and 8.6%, respectively, p>0.05). There was no significant effect (p>0.05) of donor cell type (cumulus and oviduct cells) on the rates of fusion (65.0% and 52.5%), cleavage (84.6% and 86.5%), development to the morula (35.9% and 44.2%), and blastocyst (15.4% and 17.3%) stages when reconstructed embryos were cultured in MK1 medium. Similar results were found for the reconstructed embryos cultured in CR1aa medium. These results show that culture medium has a significant impact on the early development of reconstructed cat embryos, whereas donor cell type does not have a significant effect.

• 대한생식의학회:학술대회논문집
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• 1997.11a
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• pp.69-70
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• 1997

• Clinical and Experimental Reproductive Medicine
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• v.30 no.4
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• pp.333-340
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• 2003

Objective: This study was performed to examine the maturation and the development to the blastocyst stage of immature oocytes collected from patients with high risk of ovarian hyperstimulation syndrome (OHSS). Materials and Methods: Cumulus-oocyte complexes (COCs) were collected following only HCGpriming for non stimulated IVF-ET cycles of the patients. At the time of oocyte collection, COCs were classified into three groups in accordance with their appearance (Group I: oocytes with dispersed cumulus cells; Group II: oocytes with compacted cumulus cells; Group III: oocytes with sparse cumulus cells). The in vitro maturation and blastocyst development rates of the COCs were compared among these groups. From August 2001 to June 2002, 48 IVM/IVF-ET cycles from 42 patients (mean age: $32.4{\pm}3.8$ years) were performed. To prevent the occurrence of OHSS, the patients were primed with 10, 000 IU HCG alone 36 h before oocyte collection without gonadotropin stimulation. Oocytes were aspirated on cycle days from 7 to 13. The normal COCs were classified into three groups according to their appearance. The aspirated immature oocytes were cultured in YS maturation medium containing 30% (v/v) human follicular fluid (HFF), 1 IU/ml FSH, 10 IU/ml HCG and 10 ng/ml rhEGF. Fertilization was induced by intracytoplasmic sperm injection (ICSI). All zygotes were co-cultured with cumulus cells in $10{\mu}l$ YS medium containing 10% HFF until day 7 after oocyte collection. Blastocyst transfer was performed on day 5 after ICSI. Results: Th e mean number of oocytes cultured in the IVM/IVF cycles was $24.7{\pm}10.6$. Of 1185 COCs, those assigned to Group I, II and III were 470 (39.7%), 414 (35.0%) and 301 (25.4%), respectively. The maturation rate (94.5%, 444/470, p<0.05) in Group I was significantly higher than those of Group II (62.8%, 260/414) and Group III (73.1%, 220/301). Especially, 30.9% of COCs in Group I (145/470) was matured on the day of oocyte aspiration. There were no differences in the rates of fertilization and cleavage among the three groups. The development rate to the blastocyst stage in Group I (54.6%, 206/377, p<0.05) was also significantly higher than those in Group II (33.0%, 68/206) and Group III (30.1%, 52/173). Twenty-four clinical pregnancies (50.0%) was obtained and 22 pregnancies (45.8%) are ongoing. Implantation rate in the present study was 24.6%. Conclusion: These results suggest that there is a positive correlation between the appearance of COCs and the developmental competence of the immature oocytes in non stimulated IVM/IVF cycles.

• Korean Journal of Animal Reproduction
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• v.16 no.1
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• pp.7-14
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• 1992

These experiments were designed to investigate the effects of glycosaminoglycans(GAGs), which maybe produced by cumulus cells, on in vitro maturation process of bovine follicular oocytes. The rates of in vitro maturation of bovine oocytes were examined after incubating with the various concentrations of a hyaluronic acid, chondroitin sulfate, or heparin for 26 hours. The results obatained in these experiments were summarized as follows : 1. When the cumulus or removed oocytes treated with hyaluronic acid(200, 400, 800 and 1, 600$\mu\textrm{g}$/ml), the maturation rates of cumulus-removed oocytes were 48.6, 59.4, 68.4 and 61.3% respectively. Especially, at a concentration of 800$\mu\textrm{g}$/ml, the maturation rates(68.8%) of cumulus-removed oocytes were slightly lower than that(78.3%) of cumulus-enclosed oocytes. However, the treatment of hyaluronic acid showed significantly higher maturation rates compared to that(48.4%) of control group of cumulus-removed oocytes(p<0.05). Therefore, these results suggest thate hyaluronic acid had a beneficial effect on maturation of bovine follicular oocytes. 2. The maturation rate of cumulus-free oocytes treated with chondroitin sulfate(200, 400, 800 and 1, 600$\mu\textrm{g}$/ml) was 52.3, 56.1, 55.9 and 52.2% respectively. These rates were not different from those(52.9%) of control group. However, these rates in cumulus-free oocytes were significantly lower than that(79.0%) in cumulus-enclosed oocytes(p<0.01). Therefore, these results suggest that chondroitin sulfate do not affect on the maturation of oocytes. 3. In cumulus-free oocytes cultured with different heparin concentration, the maturation rates were ranged from 48.5 to 52.1%, showing no differencies from that(50.7%) of control group. However, these rates were significantly lower than 80.0% in cumulus-enlosed oocytes(p<0.01). 4. The nuclear maturaton of oocytes was increased by treatment of each of three glycosaminoglycans(hyaluronic acid, chondroitin sulfate, and heparin). In addition, the treatment of mixed together showed the significant additive effect. Hyaluronic acid was more effective than chondroitin sulfate and heparin were.