• 한국수정란이식학회지
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• 제10권3호
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• pp.183-191
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• 1995

This experiment was carried out to study the determination of survival of vitrified and thawed mammal follicular oocytes by FDA-test. Oocytes were divided into 3 groups according to attachment of cumulus cell. Group A oocytes were tightly surrounded by cumulus cell, group B oocytes were partially surrounded by cumulus cell, and group C oocytes were poorly surrounded by cumulus cell. Vitrification solution developed by our previous study (Kim et al, 1992) which consisted of permeable agent (20 % glycerol + 10 % ethylene glycol) and nonpermeable agent (30 % Ficoll + 10 % sucrose). Oocytes (7~10) loaded into 0.25 ml straw after 10 min equilibration were plunged into liquid nitrogen (- 196$^{\circ}C$) directly. The FDA-score of vitrified and thawed group A oocytes was higher in rat (4.2) than in rabbit (3.9), cow (3.8), mouse (3.4) and porcine (2.4), however that of cumulus cell was higher in rabbit (4.7) than in rat (4.1), cow (2.9), porcine (2.6) and mouse (1.4). The FDA-score of vitrified and thawed group B oocytes were 3.1 (cow), 2.9 (rabbit), 2.9 (mouse), 2.6 (rat) and 2.5 (porcine), respectively. However that of cumulus cell was higher in rabbit (3.7) than in porcine (2.6), rat (2.3), cow (1.7) and mouse (0.3). The FDA-score of vitrified and thawed group C oocytes was higher in mouse (4.1) than in cow (2.9), rabbit (2.6), rat (1.3) and porcine (1.1). As shown in the above results, The survival rates of oocytes were higher in group A than in group B and C except in mouse and cow. These results suggest that the survival of cumulus cell as well as follicular oocytes can be reliably judged by their fluorescence with FDA-test.

• Clinical and Experimental Reproductive Medicine
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• 제25권1호
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• pp.9-16
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• 1998

This study was performed to investigate the stimulating effect on oocyte maturation and cumulus cell expansion in TC199 media by human cord serum (HCS) supplementation. Immature mouse oocyte cumulus complexes (OCCs) were cultured in TC199 media supplemented with bovine serum albumin (BSA), HCS and human chorionic gonadotropin (hCG) instead of luteinizing hormone (LH) respectively, and the expression of cumulus expansion and oocyte maturation were observed. After 4hr and 24hr culture with or without OCCs, media containing 0.4% BSA, 10% HCS and 10 IV hCG respectively were collected and analyzed for changing concentrations of estradiol $(E_2)$, progesterone $(P_4)$, testosterone (T), and $PGF_{2\alpha}$. There were no elevation of $E_2$, T, and $PGF_{2\alpha}$ by OCCs culture, but minute elevation of $P_4$ level by 24hr OCCs culture in hCG supplementation (p=0.048). The stimulating pattern of cumulus expansion of OCCs by HCS and hCG supplementation was similar to our previously report using Ham's F-10 media, however oocyte maturation rates after 24hr OCCs culture in all media were increased by $20\sim30%$ compared to Ham's F-10 media. These results suggest that LH in HCS induce cumulus expansion probably by $P_4$ secretion of OCCs, and TC199 is efficient media for immature mouse oocyte maturation.

• Asian-Australasian Journal of Animal Sciences
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• 제2권4호
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• pp.595-598
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• 1989

Bovine embryos/ova obtained from in-vitro fertilization were either co-cultured on a monolayer of bovine cumulus cells or cultured in medium alone. Embryos/ova co-cultured with cumulus cells developed to 8-cell (30.9%), morula (29.8%) and blastocyst stages (26.6%) after 3-4, 5-6, and 7-8 days of culture, respectively, while embryos/ova cultured in medium alone failed to develop beyond 8-cell (0-13.3%), morula (0-1.5%) and blastocyst stages (0%). The results of this study demonstrated the beneficial effect of cumulus cells on the development of bovine embryos.

• 한국발생생물학회지:발생과생식
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• 제20권4호
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• pp.359-365
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• 2016

Intact germinal vesicle (GV) arrest and release are essential for maintaining the fertility of mammals inducing human. Intact germinal vesicle release, maturation of oocytes is maintained by very complex procedures along with folliculogenesis and is a critical step for embryonic development. Cyclic guanosine monophosphate (cGMP) has been suggested a key factor for meiotic arrest but so far its mechanisms are controversy. In this study we examine the effects of cGMP on germinal vesicle breakdown in cumulus-enclosed oocytes and denuded oocytes. Spontaneous maturation was inhibited by a cGMP agonist, 8-Br-cGMP with concentration dependent manners both in cumulus-enclosed oocytes and denuded oocytes. The inhibitory effect was more severe in denuded oocytes than cumulus-enclosed oocytes. The Rp-8-Br-cGMP and Rp-pCPT-8-Br-cGMP did not severely block GVB compared to 8-Br-cGMP. The spontaneous GVB inhibitory effects were different by the existence of cumulus. Based on them it is suggested that the cumulus modulates the role of cGMP in GV arrest.

• 한국가축번식학회지
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• 제22권1호
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• pp.73-80
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• 1998

본 연구는 돼지난자의 체외성숙에 미치는 transforming growth factor $\beta$(TGF $\beta$)와 난수세포의 역할에 대하여 검토하였다. 난자의 체외성숙시 TGF $\beta$를 여러 농도에서 첨가한 경우 metaphase II로 성숙한 난자의 비율은 52~69%로 유의적인 차이는 나타내지 않았다. 성숙배양 24시간에서 난구세포의 유무에 관계없이 TGF $\beta$가 무첨가된 배양액 내에서는 성숙된 난자가 관찰되지 않았으나 TGF $\beta$첨가(5 및 4%)의 경우 성숙난자가 관찰되었다. 그러나, 성숙배양 48시간후 난자의 성숙율은 난구세포가 부착되어 있는 경우 TGF $\beta$첨가(70%)가 무첨가(52%)에 비해 높았으며, 난구세포를 세포를 제거한 경우 (35 및 26%)에 비해 유의적으로 높은 성숙율을 나타냈다(P<0.05). 한편, 나구세포가 부착된 난자의 성숙배양시 TGF $\beta$의 첨가시기에 의한 성숙율(54~71%)에는 큰 차이가 없었으나, 난구세포를 제거한 경우 전반기(59%) 또는 후반기(57%) 24시간 동안 TGF $\beta$를 첨가한 경우 48시간 동안 계속하여 첨가(27%) 또는 무첨가(38%)에 비하여 유의적으로 높은 성숙을 나타냈다(P<0.05). 이와 같은 결과는 난구세포가 돼지난자의 체외성숙시 필수적이지만, TGF $\beta$는 난구세포를 제거한 경우 난자의 성숙에 계속적인 역할을 하지 않는 것으로 추측된다.

• Asian-Australasian Journal of Animal Sciences
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• 제21권7호
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• pp.972-979
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• 2008

It has been recently shown that expression of the LH receptor (LHR) in cumulus cells is related with FSH-induced meiotic resumption of mouse cumulus enclosed oocytes (CEOs). However, to date, it is still unclear whether LHR expression in cumulus cells plays a key role during FSH-induced oocyte maturation. The purpose of this study was to characterize the functional role of LHRs in cumulus cells. CEOs were isolated from eCG-primed preovulatory follicles and cultured in hypoxanthine (HX) arrested medium. LHR protein expression in cumulus cells was time-dependent increasing during the process of FSH-induced oocyte maturation. While the sense oligodeoxynucleotide (ODN) had no effect, antisense ODN inhibited FSH-induced LHR expression and meiotic resumption. Moreover, this antisense ODN against LHR could inhibit FSH-induced mitogen-activated protein kinase (MAPK) phosphorylation. This study suggested that LHR expression in cumulus cells is involved in FSH-induced oocyte meiotic resumption, which process is possibly regulated by MAPK cascade.

• 한국동물생명공학회지
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• 제34권2호
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• pp.86-92
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• 2019

The aim of this study was to investigate effects of hyaluronidase during IVM on oocyte maturation, oxidative stress status, expression of cumulus expansion-related (PTX, pentraxin; GJA1, gap junction protein alpha 1; PTGS2, prostaglandin-endoperoxide synthase 2) and fatty acid metabolism-related (FADS1, delta-6 desaturase; FADS2, delta-5 desaturase; PPARα, peroxisome proliferator-activated receptor-alpha) mRNA, and embryonic development of porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated with 0.1 mg/mL hyaluronidase for 44 h. Cumulus expansion was measured at 22 h after maturation. At 44 h after maturation, nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured. Gene expression in cumulus cells was analyzed using real time PCR. The cleavage rate and blastocyst formation were evaluated at Day 2 and 7 after insemination. In results, expansion of cumulus cells was suppressed by treatment of hyaluronidase at 22 h after maturation. Intracellular GSH level was reduced by hyaluronidase treatment (p < 0.05). On the other hand, hyaluronidase increased ROS levels in oocytes (p < 0.05). Only PTGS2 mRNA was enhanced in COCs by hyaluronidase (p < 0.05). Population of oocytes reached at metaphase II stage was higher in control group than hyaluronidase treated group (p < 0.05). Both of cleavage rate and blastocyst formation were higher in control group than hyaluronidase group (p < 0.05). Our present results showed that developmental competence of porcine oocytes could be reduce by hyaluronidase via inducing oxidative stress during maturation process and it might be associated with prostaglandin synthesis. Therefore, we suggest that suppression of cumulus expansion of COCs could induce oxidative stress and decrease nuclear maturation via reduction of GSH synthesis and it caused to decrease developmental competence of mammalian oocytes.

• 한국가축번식학회지
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• 제17권2호
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• pp.133-139
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• 1993

The studies were carried out to investigate the effects of co-culture with cumulus cell, oviduct epithelial cells and uterine endometrial cells on the in-vitro fertilization and cleavage rate of porcine follicular oocytes. The ovaries were obtained from slaughtered swine. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible follicles of diameter 3~5 mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% FCS 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 12~18 hrs with motile capacitated sperm by preincubation. The results obtained in these experiments were summarized as follows : 1. The in-vitro maturation and fertilization rate of porcine oocytes co-cultured with cumulus cells in TCM-199 meidum were 64.6%~74.5% and 37.5%~55.3%, respectively. And in-vitro fertilization rate of cumulus-enclosed oocytes(51.5%) were significantly(p<0.05) higher than cumulus-denuded oocytes(21.7%). 2. The in-vitro maturation and fertilization rate of porcine oocytes co-cultured with 1$\times$104 cells/ml, 1$\times$106 cells/ml, 1$\times$108 cells/ml and 1$\times$1015 cells/ml oviduct epithelial cells in TCM-199 medium were 53.5% and 37.2%, 61.7% and 46.8%, 54.5% and 31.8%, 42.2% and 26.7%, respectively. 3. The in-vintro maturation and fertilization rate of porcine oocytes co-cultured with 1$\times$106/ml, 1$\times$108/ml, 1$\times$1015/ml uterine endometrial cells in TCM-199 medium were 54.3% and 39.1%, 58.3% and 43.8%, 55.5% and 33.3%, and 45.7% and 30.4%, respectively. 4. When the in-vitro fertilized oocytes were co-cultured with porcine cumulus cells, ovdiduct epithelial cells and uterine endometrial cells, the development rate to the blastocyst stage was 9.5%, 10.7% and 11.8%, respectively and the rates were higher than that of control, 2.1%(p<0.05).

• Clinical and Experimental Reproductive Medicine
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• 제46권2호
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• pp.76-86
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• 2019

Objective: This study was performed to explore the possibility that each oocyte and its surrounding cumulus cells might have different genetic expression patterns that could affect human reproduction. Methods: Differential gene expression analysis was performed for 10 clusters of cumulus cells obtained from 10 cumulus-oocyte complexes from 10 patients. Same procedures related to oocyte maturation, microinjection, and microarray analyses were performed for each group of cumulus cells. Two differential gene expression analyses were performed: one for the outcome of clinical pregnancy and one for the outcome of live birth. Results: Significant genes resulting from these analyses were selected and the top 20 affected pathways in each group were analyzed. Circadian entrainment is determined to be the most affected pathway for clinical pregnancy, and proteoglycans in cancer pathway is the most affected pathway for live birth. Circadian entrainment is also amongst the 12 pathways that are found to be in top 20 affected pathways for both outcomes, and has both lowest p-value and highest number of times found count. Conclusion: Although further confirmatory studies are necessary, findings of this study suggest that these pathways, especially circadian entrainment in cumulus cells, may be essential for embryo development and pregnancy.

• 한국가축번식학회지
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• 제23권2호
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• pp.149-157
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• 1999

본 연구는 체외에서 돼지정자의 난자 내 침입에 있어서 난구세포의 기능적인 역할을 검토하기 위하여 실시하였다. 체외수정 시 정자침입율은 난구세포 부착 (61%) 난자가 제거 (25%) 된 난자에 비해 높았으나 (P＜0.001), 다정자침입에는 영향을 미치지 않았다. 한편 체외수정시 hyaluronidase 를 0, 0.01, 0.1 및 1.0mg/$m\ell$ 농도로 첨가된 배양액내에서 난구세포가 부착된 난자내의 정자침입률은 각각 61, 56, 66 및 39%로 이들 세포를 제거한 난자에서의 침입률 34, 35, 30 및 27%에 비해 높았다. 그러나 다정자침입률은 hyaluronidase의 농도에 관계없이 난구세포를 제거한 난자에서 낮았으며, hyaluronidase의 농도가 높아지면서 다정자침입률이 낮아지는 경향을 나타냈다. 또한 hyaluronidase를 첨가한 배양액내에서 수정후 16 및 24시간에서의 정자침입률은 난구세포를 제거한 경우 (25 및 31%) 보다 난구세포가 부착된 난자 (48 및 62%)에서 높았으며 (P＜0.05), 다정자침입률은 난구세포 제거 시 유의적으로 낮게 나타났다 (P＜0.05). 한편, 난자로부터 채취한 난구세포를 여러 농도로 첨가한 후 난구세포 제거난자를 이용하여 체외수정한 결과 hyal uronidase 첨가보다는 무첨가시 정자의 침입률과 다정자 침입률이 낮게 나타났다. 본 연구의 결과로부터 난구세포는 정자의 침입에 효과적으로 작용하였으며, hyaluronidase의 첨가와 난구세포수의 조절이 정자의 침입과 다정자침입에 영향을 미치는 것으로 추측되었다.