• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.647-659
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• 1997

To overcome the difficulties of collecting and culture of primary cell from genital tract on embryonic development, the present study was carried out to investigate the critical effect of cell lines, such as BRL and Vero cell and its conditioned medium on the development of early Korean native cattle embryos in vitro. Oocytes collected from slaughterhouse ovaries were matured in TCM199 containing FSH, estradiol-$17{\beta}$ and FBS with granulosa cell monolayer for 24 hours and then fertilized in vitro using frozen-thawed, heparin-treated spermatozoa in TALP for 30 hours. And then early embryos (1-2cell) were cultured in TCM199 containing 10% FBS with BOEC, Granulosa, BRL, Vera cell monolayers and conditioned medium for 2~3 days. Development to morulae and blastocysts were recorded, also examined the number of blastomeres presented a valuable parameter for the evaluation of embryonic development. The early cleavage rates of in vitro fertilized embryos co-cultured, there was no differences between primary cell and cell lines(p<0.05). The rate of development to the later stage, coculture of BRL cell was significantly higher than that of the primary cell(p<0.05). The rates of development to morula and blastocyst were significantly higher in vero cell than BRL, Granulosa, Oviduct epithelial cell conditioned medium. In the result of effect of serum on development of early bovine embryos, the use of media containing serum were significantly higher than the use of not containing one on development of early and later stage of embryos. The result of number of blastomeres in blastocysts, there is no differences between primary cell and cell lines. The blastocysts from coculture were higher than from conditioned medium in blastomere cells. In summary, these experments have proved that the culture system in TCM199 with BRL, Vero cell monolayers is effective on in vitro development of early bovine embryos, In addition, it is effective to development of bovine embryos that containing serum in conditioned medium, or in co-culture rather than in conditioned medium alone. The use of cell lines opponent to primary cells is effective in bovine embryo culture.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.3
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• pp.407-417
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• 1999

Objective: Mammalian follicle cells are the most important somatic cells which help oocytes grow, mature and ovulate and thus are believed to provide oocytes with various functional and structural components. In the present study we have examined whether cumulus or granulosa cells might playa role in establishing the plasma membrane structure of mouse oocytes during meiotic maturation. Design: In particular the differential resistances of mouse oocytes against chymotrypsin treatment were examined following culture with or without cumulus or granulosa cells, or in these cell-conditioned media. Results: When mouse denuded oocytes, freed from their surrounding cumulus cells, were cultured in vitro for $17{\sim}18hr$ and then treated with 1% chymotrypsin, half of the oocytes underwent degeneration within 37.5 min ($t_{50}=37.5{\pm}7.5min$) after the treatment. In contrast cumulus-enclosed oocytes showed $t_{50}=207.0$. Similarly, when oocytes were co-cultured with cumulus cells which were not associated with the oocytes but present in the same medium, the $t_{50}$ of co-cultured oocytes was $177.5{\pm}13.1min$. Furthermore, when oocytes were cultured in the cumulus cell-conditioned medium, $t_{50}$ of these oocytes was $190.0{\pm}10.8min$ whereas $t_{50}$ of the oocytes cultured in M16 alone was $25.5{\pm}2.9min$. Granulosa cell-conditioned medium also increased the resistance of oocytes against chymotrypsin treatment such that $t_{50}$ of oocytes cultured in granulosa cell-conditioned medium was $152.5{\pm}19.0min$ while that of oocytes cultured in M16 alone was $70.0{\pm}8.2min$. To see what molecular components of follicle cell-conditioned medium are involved in the above effects, the granulosa cell-conditioned medium was separated into two fractions by using Microcon-10 membrane filter having a 10 kDa cut-off range. When denuded oocytes were cultured in medium containing the retentate, $t_{50}$ of the oocytes was $70.0{\pm}10.5min$. In contrast, $t_{50}$ of the denuded oocytes cultured in medium containing the filtrate was $142.0{\pm}26.5min$. $T_{50}$ of denuded oocytes cultured in medium containing both retentate and filtrate was $188.0{\pm}13.6min$. However, $t_{50}$ of denuded oocytes cultured in M16 alone was $70.0{\pm}11.0min$ and that of oocytes cultured in whole granulosa cell-conditioned medium was $156.0{\pm}27.9min$. When surface membrane proteins of oocytes were electrophoretically analyzed, no difference was found between the protein profiles of oocytes cultured in M16 alone and of those cultured in the filtrate. Conclusions: Based upon these results, it is concluded that mouse follicle cells secrete a factor(s) which enhance the resistance of mouse oocytes against a proteolytic enzyme treatment. The factor appears to be a small molecules having a molecular weight less than 10 kDa.

• Journal of Embryo Transfer
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• v.20 no.1
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• pp.71-77
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• 2005

To investigate the influence of relaxin and insulin on the ovarian steroid secretion of porcine granulosa cells, we used porcine granulosa cells partially luteinized in a primary culture and examined the production of progesterone and $17{\beta}-estradiol$. Porcine granulosa cells were cultured in the presence of serum for 48 h after attachment and subsequently in the absence of serum fur 24 h. To confirm the dose dependency of relaxin or insulin, various concentrations (10, 100, 1000 ng/ml) of relaxin or insulin were added in the medium for the last 24 h, respectively. To investigate the combinational effect of relaxin and insulin, 100 ng/ml relaxin and/or 100 ng/ml insulin were added in the medium for the last 24 h in the presence or absence of luteinizing hormone (100 ng/ml). The medium was collected and used for radioimmunoassay to measure the production of progesterone and $17{\beta}-estradiol$. Relaxin or insulin increased the production of progesterone by dose dependency, respectively while they had no effect of the production of $17{\beta}-estradiol$. Relaxin (100 ng/ml) and/or insulin (100 ng/ml) significantly increased the production of progesterone in the presence of luteinizing hormone while they had no effect of the production of $17{\beta}-estradiol$. In conclusion, relaxin and/or insulin increased the progesterone secretion of porcine granulosa-lutein cells in vitro while had no effect on the production of $17{\beta}-estradiol$ and had no synergism on the effects. The effects of relaxin and/or insulin on the production of progesterone were augmented by the presence of luteinizing hormone.

• 대한생식의학회:학술대회논문집
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• 2007.11a
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• pp.120.1-120.1
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• 2007

• 대한세포병리학회:학술대회논문집
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• 1993.06a
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• pp.22-22
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• 1993

• Proceedings of the Zoological Society Korea Conference
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• 1996.04a
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• pp.43.2-43
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• 1996

No Abstract, See Full Text

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.1
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• pp.20-33
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• 2017

Objective: Superstimulatory treatment of one-month-old lambs can achieve synchronous development of numerous growing follicles. However, these growing follicles cannot complete maturation and ovulation. Oocyte maturation and competence are acquired during follicular development, in which granulosa cells play an essential role. Methods: In this study, we applied RNA sequencing to analyze and compare gene expression between prepubertal and adult superstimulated follicle granulosa cells in sheep. Results: There were more than 300 genes that significantly differed in expression. Among these differently expressed genes, many extracellular matrix genes (EGF containing Fibulin Like Extracellular Matrix Protein 1, pentraxin 3, adrenomedullin, and osteopontin) were significantly down-regulated in the superstimulated follicles. Ingenuity pathway and gene ontology analyses revealed that processes of axonal guidance, cell proliferation and DNA replication were expressed at higher levels in the prepubertal follicles. Epidermal growth factor, T-Box protein 2 and beta-estradiol upstream regulator were predicted to be active in prepubertal follicles. By comparison, tumor protein P53 and let-7 were most active in adult follicles. Conclusion: These results may contribute to a better understanding of the mechanisms governing the development of granulosa cells in the growing follicle in prepubertal sheep.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.19
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• pp.8447-8450
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• 2014

Background: To evaluate factors for predicting the granulosa cell tumor of the ovary (GCTO) pre-operatively. Materials and Methods: This retrospective designed study was conducted on 34 women with GCTO as the study group and 76 women with benign ovarian cysts as the control group. Data were recorded from the hospital database and included age, body mass index (BMI), parity, serum estradiol ($E_2$) levels, diameter of the mass, ultrasonographic features, serum CA125 level, risk of malignancy index (RMI), duration of menopause, postoperative histopathology result, and the neutrophil/lymphocyte ratio (NLR). Results: The demographic parameters showed no statistically significant difference between the groups. Preoperative diameter of the mass, CA125, duration of menopause, and neutrophil/lymphocyte ratio were significantly different between the groups. ROC curve analysis demonstrated that diameter of the mass, serum estradiol and Ca125 levels, RMI and NLR may be discriminative factors in predicting GCTO preoperatively. Conclusions: In conclusion, we think that a careful preoperative workshop including diameter of the mass, serum estradiol ($E_2$) and Ca125 levels, RMI and NLR may predict GCTO and may prevent incomplete approaches.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.3
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• pp.340-345
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• 2002

In this experiment, oocytes were collected from goat ovaries available in slaughterhouse by follicle puncture method. Morphologically culturable type of oocytes which having compact, multilayered cumulus granulosa cell complex and evenly granulated cytoplasm, was separated under a stereozoom microscope. Oocytes were washed thoroughly in maturation medium containing TCM-199, $1{\mu}g/ml$ estradiol-$17{\beta}$, 0.5 ${\mu}g/ml$ FSH, $100{\mu}g/ml$ LH, 3 mg/ml BSA and 10% estrus goat serum. Washed oocytes were cultured into maturation medium on granulosa cell monolayer. Culture plate was then kept into $CO_2$ incubator at $38{\pm}1^{\circ}C$, maximum humidity and 5% $CO_2$ for 18 h. After maturation the oocytes were washed thoroughly with maturation medium containing polyvinyl alcohol (PVA) without serum and BSA and further cultured for 12 h for secretory proteins of oocytes. PVA medium was collected, pooled and concentrated by 5000 cut off centrisart. Secretory proteins were separated on 12.5% SDS-PAGE. A total number of 3.41 oocytes per ovary were obtained and 2.17 culturable oocytes per ovary were cultured into maturation medium. After 18 h of maturation, 4,567 oocytes (1.82 oocytes per ovary) were further cultured into serum and BSA free PVA medium for its secretory proteins. Four secretory proteins of oocytes with approximately molecular weight of 45, 55, 65 and 95 kDa were obtained on SDS-PAGE in silver staining and three proteins with approximately molecular weight of 45, 55 and 65 kDa in Coomassie brilliant blue staining. In conclusion, four secretory proteins with approximately molecular weight of 45, 55, 65 and 95 kDa was obtained from in vitro cultured oocytes of goats.

• Korean Journal of Veterinary Research
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• v.27 no.1
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• pp.141-151
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• 1987

A total of 1200 Korean native cow and 240 dairy cow genitalia were collected during the slaughtering process in Seoul and Kwang Ju abattoir and were examined from July 1985 to March 1986. Ovarian follicles were classified as cystic if the diameter was greater than 2.5cm or if follicles were multiple. In order to investigate the ovarian cysts, anatomical and histological examinations were performed. In addition progesterone and estrogen level in different types of cystic follicular fluid and serum were measured by radioimmunoassay. The results were summerized as follows: 1. The incidences of ovarian cysts were 2.0% in Korean native cow and 7.9% in dairy cow. 2. In distribution of cysts in the left, right and both ovaries, the most encountered ovary was right one. The frequency was 45.8% in right ovaries, 33.4% in left ovaries and 20.8% in both ovaries in Korean native cow. On the contrary the frequency was 42.1% in right ovaries, 31.8% in both ovaries and 26.3% in left ovaries in dairy cow. 3. Six speciemens (25.0%) of Korean native cow and six specimens (31.6%) of dairy cow were associated with corpora lutes in both ovaries. 4. The luteinization of theca layer was most significant in the group 2Aa (71.4%) and 2Ba (38.5%) which associated with no granulosa cell and corpora lutea in the same cystic ovaries. 5. Correlation of progesterone concentration between cystic fluid and serum was found only in the group 2Aa and 2Ab (r=0.86). Progesterone and estrogen concentrations in cystic fluid were closely related to the degree of degeneration of granulosa cell layer. The cystic follicles that consist of thickened theca and degenerated granulosa cell layers contained a large amount of progesterone, and small amount of estrogen. In conclusion, various types of ovarian cysts with various levels of progesterone and estrogen were observed in Korean native cow.