• Korean Journal of Animal Reproduction
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• v.17 no.3
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• pp.257-262
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• 1993

Various tissue and follicular components were analyzed for the determination of lactate dehydrogenase(LDH) isozyme patterns by electrophoretic technique with chromogen reaction in the pig. Optimum conditions for the tissue homogenate and the storage were finally established. Small quantities of follicular components were analysed for typing of LDH isozymes by microelectrophoresis. Microelectrophoretic analysis showed that only LDH-1 was visible in the oocytes, all isozymes in cumulus masses, and LDH-1, 2 and 3 in follicular fluid. The results provide critical information on the LDH activity of various tissues and follicular components. Furthermore, t he developed methods should be useful the analysis of LDH in the small quantity of samples, especially in the oocyte, and easily applicable to the oocyte and early embryos of other domestic species.

• Molecular & Cellular Toxicology
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• v.4 no.3
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• pp.253-259
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• 2008

Human follicular fluid (HFF) is the in vivo microenvironment for oocyte maturation and includes a variety of proteins that could be involved in oocyte development and fertilization. We therefore used a proteomic approach to identify new HFF proteins. HFF from mature human follicles was obtained from five women following oocyte collection for in vitro fertilization (IVF). Ethanol-precipitated HFF run on two-dimensional gel electrophoresis (2DE) produced approximately 250 Coomassie brilliant blue-stained spots, 64 of which were identified using matrix-assisted laser desorption/ionization-mass spectrometry (MALDIMS). In this study, several proteins including complement factor H, inter-${\alpha}$ (globulin) inhibitor H4, inter-${\alpha}$-trypsin inhibitor heavy chain H4 precursor, human zinc-${\alpha}$-2-glycoprotein chain B, PRO2619, PRO02044, and complex-forming glycoprotein HC were new proteins that have not been previously reported in HFF using proteomic methods. Additionally, we identified alloalbumin venezia for the first time from trichloroacetic acid (TCA)-precipitated HFF. These HFF proteins could serve as new biomarkers for important human reproductive processes.

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.177-184
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• 1999

This study was conducted to find out the effect of follicle size and oocyte type on in vitro maturation of poricine follicular oocytes. TCM-HEPEAS medium was used to basic medium, and the oocyte matured in vitro was stained with the Rapid staining method. The results obtained were summarized as follows; 1. The number of follicles an ovary was 20.5. The number of A-and B-typed oocytes an ovary was 2.34. The proportion of A-and b-types oocytes was 40% of the recovery oocytes. 2. Cumulus expanison indexes(CEI) by the follicle size were 1.62∼2.34(<2mm), 1.27∼2.28(2∼5mm) and 1.46∼2.75(>5mm). It was no differ to maturation rate by the follicle size. 3. The degree of oocyte maturation based on oocyte type did not differ for B-and C-typed oocyted but the index of oocyte type A was higher than that of b-and C-typed oocytes. 4. When follicluar oocytes were cultured for 42 hours, the proportion of the Met-II(second metaphase) stage were 22.5% (degree 1), 35.4%(degree 2) and 65.5% (degree 3).

• Korean Journal of Animal Reproduction
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• v.15 no.3
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• pp.221-224
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• 1991

This study was undertaken to evaluate the effect of rabbit peritioneal fluid(rPF) on in vitro maturtion of porcine follicular oocytes. From does 20h after hCG injection, rPF was aspirated aseptically at laparatomy, and then centrifuged, filtrated, and preincubated immediately for 12h. Porcine follicular oocytes isolated from ovaries of slaughtered animals were incubated in TCM-HEPES＋10% FCS, TCM-HEPES＋rPF(v/v, 50/50), or rPE only and examined the nuclear maturation after aceto-orcein or hochest staining. After identifying the optimal incubation time, this experiment was repeated for 5 times. Under the TCM-HEPES containing hormones and serum codition, the time range of porcine follicular oocyte maturation was 38 to 44 hours and the optimal time of maturation of follicular oocyte in vitro was 42 hour cultivation, respectively. The maturatin rates(89.4% and 92.7%) of porcine follicular oocytes cultured in the media with 50% rPF or only rPF were signifciantly higher thanthat (84.6%) of oocytes cultured with TCM-HEPES, respectively. These results suggest that the unknown components(s) of rPF promoted in vitro maturation of porcine follicular oocytes.

• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.267-273
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• 2004

Maturation of oocytes is maintained by complex procedures along with follicular genesis and is a critical step for embryonic development. Purine known as an oocyte maturation regulator is present in follicular fluid. In this study, the roles of guanosine as a strong inhibitor of GVBD and a modulator of cyclic GMP concentration in ooyctes were revealed. Denuded immature oocytes were treated with guanosine, and the maturation rates and cGMP concentration of oocytes were measured. GVBD was blocked in a concentration dependent manner by guanosine, but this effect was reversible. However, GVBD was lagged yet not significant by adenosine. Both guanosine and adenosine modified cGMP concentration in oocytes. The characteristic of the guanosine-treated oocyte was significantly higher cGMP compared with the adenosine-treated oocyes at initial time of the maturation. Based these results, guanosine may be a strong and reversible GVBD inhibitor. Although the precise mechanism of guanosine presently is unclear, the results suggest that guanosine may lead the accumulation of cGMP in oocyte cytoplasm, which in turn suppresses GVBD.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.179-187
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• 1998

In mammal, lots of follicles start simultaneously their growth but only a few oocytes are ovulated in every sexual cycles. Most of matured and grown oocytes are destined to degenerate by atresia. However, the molecular and physiological mechanisms are not elucidated yet. The present study was designed to establish an induction method of follicular atresia with ketamine or pentobarbitone and evaluate the effect of these anesthetics on oocyte maturation and granulosa cell apoptosis of the mouse ovarian follicle. The percentages of degenerated oocyte and apoptotic granulosa cell in ketamine treated groups were significantly higher than that in controls (58.9% vs 33.5%, p<0.01, degeneration; 44.9% vs 26.6%, p<0.01, apotosis). Futhermore, it was revealed that the concentrations of progesterone in both groups were markedly higher than that in control. In cunclusion, it is considered that ketamine induce an atresia as pentobarbitone, and may be useful for inducing follicular atresia.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.111-116
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• 1997

We determined the effects of follicular fluid fractions in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on number of cells in blastocysts following culture. Follicular fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. Follicular fluid was separated into different molecular weight fractions by untrafiltration through a membrane using a centrifuge at 500$\times$g, for 2h. For the maturation medium, follicular fluid fractions (30%, v/v), whole fluid (30%) or PVP(3mg/ml) were added to TCM 199(0.1$\mu\textrm{g}$/ml estradiol-17$\beta$, 100IU hCG). After maturation for 24h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 days after fertilization. There were no differences in maturation rates or fertilization rates among any maturation conditions. The rates of development to >2-cell stage of the oocytes were significantly decreased when fraction of follicular fluid below 10,000 MW were added into maturation medium, compared with control and fraction above 10,000 MW(26.0% vs 40.8% to 64.0%, respectveily. p<0.01). Likewise, the rates of development to blastocysts of fertilized oocytes were significantly decreased in maturation medium containing fraction of follicular fluid (<10,000 MW). The average cell number of blastocysts derived from oocytes that matured in the fraction(>10,000 MW) of follicular fluid was 154.7$\pm$13.7. These embryos contained more cells than those matured in whole follicular fluid, or the fraction(<10, 000 MW) of follicular fluid or control(107.0$\pm$8.4, 91.8$\pm$11.8 and 95.8$\pm$6.2, respectively). In conclusion, we found that fractions of follicular fluid contained factors stimulating or inhibiting oocyte cytoplasmic matruation. These suggest that a factor(s) inducing cytoplasmic maturation of oocytes may exist in >10,000 MW fraction of follicular fluid.

• 대한생식의학회:학술대회논문집
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• 1999.11a
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• pp.83.1-83.1
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• 1999

• Korean Journal of Animal Reproduction
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• v.19 no.2
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• pp.105-117
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• 1995

This study was carried out to investigate the histomorphological changes and the electrophoretic patterns of egg components, obtained from 100 of 1-year-old female catfish(Silurus asotus). Especially, the light microscopic and ultrastructural changes of ooplasm and follicular membranes of oocytes, were observed by light and transmission electron microscope. All data were collected from October in 1992 to May in 1993. The size of the nucleoli and number of the yolk granules increased as the oocyte grew. Yolk granules were deposited in the oocyte as fluid. Due to the presence of large early and late maturing oocytes, their ovaries were large, transparent, granular, and greenish in color. As the percentages of fish in LMO and RO stage increased from March to April, mean of GSI values(19.95%) increased. Follicle cells such as granulosa cell and thecal cell change a squamous into cuboid shape in LPO and EMO stage. Processes, microvilli, from the granulosa cells and from the oocyte grow and make contact with each other in the pore canals of the zona radiata during vitellogenesis, but are withdrawn as the zona radiate becomes more compact and devoid of pore canals during oocyte maturation. The electrophoretic pattern of major band in mature stage was much thicker(21k, 24k, 32k, 45k, 67∼110k, 170k dalton) than that in previtellogenic phase.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.145-150
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• 2009

This study was conducted to examine the effects of human follicular fluid and gonadotropin (FSH+HCG+rhEGF) on in vitro maturation, fertilization and development of human immature oocytes. Cumulus-oocyte complexes (COCs) were collected following for in vitro fertilization and embryo transfer (IVF-ET) cycles of the patients. At the time of oocytes collection, oocytes were classified into MII, MI and GV in accordance with their appearance (MII: Fully mature oocyte at metaphase II of meiosis; MI: Nearly mature oocytes at metaphase I of meiosis; GV: Immature oocytes at prophase I of meiosis). After controlled ovarian stimulation using gonadotropin(FSH) and human chorionic gonadotropin (HCG) in 70 ICSI cycles, 158 MI to MII matured oocytes were intracytoplasmic sperm injection (ICSI) ${\sim}4$ h after in vitro culture and 553 MII oocytes were ICSI after denudation. The aspirated MI and GV oocytes were cultured in culture medium containing 10% (v/v) serum protein substitute (SPS), 10% (v/v) human follicular fluid (hFF) and 10% (v/v) serum protein substitute (SPS)+1 IU/ml FSH+10 IU/ml HCG+10 ng/ml recombinant human epidermal growth factor (rhEGF). The maturation rate of immature oocytes was similar among the three group. When maturation medium was supplemented with 10% SPS, 10% hFF or gonadotropins, the fertilization rate of in vitro matured oocytes was higher in 10% SPS (80.0%), but there was no statistical significance (78.2%; hFF, 76.9%; gonadotropin, p>0.05). The development rate of human embryos developed to $6{\sim}8$ cells were not significant difference in the medium containing SPS, hFF and gonadotropins (65.6%, 65.9% and 66.7%). The results of these study suggest that human follicular fluid and gonadotropins supplemented in the culture medium was not effected on the in vitro maturation, fertilization and development of human immature oocytes.