• Korean Journal of Animal Reproduction
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• v.17 no.3
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• pp.249-255
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• 1993

Canine follicular oocytes were used to establish a reliable system for maturation and fertilization in vitro. Ovaries were obtained from either slaughter house or hormone-primed bitches of mixed breeds. The oocytes were recovered by mincing the ovaries in M2＋BSA. Good quality of oocyte-cumulus complexes (OCCs) were selected and cultured in TCM 199 containing 15% fetal calf serum(FCS) for 24~56 h in an atmosphere of 5% CO2 at 39$^{\circ}C$. Maturation rate of follicular oocytes was >87% showing metaphase I. Unlike other domestic animals the cumulus expansion did not occur fully in canine OCCs although minimum expansion was found between the cumulus cells and corona radiata cells, the clear nuclear morphology was presented for the first time by rapid staining. The IVM system used in this study may be useful to obtain fully maturated metaphase I oocyte in dog.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.73-83
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• 1994

The objective of this study was to develop an effective in vitro production system capable of obtaining more porcine embryos from immature oocytes. These experiments were thus conducted to examine the effect of oocytes type and maturation time on the in vitro maturation(IVM) and fertilization(IVF) of oocytes and the in vitro development (IVD)of IVF embryos. 1. The degree of oocyte maturation based on cumulus expansion index(GEI) did not differ for A- and B-typed oocytes but the index of oocyte type C was lower(P<0.05) than that of other oocyte types. 2. When the oocytes of type A and B were matured for 36, 42 and 48hrs, the GEl was not different between the 36- and 42-h maturation but the GEl after 48hrs was greatly lower(P<0.05) than that of other maturation times. 3. The highest cleavage rate(48.6%) of IVF oocytes was obtained from A typed oocytes and 42-h maturation but the developmental potential based on cleavage index was the highest when B-typed oocytes were matured for 42hrs.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.31-41
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• 1994

핵 이식 기술을 이용한 cloning 송아지 생산이 처음 보고(Prather et al., 1987) 된 후, 소 수정란 Cloning에 대한 많은 연구가 분자 생물학 등 여러 분야에서 꾸준히 계속되고 있다. 이 기술은 빈우의 번식 능력을 향상시켜 유전적 개량량을 증대할 수 있는 번식과 육종을 위한 도구로써 많은 잠재력을 지니고 있다. 최근 핵 이식 기술을 이용하여 유전적으로 우수한 빈우로부터 수천개의 수정란을 생산하여, 이들 수정란에게 생산된 송아지가 번식 축군으로 공시되어 있으므로, 그 결과가 주목되나 아직까지는 비용이 많이 들고 송아지 생산 효율이 저조하므로, 가까운 장래에 일반 양축가에 이용될 가능성이 낮다. 그러나 이 기술의 실용화를 위하여 선결되어야 할 많은 문제점들 중, 지난 몇 년 동안 많은 연구기관에서 수행된 활발한 연구의 결실로써, 난포란 제핵, cell fusion 과 oocyte activation의 방법등 주요 장애 요인들이 점차 극복되면서 실용화를 위한 접근이 예견되어지며, 구미의 일부 개량 기관에서는 이를 상업화 하기 위한 여건을 다지고 있다. 그러므로 이 Review에서는 fllicular dynamics, 난포란의 성숙, cell cycle, 난포란 제핵, cell fusion과 oocyte activation, 이식후 핵의 remodeling과 reprogramming에 대한 현재까지의 보고된 자료를 기초로 그 기본 원리를 재고하는데 초점을 맞추었다.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.113-119
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• 2004

The purpose of the present study was to examine whether collection time affects results of oocyte recovery from superovulated goats. Fiftyty-one mature Korean native goats, maintained in a pen under natural day length and fed hay ad libitum, were pretreated with progestagen impregnated CIDR for 10 days and then the goats were divided into two groups. One group of the goats received a single intramuscular injection of 1,000 IU PMSG on Day 8 of CIDR insertion. The other group of the goats received twice daily intramuscular injections of a total of 70 mg FSH for 3 days from Day 8 of CIDR. All the gonadotropin treated goats were injected with 10 mg $PGF_{2\alpha}$ on Day 8 and 400 IU hCG in the afternoon on Day 10. For oocyte recovery, donor goats were fasted 24 h before operation. Anesthesia was induced by intravenous injection of 2% xylazine(0.2 mg/kg body weight) and ketamin(11 mg/kg body weight). In vivo oocytes were recovered by follicle aspiration or oviduct flushing at 29 to 34, 35 to 40 and 41 to 50 h after hCG injection through mid-ventral incision. There was no significant difference in the mean number of CL and oocytes recovered. Oocyte collection at 29 to 40 h after hCG increased(P<0.05) the recovery rate of ovulated oocytes in oviducts compared to 41 to 50 h. The same results were also observed in the recovery of follicular oocytes. Oocyte grade was not affected by collection time. When oocytes were collected from follicular oocytes at 41 to 50 h after hCG, the recovery rate of Grade II oocytes was the lowest(P<0.05). From these results, it is suggested that oocyte recovery at 35 to 40 h after hCG will be successful for further use.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.3
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• pp.187-197
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• 2009

Objective: Phosphorylation and dephosphorylation of proteins are important in regulating cellular signaling pathways. Bead-based multiplex phosphorylation assay was conducted to detect the phosphorylation of seven proteins to maximize the information obtained from a single lysate of stage-specific mouse oocytes at a time. Methods: Cumulus-oocyte complexes (COCs) were cultured for 2 h, 8 h, and 16 h, respectively to address phosphorylation status of seven target proteins during oocyte maturation process. We analyzed the changes in phosphorylation at germinal vesicle (GV, 0 h), germinal vesicle breakdown (GVBD, 2 h), metaphase I (MI, 8 h), and metaphase II (MII, 16 h in vitro or in vivo) mouse oocytes by using Bio-Plex phosphoprotein assay system. We chose seven target proteins, namely, three mitogen-activated protein kinases (MAPKs), ERK1/2, JNK, and p38 MAPK, and other 4 well known signaling molecules, Akt, GSK-$3{\alpha}/{\beta}$, $I{\kappa}B{\alpha}$, and STAT3 to measure their phosphorylation status. Western blot analysis and kinase inhibitor treatment for ERK1/2, JNK, and Akt during in vitro maturation of oocytes were conducted for the confirmation. Results: Phosphorylation of ERK1/2, JNK, p38 MAPK and STAT3 was increased over 3 folds up to 20 folds, while phosphorylation of the other three signal molecules, Akt, GSK-$3{\alpha}/{\beta}$, and $I{\kapa}B{\alpha}$ was less than 3 folds. All of these results except for Akt were statistically significant (p<0.05). Conclusion: This is the first report on the new and valuable method measuring many phosphoproteins simultaneously in one minute sample such as oocyte lysates. All of the three MAPKs, ERK1/2, JNK, and p38 MAPK are involved in the process of mouse oocyte maturation. In addition, STAT3 might be important regulator of oocyte maturation, while Akt phosphorylation at Serine 473 may not be involved in the regulation of oocyte maturation.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.35-41
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• 2014

Laparoscopic ovum pick-up (LOPU) is a convenient method for collecting oocytes in small ruminants. LOPU has the advantage of being a less invasive means of oocyte collection, thereby allowing for a repeated usage of the oocyte donor animals. A total of 25 Korean black goats were used in the winter season (December to February) and LOPU was applied to the goats which had been treated for superovulation more than two times during the last twelve months. Estrus was synchronized with an intravaginal insert containing 0.3 g progesterone for 10 to 12 days. Ovaries were hyperstimulated with eCG 1,000 IU oneshot, FSH with eCG (50 mg / 1,000 IU; 70 mg / 500 IU; 70 mg / 1,000 IU) oneshot or FSH multiple-shot with eCG oneshot ($20mg{\times}6/300IU$) given intramuscularly 72 h prior to LOPU. For these groups, the number of follicles (mean ${\pm}$ SEM) observed which developed to larger than 2 mm in diameter were $1.6{\pm}2.5$, $4.3{\pm}3.1$, $5.5{\pm}4.2$, $6.6{\pm}2.1$ and $8.8{\pm}7.8$, respectively. Oocytes were aspirated by using OPU needles and a vacuum pump. The overall oocyte retrieval rates were 41.4%. Oocytes were matured in TCM-199 supplemented with 10% (w/v) bovine serum albumin + $10{\mu}g/ml$ FSH + $1{\mu}g/ml$ $17{\beta}$-estradiol for 27 h at $39^{\circ}C$ in 5% $CO_2$ in air. Oocytes were parthenogenetically activated by ionomycin combined with 6-diethylaminopurine (6-DMAP). Total oocyte maturation and cleavage rate were 67.3% and 78.8%, respectively. In summary, LOPU is a useful oocyte collection method in Korean black goats that can provide immature oocytes for transgenesis or nuclear transfer.

• Development and Reproduction
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• v.10 no.2
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• pp.115-125
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• 2006

The present experiment was performed to confirm the effects of selenium on maturation and viability of mouse oocyte. Maturation of oocytes was observed by microscope, Germinal vesicle breakdown(GVBD) and polar body formation(PB) were confirmed at 2.5, 13 hours after in vitro culture. Viability of oocytes was observed by microscope. Normal and abnormal oocytes were distinguished by morphological change in vitro culture for 72 hours. Glutathione(GSH) content of collected oocytes from individual stage also was measured by glutathione assay using spectrophotometer. The results obtained were as follows; The low concentration of selenium($0.005\;{\mu}g/mL{\sim}0.5\;{\mu}g/mL$) increased the maturation rate of germinal vesicle(GV) oocytes to GVBD and PB oocytes. The high concentration of selenium($5\;{\mu}g/mL$) decreased the maturation rate. The low concentration of selenium increased the viability rate of PB oocytes. The high concentration of selenium did not affect the viability rate. The low concentration of selenium increased the GSH content in PB oocytes. The high concentration of selenium decreased GSH content. GSH content in PB oocyte was much higher than that in GVBD oocyte. The results indicate that the low concentration of selenium increases the maturation rate by helping quality elevation of oocyte and minimizing damages of oxidative stress generated from metabolism process. The low concentration of selenium also increases the viability rate by increasing GSH content.

• Korean Journal of Ichthyology
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• v.19 no.2
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• pp.112-119
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• 2007

A histological study on development and transformation of the oocyte' follicle cell for Korean four sillurid fishes, Liobagrus obesus, L. mediadiposalis, Pseudobagrus koreanus, and P. brevicorpus was performed by light and electron microscopes. The follicular layer surrounding the oocyte consisted of an outer theca cell and an inner follicle cell (granulosa cell). The follicle cells of the oocyte were flatten cells at early oocyte but during vitellogenesis they were transformed it to a single layer of cuboidal cell, then to a single columnar cell layer, and finally to a layer covered with a substance secreted by themselves. Although the development and transformation of the follicle cells was similar to four species, the secreted materials, called an adhesive membrane, were divided into two types in its appearance and nature. Firstly, a jelly coat-like type was found in L. obesus and L. mediadiposalis, which they are presumed to be polysaccharides and mucoproteins in its nature and secondly, a granular type in P. koreanus and P. brevicopus, being mucoprotein. A zona radiata with about $0.6{\sim}3.1{\mu}m$ thin was present below the adhesive material secreted by the transformed-follicle cell's activity. The zona radiata was composed of two layers, a thin externa and a thick interna.

• Journal of Animal Science and Technology
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• v.48 no.3
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• pp.353-360
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• 2006

This study was performed to investigate the effects of artificial stimulation on the increase of the oocyte activation, to evaluate the expression of cyclin B1 protein levels in enucleated mouse oocytes, and to investigate correlation between the oocyte activation and the cyclin B1 protein levels. The oocyte activation was induced by 7% ethanol (EtOH) or 10μg/ml Ca-ionophore with or without 10μg/ml cycloheximide (CH). The activation rate was significantly higher in both single (p<0.05) and combined (p<0.01) stimulated groups compared to control group. The cyclin B1 protein level was significantly reduced in both stimulated groups (p<0.05), except for EtOH+CH treatment group. The expression of cyclin B1 protein showed a higher negative correlation with activation rate in EtOH+CH (r=0.61, p<0.05) and Ca+CH (r=0.86, p<0.01) stimulation groups, but not in a both single stimulation groups. Taken together, it can be suggested that single (EtOH and Ca- ionophore) and combined (EtOH+CH and Ca+CH) stimulation increases the oocyte activation, especially combined stimulation, because it induces the degradation of cyclin B1 protein after artificial stimulation treatments in mouse oocytes.

• Development and Reproduction
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• v.6 no.1
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• pp.17-23
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• 2002

In oocyte retrieval, a vein anesthetic drug is commonly used for induction and maintenance of general anesthesia. Propofol and Thiopental sodium are frequently used for ultrasound-guided transvaginal oocyte retrieval. The present study aimed to assess the effects of Propofol and Thiopental on in vitro fertilization(IVF). Immature porcine oocytes were exposed to various concentrations ot Propofo1 and Thiopental sodium. The rates of oocyte maturation, fertilization and development were observed. The parthenogenetic effects of the anesthetics were also evaluated. The rate of oocyte maturation after exposure to high concentrations of the anesthetics for long time was significantly higher than that of the control. But the rate of fertilization after long-time exposure to the high concentration of the anesthetic drugs was significantly lower than that of the control. The results support that Propofo1 serves like other anesthetics described, as a parthenogenetic activator. Oocytes exposed to Thiopental sodium showed decreased rates of maturation and fertilization. These results suggest that usage of optimum concentration of anesthetic drug is important in increasing the rates of oocyte maturation, fertilization and development in IVF.