• Clinical and Experimental Reproductive Medicine
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• 제18권1호
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• pp.81-87
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• 1991

There studies were carried for evaluation of the efficiency of freezing of hamster oocytes for use in a human sperm penetration assay. The hamster oocytes fully equilibrated in various cryoprotectant agents and inseminated with human sperm. After insemination with hamster oocytes, there was no difference in penetrated rates. Cumulus free oocytes equilibrated in 1.5M various cryoprotective agents and slowely cooled to temperature $-30^{\circ}C$ before rapid cooling and storage in nitrozen tank. After rapid thawing, survival rates of frozen oocytes according to cryo-protective agents were examined and the human sperm penetration assay with zona free hamster oocytes was conducted. 1. Survival rates of oocytes after cryoprotectants exposure have no significant difference (range 88-91%) and peneration rate was 51.1%. 2. Recovery and survival rate of frozen-thawed oocytes were 85.1 and 66.8%. There was no significant difference on cryoprotective agents. 3. Penetration rates of the frozen-thawed and intact oocytes were 69.0 and 77.0%, respectively. 4. Hamster oocytes cryopreservation provides a convenient way of supplying and trans-porting hamster oocytes for the assessment of the fertilizing potential of human spermatozoa.

• 한국임상수의학회지
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• 제27권1호
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• pp.46-49
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• 2010

In this study, we investigated the number of follicles, oocyte recovery rate and oocyte competence after in vitro maturation according to the size of follicle. And equine oocyte competence after in vitro maturation was investigated in terms of the diameter of follicle with criteria of maturation: nuclear stage after Hoechst staining. The average number of follicles per ovary with middle size (11-20 mm, 2.68) was higher than those of small (5-10 mm, 0.74) and large size follicle (> 21 mm, 1.63), therefore medium follicle (53.1%) had higher proportion than other size of follicles. The average numbers of follicle per ovary was 5.05. The rate of oocyte recovery in small (54.5%) and middle follicle (50%) was higher than that in large follicle (40.9%). After culture for 48 h in Medium 199, 50%, 45.5%, and 44.4% of oocytes from the follicles with diameters of 5-10, 11-20, > 21 mm, respectively reached the metaphase II stage. This is the first report showing number of follicle, oocyte recovery rate according to follicular size, and in vitro oocyte maturation in Jeju mare in Korea. To fulfill in vitro equine embryo production, further studies such as the seasonal effect, in vitro fertilization etc is need.

• 한국임상수의학회지
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• 제32권6호
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• pp.536-539
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• 2015

한살령의 잡종 암캐에서 체내 성숙된 난자를 회수하기 위한 실험이 진행되었다. 혈청 프로게스테론 농도를 이용하여 배란 후 약 72시간 뒤에 개복술을 시행하였고, 비정상적으로 확장된 왼쪽 자궁각이 발견되었다. 양쪽 난소는 각각 여덟 개의 황체를 가지고 있었고, 배지를 난관에 관류시켜서 총 16개의 난자를 회수하였다. 모든 난자는 극체가 돌출되어 있었고, 25 um 미만의 난황주위 공간과 제 2중기의 핵을 가지고 있었다. 본 증례는 자궁수종을 가진 암캐에서 체내 성숙된 난자를 확인한 최초의 연구이며, 생식기 관련 질병을 가진 개과 동물에서 회수된 난자가 보조 생식 기술에 사용될 수 있음을 의미한다.

• 한국가축번식학회지
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• 제24권1호
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• pp.83-88
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• 2000

본 연구는 한우에 있어서 초음파기기를 이용하여 반복적으로 생체내 난자를 회수시 난자회수에 미치는 영향과 시술후의 난소 유착과 같은 부작용을 조사하기 위하여 실시한 결과는 다음과 같다. 1. 난포란의 채란빈도에 따른 채란수에서는 주 2회 시술시 난포수는 8.7$\pm$4.2개로 주 1회 시술의 10.2$\pm$6.1개보다 다소 적었으나 유의적인 차이는 인정되지 않았으며, 채란수도 각각 4.1$\pm$3.4개 및 4.3$\pm$2.9개로 유의적인 차이가 인정이 되지 않았다(P＜0.05). 2. 반복적으로 9회 난포란을 채란한 경우 40개의 난소중 8개 (20%)가 유착이 발생하였으며 1~3 채란시 3개 (37.5%), 4~6 채란시 4개 (50%) 7~9회 채란시 1개(12.5%)가 유착이 발생하였다. 3. 반복적인 난포란 채란후 난소의 유착 여부에 따른 공란우의 발정주기를 조사한 결과 난소 유착을 보인 공란우의 8두중 7두(87.5%) 25일 이상의 장발정주기를 보였으며, 유착이 일어나지 않은 12두 중 정상 발정주기는 6두(50.0%), 단 발정주기는 1두(8.3%), 장발정주기는 5두(41.7%)였다.

• Clinical and Experimental Reproductive Medicine
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• 제50권1호
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• pp.1-11
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• 2023

In reproduction, mitochondria produce bioenergy, help to synthesize biomolecules, and support the ovaries, oogenesis, and preimplantation embryos, thereby facilitating healthy live births. However, the regulatory mechanism of mitochondria in oocytes and embryos during oogenesis and embryo development has not been clearly elucidated. The functional activity of mitochondria is crucial for determining the quality of oocytes and embryos; therefore, the underlying mechanism must be better understood. In this review, we summarize the specific role of mitochondria in reproduction in oocytes and embryos. We also briefly discuss the recovery of mitochondrial function in gametes and zygotes. First, we introduce the general characteristics of mitochondria in cells, including their roles in adenosine triphosphate and reactive oxygen species production, calcium homeostasis, and programmed cell death. Second, we present the unique characteristics of mitochondria in female reproduction, covering the bottleneck theory, mitochondrial shape, and mitochondrial metabolic pathways during oogenesis and preimplantation embryo development. Mitochondrial dysfunction is associated with ovarian aging, a diminished ovarian reserve, a poor ovarian response, and several reproduction problems in gametes and zygotes, such as aneuploidy and genetic disorders. Finally, we briefly describe which factors are involved in mitochondrial dysfunction and how mitochondrial function can be recovered in reproduction. We hope to provide a new viewpoint regarding factors that can overcome mitochondrial dysfunction in the field of reproductive medicine.

• 한국동물위생학회지
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• 제44권4호
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• pp.227-237
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• 2021

Artificial insemination of Korean native cattle (KNC) is the predominant method for breed improvement. However, industrialization of embryo production and transfer is necessary to utilize the genetic potential of KNC. The aim of this study was to examine associations between KNC donor cows and ovum pick-up (OPU) conditions, in-vivo oocyte recovery, and embryo development. Oocyte recovery and blastocyst development rates were higher at 50 and 60 mmHg OPU vacuum pressure than at 40 mmHg, which was, however, not significant. Regarding follicle growth, injection of 500 ㎍ GnRH 36 hours before OPU significantly increased the number of OPU oocytes from an average of 4.6 to 7.6 (P<0.05); no significant difference in embryo development rates was observed. Significant differences were observed in the numbers of OPU oocytes, embryo development rates, and transplantable blastocysts per individual among nine KNC donors (P<0.05). Furthermore, although there was no difference in OPU oocyte recovery intervals in approximately 2~8 weeks, the number of recovered oocytes significantly decreased at the 12-week interval (P<0.05); there was no difference in embryo development rates. The number of oocytes and embryonic development rates only tended to decrease until the seventh OPU session, but decreased significantly until the eighth session (P<0.05). The average pregnancy rate after transfer of OPU-derived in-vitro embryos into recipient cows was 41.8%. To improve the efficiency of OPU egg recovery and in-vitro embryo production, considering KNC donor characteristics, vacuum pressure of 60 mmHg, GnRH pretreatment to induce follicle growth, and effective OPU egg recovery up to seven times at intervals of 2~4 weeks appears to be most suitable. This study may facilitate the industrialization of KNC embryo production and transfer using high-quality cows.

• 한국수정란이식학회지
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• 제10권1호
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• pp.45-51
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• 1995

In order to determine the optimum condition and timing for in vitro maturation of oocytes to metaphase of meiosis II (M II), the immatured follicular oocytes were recovered by puncturing the large(1.0~1.5 mm in diameter) and small(<1.0 mm in diameter) follicles in the ovaries of rabbits treated intramuscularly with a single dose of 100 TU PMSG 68 hours previously. The follicular oocytes were classified into three grades by the attachment of cumulus cells. The Grade I and II follicular oocytes from large follicles were cultured in BO-DM medium with 10% FCS, 35 $\mu$g /nl of FSH, 10 $\mu$g /ml of LH and 1 $\mu$g /ml of estradiol-17$\beta$ at 39t in a 5% $CO_2$ incubator for 11 to 23 hours. In 3 hours interval during the culture period, the oocytes were harvested and their cumulus cells were removed with hyaluronidase. The denuded oocytes were stained with Hoechst 33342 dye and their meiotic status and extrusion of the first polar body (PB) were examined under a fluorescence microscope. Also the fragmentation of the first PB and the distance between the first PB and nucleus were examined. The results obtained were as follows: 1. The mean recovery rate of follicular oocytes from the large and small follicles was 59. 9 and 31.3%, respectively. The mean number of oocytes recovered per rabbit and the Grade I percentage were 14.6 and 94.4% in large follicles, but 2.1 and 61.1% in small follicles, respectively. All the parameters examined were different significantly (p<0.05) between both the folliclular size. 2. Most of the follicular oocytes(86.8%) were matured in vitro to M II phase in 14 hours in Grade I oocytes, but the significantly(p<0.05) less oocytes(45.5%) were matured in Grade II oocytes. 3. The first PB was extruded in most of the oocytes(94.7%) in 14 hours of culture with the fragmentation rate of 29.6%, but the fragmentation rate of the first PB increased significantly (p<0.05) as the culture period for maturation was longer to 20 hours(63.5%). 4. The distance between the first PB and nucleus was increased linearly (p<0.05) as the maturation time passed from 14(7.1$\mu$rn) to 23 hours(58.4$\mu$m). 5. From the above results it was concluded that the optimum time for in vitro maturation culture might be 14 hours in the follicular oocytes from rabbit primed with PMSG for 68 hours, expecially when these follicular oocytes were used for recipient cytoplasms in embryo cloning.

• 한국수정란이식학회지
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• 제33권4호
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• pp.229-235
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• 2018

Polo-like kinase 1 (Plk1) has been known to be a critical element in cell division including centrosome maturation, cytokinesis and spindle formation in somatic, cancer, and mammalian embryonic cells. In particular, Plk1 is highly expressed in cancer cells. Plk1 inhibitors, such as BI2536, have been widely used to prevent cell division as an anticancer drug. In this study, the fertilized murine oocytes were treated with BI2536 for 30 min after recovery from the oviduct to investigate the effect of down-regulation of Plk1 in the in vivo-fertilized murine embryos. Then, the localization and expression of Plk1 was observed by immunofluorescence staining. The sperm which had entered into the oocyte cytoplasm did not form male pronuclei in BI2536-treated oocytes. The BI2536-treated oocytes showed significantly lower expression of Plk1 than non-treated control group. In addition, alpha-tubulin and Plk1 gathered around sperm head in non-treated oocytes, while BI2536-treated oocytes did not show this phenomenon. The present study demonstrates that the Plk1 inhibitor, BI2536, hinders fertilization by inhibiting the formation of murine male pronucleus.

• 한국수정란이식학회지
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• 제30권4호
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• pp.327-333
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• 2015

The objectives of the present study were to select an effective basic medium including its hormone and protein supplementation for IVM of oocytes of indigenous zebu cows. The ovaries of cows were collected from slaughter house and the follicular fluid was aspirated from 2 to 8 mm diameter follicles. The COCs with more than 3 cumulus cell layers and homogenous cytoplasm were selected for maturation. The oocytes were matured in media for 24 hrs at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The maturation of oocytes was evaluated by examining the presence of first polar body under microscope. An efficient basic medium was determined after culturing COCs in either TCM 199 or SOF medium in Experiment 1. An efficient hormone supplementation was determined after culturing COCs in either FSH or gonadotrophin supplemented TCM 199 in Experiment 2. An efficient protein supplementation was determined after culturing COCs in either FBS or Oestrous cow serum (OCS) supplemented TCM 199 in Experiment 3. The oocyte recovery rate per ovary was 3.35. The overall rate of IVM was 74.6%. The maturation rate was $75.5{\pm}3.9$ and $62.2{\pm}20.2%$ in TCM and SOF medium, respectively (P>0.05). The maturation rate of oocytes was significantly higher ($76.6{\pm}13.2%$) in FSH supplemented medium than gonadotrphin supplemented counterpart ($69.7{\pm}10.8%$) (P<0.05). The maturation rates of oocytes were $81.7{\pm}12.9$ and $85.7{\pm}12.7%$ in medium supplemented with FBS and OCS, respectively (P>0.05). In conclusions, both TCM 199 and SOF supplemented with either FBS or OCS, and FSH may be used as medium for IVM of indigenous zebu oocytes in Bangladesh.

• 한국수정란이식학회지
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• 제11권3호
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• pp.249-258
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• 1996

To improve the efficiency of in vitro production of embryos with follicular oocytes in Korean Native cows, the recovery rates, in vitro maturation, fertilization and development, and the time required for collecting and processing oocytes by aspiration with or without slicing were evaluated comparatively. The ovaries were obtained from a local abattoir and placed in physiological saline at 25~28$^{\circ}C$ and brought to the laboratory within 3 hrs. The oocytes were collected by aspiration of follicles(2~6mm) with or without slicing ovaries after aspiration, and classified into Grade I, Grade II, Denuded, Expanded oocytes by the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules. Also the time required for each step of collecting and processing oocytes were measured. The cumulus cells were removed in some Grade I oocytes to measure their size and nuclear configuration before and after in vitro maturation. The Grade I oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35$\mu$g /ml FSH, 10$\mu$g /ml LH, 1 $\mu$g /ml at 39$^{\circ}C$ under 5% C02 in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24hrs. and then the zygotes were cocultured in vitro (IVC) with bovine oviductal epithelial cells for 10 days. The results obtained were as follows: The number of oocytes recovered per ovary was averaged 6.6 by aspiration and 11.2 by slicing post aspiration, which summed to 17.8. The number of Grade I oocytes recovered per ovary was averaged 3.1 by aspiration and 3.6 by slicing, which summed to 6.7. The percentage of Grade I to total oocytes recovered was significantly(P<0.05) higher as 48.0 % in aspiration than 31.6% in slicing post aspiration. The time requlred for recovering a Grade I oocyte by aspiration and slicing was 1.1 and 2.5 min, respectively. The mean diameter of Grade I oocytes by aspiration and slicing was similar as 148.7 and 151.5$\mu$m, respectively. The percentage of Metaphase II stage oocytes after IVM for 24 hours was significantly (P