• 한국수정란이식학회지
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• 제24권3호
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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.

• Reproductive and Developmental Biology
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• 제34권4호
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• pp.275-281
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• 2010

The aim of this study was to investigate what protein(s) of porcine epididymal fluid (pEF) are able to enhance the nuclear maturation of porcine germinal vesicle (GV) oocytes in vitro. Proteins of pEF were fractionated by affinity, ion exchange, and gel filtration chromatography. Porcine cumulus-oocytes complexes (COC) from follicles were cultured in tissue culture medium (TCM 199) containing various fractions obtained by chromatography. Porcine COCs were also cultured in TCM 199 containing various meiosis inhibitors and pEF. After 24 or 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. When porcine COCs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than 80% of oocytes were unable to resume meiosis. However, porcine COCs supplemented with pEF were able to overcome the inhibitory effect of dbcAMP and Fo. Maturation rate of oocytes was significantly (p<0.05) increased in the media supplemented with cationic protein(s) during in vitro maturation than in those with anionic protein(s) (44.1% vs 20.0%). When oocytes were cultured in the TCM 199 with fractions obtained by gel filtration, the maturation rate of oocytes was significantly (p<0.05) higher in fraction 11 containing 18 kDa than other fractions. The present study suggests that 1) dbcAMP and Fo prevent the spontaneous maturation of oocyte after isolation from follicles, and that pEF contain a substance(s) that improves meiosis resumption in vitro of porcine COCs, 2) cationic 18 kDa protein(s) are responsible for promotion of Mil stage.

• Reproductive and Developmental Biology
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• 제34권3호
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• pp.169-173
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• 2010

Correlations between cumulus cells and germinal vesicle (GV) chromatin configuration were examined in porcine oocytes. Cumulus-oocyte complexes (COCs) were collected from 2~6 mm follicles and divided into three categories according to cumulus cell morphology. "A" group was compacted COCs with more than three cumulus cell layers. "B" group was COCs with less cumulus cell layers than "A" group. "C" group was COCs with one or less layer of cumulus cells. Cumulus cells were removed 0.1% hyaluronidase, and denuded oocytes were stained with Hoechst 33342. GV chromatin configuration was classified into GV-Con and GV-Dis. GV-Con meant that a nucleus was surrounded by condensed chromatin in a ring. GV-Dis meant that filamentous chromatin clumps were distributed in nucleus. The proportion (80.2%) of GV-Con in "A" group was significantly higher than "B" (62.0%) or "C" (44.9%). The proportion (55.1%) of GV-Dis in "C" group was significantly higher than "A" (19.8%) or "B" (38.0%). The meiotic competence of COCs was examined after 44 h culture. The proportion (90.0%) of oocytes reaching to metaphase II (M-II) in "A" group was significantly higher than "B" (76.5%) or "C" (45.5%). In conclusion, oocytes with good quality cumulus cell layers are synchronized early GV stage, and early GV stage is important for meiotic competence in pigs.

• 한국수정란이식학회지
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• 제11권3호
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• pp.241-248
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• 1996

This study was conducted to improve the production efficiency of in vitro produced (IVP) embryos in Korean Native cows. The optimal conditions and procedures for in vitro maturation(IVM), in vitro fertilization(IVF) and in vitro culture(IVC) of bovine follicular oocytes and IVP embryos were evaluated. Immature follicular oocytes were collected fiom the follicles of bovine ovaries obtained from abattoirs. The oocytes of Grade I and II for IVM were cocultured with monolayered bovine oviductal epithelial cells(BOEG) or granulosa cells in TCM-199 solution supplemented with follicle stimulating hormone, lutenizing hormone, estradiol-17$\beta$ and heat inactivated fetal calf serum at 39$^{\circ}C$ under 5% $CO_2$ in air for 14 to 24 hours. Most of the oocytes(93%) matured to metaphase II in 24 hours. The cocultured IVM oocytes were fertilized in vitro at significantly(P<0.05) higher rate with BOEC(83.8%) and with granulosa cells(84.6%) than the non-cocultured IVM oocytes(73.6%). The IVM-IVF embryos developed to morula and blastocyst at significantly(P<0.05) higher rate in coculture with BOEC(41.2%) than with granulosa cells(23.1%) or conditioned medium(23.4%).

• Journal of Plant Biology
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• 제30권4호
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• pp.225-247
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• 1987

Meiosis and postmeiotic mitosis in Boletus rubinellus were examined ultrastructurally. Meriosis occurred at the apex of the basidium. A sausage-shaped spindle pole body(SPB) was observed along with the presence of synaptonemal complexes during pachytene and a diglobular SPB was present on late pachytene or diplotene nuclei. During metaphase I, the monoglobular SPB at the spindle pole was surrounded bya membrane and the nuclear enveloope was discontinuous. At anaphase I, the chromosomes became better defined and formed a central spindle. The nucleolus was extruded from the nucleus. During anaphase I, the SPB was excluded from the chromosomal region by a membrane and both poles were fully separated to opposite sides of the basidial wall. In meiosis II, the two nuclei divided synchronously and the spindles were parallel. The spindles were smaller than in meiosis I, while the SPB was approximately the same size as that of the similar stage in meiosis I. During anaphasetelophase II, the SPB was surrounded by a cap of endoplasmic reticulum (ER) that delimited it from the spindle. The postmeiotic interphase nuclei migrated to the mid-region of the basidium before migration to the spores. The SPB at this stage was diglobular. A postmeiotic mitosis occurred within the basidiospore, and the plane of the spindle was obique to the long axis of the spore. The spindle and SPB were smaller than at meiosis I and there were fewer nonchromosomal microtubules. At anaphase, the nucleolus was present inside the nuclear envelope but lateral to the spindle.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2006년도 The 6th Intermational Symposium on Developmental Biotechnology
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• pp.108-108
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• 2006

• 한국발생생물학회지:발생과생식
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• 제23권1호
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• pp.1-9
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• 2019

The ability of oocytes to undergo normal fertilization and embryo development is acquired during oocyte maturation which is transition from the germinal vesicle stage (GV), germinal vesicle breakdown (GVBD) to metaphase of meiosis II (MII). Part of this process includes redistribution of inositol 1, 4, 5-triphosphate receptor (IP3R), a predominant $Ca^{2+}$ channel on the endoplasmic reticulum membrane. Type 1 IP3R (IP3R1) is expressed in mouse oocytes dominantly. At GV stage, IP3R1 are arranged as a network throughout the cytoplasm with minute accumulation around the nucleus. At MII stage, IP3R1 diffuses to the entire cytoplasm in a more reticular manner, and obvious clusters of IP3R1 are observed at the cortex of the egg. This structural reorganization provides acquisition of $[Ca^{2+}]_i$ oscillatory activity during fertilization. In this review, general properties of IP3R1 in somatic cells and mammalian oocyte are introduced.

• Journal of Veterinary Science
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• 제21권3호
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• pp.48.1-48.18
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• 2020

Background: Mature oocytes at the metaphase II status (MII-stage oocytes) played an important role in assisted reproductive technology in non-human primates. Objectives: In order to improve the proportion of MII-stage oocytes retrieval, three different superovulation protocols were performed on 24 female cynomolgus monkeys. Methods: All the monkeys received once-daily injection of follicle-stimulating hormone (25 international unit [IU]) on day 3 of the menstruation, 3-day intervals, twice daily for 8-12 days until the time of human chorionic gonadotropin (1,500 IU) injection, on the 14-17th day of menstruation collecting oocytes. The difference between protocol I and protocol II was that 0.1 mg the gonadotropin-releasing hormone agonist was injected on day 1 of the menstruation, while the difference between personalized superovulation protocol and protocol II was that oocytes could be collected on the 14-17th day of menstrual cycle according to the length of each monkey. Results: The total number of oocytes harvested using the personalized superovulation protocol was much higher than that using protocol I (p < 0.05), and the proportion of MII-stage oocytes was significantly greater than that from either superovulation protocol I or II (p < 0.001 and p < 0.01 respectively), while the proportion of immature oocytes at the germinal vesicle was less than that from superovulation protocol I (p < 0.05). Conclusions: The personalized superovulation protocol could increase the rate of MII-stage oocytes acquired, and successfully develop into embryos after intracytoplasmic sperm injection, and eventually generated fetus.

• 한국가축번식학회지
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• 제22권4호
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• pp.319-329
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• 1998

본 연구는 돼지 난포란의 동결시기를 결정하고자 미성숙, 성숙중, 생숙후의 3 가지 성숙단계의 난포란을 동해방지제에 노출하거나 동결, 융해하여 체외성숙율과 체외발생율을 대조구와 비교 조사하였다. 그 결과를 요약하면 다음과 같다. 1. 각각 0, 24, 44 시간 동안 성숙배양한 난포란을 동해방지제에 노출한 후 추가배양했을 때 난포란의 성숙율은 각각 45.2, 45.0, 50.3%으로 무처리구 55.0% 와 비슷하였다. 2. 각각 0, 24, 44 시간 동안 성숙배양 한 난포란을 동해방지제에 노출한 후 체외수정하고 배양했을 때 난자의 발달율은 각각 18.6, 19.7, 47.6% 로 0 시간과 24 시간에서는 무처리구 50.9% 보다 유의하게 낮은 배발달율을 나타내었으나 44 시간에서는 차이가 나타나지 않았다. 3. 각각 0, 24, 44 시간 동안 성숙배양한 난포란을 일단계 초자화동결법 (one-step vitrification)으로 추가배양했을 때 난포란의 동결, 융해 후 성숙율은 각각 4.3, 7.1, 46.7%로 무처리구 62.4% 보다 유의하게 낮은 성숙율을 나타내었다. 4. 각각 0, 24, 44 시간 동안 성숙배양한 난포란을 일단계 초자화동결법 (one-step vitrification)으로 동결, 융해 후 체외수정하고 배양했을 때 난자의 발달율은 각각 2.5, 2.4, 10.2%로 무처리구 49.6% 보다 유의하게 낮은 성숙율을 나타내었다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.28-28
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• 2001

The objective of this study was to the effect on subsequent development of EGF present in defined medium during bovine 1)oocyte maturation or 2)embryo culture. The presence of EGF during IVM, irrespective of concentration(1, 10, 100ng/$m\ell$), stimulated cumulus expansion and significantly increased the proportion of oocytes attaining metaphaseII, the rate of cleavage, and develop to blastocyst. 1. In the group of EGF-added medium(1, 10, 100ng/$m\ell$), nuclear maturation rate for in vitro maturation was 91% to 92% but was not significantly higher than control group(87%). 2. For in vitro maturation, in the group of EGF-added medium(1, 10, 100ng/$m\ell$)the rate of cumulus cell expansion degree 2 ranged from 81% to 87%, which was significantly higher than the control group(medium with EGF not added). The rate of in vitro fertilization, developing to 2-to 4- cell stage, was 76% to 80%, which was also significantly higher(p<0.05)than control group(62%). 3. For in vitro maturation, in the group of EGF added in medium(1, 10, 100ng/$m\ell$)the development rate to blastocyst was 24.3% to 27%, which was significantly higher than control group(13.7%). The total cleavage rate in the group of EGF-added medium was 77% to 82%, which was higher than control group. 4. The development rate to blastocyst for 6 days of cultivation and the hatching blastocyst were 30.6% and 59.1%, respectively, in the group of 100ng/$m\ell$ of EGF, which were significantly higher(p<0.05)than control group(14.0% and 24%, respectively), The numbers of cells in blastocyst were 140.2 and 148, respectively, in 10ng/$m\ell$ and 100ng/$m\ell$ of EGF-added medium, which were higher than 108.5 in control group. 5. The development rate of in vitro fertilized embryos to blastocyst in 10ng/$m\ell$ of EGF-added medium co-cultured with somatic cell was 28%, which was significantly higher(p<0.05)than control group(11.8%). The numbers of cells in blastocyst were 141.6 for EGF-added medium and 145 for EGF+co-culture group, which were higher than control(101.6)and medium co-cultured with somatic cells(110.6). These results showed that in vitro maturation and fertilization, EGF was found a significant effect of increase of development rate to blastocyst and cell number.