• Clinical and Experimental Reproductive Medicine
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• 제45권4호
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• pp.189-194
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• 2018

Objective: The aim of this study was to compare the rate of maturation, fertilization, and embryo development of in vitro-matured human oocytes derived from pregnant and non-pregnant women. Methods: Immature oocytes were obtained by needle aspiration from 49 pregnant women (group 1) who underwent a cesarean section at term and 77 non-pregnant women (group 2) who underwent a gynecological operation during the same period (8 months). Healthy immature oocytes (530 in group 1 and 539 in group 2) were cultured and assessed for maturation 36 hours later. Mature oocytes were inseminated by intracytoplasmic sperm injection and cultured up to 144 hours. Results: The percentage of degenerated oocytes was significantly higher (12.1% vs. 6.3%; p<0.001) in group 1 than in group 2. There was no significant difference in the maturation rate (66.8% vs. 68.1%; p=0.698), fertilization rate (66.7% vs. 67.6%; p=0.857), or the rate of formation of good-quality blastocysts (46.2% vs. 47.2%; p=0.898) in oocytes obtained from pregnant and non-pregnant women. Conclusion: The developmental competence of immature oocytes did not differ between pregnant and non-pregnant women.

• Reproductive and Developmental Biology
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• 제35권4호
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• pp.469-473
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• 2011

These study was carried out to investigate the effects of the supplementation with sodium nitroprusside (SN) and nitric oxide (NO) of canine oocytes on IVM rates. Oocytes were incubated in TCM-199 supplement with at 0.03~0.10 mM SN and 0.3~1.0 mM NO for 48 hrs. Oocytes were transferred to 50 ul drops of maturation medium covered mineral oil and cultured in a $CO_2$ incubator (5% $CO_2$, 95% air, $38^{\circ}C$). The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03, 0.05, 0.07, 0.10 mM SN were $25.9{\pm}3.5%$, $36.4{\pm}3.2%$, $33.3{\pm}3.5%$, $28.8{\m}3.2%$, respectively. The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03~0.07 mM SN were significantly increased compare to the control ($26.0{\pm}2.2%$). The in vitro maturation rates of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.3, 0.5, 0.7, 1.0 mM NO were $28.0{\pm}4.2%$, $36.5{\pm}3.6%$, $30.0{\pm}3.8%$, $19.2{\pm}3.5%$, respectively. The in vitro maturation rate of oocytes in TCM-199 medium supplemented with 0.3 and 0.5 mM NO were significantly increased compare to the control ($26.0{\pm}2.2%$). The in vitro maturation rates of oocytes cultured for 12~48 hrs in TCM-199 medium supplement with 0.05 mM SN were $26.0{\pm}3.2%$, $28.0{\pm}3.4%$, $38.0{\pm}3.2%$, respectively. The in vitro maturation rate of oocytes cultured for 12~48 hrs in TCM-199 medium supplement with 0.5 mM NO were $22.0{\pm}3.0%$, $30.0{\pm}3.8%$, $36.0{\pm}4.2%$, respectively. These result was significantly increased compare to the control.

• 한국수정란이식학회지
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• 제29권3호
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• pp.201-206
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• 2014

The objectives of the study were to determine an effective culture dish, culture duration and protein supplementation in medium for in vitro maturation (IVM) of oocytes of native zebu cows in Bangladesh. The ovaries of cows were collected from local slaughterhouse followed by aspiration of follicular fluid. The cumulus-oocyte-complexes (COCs) with more than 3 compact cumulus cell layers were cultured in tissue culture medium (TCM) 199 for maturation. The maturation of oocytes was determined by observing polar body under microscope. To determine an effective culture dish, 130 COCs derived from 48 ovaries in a well of 4-well dish and 102 COCs derived from 36 ovaries in drops covered with mineral oil within 35 mm petri dish were cultured for 24 hours. The rate of maturation of oocytes did not vary between 4-well dish ($51.3{\pm}15.0%$) and drops in petri dish ($52.4{\pm}11.6%$). To determine the effective culture duration, 185 COCs derived from 62 ovaries were cultured in drops for 18, 21, 24 and 27 hours. The rate of maturation of occytes ranged from $51.9{\pm}9.4%$ (18 hours) to $59.0{\pm}17.1%$ (27 hours) and the difference in maturation rate among different culture durations was not significant (P>0.05). To determine an effective protein supplementation, 63 oocytes from 19 ovaries were cultured separately in TCM 199 supplemented with either fetal bovine serum (FBS) or bovine serum albumin (BSA). The rate of maturation was significantly (P<0.01) higher in medium supplemented with FBS ($55.63{\pm}16.19%$) than that of BSA ($14.82{\pm}9.36%$). In conclusion, COCs of native zebu cows can be cultured for IVM either in 4-well culture dish or droplets in petri dish for 18 to 27 hours in medium supplemented with FBS.

• 한국가축번식학회지
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• 제19권3호
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• pp.181-189
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• 1995

In this study, methods on fabrication of microtool and setting of micromanipulator were examined and relationship between first polar body extrusion rate and maturation time of follicular oocyte, enulceation rae and repetition of trial, and enucleation rate and maturation period were investigated. The results are as follows: 1. Suitable outside diameter of micropipette tube was 1mm. Holding pipette with less than diameter of oocyte was fitred for manipulation, and zona dissection needle was easily operated when its sharp-point had diameter of about 8 ${\mu}{\textrm}{m}$ and length of 300${\mu}{\textrm}{m}$. The injection pipette with 20~35${\mu}{\textrm}{m}$ outside diameter was adequate for injection of blastomere into perivitelline space. 2. Separation of blastomere was effective when zona pellucida had cut with zonadissection needle and the embryo was pipetted gently with the pipette that had narrower diameter than that of embryo until separation of blastomeres had completed. 3. The extrusion rate of first polar body was 78% during 20~24% hours incubation for maturation. 4. According to repetitions of micromanipulation, the enucleation rate was increased to 85% and the time required for enucleation of a oocyte was shortened to 3 min. 5. The extrusion rate of first polar body and enucleation rate were 82 and 76% respectively, in the group of the oocytes cultured for 22 hours. However in the group cultured for 24 hours, the extrusion rate of first polar body and enucleation rate were 53 and 100% respectively.

• 한국수정란이식학회지
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• 제7권1호
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• pp.41-47
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• 1992

1. In vitro system, LR and FSR accelerated and facilitated meiotic progression, and LH selectively improved cytoplasmic maturation which is required to promote the formation of a male pronucleus. 2. Caffeine (2mM) in the fetilization medium was required not only for inducing zona penetrating ability of boar also for developing to the male pronucleus of the penetrat- ing spermatozoa in vitro. 3. The germinal vesicle (GV)stage was observed for the first 17.6 hr;germinal vesicle break-down (GVBD)stage between 17.6~26.4 hr ;metaphase I (M-I)from 26.4 - 30. 9hr;anaphase I(A-I)ranged from 30. 9~33.4hr;telophase I(T-I) at 33.4~34.4hr; and metaphase II(M-II) at 34.4-48hr. 4. The addition of 10%(v /v) pig follicular fluid (pFF) to maturation media significantly increased the rate of nuclear maturation of pig oocytes (p<0.01), whereas the rate of nuclear maturation of pig oocytes among three different media did not differ. 5. The presence of a primary culture of POEC promotes in vitro development of early cleavage stage pig embryos.

• 한국발생생물학회지:발생과생식
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• 제10권2호
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• pp.115-125
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• 2006

본 실험은 생쥐 난자의 성숙과 생존에 미치는 selenium의 영향을 알아보고자 수행하였다. 난자의 성숙은 현미경을 통해 관찰하였으며, 핵막 붕괴(germinal vesicle breakdown, GVBD)와 극체 형성(polar body formation, PB)은 체외 배양 시작 후 각각 2.5, 13시간에 확인하였다. 난자의 생존은 72 시간동안 체외 배양하면서 형태학적 차이로 정상 난자와 비정상 난자를 판별하였다. 또한 각 단계별로 수집된 난자의 glutathione(GSH) 함량은 spectrophotometer를 사용하여 glutathione assay로 측정하였다. 결과는 다음과 같다; 저농도의 selenium($0.005\;{\mu}g/mL{\sim}0.5\;{\mu}g/mL$)은 핵막 붕괴율과 극체 형성률을 증가시켰지만, 고농도의 selenium($5\;{\mu}g/mL$)은 감소시켰다. 저농도의 selenium은 극체 형성 시기 난자의 생존율을 증가시켰지만, 고농도의 selenium은 대조군과 별 차이가 없었다. 저농도의 selenium은 난자내 GSH 함량을 높게 유지시켰지만, 고농도의 selenium은 GSH 함량을 감소시켰다. 또한 극체 형성 시기 난자내 GSH 함량은 핵막 붕괴 시기 난자에 비해 높았다. 본 실험으로 볼 때, 저농도의 selenium은 대사과정에서 발생하는 oxidative stress에 의한 손상을 감소시킴으로써 난자의 질적 향상을 도우며, 성숙률을 증가시키는 것으로 사료된다. 또한 저농도의 selenium은 난자내 GSH 함량을 증가시켜 극체 형성 난자의 생존율을 증가시키는 것으로 사료된다.

• 한국발생생물학회지:발생과생식
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• 제20권2호
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• pp.127-133
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• 2016

Purine metabolism is known factor for nuclear maturation of oocytes through both follicle cells and oocyte itself. However, it is largely unknown the roles of purine metabolism in the oocyte competence for fertilization and early development. In this study, the effects of adenosine in oocyte competence for development were examined using adenosine and its synthetic inhibitors. Adenosine treatment from GV intact stage for 18 hr (fGV) caused of decrease the fertilization rate but of increase the cleavage rate compared from the other stage treatment groups. Hadacidin did not effect on fertilization rate but increased cleavage rate without stage specificity. Adenosine did not block the effects of hadacidin with the exception of fGV group. Inhibition of purine synthetic pathways the fertilization rate was decreased in the fGV and fGVB groups but increased in the fMII group. Exogenous adenosine caused of decrease fertilization rate in the fGVB group but increase in the fMII group. Cleavage rate was dramatically increased in the adenosine treatment with synthetic inhibitors. It means that the metabolism of purine has stage specific effects on fertilization and cleavage. Exogenous adenosine had only can improve oocyte developmental competence when it treated at GV intact stage. On the other hand, endogenous synthesis in all maturation stage cause of increase the cleavage rate without effects on fertilization. These data suggest that adenosine at GV stage as a paracrine fashion and inhibitions of endogenous adenosine in all stage improve oocyte developmental competence.

• 한국발생생물학회지:발생과생식
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• 제21권2호
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• pp.205-213
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• 2017

The aim of this study was to determine the effect of additional alpha-linolenic acid (ALA) supplementation during in vitro maturation (IVM) and culture (IVC) on nucleic maturation and embryo development of pigs. Cumulus-oocyte complexes (COCs) were incubated in IVM medium containing different concentration of ALA (25, 50 and $100{\mu}M$) for 44 h. After in vitro maturation, nuclear maturation of oocytes were evaluated by aceto-orcein stain. Mature oocytes with $50{\mu}M$ ALA were fertilized and cultured in IVC medium with ALA (25, 50 and $100{\mu}M$) during early-embryogenesis (48 hours after fertilization). Then, embryos were cultured with $25{\mu}M$ ALA during early embryogenesis and/or late embryogenesis (120 hours after early-embryogenesis). In results, oocyte maturation were significantly increased by $50{\mu}M$ ALA treatment groups compared with control groups (p<0.05). Treatment of $25{\mu}M$ ALA during early-embryogenesis enhanced cleavage rate of embryo compared with other groups (p<0.05), whereas formation and total cell number of blastocyst had no significant difference. Similarly, cleavage rate of embryos were increased by $25{\mu}M$ ALA supplement during early- or late-embryogenesis than ALA treatment both stage of embryogenesis (p<0.05), but did not influence to blastocyst formation. Interestingly, total cell number of blastocyst were enhanced in ALA treatment group during early-embryogenesis. These findings indicated that ALA supplement enhance the nuclear maturation of oocyte and embryo development, however, excessive ALA could negatively influence. Therefore, we suggest that ALA is used for improvement of in vitro production of mammalian embryo and further study regarding with functional mechanism of ALA is needed.

• 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.

• 한국가축번식학회지
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• 제21권1호
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• pp.19-23
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• 1997

This present study was carried out to examine the effect of maturation media and liquid boar semen on in vitro maturation and feritilization of pig oocytes. The results obtained were as follows : When the oocytes were cultured for 36∼42 hours in mTCM-199, Waymouth MB 725/1 and mTLP-PVA medium, the maturation rates were 90%, 92% and 88%, respectively. The sperm penetration rates of pig oocyte matured in vitro were 87%(mTCM-199), 90%(Waymouth MB 725/1) and 86%(mTLP-PVA), respectively. The rates of nuclear maturation and fertilization of pig oocytes among three different media did not differ. However, the rate of male pronucleus formation of pig oocytes was significantly higher in pig oocytes matured in Waymouth MB 725/1(91%) than oocytes matured in mTCM-199(66%) and mTLP-PVA(62%) medium (P<0.05). When the collected sperm-rich fraction without diluent was used fro in vitro fertilization in mTCM-199 fertilization medium, the fertilization rate was 87.9%. However, when the liquid boar semen diluted with B tschwiler diluent was used at day 3 and 5 after dilution, the fertilization rate was 40.8% and 0.0%, respectively.