• 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.

• Archives of Pharmacal Research
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• 제27권11호
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• pp.1168-1176
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• 2004

Exposure to stress is known to precipitate or exacerbate many reproductive dysfunctions such as dysmenorrhea and infertility. Abnormalities of the reproductive system, as shown by reduced ovulation, fertilization and early embryonic development, are frequently seen in dysmenorrhea and infertility. It has been generally accepted that Gamisoyosan (GSS) is a useful prescription for treating insomnia, dysmenorrhea and infertility induced by a stress. Also GSS has been used traditionally to improve systemic circulation and biological energy production. Based on these, this study investigates whether GSS improved ovarian dysfunction caused by stress in mice. Mice were subjected to stress by electric shock on the foot for 30 min daily for a week and treated with GSS at 500 / body weight per day for one week. Thereafter, changes body weight, adrenal weight, ovulation rate, in vitro and in vivo fertilization, embryonic development and estradiol concentrations were measured. GSS markedly increased the body weight of mice with stress, but not normal mice. The administration of GSS caused a reduction in adrenal weight in stressed mice. GSS also had significant positive effects on ovulation rate, estradiol production, in vivo and in vitro fertilization rates and embryonic development. These results indicate that GSS can improve the reproductive dysfunctions caused by stress, and these may production biological energy.

• 한국수정란이식학회지
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• 제14권1호
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• pp.23-29
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• 1999

The objectives of the study were to establish sperm separation method and duration of insemination for bovine IVF. Oocytes from slaughterhouse ovaries were matured and fertilized using general protocol. After 18 or 42 h of insemination, six to ten embryos were placed into a 30${mu}ell$ drop of each medium, and the embryos were examined 7~10d post in semination without medium renewal. First, we compared Percoll gradient will swim-up technique for sperm separation. There was no difference in cleavage rates between them, but the development rates over morula stage of oocytes fertilized with sperm separated by Percoll gradient was significantly higher than that sperm selected by swim-up technique (p<0.05). Second, we evaluated development of bovine embryos derived from the IVF procedure with different durations(18 vs 42 h) of fertilization. There was also no difference in cleavage rates, but the development to blastocyst stage of oocytes exposed in cleavage rates, but the development to blastocyst stage of oocytes exposed to sperm for 42 h was significantly higher than that exposed for 18 h (p<0.05). In conclusion, Percoll gradient can be used for sperm selecton, improving of embryonic development. Also, 42h of IVF may improve the development of bovine embryos.

• 한국가축번식학회지
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• 제21권3호
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• pp.247-253
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• 1997

본 연구는 돼지난포란의 체외수정시 정소상체 미부정자의 형태학적 정상정자 비율에 따른 수정율과 배발달율을 조사하고자 실시하였다. 그 결과는 다음과 같다. 1. 정소상체 미부정자의 형태학적 정상정자 비율을 10%, 10~30%, 50%로 구분하여 수정율과 배발달율을 조사하였던 바, 50%에서 각각 64%, 26%로 나타나 다른 두 군보다 현저히 높게 나타났다 ( 10%: 27%, 6%, 10~30%: 36%, 5%) (p<0.01). 2. 50% 이상의 정상정자를 가진 정소상체 미부정자를 100% (5$\times$105 cells/ml)로 조정하여 수정하였던 바, 정소상체 미부정자군의 수정율과 배발달율 (63%, 27%)은 동결 융해된 사출정자군 (56%, 35%)과 유사한 결과를 나타냈다. 3. 또한, 정소상체 미부정자군에서 난자와 정자의 비율 (1:6000, 1:6650, 1:7700, 1:10000)에 따라 수정률과 배발달율을 조사하였던 바, 난자당 정상정자 비율이 1:6000 (68%, 32%), 1:6650 (89%, 31%)일 때 가장 높은 수정율과 배발달율을 나타내었다. 따라서, 정소상체 미부정자의 형태학적 정상정자 비율이 50%일 때, 동결 융해된 사출정자와 유사한 배발달을 얻을 수 있으며, 이러한 결과로 미루어 볼 때, 정소상체 미부정자를 체외수정에 사용함에 있어서 정자의 형태학적 평가가 선행되어진다면 더욱더 효과적인 배발달을 유기할 수 있을 것으로 사료된다.

• 환경생물
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• 제30권2호
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• pp.128-135
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• 2012

본 연구는 말똥성게(Hemicentrotus pulcherrimus)의 생식세포 및 pluteus 유생을 이용하여 중금속인 납(lead, Pb)과 아연(zinc, Zn)의 독성을 조사였다. H. pulcherrimus 배우자 및 배아에 미치는 Pb과 Zn의 독성은 각각 31, 63, 125, 250, 500 ppb 및 16, 31, 63, 125, 250 ppb의 농도에서 조사하였다. 0.5 M KCl 용액을 이용하여 방란 및 방정을 유도하였고, 수정률 및 정상 배아발생률의 조사는 수정 후 각각 10분 및 64시간째 관찰하여 시행하였다. Pb 노출 시 수정률은 대조군과 비교하여 유의적인 변화가 없었다. 그러나 정상 배아발생률은 농도가 높을수록 농도의존적으로 유의적인 감소를 보였다. Zn을 노출시켰을 경우 수정률과 정상 배아발생률은 농도가 높을수록 농도의존적인 유의적 감소를 나타냈다. H. pulcherrimus의 정상 배아 발생에 대한 독성치는 각각 Pb (반수 영향농도 ($EC_{50}$) 45.13 ppb, 95% Cl 40.12~50.05 ppb), Zn (반수영향농도 ($EC_{50}$) 19.82 ppb, 95% Cl 18.26~21.31 ppb)로 나타났다. 또한 Pb과 Zn의 무영향농도(NOEC)는 각각 <31.25 ppb 및 <15.63 ppb로 나타났고, 최소영향농도(LOEC)는 31.25 및 15.63 ppb로 나타났다. 본 연구 결과, H. pulcherrimus의 초기 배아발생 과정은 Pb과 Zn 등의 중금속에 높은 민감성을 보인다. 따라서 H. pulcherrimus는 해양생태계 위해 평가를 위한 시험생물로서 사용이 가능하다고 사료된다.

• 한국수정란이식학회지
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• 제12권2호
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• pp.181-188
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• 1997

This study was carried out to determine the effect of bovine follicular fluid(bFF), hormones, and fetal bovine serum(FBS) supplemented in the medium on the in vitro fertilization and development of bovine embryos. The ovaries were obtained from a local abattoir and placed in physiological saline kept at 30~32˚C and brought to the laboratory within 3~4 hours. The oocytes and follicular fluid were collected by aspiration from visible follicles, and the oocytes of grades I on the basis of the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules were selected and used for maturation. The basal media used for oocyte maturation, fertilization and embryo development in vitro were Ham' F-10, TALP and TCM-199, respectively. The hormones supplemented in maturation medium were consisted of 35 pg /ml FSH, 10 pg /ml LH and 1 pg/mi estradiol-l7$\beta$. The bFF collected from 5~9 mm follicles was centrifuged, filtered and inactivated by heat-treatment at 56˚C for 30 min. FBS also was inactivated with the same method and kept at -20˚C until use. The embryos were co-cultured with the monolayer of bovine oviductal epithelial cells at 39˚C under 5% $CO_2$ in air for 9 days. The results obtained were summarized as follows: The fertilization rate of oocytes was found 87.4% from 10% FBS and hormones treatment for IVM, and 37.1% of these TVF embryos were developed to blastocyst stage in 10% FBS groups. Compared with this control system, the fertilization rate was decreased significantly(P<0.05) in the maturation without either FBS or hormones. These IVF embryos were developed to morula stage at the similar rate, but to blastocyst at significantly(P<0.05) lower rate in the embryo culture with or without FBS supplementation. The fertilization rate(82.9%) in hormones and 10% inactivated bFF was similar with 10% FBS and hormone groups(87.4%), but decreased significantly(P<0.05) in 20 or 30% bFF (61.0 or 66.0%), respectively. In vitro developmental competence to blastocyst stage in 10% FBS and 20% inactivated bFF(37.1% and 31.4%) was higher than in 10 or 30% inactivated bFF(20.0 or 19.2%) or 10, 20 and 30% fresh bFF(19.1, 21.0 and 17.5%) The results indicated that the in vitro fertillzation and development rate of the embryos should be improved in 10% FBS or 20% inactivated culture system and 20% inactivated bFF might be available economically for bovine oocyte maturation and embryo culture instead of fetal bovine serum.

• Clinical and Experimental Reproductive Medicine
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• 제42권3호
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• pp.77-85
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• 2015

One of the key factors of early development is the specification of competence between the oocyte and the sperm, which occurs during gametogenesis. However, the starting point, growth, and maturation for acquiring competence during spermatogenesis and oogenesis in mammals are very different. Spermatogenesis includes spermiogenesis, but such a metamorphosis is not observed during oogenesis. Glycosylation, a ubiquitous modification, is a preliminary requisite for distribution of the structural and functional components of spermatids for metamorphosis. In addition, glycosylation using epididymal or female genital secretory glycans is an important process for the sperm maturation, the acquisition of the potential for fertilization, and the acceleration of early embryo development. However, nonemzymatic unexpected covalent bonding of a carbohydrate and malglycosylation can result in falling fertility rates as shown in the diabetic male. So far, glycosylation during spermatogenesis and the dynamics of the plasma membrane in the process of capacitation and fertilization have been evaluated, and a powerful role of glycosylation in spermatogenesis and early development is also suggested by structural bioinformatics, functional genomics, and functional proteomics. Further understanding of glycosylation is needed to provide a better understanding of fertilization and embryo development and for the development of new diagnostic and therapeutic tools for infertility.

• Environmental Analysis Health and Toxicology
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• 제20권1호
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• pp.57-65
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• 2005

Effects of five polycyclic aromatic hydrocarbon (PAHs) constituents (naphthalene, fluorine, fluoranthene, benzo(a)pyrene, pyrene) on fertilization and early development of sea urchin egg, sperm and fertilized egg were investigated. The eggs, sperm and fertilized eggs were exposed to several concentrations of PAHs (1, 10, 100, 1000 and 10000㎍/L). The rate of fertilization and hatching decreased when the eggs and sperm were exposed to aqueous solution of PAHs. Also, Exposure of fertilized eggs with each PAHs did decrease survival and hatching rate. Concentration-dependent toxic effects on the rate of fertilization, hatching, survival and abnormality in A. crassispina were observed following exposure to PAHs (1, 10, 100, 1000 and 10000㎍/L). These data show that PAHs exposure decreased in fertilization success of sea urchin egg and sperm and producted abnormal embryo. It is plausible to suggest that PAHs had the potential to significantly reduce coastal recruitment of sea urchin.

• Asian-Australasian Journal of Animal Sciences
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• 제23권7호
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• pp.873-879
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• 2010

We investigated the effect of various concentrations of taurine during in vitro fertilization (IVF) on the embryonic development up to the blastocyst stage of bovine oocytes fertilized with three different Japanese Black bulls (Bull A, B and C). In vitro matured oocytes were fertilized with various concentrations of taurine (0, 1, 10, 50 and 100 mM) in the presence of 2.5 or 5.0 mM caffeine plus $25{\mu}g$/ml heparin (CH) for 6 hr or $100{\mu}g$/ml heparin (H) for $24{\pm}2$ h. After IVF, the cleavage rates from the 2 to 16 cell stage determined at 3 days and the development rates up to the blastocyst stage determined at 7-8 days from the onset of IVF were assessed. Although the cleavage rates for the taurine concentration groups were not significantly increased in any of the three bulls in the CH groups, the development rates up to the blastocyst stage of the 50 mM taurine group of Bulls A and B, and of the 1 to 50 mM groups of Bull C were increased (p<0.05) compared to those of the control (0 mM taurine) groups. On the other hand, none of the bulls in the H groups showed any significant increase either in the cleavage rates or blastocyst formation rates in any taurine concentrations groups compared with those of the control groups. These results indicate that the addition of 50 mM taurine to a fertilization medium containing caffeine and heparin may stimulate embryonic development up to the blastocyst stage when fertilized with different bull semen.

• 한국발생생물학회지:발생과생식
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• 제6권1호
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• pp.17-23
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• 2002

체외수정의 성공률에 있어서 배란되기 직전의 충분히 성숙된 난자를 채취하는 것이 중요하며, 최근에는 질 벽을 통하여 초음파를 이용하는 방법을 많이 사용하고 있다. 난자 채취 시술 시 정맥 마취제를 투여하는데 최근에는 수술회복이 빠르고 오심이나 구토 등의 부작용이 적은 Propofol(2,6-disoprooylphenol)과 Thiopental sodium을 사용한다. 이들 마취제가 마취 투여 시간과 양에 따라 난자의 성숙률과 수정률, 발생률에 어떠한 영향을 미치는지 조사하고자 하였다. 돼지에서 추출한 성숙 전 단계의 난자를 마취제인 Propofo1 및 Thiopental sodium의 다양한 농도와 시간에 노출시킨 후 난자 성숙를을 조사하였으며 돼지 정자와 체외수정시킨 후 수정률과 발생률을 관찰하였다. 또한, 수정 단계 없이 단독 발생하는 처녀생식에 대한 영향도 조사하였다. Propofol에 단시간 노출된 난자는 대조군과 큰 차이가 없었으나 고농도에서 장시간 노출 시 성숙률이 현저히 높게 나타났다. 그러나 수정률은 높은 농도에 장시간 노출할 경우 대조군보다 낮아졌는데, 이와 같은 일시적인 난자 성숙은 Propofol이 처녀생식 유발물질이기 때문으로 보여진다. Thiopental sodium은 저농도 단시간 처리 시 난자의 성숙률과 수정률을 모두 감소시켰다. 이상의 결과로 볼 때, 각 마취제의 최적농도와 투여시간을 결정하여 이러한 영향을 최소화시키는 것이 체외수정의 성공률과 밀접한 관계가 있는 것으로 생각된다.