• 한국수정란이식학회지
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• 제12권3호
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• pp.283-291
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• 1997

In vitro development of bovine embryos is affected by many factors such as energy substrates, amino acids, and some growth factors. It has been reported that mRNA of insulin, PDGF and their receptors are detected in cow embryos, and that some chelating agents such as EDTA and transferrin have beneficial role on mouse and bovine embryos. The author hypothesized that insulin, transferrin arid PDGF added to a culture medium increase in vitro development of bovine embryos by chelating toxic substance(s) or increasing cell growth and metabolism. Immature oocytes from slaughtered ovaries of Holstein cows and heifers were matured for 24 hours in a TCM199 containing 10% fetal calf serum, FSH, LH and estradiol with granulosa cells in vitro. Matured oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Embryos cleaved to 2- to 4-cell at 30 hours after IVF were selected and cultured in a 30-$\mu$l drop of a synthetic oviduct fluid medium (SOFM) containing 0.8% BSA, Minimum Essential Medium essential and non-essential amino acids, and insulin, transferrin or PDGF for 9 days. Supplementation of a SOFM with insulin, and /or transferrin did not increase develop-mental rate to expanding and hatching blastocyst of 2- to 4-cell bovine embryos compared with control. The highest developmental rate to hatching blastocyst was shown when PDGF was added at the concentration of 10 ng /ml among the supplementing doses tested in the present study (p<0.05). Addition of PDGF without insulin to a SOFM could not increase embrye development, but combined addition of PDGF with insulin significantly increased (p<0.05) embryo development to hatching blastocyst (50%) compared with control (38%). In conclusion, insulin and PDGF supplemented to a SOFM may act synergistically and have beneficial effect on in vitro development of 2- to 4-cell bovine embryos matured and fertilized in vitro.

• 한국가축번식학회지
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• 제17권3호
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• pp.183-191
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• 1993

These experiments were carried out to investigate whether the enzyme is involved in zona hardening during normal activatin of the oocytes by sperm, and demonstrate peroxidase activity during in vitro fertilization of oocytes treated with peroxidase inhibitors(250 $\mu$M phenylhydrazine, 28mM sodium sulfite, 350mM glycine ethyl ester and 50mM sodium azide) and tyrosine analogue(12.5mM tyramine). Also, zona soluble properties of the ovarian oocytes incubated for 0, 5, 10 and 15 hr in the presence of pheylhydrazine or tyramine were studied by using $\alpha$-chymotrypsin. The results obtained from these experiments were summarized as follows ; 1. The rates of fertilizatin in control oocytes and oocytes treated with phenylhydrazine or tyramine were 69.8%, 62.3% and 88.2%, respectively. However in vitro fertilization in oocytes treated with three different peroxidase inhibitors, sodium sulfite, glycine ethyl ester and sodium azide, were not induced. The oocytes treated with phenylhydrazine had no significant effect on in vitro fertilization rate as compared to control. However there was a significantly different in fertilization between tyramine treated group and control group(P<0.01). 2. The zona solubility(t50) of control and fertilized oocytes in culture treated with phenylhydrazine or tyramine were 30.7, 26.0 and 16.3 min., respectively. Phenylhydrazine treated group and tyramine treated group had effect on inhibition of zona hardening as compared to control group. These results suggest that ovoperoxidase is involved in zona hardening during normal activation of the oocytes by sperm. 3. t50 of control oocytes and ovarian oocytes treated with phenylhydrazine or tyramine for 5, 10 and 15 hr in vitro were 14.0, 26.2 and 32.0 min., 14.5, 26.9 and 30.2 min., and 14.0, 24.3 and 31.2 min., respectively. These results suggest that zona hardening in ovarian oocytes matured for various times in vitro cannot be inhibited by peroxidase inhibitors and tyrosine analogue, that the spontaneous zona hardening incultured ovarian oocytes is not caused by the secretory products of cortical granules released during the cortical reaction, ovoperoxidase.

• 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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• 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%).

• 한국동물생명공학회지
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• 제36권4호
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• pp.183-188
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• 2021

In vitro maturation (IVM) of oocytes is the procedure where the immature oocytes are cultivated in a laboratory until they are mature. Since IVM oocytes generally have low developmental competence as compared to those matured in vivo, development of an optimal IVM culture system by fine-tuning culture conditions is crucial to maintain high quality. In-depth knowledge and a deep understanding of the in vivo physiology of oocyte maturation are pre-requisites to accomplish this. Within ovarian follicles, various signaling pathways that drive oocyte development and maturation regulate interaction between oocytes and surrounding somatic cells. This review discusses the sonic hedgehog (SHH) signaling pathway, which has been demonstrated to be intimately involved in folliculogenesis and oocyte maturation. Advances in elucidating the role of the SHH signaling pathway in oocyte maturation will aid attempts to improve the current inferior in vitro oocyte maturation system.

• Clinical and Experimental Reproductive Medicine
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• 제36권4호
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• pp.249-254
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• 2009

목 적: 고식적 체외수정시술과 세포질내정자주입법을 사용하여 체외성숙 난자를 수정시키고 수정률과 배발달율을 비교하고자 하였다. 연구방법: 2007년 1월부터 2008년 8월까지 난소과자극과 체외수정시술을 받은 59명의 여성에서 135개의 배아소포 단계의 미성숙난자만을 얻어 75 mIU/mL rFSH, 0.5 IU/mL rhCG, 10 ng/mL rEGF가 포함된 체외성숙용 배양액에서 최대 48시간까지 배양하였다. 체외성숙된 난자는 고식적 체외수정방법 (n=41) 또는 세포질내정자주입법 (n=94)을 이용하여 수정시켰으며 이후 배발달율을 관찰하였다. 결 과: 고식적 체외수정군과 세포질내정자주입군에서 체외성숙률은 각각 51.2%와 59.6%였고, 수정률은 각각 71.4%와 80.4%로 두 군간에 통계적으로 유의한 차이는 없었다. 배발달율도 두 군에서 비슷하게 관찰되었다. 결 론: 난소과자극을 통해 얻은 미성숙난자는 고식적 체외수정법을 사용하더라도 세포질내정자주입법과 동등한 수정률과 배발달율을 보임을 확인하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제4권3호
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• pp.251-254
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• 1991

Immobilized (killed) mouse spermatozoa or sperm head were microinjected into mouse oocytes matured in vivo and cultured for 72h in vitro. When non-capacitated spermatozoon was injected, oocytes that developed to $${\geq_-}$$ 2-cell and $${\geq_-}$$ 4-cell was 27.8 (15/54) and 3.7% (2/54), respectively. When non-capacitated sperm head was injected. development to $${\geq_-}$$ 2-cell and $${\geq_-}$$ 4-cell was 21.3 (16/75) and 8.0% (6/75), respectively. When capacitated spermatozoon was injected, development to $${\geq_-}$$ 2-cell and $${\geq_-}$$ 4-cell was 21.4 (15/70) and 4.3% (3/70), respectively. When capacitated sperm head was injected, development to $${\geq_-}$$ 2-cell and $${\geq_-}$$ 4-cell was 29.9 (35/117) and 10.3% (12/117), respectively. In contrast, none developed beyond 4-cell in the sham-operated group. The results of this study demonstrated that mouse oocytes matured in vivo can undergo normal appearing cleavage to 4-cell stage by dead-sperm injection. Sperm treatment prior to injection did not affect the ability of mouse oocytes to cleave in vitro.

• 한국가축번식학회지
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• 제14권4호
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• pp.245-252
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• 1990

This study was carried out to investigate the factors affecting fertilization in vitro of follicular oocytes with frozen-thawed spermatozoa in Korean Native Cattle. The bovine ovaries were obtained at a slaughter house and the follicular oocytes were recovered by aspirating the follicular fluid from the visible follicles of 3~6mm. The bovine oocytes were matured in vitro for 20~24 hours in TCM-199 containing FCS and hormones. The matured oocytes were fertilized in vitro using Percoll-separated frozen-thawed spermatozoa in BO solution. The effects of dilution and fertilization media, capacitating method, concentration of inseminated sperm and time after insemination of fertilization, were observed. The results obtained are summarized as follows : 1. The fertilization rate of frozen-thawed sperm inseminated in BO solution with caffeine and heparin together(56.4%) was higher than that of sperm inseminated in BO solution with either caffeine(10.5%) or heparin(8.9%) and without both caffeine and heparin(0%)(P<0.05). 2. The fertilization rate(56.3%) of frozen-thawed sperm inseminated in BO solution with both caffeine and heparin without preincubation was higher than that of sperm preincubated(2.9%)(P<0.05). 3. The fertilization with high concentration of frozen-thawed sperm(1.4~1.8$\times$107cells/ml) in BO solution containing caffeine and heparin resulted in higher fertilization rate, 76.7%, than the low concentration of sperm(0.8~1.0$\times$107cells/ml), 32.7%(P<0.01). 4. When the oocytes were inseminated with frozen-thawed sperm in BO solution containing caffeine and heparin without preincubation, fertilization rate increased by time and the rates were 5.9, 46.0 and 59.4% at 8, 16 and 24 hours, respectively.

• 한국가축번식학회지
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• 제23권2호
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• pp.133-139
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• 1999

본 연구는 생쥐 미성숙란올 초자화 동결-융해하였올 때, 체외/체내 배발달능을 검토하고자 실시하였다. 생쥐 미성숙란은 동해제인 EFS40(40% ethylene glycol, 18% ficoll, 0.5 M sucrose)으로 초자화동결되었으며, 융해 후 16 시간동안 체외성숙을 유도하여, 제 1 극체가 나타난 성숙된 난자를 1~2$\times$$10^{6}$$m\ell$ 농도의 정자로 체외수정시킨 다음, 난할율 ($\geq$ 2- 세포기)과 체외 / 체내 발달율을 조사하였다. 쥐 미성숙란을 초자화 동결 융해하였던 군 (63.1%)의 체외성숙율은 동해제 노출군 (67.5%)과 대조군(66.3%)에 유사하게 나타났으나, 초자화 동결군의 난할율과 배반포형성율 (64.9, 59.0%)은 동해제노출군 (83.7, 74.7%)과 대조군 (90.7, 83.7%) 에 비해 유의하게 감소하였다 (p＜0.05). 그러나, 초자화 동결 융해하였던 생쥐 미성숙란으로부터 얻어진 배반포기배를 가임신 생쥐에 이식하였을 때, 체내발달율인 전체착상율 (31.3%)과 착상된 배로부터 발달된 산자형성율 (66.7%)은 대조군의 결과 (40.8%, 58.1%)와 각각 비교하였을 때 유의차가 인정되지 않았다. 따라서, 생쥐 미성숙란을 초자화 동결-융해하였을 때, 체외발달율은 유의하게 감소하였지만 생성된 배반포기배로부터의 산자발달율은 대조군과 유사하게 나타나, EFS40을 이용한 초자화 동결 방법은 생쥐 미성숙란 동결에 유용하게 이용될 수 있다는 것을 알 수 있었다.

• 한국가축번식학회지
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• 제16권3호
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• pp.239-246
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• 1992

미성숙 나포란을 20% FCS가 첨가된 TCM-199으로써 24시간 동안 5% CO2, 8% O2의 공기 조건하에서 체외성숙시킨 다음 난자의 위란강내로 정자를 미세주입하여 체외수정을 실시하였다. 미세주입 하기전 정액을 0.75% BSA가 첨가된 Ham's F-10 배양액으로 2시간 동안 5% CO2, 8% O2의 공기 조건하에서 배양함으로써 수정능 획득을 하도록하였으며 이어 30분 동안 12mM의 dbcGMP와 10mM의 imidazol이 함유된 Ham's F-10 배양액으로 배양함으로써 첨체반응을 유도하였다. 본 실험에서 정자의 미세주입 후 제 2극체와 전핵형성이 일어난 난자의 비율은 각각 9.5, 5.4%였으며 2세포기 이후로 발달한 난자의 비율은 4.1%이었다.