• 한국수정란이식학회지
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• 제10권2호
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• pp.153-161
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• 1995

The present study was performed to investigate the effects of caffeine and heparin on capacitation and acrosome reaction of bovine spermatozoa, effects of antisperm antibodies on acrosome reaction of bovine spermatozoa. The rates of acrosome reaction in control group, caffeine treated group, heparin treated group, caffeine-heparin complex treated group were 40.3, 54.3, 63.3, 72.3%, respectively and there were significant differences among the groups(p<0.01), especially higher in caffeine-heparin complex treated group than the others. The rates of acrosome reaction of antisperm antibodies serum supplemented groups(5, 10 and 20%) were 60.4, 48.9 and 37.1%, respectively and there were significant differences among the groups(p<0.0l), and the more increases in serum concentrations, the more decreases in acrosome reaction, but this phenomenon was not seen in fetal calf serum supplemented group and heifer serum group. When the serum concentration was 5%, the rates of acrosome reactions were significantly lower in fetal calf serum supplemented group than heifer serum group and in antisperm antibodies serum group(p<0.01), and there were no significant differences between heifer serum group and antisperm antibodies serum group(p<0.01). When the serum conecntrations were 10%, 20%, the rates of acrosome reactions were significantly lower in antisperm antibodies serum supplemented group than in fetal calf serum group and in geifer serum group(p<0.01), and there were no significant differences between fetal calf serum group and heifer serum group(p<0.01). These results indicate that caffeine-heparin complex treatment is very effective for inducing acrosome reaction of bovine spermatozoa and that antisperm antibodies block acrosome reaction.

• 한국가축번식학회지
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• 제13권2호
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• pp.105-112
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• 1989

These experiments were carried out to obtain the basic information for in vitro fertilization and development of bovine follicular oocytes matured in vitro. The bovine ovaries were obtained at a slaughter house and the follicular oocytes surrounded by cumulus cells were collected by puncturing follicles with 2-6mm of diameter. Bovine oocytes were matured in vitro for 24-26 hours in a CO2 incubator with 5% CO2 in air at 39$^{\circ}C$. The medium used for maturation was TCM199 supplemented with hormones, pyruvate, FBS and antibiotics. Epididymal spermatozoa were capacitated by in vitro culture for 2-3 hours in BO solution containing bovine serum albumin(5mg/ml) and caffein(2.5mM). Insemination was made by introducing about 10-15 matured oocytes into the suspension of capacitated spermatozoa. Six hour after inseminatin the eggs were transferred to TCM 199 supplemented with FBS(10%) for in vitro development. The results obtained in these experiments were summarized as follows : 1. The maturation rate of oocytes following incubation for 24-26 hours was 78.4%(228/291). 2. Of total 250 oocytes, 172 embryos extruded 2nd polar body following in vitro culture with spermatozoa for 20 hours, and the rates of embryos developed to 2-, 4-, 8-, 16-cells and morula or early blastocyst were 64.0, 39.2, 22.0, 15.2 and 11.2%, respectively. 3. The time needed for development to 2-, 4-. 8-, 16-cell stage and morula was 42.5$\pm$5.4, 58.0$\pm$9.2, 74.4$\pm$11.5, 96.1$\pm$13.4 and 119.0$\pm$18.2 hours, respectively.

• 한국가축번식학회지
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• 제17권4호
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• pp.339-346
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• 1994

정자의 구조와 기능을 이해하기 위해서는 정자세포구성분의 분리가 필요하다. 본 실험은 동결융해된 한우정액에 다양한 물리·화학적 처리를 하여 효과적인 정자세포구성분의 분리방법을 확립하고자 실시하였다. 물리적 분리방법으로는 vortexing 처리, 26 gauge needle 또는 strained 26 gauge needle을 1ml 주사기에 부착시킨 후 반복된 pumping, 동결보존액없이 반복된 동결융해처리등을 시행하였다. 또한, 화학적인 분리를 위해 trypsin, dithiothreitol, sodium dodecylsulfate, mercaptoethanol 등을 사용하였다. 모든 처리구중에서 가장 효과적인 정자두부와 미부의 절단을 strained 26 gauge needle이 부착된 주사기를 사용한 반복된 pumping에 의해 얻어졌다. 이러한 처리에 의해 95∼100%의 높은 정자구성분의 분리가 이루어졌다. 분리된 정자구성분의 두부표면의 보존여부를 알아보기 위해 250g/ml FITC-UEA 1 염색을 실시하였지만 특별한 두부표면변화는 관찰되지 않았다. 다른 물리적 처리방법들도 높은 정자구성분의 분리결과를 보여주었지만, strained 26 gauge needle를 사용한 방법에 비해서는 분리효율, 시간등 여러면에서 비효율적이었다. 화학적 처리에 의한 정자구성분의 분리결과는 물리적 처리에 비해 효과적이지 못했다. 분리된 정자두부와 미부를 각각 회수하기 위해 sucrose 용액을 2M, 1M, 0.5M, 0.25M 순으로 시험관에 넣은 후 1,000rpm에서 15분간 원심분리한 결과, 1M과 2M의 경계부분에 형성된 정자두부층을 얻을 수 있었다. 정자구성분의 효과적이고 간편한 분리방법이 본 실험에 의해 확립되어졌으며, 위의 방법에 의해 분리, 회수된 정자구성분은 생화학적 연구, 난자활성화기작의 이해등 다양한 응용연구의 기초자료로서 이용될 수 있을 것이다.

• Reproductive and Developmental Biology
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• 제37권4호
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• pp.169-174
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• 2013

Sperm capacitation refers to polymerization of filamentous (F)-actin from globular (G)-actin. While the role of actin-related protein 2/3 (Arp2/3) complex in actin polymerization is well appreciated, the underlying mechanism(s) and its relationship with capacitation are poorly understood. Therefore, to evaluate the potential role of Arp2/3 complex on capacitation, bovine spermatozoa were incubated with multiple doses (1, 10 and $100{\mu}M$) of CK-636, an inhibitor of Arp2/3 complex with heparin. The cellular localization of the Arp2/3 complex in spermatozoa was identified by immunohistochemistry, whereas western blot was also applied to detect the protein tyrosine phosphorylation of sperm proteins. Additionally, sperm motility and kinematic parameters were evaluated using a computer-assisted sperm analysis system. CK-636 resulted in significant changes in the ratio of Arp2/3 complex localization between acrosome and equatorial region of the spermatozoa. Short-term exposure of spermatozoa to $100{\mu}M$ of CK-636 significantly decreased sperm motility, however a non-detectable effect on protein tyrosine phosphorylation was observed during capacitation. On the basis of these results, we propose that Arp2/3 complex is associated with morphological changes during capacitation and compromised sperm motility.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.52-52
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• 2002

The objective of this study was to determine the developmental competence of in vitro matured bovine oocytes after intracytoplasmic sperm injection(ICSI) with frozen-thawed epididymal spermatozoa. The ovaries were obtained from slaughtered Korean native cows. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with frozen-thawed epididymal spermatozoa. After ICSI, one group of oocytes was activated with 7% ethanol for 5 min, and second group was not activated. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24-30 hrs in a incubator with 5% CO₂ in air at 38.5℃. (omitted)

• 한국가축번식학회지
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• 제16권1호
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• pp.15-20
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• 1992

These experiments were undertaken to investigate the rate of in vitro fertilization of bovine follicular oocytes treated with glycosaminoglycans(GAGs). Bovine follicular oocytes were obtained from the ovary of slaughtered animal and matured in media containing the various concentrations of hydluronic acid, chondroitin sulfate or heparin for 26 hours. Epididymal spermatozoa were capacitated and insemination was made by introducing about 10~15 matured oocytes into the suspension of spermatozoa. Six hour after insemination the eggs were transferred to TCM-199 supplemented with FCS(10%) and then examined the embryo development. After in vitro insemination, percentages of ova fertilized were 61.3 or 48.3%, respectively, for the cumulus intact or removed in the percentages of GAGs. However, in case of cumulus-free oocytes treated with GAGs, the fertilization rates were 58.8, 62.1, 58.8, and 61.8%, respectively, showing significant effect compared to 48.3% in cumulus-free oocytes. Our findings suggest that cnondroitin sulfate and heparin are superior to hyaluronic acid in the fertilizatin and pronuclear formation of bovine oocytes.

• 한국가축번식학회지
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• 제17권1호
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• pp.69-74
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• 1993

This study was investigated to examine the effect of conditioned medium from bovine oviductal cell(BOEC) in the co-culture system with BOEC on in vitro development of in vitro produced bovine embryos. Oocyte-cumulus complexes were cultured for 24 hrs in TCM-199 supplemented with 10% fetal calf serum, 1$\mu\textrm{g}$/ml FSH and 21U hCG, 1$\mu\textrm{g}$/ml oestradiol-17$\beta$ at 39$^{\circ}C$ under 5% CO2 in air. In vitro fertilization was performed with epididymal sperm and heparin (10$\mu\textrm{g}$/ml, 15min.) or caffeine(2.5mM)-treated spermatozoa. Oocytes were incubated with 1$\times$106 spermatozoa/ml for 18 hrs and then cultured in various culture system for 7 days. The development rates of 16-cell or blastocyst stages were recorded on 4, 7 days, respectively, after incubating. The proportions ofembryonic development into molulae and blastocysts were higher in cumulus cell co-culture(23.4%) and BOEC co-culture(34.3%) than in M199-FCS(6.1%). Similarily, the development rates into molulae and blastocysts were significantly higher in BOEC-conditioned medium than those in M199-FCS. Therefore, it is suggested that BOEC co-culture and BOEC conditioned medium increase significantly the development of in vitro produced bovine embryos in in vitro system.

• 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.

• 한국가축번식학회지
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• 제13권1호
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• pp.32-39
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• 1989

The present studies were conducted to investigate the possibility of capacitation of bull spermatozoa in the uteri isolated from estrous hamsters and to investigate the possibility of in vitro fertilization of follicular oocytes matured in culture, using ejaculat spermatozoa preincubated in the uteri isolated from estrous hamsters. The follicuar oocytes matured in culture were not fertilized after insemination spermatozoa preincubated for 3 and 5.5-7 h in the uteri isolated from estrou hamsters, but 5, 77, 85 and 50-67% of those oocytes were fertilized by spermatozoa preincubated for 3.5, 4, 4.5 and 5h in those uteri, respectively.

• 한국가축번식학회지
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• 제15권2호
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• pp.125-132
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• 1991

The studies on the carried out to investigate the effects of capacitation method of spermatozoa on the in vitro fertilization and cleavage rate of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible of diameter 3~5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and FCS for 24~48hrs in an incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18hrs with motile capacitated sperm by preincubation of mKRB, treatment of HIS(high strength ion), Ca-IA(Inophore A), BFF(bovine follicular fluids) and heparin. The results obtained in these experiments were summarized as follows : 1. The in vitro fertilizatin and cleavage rate offollicular oocytes fertilized with capacitated spermatozoas in BO solution by preincubation of mKRB, treatment of HIS, Ca-IA, BFF and heparin method were 53.1%, 33.9%, 50.8%, 48.1%, 58.8% and 28.1%, 17.7%, 26.2%, 22.8%, 32.8%, respectively. And the fertilization and cleavage rate of heparin method was of highest of all. 2. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solutin by both caffeine, BSA and heparin methods were 65.8%, 70.3% and 40.8%, 47.3%, respectively, and those rates were higher treatment of heparin＋BSA, heparin＋caffeine than treatment of heparin. 3. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoa in BO solution with heparin concentrations of 2, 5, 10, 20, 40$\mu\textrm{g}$/ml were 50.0%, 54.7%, 58.1%, 51.7% and 27.9%, 32.8%, 37.1%, 30.0%, respectively. And the fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution with 10$\mu\textrm{g}$/ml of heparin was the highest of all. 4. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution containing heparin with caffeine concentraton of 10, 20, 30, 40$\mu\textrm{g}$/ml were 71.4%, 74.3% and 70.6%, 70.0% and 45.7%, 47.3%, 44.1%, 41.4%, respectively. The fertilization and cleavage rate of spermatozoa fertilized in BO solution with caffeine and heparin together(70.3~74.3%) was higher than that of spermatozoa fertilized in BO solution with heparin(58.8%). 5. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution containing heparin with BSA concentration of 5, 10, 20, 30$\mu\textrm{g}$/ml were 63.6%, 62.9%, 66.7%, 60.3% and 44.1%, 43.5%, 48.5%, 42.7%, respectively. The fertilization and cleavage rate of spermatozoa fertilized in BO solution with BSA and heparin together(60.3~66.73%) was higher than that of spermatozoa fertilized in BO solution with heparin(58.8%).