• 한국발생생물학회지:발생과생식
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• 제24권4호
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• pp.297-306
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• 2020

Repetitive changes in the intracellular calcium concentration ([Ca²⁺]i) triggers egg activation, including cortical granule exocytosis, resumption of second meiosis, block to polyspermy, and initiating embryonic development. [Ca²⁺]i oscillations that continue for several hours, are required for the early events of egg activation and possibly connected to further development to the blastocyst stage. The sources of Ca²⁺ ion elevation during [Ca²⁺]i oscillations are Ca²⁺ release from endoplasmic reticulum through inositol 1,4,5 tri-phosphate receptor and Ca²⁺ ion influx through Ca²⁺ channel on the plasma membrane. Ca²⁺ channels have been characterized into voltage-dependent Ca²⁺ channels (VDCCs), ligand-gated Ca²⁺ channel, and leak-channel. VDCCs expressed on muscle cell or neuron is specified into L, T, N, P, Q, and R type VDCs by their activation threshold or their sensitivity to peptide toxins isolated from cone snails and spiders. The present study was aimed to investigate the localization pattern of N and P/Q type voltage-dependent calcium channels in mouse eggs and the role in fertilization. [Ca²⁺]i oscillation was observed in a Ca²⁺ contained medium with sperm factor or adenophostin A injection but disappeared in Ca²⁺ free medium. Ca²⁺ influx was decreased by Lat A. N-VDCC specific inhibitor, ω-Conotoxin CVIIA induced abnormal [Ca²⁺]i oscillation profiles in SrCl₂ treatment. N or P/Q type VDC were distributed on the plasma membrane in cortical cluster form, not in the cytoplasm. Ca²⁺ influx is essential for [Ca²⁺]i oscillation during mammalian fertilization. This Ca²⁺ influx might be controlled through the N or P/Q type VDCCs. Abnormal VDCCs expression of eggs could be tested in fertilization failure or low fertilization eggs in subfertility women.

• 한국수정란이식학회지
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• 제12권1호
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• pp.27-36
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• 1997

The studies were carried out to investigate the effects of co-culture with cumulus cells and oviduct epithelial cells on the in vitro fertilization and cleavage rate of bovine follicular cocytes and to determine the optimum thawing temperature and equilibration time on in vitro developmental rate of frozen bovine embryos. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes were cultured in TGM-199 medium containing 10 IU /ml의 PM SG, 10 IU /ml의 hCG, ip g/ml의 $\beta$-estradiol and 10% FCS for 24~48 hrs in incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. The bovine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquld nitrogen and thawed in 3$0^{\circ}C$ water. Survival rate was defined as developmental rate on in vitro culture or FDA-test. The results are sunanarized as followes :1. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with cumulus cells in TCM499 medium were 75.0~76.8% and 17.3~27.6%, respect-ively. And in-vitro fertilization rates of cumulus-enclosed oocytes(55.4%)were significantly(p<0.05) higher than cumulus-denuded oocytes (23.1%). 2. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with l$\times$ l04cells /ml, 1 x l06cells /ml, lx l08cells /ml and 1 x l015cells /ml oviduct epithelial cells in TCM-199 medium were 74.5~77.8% and 15.7~21.20 respectively.3. The in-vitro fertilization and in vitro developmental rates of bovine oocytes cocultured in '1CM-199 media containing PMSG, hCG, PMSG+hCG. PMSG+$\beta$-estradiol, hCG+$\beta$-estradiol 0 to 40 hrs after insemination were 74.0~77.4% and l8.9~23.l%, re-spectiv ely.4.The survival rates of bovine embryos thawed after rapid freezing in the freezing medium containing a various concentration of sucrose added 1.5M and 2.OM glycerol,DMSO and propanediol were 23.5~31.4% and 20.6~34.l%, respectively. 5. The temperature thawed at 3$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 2$0^{\circ}C$ and 35$^{\circ}C$.6. The equilibration time on the survival rates of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (10~20min.). (Key words : bovine embryos, co-culture, freezing, in vitro development)

• 한국가축번식학회지
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• 제17권2호
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• pp.127-132
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• 1993

Frozen semen obtained from 5 different Korean Native Cattle were used for in vitro fertilization of oocytes. The proportions(3∼11%) of oocytes penetrated in the basic medium with 13.9 mM glucose were very low in different semen. However, the penetration rates of oocytes in the presence of caffeine and heparin increased to 68∼84%. The proportions(10∼52%) of oocytes penetrated in the presence of caffeine alone were low at various glucose concentrations(0∼27.8 mM). In the medium with heparin, significantly(P<0.05 at least) higher penetration rate was obtained in the absence(94%) than in the presence(28∼45%) of 0∼27.8 mM glucose concentrations. The medium with caffeine and heparin, however, produced similar penetration rates(84∼97%) regardless of the various glucose concentrations.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.134-134
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• 2003

Bovine embryos produced by in vitro maturation, feretilization and development was examined for presevation and transfer. The fertilization medium used BO medium with 5 mM/$m\ell$ caffeine and 10$\mu\textrm{g}$/$m\ell$ heparin and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to 1$\times$$10^{6}$ cells/$m\ell$ motile sperm during fertilization in vitro. At 8~10 hrs after insemination, the oocytes were transferred into CR1aa medium and cultured for 7 days. Embryos were preserved by vitrification method for transfer. When the embryos of early, blastocyst and expanded blastocyst stages were frozen-thawed, the proportions of embryos with normal morphology 83.6, 88.1 and 85.2%. (중략)

• 한국가축번식학회지
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• 제10권2호
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• pp.204-210
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• 1986

As a part of in vitro fertilization(IVF) for farm animals, IVF experiment was conducted using New Zealand white rabbits with their sperm capacitated in vivo. The effect of uterine conditions on sperm capacitation and effect of sperm concentration and fertilization media on IVF rate and implantation of in vitro fertilized ova were studied. The results obtained are summarized as follows; 1. Acrosomal reaction, noted after staining, of sperm recovered from ligated and intact uterus of capacitators was 83.0% and 65.7%, respectively. 2. IVF rate of ova inseminated with sperm from ligated uterus tended to be higher in DM or with higher concentration of sperm than in the modified F12 medium or with lower sperm concentration. Cleavage rate of fertilized ova for 48hr in DM was 31.5% for 106/ml and 30.0% for 104/ml of sperm and that in modified F12 medium was 26.0% for 106/ml and 22.3% for 104/ml of sperm. 3. Using the sperm from intact uterus, cleavage rate of fertilized ova showed same tendency as those shown with ligated uterus. The rate was 82.0% for 106/ml and 66.5% for 104/ml of sperm in DM and was 69.0% for 106/ml and 56.5% for 104/ml of sperm in the medium. 4. When normal ova up to 48hr after IVF were cultured for 4 days in either DM or modified F12 medium, ova developed to blastocyst stage showed higher rate in the groups of higher sperm concentration in the both media. The rate was 80.9% and 60.0% for 106/ml and 104/ml of sperm in DM and 91.7% and 71.4% for 106/ml and 104/ml of sperm in the modified F12 medium, respectively. 5. Rate of implantation after transfer of 4- or 8-cell embryos was 36.8%.

• 한국수정란이식학회지
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• 제28권1호
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• pp.41-48
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• 2013

This study examined the motility of either the unattached(upper) or attached(lower) Hanwoo sperm to bovine oviduct epithelial cell(BOEC) monolayers to determine whether there are any changes in their motility during co-culture. The cleavage and blastocyst development rate were compared among different preincubation methods in-vitro, after oocytes were fertilized in-vitro with Hanwoo sperm on BOEC monolayers. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 5 hours and 6 hours (p<0.05) of incubation, in sperm treatment medium without heparin and caffeine. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 3 hours (p<0.05) and 6 hours (p<0.01), in sperm treatment medium containing heparin and caffeine. The motility of the attached( lower) sperm was significantly higher than the unattached(upper) sperm during co-culture with BOEC at all times(p<0.01 or p<0.05), except for 6 hours. After Hanwoo oocytes were fertilized in-vitro with the sperm that had been co-cultured with BOEC in sperm treatment medium containing heparin and caffeine, we determined the cleavage and blastocyst development rate, according to the preincubation methods. Both the cleavage and blastocyst development rate from 2 hour preincubation group were the highest, but significant difference was not recognized. These results show that BOEC plays an important role on sperm hyperactivation related to capacitation regardless of heparin and caffeine in sperm treatment medium. However, oviduct epithelial cell had no significant effect on the development of embryos after in-vitro fertilization in the presence of added heparin and caffeine in sperm treatment medium.

• 한국가축번식학회지
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• 제27권3호
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• pp.215-223
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• 2003

본 연구는 체외성숙된 돼지난포란을 액상정액으로 수정시 수정시간과 배양배지가 난포란의 발달에 미치는 영향을 조사하기 위하여 실시하였다. 정자농후정액 (30∼60 ml)을 채취하여 실온에서 2시간 정도 서서히 냉각시킨 후, 정액을 15 ml 튜브에 담아 800${\times}$g로 10분간 원심분리하였다. 상층액은 버리고 하부의 정자는 5 ml LEN 희석액으로 1${\times}$$10^{9}$ 전자/ml가 되도록 재희석하였다. 희석된 정액은 4$^{\circ}C$ 냉장고에 보존하였다. 미성숙 난모세포의 성숙에 사용된 배지는 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin B$_{12}$ , 25 mM HEPES, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml PMSG, 10 IU/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate그리고 10% pFF를 첨가한 TCM-199 배지였다. 22시간 성숙 배양한 후 난모세포는 cysteamine과 hormone들을 배제한 후 38.5$^{\circ}C$, 5% $CO_2$ incubator에서 22시간 더 성숙시켰다. 성숙된 난모세포는 채취 후 2일간 4$^{\circ}C$에 보존된 액상정액으로 수정되었다. 난모세포는 500 $\mu$l mTBM 수정 배지에서 1${\times}$$10^{6}$ 정자/ml의 농도로 1, 3, 6 그리고 9시간 동안 수정시켰다. 그 후 난모세포는 500 $\mu$l NCSU-23, Hopes buffered NCSU-23, PZM-3 그리고 PZM-4 배양배지에 옮겨서 6, 48 그리고 144시간을 더 배양하였다. 정자침투율, 웅성전핵형성율 그리고 난모세포의 난할율은 6 및 9시간 수정시간에서 1 및 3시간 수정시간 보다 높았다. 6시간 수정시 배반포형성율 (33.6%)은 1, 3 그리고 9시간 수정시 배반포형성율 (11.4, 23.0 그리고 29.6%) 보다 높았다. 배반포의 평균세포수는 6, 9, 3 그리고 1시간 수정시 각각 32.9, 27.6, 26.3 그리고 24.4개 였다. 분할된 난모세포의 배반포형성율 그리고 배반포의 평균세포수는 NCSU-23, PZM-3 그리고 PZM-4 배양배지보다 HEPES buffered NCSU-23 배양배지가 우수하였다. 결론적으로 4$^{\circ}C$ 보존 돼지액상정액은 체외성숙된 돼지 난모세포의 체외수정에 사용될 수 있음이 입증되었다. 또한 체외성숙된 돼지 난모세포는 500 $\mu$l mTBM 수정배지에서 1${\times}$$10^{6}$ 정자/ml로 6시간 공배양시키는 것이 바람직하며, HEPES buffered NCSU-23 배양배지에서 배양하는 것이 좋다는 결과를 얻었다.

• Clinical and Experimental Reproductive Medicine
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• 제12권2호
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• pp.81-88
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• 1985

• 농업과학연구
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• 제18권2호
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• pp.114-118
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• 1991

본(本) 연구(硏究)는 생체(生體) 및 실험실(實驗室)에서 성숙(成熟)된 돼지 난모세포(卵母細胞)의 체외수정(體外受精) 및 배양(培養)에 적합(適合)한 배지(培地)를 찾기 위하여 실시(實施)한 바, 그 얻어진 결과(結果)는 다음과 같다. 생체내(生體內)에서 성숙(成熟)된 난모세포(卵母細胞)에 대하여 10% FCS을 함유(含有)한 M199수정배지(受精培地)와 1% BSA를 함유(含有)한 TL Hepes 수정배지(受精培地)를 비교(比較)한 결과(結果), 수정율(受精率)은 TL Hepes 수정배지(受精培地)가 M199 수정배지(受精培地)보다 우수(優秀)하였으나, 다정자침입율(多精子侵入率)이 다소 좋지 않은 편이었다. 체외수정후(體外受精後) TL Hepes 세척(洗滌) 및 배양배지(培養培地)에서 48시간(時間) 배양(培養)한 결과(結果), 배양(培養)된 난자(卵子) 53개중(個中) 39개(個) (73.6%)가 분할(分割)되었으며, 분할(分割)된 수정란(受精卵) 39개중(個中) 31개(個) (79.5%)가 2~8세포기(細胞期)까지 균등(均等)하게 분할(分割)되었다. 미성숙난포란(未成熟卵胞卵)을 Waymouth 성숙배지(成熟培地)에서 배양(培養)했을 때가 TL Hepes성숙배지(成熟培地)에서 배양(培養)했을 때 보다 수정후(受精後) 더 많은 확장(擴張)된 정자두부(精子頭部)를 유도(誘導)할 수 있었으나, 대부분 다정자침입(多精子侵入)이 되었다. 48시간(時間) TL Hepes 세척(洗滌) 및 배양배지(培養培地)에서 배양(培養)한 결과(結果) 4세포기(細胞期)까지 발육(發育)을 시킬 수가 없었다.

• 한국산학기술학회논문지
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• 제17권4호
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• pp.692-697
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• 2016

해수산 rotifer Brachionus rotundiformis의 내구란 대량생산을 위한 효과적인 유성생식자극요인을 구명하기 위해 염분자극과 기사용배양수의 효과를 9일 동안 조사하였다. 각 염분에 노출된 후 모든 실험구에서 배양 3일째부터 유성생식률이 증가하기 시작했으며 이후 배양 4-5일째 이후 감소하는 경향을 보였다. 내구란은 10‰, 15‰, 20‰, 25‰ 실험구에서 배양 4일째에 출현하였으며 특히 20, 25‰ 실험구는 배양 6일째 수정률이 각각 33.3%와 31.3%로 다른 실험구에 비해 높게 나타났다. 9일간 평균 유성생식률은 10‰와 20‰ 실험구에서 각각 25.3%와 20.2%로 높게 나타났다. 수정률은 25‰ 실험구에서 14.0%로 가장 높았다. 각 기사용배지농도에 노출된 후 모든 실험구의 유성생식률은 염분실험결과와 유사한 경향을 보였고 내구란은 0%과 25% 기사용배지에 노출되었을 때 만 출현하였다. 평균수정률은 모든 실험구가 차이를 보이지 않은 반면, 유성생식률은 25% 실험구가 22.3%로 대조구인 0%에 비해 높게 나타났다. 본 연구결과를 종합해보면, B. rotundiformis의 내구란 대량생산을 위한 효과적인 유성생식유발요인으로 기배양수자극보다 염분자극이 더 효과적이며 최적 염분은 비교적 높은 유성생식률과 수정률을 유도한 20-25‰로 판단된다. 본 연구결과는 차후 rotifer 내구란 대량생산을 위한 핵심적인 기초자료로 활용될 수 있을 것으로 판단된다.