• 대한생식의학회:학술대회논문집
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• 대한불임학회 2006년도 The 5th Biannual Meeting of Pacific Rim Society for Fertility and Sterility
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• pp.118-121
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• 2006

• 대한수의학회지
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• 제63권4호
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• pp.40.1-40.7
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• 2023

To optimize the most efficient method for porcine in vitro maturation (IVM), we compared the effects of supplementing extracellular vesicles (EVs) derived from porcine follicular fluid (pFF). The cumulus oocyte complexes were grouped into 4 groups with different supplementations as following: pFF (G1), pFF-depleted EVs (G2), EVs (G3) and control (G4) groups. After IVM with different supplementations, maturation rates and the developmental competences of porcine oocytes and blastocyst development were investigated. Additionally, glutathione (GSH) and reactive oxygen species (ROS) levels were measured in mature oocytes. The EVs were isolated and characterized with cryo-TEM and nanoparticle tracking analysis. The pFF significantly affected the maturation rate, whereas the presence of EVs did not show notable difference in the maturation rates. Although there were numerical increases in the measured parameters in EV and pFF-depleted EVs groups, no significant differences were observed between them. The EV group showed similar oocyte maturation rate for both positive and negative control groups. The GSH was not different among the groups, but ROS levels were significantly lower in pFF-supplemented group when compared with other groups with the highest level in the control group. G2 group wasn't significantly different G1 and G3 group. G3 group wasn't significantly different from G2 and G4 group. This suggests that EVs in IVM medium which probably effected partially to protect against oxidative stress and potentially enhance the quality of oocytes. This study indicates that the EVs in pFF play a significant role in improving the efficiency of oocyte maturation in porcine.

• Reproductive and Developmental Biology
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• 제29권4호
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• pp.223-227
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• 2005

In vitro maturation of porcine immature cumulus-enclosed oocytes can be enhanced by co-incubation with spermatozoa even before fertilization. The aim of this study was to determine whether the addition of spermatozoa into the culture medium can stimulate the meiosis resumption of porcine cumulus-enclosed oocytes arrested at germinal vesicle (GV). Cumulus-enclosed oocytes (CEOs) were collected from follicles of 3 to 5mm diameter. Porcine CEOs were cultured in tissue culture medium containing various meiosis inhibitors and spermatozoa. Oocytes were examined for evidence of GV and GV breakdown after 24 h culture. After 24 h culture $43.8\%$ of oocytes cultured in only TCM 199 remained at GV stage whereas $56.2\%$ of oocytes were able to resume meiosis. When porcine CEOs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than $90\%$ of oocytes were not able to resume meiosis. However, co-culture of porcine CEOs with spermatozoa was able to overcome the inhibitory effect of dbcAMP and Fo. Irrespective of the presence of 3-isobutyl-1-methylxanthine (IBMX), no difference was observed in the proportion of oocyte reached germinal vesicle breakdown (GVBD). The present study suggests that dbcAMP and Fo prevent the spontaneous maturation of competent oocyte in culture after isolation from follicles and that mammalian spermatozoa contain a substance(s) that improves meiosis resumption in vitro of porcine cumulus-enclosed oocytes.

• 한국수정란이식학회지
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• 제11권1호
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• pp.15-26
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• 1996

This study was conducted to improve the in vitro maturation(JVM), in vitro fertilization (IVF) and in vitro developmental capacity of oocytes derived from slaughtered Korean native cattle. The recoverd oocytes, obtained from a local slaughter house, were used completely surrounded by at least 3 layers of cumulus cells in combination with a homogeneous cytoplasmic pigmentation. In vitro maturation was induced in TCM-199 or Ham's F-10 supplemented with LH(1O $\mu$g/rnl), FSH(35 $\mu$g/ml), estradiol-17$\beta$(1 $\mu$g/ml) at 39$^{\circ}C$ under 5% $CO_2$ in air for 24 hours. Sperm from caudal epididyrnis and previously matured cumulus-oocytes complexes were cultured for 24 hours in 100 $\mu$l droplets of fertilization media under paraffin oil. The zygotes were cultured with media(TCM-199 with bovine oviductal epithelial cells or CRlaa) for 7 to 10 days. The cleavage rate of IVM-IVF oocytes was significantly (P<0.05) higher following maturation using Ham's F-10 (59.9%) than TCM-199 (51.6%). Development to the blastocysts among cleaved embryos was not signficantly different between maturation media: Ham's F-10 (16.0%) and TCM-199(11.9%). However, the hatching rate was affected significantly (P<0.05) on rnaturation media as 62.9% in Ham's F-10, compared with 41.2% in TCM-199. The cleavage rate of IVM-IVF oocytes was significantly (P<0.05) higher following IVF using m-TALP medium (80.1%) than BO medium (51.6%). The percentage of in vitro developed blastocysts among cleaved embryos was not signficantly different between fertimization media: BO (11.7%) and m-TALP (17.6%). The cleavage and the developmental rate to the blastocysts after IVF in m-TALP or condition medium(CM) with or without oviduct epithelial cell monolayer(OECM) was similar(80.1% and 17.6% in m-TALP, 83.8% and 19.4% in M-TALP with OECM. 82.9% and 18.9% in CM, 87.6% and 16.0% in CM with OECM, respectively). The percentage of in vitro developed blastocysts among cleaved embryos was significantly (P<0.05) higher in TCM-199 medium co-cul tured with bovine oviduatal epithelial cell monolayers(35.2%) than CRlaa medium(1.9%). These results stggest that the most transferable IVF embryos could be produced from Ham's F-10, m-TALP and TCM-199 medium with bovine oviductal epithelial cell monolayers for IVM, IVF and IVC, respectively.

• 한국가축번식학회지
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• 제23권3호
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• pp.263-270
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• 1999

본 연구는 체외성숙배 양액에 L-ascorbic acid 와 selenium을 첨가 배양, 돼지 미성숙 난포란의 체외성숙, 체외수정 및 체외배발달에 미치는 영향을 검토코자 수행하였다. 배양액내 L-ascorbic acid를 0. 62.5. 100 그리고 30 $\mu$Ml 첨가 40~44시간동안 배양한 성적은 난핵포 붕괴율이 각각 86.8%, 92.9%, 91.7%, 그리고 92.6%였으며 핵성숙율은 각각 44.7%. 57.1%, 52.8%, 그리고 53.7%로 첨가구에서 유의적으로 높았다 (p＜0.05). 체외성숙배양액내 L-ascorbic acid와 selenium을 첨가 배양 후 체외 수정 유기 결과, 웅성전핵 형성율은 유의적으로 높았고 (p＜0.05) 다정자 침입율은 유의적으로 낮게 나타났으며 (p＜0.05), 체외수정 후 난할율, 상실배와 배반포배 발달율도 첨가구에서 유의적으로 높게 나타났다 (p＜0.05). 이러한 결과는 체외성숙 배양액내 L-ascorbic acid와 selenium을 첨가 배양했을 때 돼지 미성숙난포란의 체외 성숙율, 웅성 전핵 형성율 그리고 돼지 체외수정란의 배발달율을 향상 시킬 수 있으리라 사료된다.

• 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%).

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.47-47
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• 2003

The present study was conducted to determine the expression of the antioxidant enzyme(CuZn-SOD, Mn-SOD and GPX and apoptosis gene(caspase-3) for in vitro culture in in vitro maturation and in vitro fertilization(IVM/IVF) embryos in porcine. Porcine embryos derived from IVM/IVF were cultured in NCSU23 medium under 5% $CO_2$ in air at 38.5$^{\circ}C$. The patterns of gene expression for several antioxidant enzyme and apoptosis genes during preimplantion porcine embryo development were examined by the modified semi-quantitative single cell reverse transcriptase- polymerase chain reaction (RT-PCR). Preimplantation porcine embryos produced by IVM/IVF have expressed mRNAs for CuZn-SOD and GPX, whereas transcripts for Mn-SOD have not detected at any developmental stages. Expression of caspase-3 mRNA was detected at 2 cell, 8 cell, 16 cell and morula stages. The fas ligand transcripts were detected in porcine blastocyst. These results suggest that various antioxidant enzymes and apoptosis genes play crucial roles in in vitro culture of porcine IVM/IVF embryos.

• 한국가축번식학회지
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• 제21권4호
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• pp.355-362
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• 1997

This study was undertaken in an effort to product embryos through in vitro maturation(IVM), in vitro fertilization(IVF) and in vitro culture(IVC) after cryopreservation of immature and mature porcine oocytes. The experiments were conducted to investigate IVM rate of oocytes frozen with 3 different cryoprotectants and to examine IVF and IVC of frozen-thawed oocytes. The CEI(cumulus cells expansion index) after IVM of frozen-thawed immature oocytes was higher in oocytes frozen with PG＋PEG(propylene glycol plus polyethylene glycol) than those frozen with single cryoprotectant and this index was almost 90% of unfrozen oocyte's index(2.39 vs. 2.66). The IVF rate of all frozen oocytes was very low(68% of unfrozen oocytes) and the IVF rate of frozen immature oocytes was slightly higher than that of frozen mature oocytes(39.0% vs. 34.4%), but polyspermic penetration was higher in frozen immature oocytes(21.9% vs. 19.1%). The cleavage rate after IVF of frozen-thawed oocytes was 9.3% for frozen mature oocytes and 11.3% for frozen immature oocytes and this rate was significantly lower(P<0.05) than that of control(60.7%). The development to 8-cell stage was greatly lower in frozen mature oocytes than in frozen immature oocytes. The results indicate that the use of PG plus PEG as cryoprotectant may be very effective for vitrification of porcine oocytes and the frozen-thawed immature porcine oocytes can be used fro in vitro embryo production based on IVM, IVF and IVC system.

• 한국가축번식학회지
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• 제19권4호
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• pp.323-329
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• 1996

미성숙난자 성장과 핵성숙을 성공적으로 유도할 수 있는 체외 배양체계의 개발은 난자발생과정 등 기초 생리학 연구에 도움을 줄 뿐만 아니라, 산업적으로 필요한 난자를 공급해 줄 수 있다는 데 그 의의가 있다. 최근 보고에 따르면 성장 중에 있는 생쥐 난포란은 체외배양과 체외수정과정을 거쳐 산자 생산가지 가능하지만 돼지 난포란 성숙을 위해서는 좀더 고려해야 할 사항들이 있다. 즉, 난성숙 배양액에 낮은 농도의 FSH 첨가는 난자와 과립세포의 생존성을 유지시키고, Hypoxanthine의 첨가는 난성숙율의 향상에 도움을 준다고 한다. 따라서 금후 생쥐난자에서 얻어진 결과들을 기초로 하여 돼지 난포란의 성장과 성숙에 적합한 배양체계의 개발연구에서 괄목할만한 진전이 있다면 대가축들의 난포란 성숙기술을 개발하는데 큰 도움을 줄 것이라고 본다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2005년도 제5회 발생공학 국제심포지엄 및 학술대회
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• pp.87-88
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• 2005