• 제목/요약/키워드: Embryo-fetal development study

검색결과 96건 처리시간 0.023초

돼지 난포란 유래 체외수정란 생산에 대한 제요인의 영향 IV. 체외발달 배양액의 종류와 배양액 교체가 체외발달에 미치는 영향 (Effects of Some Factors on In Vitro Production of Embryos from Antral Follicle-Derived Porcine Oocytes IV. Effects of Development Media and Those Change on In Vitro Development)

  • 연성흠;최선호;조창연;한만희;손동수;이규승
    • 한국수정란이식학회지
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    • 제19권3호
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    • pp.275-282
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    • 2004
  • 본 연구는 체외성숙/체외수정 유래의 돼지 난자를 이용하여 체외발달시 배양액의 종류나 교체에 따른 영향을 구명하고자 수행하였다. mNCSU-23에서 체외성숙시킨 다음 mTBM에서 체외수정시킨 난자를 목적에 따라 두 가지로 나누어 실험한 결과는 다음과 같다. 1. 체외성숙/체외수정란을 NCSU-23에서 배양액 교체없이 7일 동안 배양하거나 CZB에서 4일 배양한 다음 Pig-MEM으로 옮겨서 나머지 3일간 배양한 결과, 난분할율은 배양액간 차이를 보이지 않은 반면, 추정수정란대 배반포 발달을(P<0.05)과 분할란대 배반포 발달율은 NCSU-23에서 배양된 것이 유의적으로 높았다(P<0.01). 그러나 배 반포의 ICM 세포수, TE 세포수 및 총세포수에서는 모두 차이가 없었다. 2. 체외성숙/체외수정란를 NCSU-23에서 배양액 교체없이 7일 동안 배양하거나 체외배양 5일째에 신선한 동일 배양액이나 0.4% BSA를 10% FBS로 대체한 배양액(mNCSU-23F)으로 완전히 교체하여 배양한 결과, 난분할율, 배반포발달율, 배반포의 ICM 세포수, TE 세포수 및 총세포수 모두 처리간 유의적인 차이를 보이지 않았다. 결과적으로, NCSU-23이 CZB/Pig-MEM보다 체외성숙/체외수정 유래의 난자를 체외발달시키는데 적합한 것으로 사료되며, 체외발달배양 과정에 신선한 배양액이나 일부 변경된 배양액으로의 교체에 대해서는 더 많은 연구가 필요할 것으로 사료된다.

Glucose와 Phosphate가 제거된 M-TALP 배지에서의 난구세포 공배양에 의한 임신율 향상에 관한 연구 (Improvement of Pregnancy Rates by Coculture of Human Embryos with Cumulus Cells in Glucose and Phosphate Free M-TALP Media)

  • 정범식;장우현;이문희;김지연;방지호;김규현;서태광
    • Clinical and Experimental Reproductive Medicine
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    • 제26권1호
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    • pp.75-81
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    • 1999
  • The beneficial effect of glucose and phosphate ions in culture medium on the development of human embryos in vitro has not been fully elucidated. The purpose of this study was to evaluate the influence of fertilization and culture of embryos in glucose/phosphate-free m-TALP medium on pregnancy rates in IVF-ET program. The patients in 244 IVF-ET cycles received GnRH agonist + HMG regimens. A does of 10,000 IU HCG was administered when two or more dominent follicles reached 18mm in diameter. Thirty-six hours after HCG, oocytes were recovered transvaginally using ultrasound guidance. Aspirated oocytes were matured for 4 to 6 h in TCM-199 supplemented with 10% follicular fluid (FF). Insemination was carried out with 50,000 motile spermatozoa in TCM-199 + 10% FF or m-TALP + 5% FF + 5% fetal cord serum (FCS) according to experimental design. After 6 h, oocytes were washed 3 to 4 times and cultured in each fresh medium. After 20 h, oocytes were freed from cumulus/corona cells and examined for the presence of pronuclei. Fertilized oocytes were transferred into each co-culture drops and cultured for further incubation. On day 3, embryo transfer was performed with grade 1 and 2 embryos. Monolayers for co-culture of embryos were prepared by plating $1{\times}10^5$ cumulus cells/ml in 10ul drop of TCM-199 + 10% FF or m-TALP + 5% FF + 5% FCS media 24 h prior to the onset of co-culture. Development to 4 to 16 cell stage was observed at 70x magnification following two days of incubation. Pregnancy was confirmed by detecting increasing serum ${\beta}$-hCG concentrations for 11 days following embryo transfer. Data were analyzed by ${\chi}^2$-test. Oocytes from 244 IVF-ET cycles were randomized. The number of cycles and mean age of patients were 97 and 147, 31.3 yrs and 31.2 yrs for TCM-199 (control) and m-TALP groups, respectively. The mean number of retrieved oocytes/cycle, fertilization rates, number of embryos transferred/ET and pregnancy rates were 11.1 and 10.3, 65.1% and 67.3%, 4.1 and 4.7, 28.9% and 43.8% for TCM-199 and m-TALP groups, respectively. Differences in the pregnancy rates were found between control and m-TALP groups (p<0.05). The pregnancy rate of patients divided according to maternal age groups of ${\leq}30$, 31-35, $36{\leq}$ were 44.4% and 49.0%, 26.1% and 41.3%, 29.2% and 41.2% for control and m-TALP groups, respectively. These data indicate that culture of human embryos in glucose/phosphate-free m-TALP medium improves pregnancy rates.

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한우 난소의 채란방법이 회수율, 배발달율 및 수태율에 미치는 영향 (Effect of Collective Methods on the Collection Efficiency, Blastocyst and Pregnancy Rate after IVP Embryo Transfer in Hanwoo)

  • 이정우;정수용;손병훈;한기호;오인석;서현준;공일근
    • 한국수정란이식학회지
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    • 제20권1호
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    • pp.55-62
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    • 2005
  • 도축장에서 회수한 한우 난소로부터 난자를 회수하기 위한 방법으로 흡입법 후 세절법과 흡입법으로 난자를 회수하여, 난자의 회수율과 채란된 난자를 체외수정 후 발달율과 수정란 이식 후 수태율에 영향을 조사한 결과는 다음과 같다. 1. 난자 회수율은 각 난소당 회수된 난자수는 흡입 후 세절법이 8.2개, 흡입법이 6.5개로서 흡입 후 세절법을 병용하는 것이 난자 회수율에서 유의적으로 많았다. 2. 채란방법에 따른 체외수정란의 분할율은 흡입 후 세절법이 $75.8\%$, 흡입법은 $84.4\%$로서 유의적이 차이를 보이지 않았다. 3. 배반포 발달율은 흡입 후 세절법이 $28.3\%$, 흡입법은 $22.8\%$로서 흡입 후 세절법이 유의적으로 높았다. 4. 난소당 배반포수에서는 흡입 후 세절법이 1.8개로서 흡입법의 1.1개보다 유의적으로 많은 배반포수를 생산할 수 있었다. 5. 채란별 수태율 조사 결과 흡입 후 세절법이 $54.4\%$, 흡입법이 $62.5\%$로서 흡입법으로 채란된 난자로부터 얻어진 수정란을 이식하였을 때 높은 수태율을 얻을 수 있었다. 6. 경산우와 처녀우에 수정란이식 후 수태율은 경산우는 흡입법 후 세절법이 $54.4\%$, 흡입법은 $62.5\%$ 나타났고, 처녀우는 흡입법 후 세절법이 $58.1\%$, 흡입법은 $68.2\%$로서 경산우와 처녀우에 관계없이 흡입법으로 채란한 난자로부터 생산된 수정란을 이식하였을 때 수태율이 유의적으로 높은 결과를 얻었다. 이상의 결과에서 채란방법에 따른 난소당 이용 가능한 난자의 회수율은 흡입 후 세절법을 이용함으로서 많은 난자와 수정란을 생산할 수 있었다. 그리하여 한정된 난소를 효율적으로 활용하기 위해서는 흡입 후 세절법을 이용하는 것이 전체적인 효율 향상에 도움이 될 것으로 판단된다.

Effect of supplement of SCM in culture medium for in vitro development of bovine in vitro fertilized oocytes

  • Sang Jun Uhm
    • 한국동물생명공학회지
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    • 제38권3호
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    • pp.143-150
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    • 2023
  • Background: The successful production of superior or transgenic offspring from in vitro produced embryos in cattle relies heavily on the quality of blastocyst stage embryos. In order to enhance the developmental competency of these embryos, a novel culture method was devised. Methods: This study utilized stem cell culture medium (SCM) from hESCs as a supplement within the culture medium for bovine in vitro produced embryos. To gauge the efficacy of this approach, in vitro fertilized embryos were subjected to culture in CR1aa medium enriched with one of three supplements: 0.3% BSA, 10% FBS, or 10% SCM. Results: The blastocyst development and hatching rates of one-cell zygotes cultured in CR1aa medium supplemented with SCM (23.9% and 10.2%) surpassed those cultured in CR1aa medium supplemented with BSA (9.3% and 0.0%) or FBS (3.1% and 0.0%) (p < 0.05). Furthermore, post-zygotic gene activation, cleaved embryos cultured in CR1aa medium supplemented with SCM (57.8% and 34.5%) exhibited notably higher rates (p < 0.05) compared to those cultured with BSA (12.9% and 0.0%) or FBS (45.7% and 22.5%) supplementation. Furthermore, the microinjection of SCM into the cytoplasm or pronucleus of fertilized zygotes resulted in elevated blastocyst development and hatching rates, particularly when the microinjected embryos were subsequently cultured in CR1aa medium supplemented with SCM from the 8-cell embryo stage onwards (p < 0.05), in contrast to those cultured with FBS supplementation. Conclusions: In conclusion, this study conclusively demonstrated that the incorporation of SCM into the culture medium significantly enhances the developmental progress of preimplantation embryos.

hFSH 유전자가 도입된 소 태아섬유아세포를 이용한 형질 전환 복제 수정란의 발달 (Development of Transgenic NT Embryos Using Bovine Fetal Fibroblasts Transfected with hFSH Gene)

  • 양병철;임기순;김동훈;민관식;윤두학;박효숙;김세웅;황인선;서진성;성환후;양보석
    • 한국수정란이식학회지
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    • 제21권1호
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    • pp.13-20
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    • 2006
  • 본 연구의 목적은 요를 통해 hFSH를 발현하는 형질 전환 소의 생산이다. 요의 분비와 관련 있는 유전자로서 mUII promoter를 사용하여 hFSH유전자를 구성했다. 태아섬유아세포(KbFF)는 임신 45일령의 태아(male)에서 채취하였다. hFSH gene은 pcDNA3(neo) vector와 같이 KbFF 세포에 electroporation 방법으로 transfection하였다. 유전자를 transfection한 세포는 G-418로 2주 동안 배양하였고, 선발된 colony는 PCR로 확인하였다. 핵이 제거된 난자는 hFSH가 transfection된 세포와 transfection 되지 않은(control) 세포를 이용하여 핵이식하였다. 48시 간 후 hFSH가 transfection된 세포는 68.7%의 수정란이 난할되었으며, 8일 후 15.7%의 수정란이 배반포로 발달하였다. 그러나 대조구에서는 67.6%가 난할되었으며, 24.5%가 배반포로 각각 발달하였다. 이들 배반포에서 apoptosis 분석 결과 hFSH 유전자가 transfection된 또는 transfection되지 않은 대조구에서 유의적인 차이는 보이지 않았다. 배반포는 53두의 수란우에 이식하여 두 마리의 산자가 생산되었으나(1.9%) hFSH가 transfection되지 않은 것으로 나타났다. 이 결과는 선발된 hFSH colony에서 transfection되지 않은 세포가 혼합되어 있었다는 것을 나타내 주고 있으며, colony의 선발과 검증에 더 많은 연구의 필요성이 있음을 나타내준다.

Effect of Taxol Pre-treatment to In Vitro Matured Bovine Oocytes on Spindle Morphology and Embryonic Development Following Vitrification

  • Park, Sang-Hyoun;Yu, Il-Jeoung
    • 한국수정란이식학회지
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    • 제23권4호
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    • pp.269-274
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    • 2008
  • The purpose of this study was to determine the effects of Taxol pre-treatment to in vitro matured bovine oocytes, and sucrose and trehalose added to vitrification solution on spindle morphology and embryonic development following cryopreservation. Bovine oocytes were collected from ovaries and matured in tissue culture medium 199 (TCM 199) supplemented with 10% Fetal Bovine Serum (FBS), 0.05ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone and $1{\mu}g/ml$ estradiol for 22h in $39^{\circ}C$, 5% $CO_2$, TCM 199-HEPES containing 20% FBS was used as basic medium (BM) to prepare vitrification solution. Oocytes were pre-treated with $1\;{\mu}M$ Taxol in maturation medium for 15 min prior to vitrification. Oocytes were exposed to 1.6 M ethylene glycol (EG) and 1.3M dimethyl sulfoxide (DMSO) in BM and then were exposed to 3.2 M EG, 2.6 M DMSO and 0.5 M sucrose in BM or 3.2 M EG, 2.6 M DMSO and 0.5 M trehalose in BM. Oocytes with cumulus cells and oocytes without cumulus cells were considered as control 1 and control 2, respectively and held in TCM 199-HEPES at $39^{\circ}C$. Oocytes were frozen using modified solid surface vitrification and were stored in cryotubes in liquid nitrogen for more than 1 week. Frozen oocytes were thawed in TCM 199-HEPES containing 0.5 M, 0.25 M and 0.1 M sucrose in BM for 2 min, respectively or 0.5 M, 0.25 M and 0.1 M trehalose in BM for 2 min, respectively. Immunoflurorescence staining of oocytes was performed to assess spindle morphology and chromosome configuration of oocytes. The rates of cleavage and blastocyst were examined following in vitro fertilization. Normal spindle morphology rate of oocytes pre-treated with Taxol prior to vitrification was not higher than that of other vitrified groups. Taxol pre-treatment did not increase cleavage and blastocyst formation rates, although control groups showed significantly higher rates (p<0.05). Percentages of normal spindle and embryonic development were not significantly different among vitrified groups regardless of type of sugar. In conclusion, Taxol pre-treatment of oocytes before cryopreservation did not reduce the damage induced by vitrification and subsequently did not improve embryonic development following vitrification. Trehalose may be used as an alternative non-permeating cryoprotectant in vitrification solution.

태아 섬유아세포로 핵치환된 소 난자의 핵의 재구성과정과 체외 배발달 (Nuclear Remodeling and In Vitro Development of Bovine Oocytes Following Nuclear Transfer of Bovine Fetal Fibroblasts)

  • Um, J. H.;S. J. Uhm;Kim, N-H;Lee, H. T.;K. S. Chung
    • 한국가축번식학회지
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    • 제24권1호
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    • pp.59-67
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    • 2000
  • 본 연구는 핵이 제거된 소난자 내에 소의 태아 섬유아세포를 이식한 후 난자의 발달능력을 조사하였다. 소 태아 섬유아세포는 45일된 응성 태아로부터 분리한후 MitoTracker로 염색하고 세포 주기를 동기화시키지 않은 세포를 핵이 제거된 소난자의 위란강 내에 이식하였다. 섬유아세포와 소난자의 복합체는 전기 자극을 주어 융합시키는 방법을 이용하였으며, 융합된 난자는 Calcium ionophore와 6-DMAP를 이용하여 난활성을 유도시킨 다음 CR1aa 에서 배양하였다. 핵치환 된 난자의 핵은 핵질 응축과정, 팽대과정, 전핵 형성과정이 일어났으며, 이러한 과정에서 재구성된 난자는 분열 과정을 거쳐 18∼26시간 사이에는 2-세포기로 발달하였다. 섬유아세포의 미토콘드리아는 핵치환시 난자 내로 이전되었는데 이것들은 난자 내에서 빠르게 사라지는 것으로 관찰되었으며, 핵융합 후 8시간째에는 섬유아세포의 미토콘드리아가 전혀 관찰되지 않았다. 2-세포기로 분열된 난자의 21% 가 이식 가능한 단계인 배반포 단계까지 발달하였다. 이러한 결과는 소의 태아 섬유아세포가 탈핵된 소난자 내에서 성공적으로 재분화과정을 거치며, 이렇게 재조합 된 수정란은 배반포 단계까지 발달이 가능하다는 것을 보여주고 있다.

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생식보조시술시 단백질원으로서 인간난포액의 적합성 및 효율성에 관한 연구;III. 인간난포액이 생식보조시술시 임신율 향상에 미치는 효과 (Studies on the Suitability and Efficiency of Human Follicular Fluid as Protein Supplement in Assisted Reproductive Technology(ART);III. Effect of Human Follicular Fluid on Improvement of Pregnancy Rates in ART)

  • 구정진;지희준;김동훈;김지연;장상식
    • Clinical and Experimental Reproductive Medicine
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    • 제23권1호
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    • pp.103-108
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    • 1996
  • Through the previous studies(I,II), it was observed that human follicular fluid(HFF) was more effective than human fetal cord serum(HFCS) on promoting meiotic resumption of oocytes and improving embryonic development of mouse in vitro. On the basis of these results, we have gradually exchanged HFCS with HFF as protein supplement in human ART. This study was performed to investigate the efficiency of HFF on improving the pregnancy rate in ART. Oocytes were retrieved transvaginally from patients treated with pituitary suppression with GnRH-agonist and ovarian stimulation with human menopausal gonadotro-pin(HMG) and pure follicle stimulating hormone(FSH). Aspirated oocytes were rinsed and cultured in TCM-199 containing HFF, and the concentrations of HFF were adjusted to 10, 20, and 30% according to the use for insemination, embryo growth and embryo transfer, respectively. As possible as, we tried to do embryo transfer into fallopian tube to mimic the coincidence of the cell stage with the place of sojourn in vivo, so we performed various ART programs(IVF & ET; in vitro fertilization, ZIFT; zygote intra fallopian-tube transfer, ZIFT & ET) according to the tubal conditions of patients. On the while, intra cytoplasmic sperm injection(ICSI) was used to assist IVF of the patients who had shown poor standard IVF results by immunological or severe male factor. Of the 255 cycles of ART programs using HFF as protein supplement, 118 cycles were turn out to be succeeded in pregnancy(46.2%, per cycle, p<0.05), while 21 pregnancies were achieved in the 69 cycles using HFCS(30.4%). The 255 cycles using HFF were subdivided into cycles with the type of ART programs, and each pregnancy rate of the ART programs were 44.7% (IVF & ET, 76/170 cycles), 53.4%(ZIFT, 31/58 cycles) and 40.7% (ZIFT & ET, 11/27 cycles), respectively. In the 61 ICSI cycles using HFF, 28 cycles succeed in pregnancy(45.9%), while 7 pregnancies were obtained in the 17 ICSI cycles using HFCS. Also the ongoing pregnancy rate in the group using HFF(78.8%, 93/118 cycles) was higher than that in the group using HFCS(61.9%). Therefore, we found that the use of HFF as protein supplement was more suitable and effective than the use of HFCS to improve the pregnancy rate in ART.

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토끼의 체외배양 난자를 이용한 핵이식으로 복제수정란 및 복제산자의 생산 (Production of cloned Rabbits Embryos and Offsprings by Nuclear Transplantation using In Vitro Matured Oocytes in Rabbits)

  • 박충생;전병균;이경미;윤희준;이효종;곽대오;최상용
    • 한국수정란이식학회지
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    • 제10권1호
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    • pp.65-72
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    • 1995
  • The purposes of this study were to produce cloned rabbit embryos and offsprings by nuclear transplantation(NT) using in vitro matured oocytes as nuclear recipient cytoplasm and to determine the effect of frozen nuclei donor embryos on the production efficiency of cloned embryos. The 8cell embryos were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline containing 10% fetal calf serum(FCS) at 40 hours after hGG injection. A portion of collected embryos were preserved at 4$^{\circ}C$ for 24 hours and a portion of them were frozen by vitrification method. The embryos used for donor nuclei were synchronized in the phase of Gi /S transition. The in vitro matured oocytes were used as recipient cytoplasm following removing the nucleus and the first polar body. The synchronized blastomeres from fresh, cooled or frozen embryos were injected into the enucleated oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.0 W /cm in 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$incubator. Following in vitro culture of the NT embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The results obtained were summarized as follows: 1. The fusion rates of the blastomeres from fresh, cooled and frozen embryos with the in vitro matured and enucleated oocytes were 100, 95.8 and 64, 3%, respectively. 2. Development in vitro to blastocyst was significantly(p<0.05) different between the cloned embryos with the blastomeres from fresh, cooled or frozen embryos as 39.0, 20. 9 and 15.7%, respectively. 3. The mean numbers of cell cycle per day during in vitro culture of cloned embryos blastomeres from fresh, cooled or frozen embryos was 1.31, 1.29 and 1.16, respectively. 4. A total of 77 nuclear transplant embryos were transferred into 6 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer.

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The Imprinted Messenger RNA Expression in Cloned Porcine Pre-implantation Embryos

  • Park, Mi-Rung;Kim, Bong-Ki;Lee, Hwi-Cheul;Lee, Poong-Yeon;Hwang, Seong-Soo;Im, Gi-Sun;Woo, Jae-Seok;Cho, Chang-Yeon;Choi, Sun-Ho;Kim, Sang-Woo;Ko, Yeoung-Gyu
    • 한국수정란이식학회지
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    • 제25권2호
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    • pp.127-131
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    • 2010
  • The objective of this study was to determine the mRNA expression patterns of several putative imprinted genes in in vivo and in vitro fertilized, parthenogenetic, and cloned porcine preimplantation embryos. Both maternally (Dlk1, IGF2, Peg1/Mest and Ndn) and paternally (IGF2r, H19 and Xist) imprinted genes were selected. We have used reverse transcription polymerase chain reaction (RT-PCR) to investigate gene expression patterns in the porcine embryos. IGF2 transcripts were detected in the most of embryos. In nuclear transfer (NT), Peg1/MEST transcripts showed fluctuating pattern. Dlk1 was only expressed partially from the morula and blastocyst stage of NT embryos. Ndn gene expression was started somewhat early for in vivo embryos. However, the expressions of maternally imprinted genes were similar in all types of blastocysts (NT, in vivo and in vitro fertilized, and parthenogenetic embryos). The IGF2R gene expression level was somewhat irregular and varied among samples. However, for the majority samples of all types of embryos, IGF2R expression was diminished after one- to two-cell stages and reappeared at the morulae or blastocyst stage embryos. H19 gene was only expressed early in parthenogenetic and in vivo embryos. For NT embryos, H19 was only expressed in blastocysts. Xist expression was detected in all blastocysts with the earliest being in vivo 8-cell stage embryos and the last one being NT blastocysts. These putative imprinted genes appeared to have stage specific expression patterns with a fluctuating pattern for some genes (Peg/Mest, IGF2r, H19). These results suggest that stage specific presence of imprinted genes can affect the embryo implantation and fetal development.