• The Korean Journal of Zoology
• /
• v.34 no.3
• /
• pp.389-393
• /
• 1991

To obtain monozygotic multiplets from 8-cell mouse embryos, we artificially constructed chimeric embryos by introducing one blastomere (donor) of 8-cell embryos of Fl hybrid (C57BL/6 X CBA) mice into 4-cell ICR mouse embryos (carrier) of which one blastomere had been previously removed with a micromanipulator. After 42 h of culture, the developmental frequency of chimeric embryos to normal morula and blastocyst was 95% (310/328). When chimeric embryos at morula or blastocvst stage were transferred to pseudopregnant mice,39%, (70/180) of them were born. Most of the offspring (56/70) were the carrier type in coat color, whereas only three of them were the donor type, of which ho were assumed to be derived from single 8-cell donor embryo. Because the two donor type mice Ivere the same sex and produced only the donor type offspring from a testcross, they are probably monozvgotic multiplets of 8-cell mouse embryos. However, since their internal chimerism was not able to be examined, it remains to be determined if their genetic constitutions are identical.

• Clinical and Experimental Reproductive Medicine
• /
• v.24 no.3
• /
• pp.347-353
• /
• 1997

This study was to test whether in vitro/in vivo survival of vitrified mouse blastocysts was influenced by culture conditions and ET method. Mouse blastocysts were obtained from in vitro fertilization and cultured for 4 days in M16 medium, and they were vitrified in EFS40 which contained 40% ethlyene glycol, 18% Ficoll and 0.5 mol sucrose in PBS. In experiment I, in vitro and in vivo survival rate of these embryos were evaluated in different culture condition after thawing. When thawed embryos were cultured in M16 medium as a control, m-CR1 medium contained 20 amino acids (2% BME amino acis and 1% MEM non-essential amino acids solution) and 4 mg/ml BSA and cumulus monolayer cell co-cultured condition in mCR1 medium (10% FBS), their in vitro survival at 24 hr after thawing was not affected by culture condition (75.6, 83.1, 82.4%). However, in vivo survival rates of implantation in m-CR1 medium (80.4%) were significantly higher than those of M16 medium (51.2%), co-culture (57.1%) condition, although there was no difference in live fetuses rates on day 15 gestation (39.0, 49.0, 38.1%). In experiment II, the in vivo development potential of embryos by ET methods was examined. When blastocysts were transferred to the day 2, 3 pseudopregnant recipient without culture soon after thawing, no pregnant recipient was obtained on the day 2 pseudopregnancy, and 50% of pregnancy rates and 15.4% of live fetus rates were obtained on the day 3 pseudopregnant recipients. These results were significantly lower than those of transferred group (day 3 pseudopregnant recipients) after culture for 16 hr post thawing (73.5, 57.1%) (p<0.05). In experiment III, to elevate usability of delayed embryos in vitro/in vivo survival of vitrified embryos (day 4 early, day 5 early and expanding blastocyst) were examined. in vivo survival rates (live fetus, total implantation) were higher in day 4 early blastocysts (33.3, 66.7%) than in day 5 expanding blastocysts (29.0, 38.7%), although the highest in vitro survival rates were obtained in the day 5 expanding brastocysts (78.3%). Therefore, these results suggest that the in vitro/in vivo survival rates of vitrified embryos could be improve by the culture condition and ET method and that the in vivo development rates of delayed embryos were decreased with longer culture duration in vitro. It means that more effective cryopreservation was obtained in day 4 early blastocysts than in day 5 expanding blastocysts.

• Reproductive and Developmental Biology
• /
• v.31 no.2
• /
• pp.109-113
• /
• 2007

This study was conducted to evaluate the effect of cytochalasin B (CB) treatment in the activation medium on the development of somatic cell nuclear transfer (SCNT) rat embryos. Fetal fibroblast cells were isolated from a Day 14.5 fetus, and the oocytes for recipient cytoplasm were recovered from 4-week old Sprague Dawley rats. After enucleation and nuclear injection, the reconstructed oocytes were immediately exposed to activation medium consisting of 10 mM $SrCl_2$ with or without CB for 4 hr, and formation of pseudo-pronucleus (PPN) was checked at 18 hr after activation. Then, they were transferred into day 1 pseudopregnant recipients (Hooded Wistar) or cultured for 5 days to check their developmental competence in vivo or in vitro. The number of PPN was not affected by CB treatment during the activation. However, CB treatment supported pre-implantation development of rat SCNT embryos. Embryos generated by the procedures of SCNT were also capable of implanting, with 1 implantation scar found from a recipient following the transfer of 87 SCNT embryos to four foster mothers. The result of the present study shows that rat SCNT embryo can develop to post-implantation stage following treatment with CB.

• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.30-30
• /
• 2003

The present study examined the developmental ability of embryonic stem (ES) cells aggregated with mouse parthenogenetic embryos. Oocytes obtained from superovulated female mouse (BCF1) were treated with 7% ethanol and 5 $\mu\textrm{g}$/$m\ell$ cytochalasin B (CB) for producing pathenotes and in vitro fertilized with fresh sperm for producing normal embryos. The reporter vector (pNeoEGFP) were inserted into ES cells (129S4/svJae) by electroporation. At the 8-cell stage, in vitro fertilized embryos and pathenotes, which the zona pellucida was removed, were co-cultured with 5~10 ES cells for 4 hr. After in vitro fertilized embryos and parthenotes aggregated with ES cells were incubated to blastocyst stage, and these blastocysts transferred into the uterus of pseudopregnant recipients. The fertilized embryos aggregated with ES cells were successfully developed to offspring, but the parthenotes aggregated with ES cells failed to develop offsprings. However, genomic DNA of ES cells was detected in the pathenogenetic fetus by polymerase chain reactions at 15 day post gestation. In this study, results indicated that parthenotes aggregated with ES cells showed possible development to fetus. In the future, this method may help to produce transgenic chimera from parthenotes aggregated with ES cells.

• Journal of Embryo Transfer
• /
• v.8 no.1
• /
• pp.13-19
• /
• 1993

These experiments were carried out to determine the effect of pregnancy in bisected embryo. The embryos of ICR mouse were microsurgically bisected at morula and blastocyst stage using microsurgical blade attached a micromanipulator. These bisected embryos without zona pellucida were cultured up to blastocyst stage and cell count and diameter of stained blastomere, and transferred pseudopregnant mice. And the development of these bisected embryos was compared with the results of production of young of the corresponding intact embryos or cell stage. When the bisected mouse embryos were cultured in vitro for 20 to 24 hours in morula stage(77.2%) or 3 to 6 hours in blastocyst stage(84.1%), them were developed to the expanded blastocyst stage. There were no significant(P<0.05) differences in the development rate of bisected embryos between in morula and blastocyst stages. The embryo size of blastocyst developed in vitro from bisected embryo was small(P<0.05)than intact embryo. However, the number of blastomeres with bisected embryo (24.7+1.3and 21.5+1.2 respectively) were significantly(P<0.05) reduced, compared with that of intacted embryos(36.3+1.1 and 41.4+1.2 respectively). When compared with the result of pregnancy rate(63.6%) after surgical transfer of bisected morulae, a similar result(65.4%) was obtained with bisected blastocyst stage(P< 0.05). However, production of youngs (38.8%) after transfer of bisected morula, a similar result (38.1%) was obtained with bisected blastocyst stage (P<0.05).

• Korean Journal of Animal Reproduction
• /
• v.16 no.3
• /
• pp.217-224
• /
• 1992

These studies were carried out ot investigate on the transferred embryo development following ultrarapid frozen for 8-cell and morula of in vitro fertilization mouse embryos. The post-thaw embryo survival was evaluated and compared by cell stage of embryos and by equilibration time before ultrarapid freezing. The results obatined were summerized as follows: 1. The effects of equilibration time of 3 vs. 6 minutes before ultrarapid freezing and after thawing on the morphological survival and the viability of 8-cell and morulas embryos were not significant. 2. When the ultrarapid frozen-thawed 32 eight-cell and 33 morula embryos, and 30 fresh blastocysts were transferred to pseudopregnant recipient mice, the number of normal offsprings produced were 9(28.1%), 14(42.4%) and 18(60.0%), respectively. From the above resutls, it was concluded that the optimal conditions of pH osmolality of the media for mouse IVF and embryo culture, and the period of sperm preincubation might be 7.1, 310 mOsm and 120 min., respectively,a nd somewhat high conception rate might be resulted from transfer of frozen embryos of morula stage and fresh embryos of blastocyst stage.

• The Korean Journal of Zoology
• /
• v.34 no.4
• /
• pp.479-490
• /
• 1991

포유류 배아의 착상기전을 규명할 목적의 일환으로 자궁내막조직의 탈락막 형성과 분화에 미치는 난소 스테로이드 호르몬의 영향과 인위적 자극등의 영향을 조사하였다. 가임신을 유도시킨 흰쥐와 정상임신군의 자궁내막조직을 착상부위와 비 착상부위로 분리하여 자궁내막조직의 분화에 지표가 되는 Alkaline phosphatase(ALPase)의 활성을 측정하였다. 가임신군에서 인위적 자극(trauma)을 가한 자궁이 자극을 받지않은(control) 자궁에 비해 Progesterone(P)만을, 또는 progesterone과 estradiol(E + P)을 동시 처리받은 실험군에서 ALPase 활성이 유의한 차이(p<0.01)로 높게 나타났다. 정상임신군에서는 착상시기인 임신 제6일군의 착상부위에서 I처리군과 P처리군의 ALPase활성이 비착상 부위에 비해 유의한 차이(P < 0.05)로 크게 나타나며, 특히 임신 제6일군에서는 착상, 비착상 부위의 P처리군의 활성이 Intact군보다 유의하게(P<0.05) 높아졌다. 정상임신 제 9일 Intact군의 ALPase활성은 임신 제3일, 6일의 Intact 군의 활성에 비하여 착상(3, 6일 :P<0.01), 비 착상부위 (3일 :P<0.05, 6일 :P<0.02)에서 다같이 유의한 차이로 높아졌다. 본 연구결과에서 배아가 분화, 발생해 감에따라 자궁내막조직 분화도 활발해지고, 착상부위 분화는 착상시기부터 현저해지며, 특히 P호르몬의 영향이 크다는 것과 배아 아닌 인위적 자극으로도 탈락막 형성 유도가 가능하다는 것을 확인할 수 있었다.

• Clinical and Experimental Reproductive Medicine
• /
• v.47 no.2
• /
• pp.85-93
• /
• 2020

The implantation process is highly complex and difficult to mimic in vitro, and a reliable experimental model of implantation has yet to be established. Many researchers have used embryo transfer (ET) to assess implantation potential; however, ET with pseudopregnant mice requires expert surgical skills and numerous sacrificial animals. To overcome those economic and ethical problems, several researchers have tried to use outgrowth models to evaluate the implantation potential of embryos. Many previous studies, as well as our experiments, have found significant correlations between blastocyst outgrowth in vitro and implantation in utero by ET. This review proposes the blastocyst outgrowth model as a possible alternative to animal experimentation involving ET in utero. In particular, the outgrowth model might be a cost- and time-effective alternative method to ET for evaluating the effectiveness of culture conditions or treatments. An advanced outgrowth model and further culture of outgrowth embryos could provide a subtle research model of peri- and postimplantation development, excluding maternal effects, and thereby could facilitate progress in assisted reproductive technologies. Recently, we found that outgrowth embryos secreted extracellular vesicles containing specific microRNAs. The function of microRNAs from outgrowth embryos should be elucidated in further researches.

• Journal of Animal Reproduction and Biotechnology
• /
• v.36 no.4
• /
• pp.247-252
• /
• 2021

Embryo transfer (ET) in the animal is an important procedure to generate genetically engineered animals and conserve genetic resources. For ET experiments in mice, pseudopregnant recipients are usually prepared with proestrus stage of females and vasectomized males. However, this conventional method is inefficient because the size of female colonies should be large to select only the proestrus stage in the estrous cycle and the surgical procedures are required to generate vasectomized males. In this study, we established a simple and efficient protocol to prepare ET recipients using the estrous synchronization with hormone injection and the mating with wild male mice. The delivery rate of ET recipients tended to be increased with estrous synchronization using hormone injection (100%) compared to the conventional method (71%). Further, natural pregnancy of the recipients, induced by mating with a wild male, significantly enhanced the birth rate of ET offspring than the conventional method (33% vs. 13%). Based on the results, we concluded that our new protocol using hormone injection to ET recipients and mating with wild males could be more efficient and simpler compared to the conventional method.

• Proceedings of the Korean Society of Developmental Biology Conference
• /
• 2003.10a
• /
• pp.121-121
• /
• 2003

The standard protocol for the production of transgenic mouse from ES-injected embryo has to process via chimera producing and several times breeding steps, In contrast, tetraploid-ES cell complementation method allows the immediate generation of targeted murine mutants from genetically modified ES cell clones. The advantage of this advanced technique is a simple and efficient without chimeric intermediates. Recently, this method has been significantly improved through the discovery that ES cells derived from hybrid strains support the development of viable ES mice more efficiently than inbred ES cells do. Therefore, the objective of this study was to generate transgenic mice overexpressing human resistin gene by using tetrapioid-ES cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR and cloned into pCR 2.1 TOPO T-vector and constructed in pCMV-Tag4C vector. Human resistin mammalian expression plasmid was transfected into D3-GL ES cells by lipofectamine 2000, and then after 8~10 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec. (fusion rate : 93.5%) and cultured upto the blastocyst stage (development rate : 94.6%). The 15~20 previously G418-selected ES cells were injected into tetraploid blastocysts, and then transferred into the uterus of E2.5d pseudopregnant recipient mice. To investigate the gestation progress, two El9.5d fetus were recovered by Casarean section and one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, this finding demonstrates that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mouse for the rapid analysis of gene function in vivo.