• 한국수정란이식학회지
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• 제12권3호
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• pp.335-341
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• 1997

The aims of this study are to establish a stable isolation method of blastomeres from bovine early embryos and examine their developmental potential in vitro Early embryos were produced by maturation and fertilizaion in vitro of bovine follicular oocytes. Blastomeres were isolated from 2~8-cell embryos in $Ca^2$+-, $Mg^2$+-free PBS+EDTA after removing the zonae pellucidae Isolated blastomeres were cultured in CZB containing BOEC for upto 240 hpi. Cleavage rates of them were 18.5%(10 /54) in 1 /2 blastomeres, 33.3%(16/48) in 1/4 blastomeres and 34.2%(14 /41) in 1/8 blastomeres, respectively. The rates of blastocystic vesicle formed were 8.7%(4 /46) in 1/2 blastomeres, 26.6% (17/64) in 1/4 blastomeres and 10.3%(8 /78) in 1/8 blastomeres, respectively. Blastomeres developed into various types of blastocystic vesicles and trophoblastic vesicles as evidenced by the Hoechst 33258 staining and morphology. This results suggest that the isolation method used and subsequent culture of isolated blastomeres from bovine early embryos should be useful to obtain extra embryonic cells for various analyses such as PCR and putative ES cell culture.

• 한국수정란이식학회지
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• 제14권3호
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• pp.195-201
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• 1999

This study was carried out to improve a technique of cloned animal prodcution by preactivation of nuclear recipient oocytes with ionomycin and 6-dimethylaminopurine (6-DMAP) in rabbits. The oocytes were collected from the oviduct of superovulated rabbit at 19∼20 hours post hCG injection. The collected oocytes were preactivated and self-enucleated by treating 5 uM ionoycin for 5 min, and 2.0 mM 6-DMAP for two hours. Microsurgical removel of the chromation complex in the second polar bodies was effectively performd and single blastomere separated from 32-cell stage rabbit embryos was injected into the perivitelline space of the enculeated recipient oocyts. Follwoing electrofusion and in vitro culture for 18 hours, the nuclear transplant(NT) embryos were transferred into the uterine horns of naturally mated or synchronized recipient does. When 32 NT embryous reconstituted with preactivated oocytes were transferred to 2 recipient does, one foster doe delivered two offspring (6.3%), while not a offspring was delivered from three foster does which received 17 NT embryos reconstituted with non-preactivated oocytes. A total of 68 NT embryos reconstituted with preactivated oocytes were transferred into the uterine horns of 7 synchronized ecipient does. Among them, two recipients were pregnant and delivered three offspring(5.9%).

• 한국동물생명공학회지
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• 제35권1호
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• pp.12-20
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• 2020

Cell adhesion plays an important role in the differentiation of the morphogenesis and the trophectoderm epithelium of the blastocyst. In the porcine embryo, CDH1 mediated adhesion initiates at compaction before blastocyst formation, regulated post-translationally via protein kinase C and other signaling molecules. Here we focus on muscle RAS oncogene homolog (M-RAS), which is the closest relative to the RAS related proteins and shares most regulatory and effector interactions. To characterize the effects of M-RAS on embryo compaction, we used gain- and loss-of-function strategies in porcine embryos, in which M-RAS gene structure and protein sequence are conserved. We showed that knockdown of M-RAS in zygotes reduced embryo development abilities and CDH1 expression. Moreover, the phosphorylation of ERK was also decreased in M-RAS KD embryos. Overexpression of M-RAS allows M-RAS KD embryos to rescue the embryo compaction and blastocyst formation. Collectively, these results highlight novel conserved and multiple effects of M-RAS during porcine embryo development.

• 한국가축번식학회지
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• 제20권3호
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• pp.307-313
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• 1996

This study was conducted to investigate the effect of S-phase synthronized nuclear transfer on the development of nuclear transplant bovine embryos. A blastomere derived from the 16~32 cell stage bovine embryos was transferred into an enucleated metaphase II(MII) oocytes or activated S-phase eggs. From the MII-phase and S-phase nuclear transfer, 6.3%(4/63) and 13.8%(9/65) of nuclear transplant embryos developed to the blastocyst stage, respectively. In the S-phase nuclear transfer, maximal proportion of embryos developed to the blastocyst stage(16.6%) was obtained after the recipient cell was activated 8 h prior to receving a donor nucleus. MII-phase nuclear transplant embryos showed the PCC state of their nuclear at 1.5~2 h after fusion, whereas, S-phase nuclear transplant embryos did not undergo PCC. The result of this study suggests that if blastomeres of unknown cell-cycle-stage are used, S-phase nuclear transplantation through the activation of enucleated oocytes prior to fusion enhances development of nuclear transplant embryos. This result also suggests that the interval time from oocyte activation to cell fusion may affect the development of nuclear transplant embryos.

• 한국수정란이식학회지
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• 제31권3호
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• pp.199-205
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• 2016

The aim of this study was to investigate the effect of uterine histotroph on embryo development and the expression of cysteine-rich protein 2 (CRP2), coatomer subunit gamma-2 (G2COP), myoglobin (MYG), vascular endothelial growth factor D (VEGFD), collagen alpha 4 chain (COL4) and galactoside 3-L-fucosyltransferase 4 (FUT4) proteins in porcine embryo during pre-implantation. Uterine histotroph (UH) was collected from uterine horn on corpus albican phase, and embryos were cultured in porcine zygote medium with UH for 168 hours. Cleavage and blastocyst formation of embryo were detected at 168 hours after in vitro fertilization. And CRP2, G2COP, MYG, VEGFD, COL4 and FUT4 proteins were observed using confocal laser microscope. In results, embryo cleavage rate was not significantly changed by UH, but blastocyst rate was significantly (P<0.05) decreased in UH-treated embryos. Moreover, CRP2, G2COP, MYG, VEGFD, COL4 and FUT4 proteins were expressed in blastomere. CRP2 in embryo was significantly overexpressed (P<0.05), but not G2COP, MYG, VEGFD, COL4 and FUT4 proteins. In summary, UH on corpus albican phase was increased CRP2 protein in embryo, and inhibited blastocyst formation in preimplantation porcine embryos, suggesting that CRP2 may play an interrupter on embryo development in pigs.

• Clinical and Experimental Reproductive Medicine
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• 제44권4호
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• pp.193-200
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• 2017

Objective: This study was conducted to investigate the efficacy of laser-assisted hatching (LAH) and various vitrification times for embryonic development and blastocyst cell numbers. Methods: First, 2-cell and 8-cell embryos were collected by flushing out the oviducts. In the control groups, they were vitrified for 8 or 10 minutes without LAH. The LAH groups underwent quarter laser zona thinning-assisted hatching before vitrification (4, 6, and 8 minutes or 4, 7, and 10 minutes, respectively). After incubation, double-immunofluorescence staining was performed. Results: The hatched blastocyst rate 72 hours after the 2-cell embryos were thawed was significantly higher in the 2LAH-ES8 group (33.3%) than in the other groups (p< 0.05). In the control-8 group ($22.1{\pm}4.6$), the cell number of the inner cell mass was higher than in the LAH groups (p< 0.05). The number of trophectoderm cells was higher in the 2LAH-ES6 group ($92.8{\pm}8.9$) than in the others (p< 0.05). The hatched blastocyst rate 48 hours after the 8-cell embryos were thawed was higher in the 8LAH-ES4 group (45.5%) than in the other groups, but not significantly. The inner cell mass cell number was highest in the 8LAH-ES7 group ($19.5{\pm}5.1$, p< 0.05). The number of trophectoderm cells was higher in the 8LAH-ES10 group ($73.2{\pm}12.1$) than in the other groups, but without statistical significance. Conclusion: When LAH was performed, 2-cell embryos with large blastomeres had a lower hatched blastocyst rate when the exposure to vitrification solution was shorter. Conversely, 8-cell embryos with small blastomere had a higher hatched blastocyst rate when the exposure to vitrification solution was shorter.

• 한국발생생물학회지:발생과생식
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• 제2권2호
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• pp.157-163
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• 1998

흰쥐의 배아발생 동안에 피질반응 후 배아 외부에 새롭게 형성된 피질과립막 (cortical granule envelope, CGE)이 존속하는지 여부와 투명대와 배아표면의 미세구조 변화를 조사하였다. 흰쥐배아의 투명대와 배아표면의 미세구조는 주사전자현미경으로 관찰하였으며, 피질과립막 형성과 분포는 Ulex europaeus agglutinin I lectin을 표지하여 형광현미경으로 관찰하였다. 배아표면은 미수정란 과는 다르게 배아표면의 미세융모가 단축된 특징을 보였고, 8-세포기 배아에서 뚜렷하게 볼 수 있는 CGE에 의해 덮여 있다. 투명대의 구조 역시 미수정난자에 존재하는 구조와는 다른 특징을 나타냈고, 특히 투명대의 섬유성 미세공 구조가 거칠어지고 수적인 감소가 나타났다. 위란강에 피질반응에 의한 피질과립막이 형성되어 배아발생 동안에 존속하였으나 수정란보다는 엷고 국소적인 분포양상을 나타내었다. 본 연구 결과를 종합해 볼 때 흰쥐 초기 배아 발생 동안에는 배아 외부에 피질과립막이 존속하고, 수정시에는 투명대 경화 뿐만 아니라 피질반응에 의해 투명대의 미세구조와 배아표면의 구조도 변화됨을 알 수 있다.

• Journal of Genetic Medicine
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• 제2권1호
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• pp.35-39
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• 1998

Duchenne/Becker muscular dystrophy are the major neuromuscular disorders with X-linked recessive inheritance. Preimplantation diagnosis of sex determination has been generally used to avoid male pregnancies with these diseases. However, in order to determine if the embryo is normal, carrier or affected regardless of the sex, there is a need for a combined analysis of specific exon on dystrophin gene as well as sex determination of embryo using the same biopsied blastomere. If the exon deletion is not determinable, further diagnosis of carrier or patient can be performed by haplotype analysis. In this study, we applied the primer extension preamplification (PEP) method, which amplifies the whole genome, in 40 cases of single amniocyte and 40 cases of chorionic villus cell. We analysed haplotypes using two (CA)n dinucleotide polymorphic markers located at the end of 5' and 3' region of the dystrophin gene. Exon 46 of dystrophin gene and DYZ3 on chromosome Y were chosen as a target sequence for coamplification PCR. Upon optimizing the conditions, the amplification rates were 91.25% (73/80) for haplotypes (92.5% in amniocyte, 90% in chorionic villus cell) and 88.75% (71/80) for coamplification (85% in amniocyte, 92.5% in chorionic villus cell). The result of the study indicates that haplotypes analysis and coamplification of dystrophin and Y-specific gene using PEP can be applied to prenatal and preimplantation diagnosis in Duchenne/Becker muscular dystrophy making it possible to determine if the fetus is a carrier or an affected one.

• Journal of Animal Science and Technology
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• 제47권3호
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• pp.317-324
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• 2005

A method for sex determination of pigs was examined using polymerase chain reaction(PCR). Sex determining region Y(SRY) gene encoded on Y chromosome plays a key role for primary male development. Zinc finger X-Y(ZFX-ZFY) gene, one of the X-V homology gene group was found on the X and Y chromosomes, respectively, We tested for molecular sexing by amplification patterns of SRY and ZF genes. Genomic DNAs from various resources including porcine hairs and semen collected from domestic pig breeds and native pigs was used for PCR assay of each gene. The amplified products for porcine SRY gene were yielded only in males but not in females. On the other hand, two differential patterns were observed in amplification of ZF gene reflecting the chromosomal dimorphism by a length polymorphism between X and Y chromosomes. Of both, a common band was detected in all individuals tested so that this band might be amplified from ZFX gene as a PCR template, but another is specific for males indicated that from ZFY. The result of PCR assay provides identical information to that from investigation of phenotypic genders of the pigs tested. We suggest that this PCR strategy to determine porcine sexes using comparison of the amplification patterns of the SRY gene specific for Y chromosome and the dimorphic ZF gene between X and Y chromosomes may be a rapid and precise method for discrimination of two sexes and applied to DNA analysis of small samples such as embryonic blastomere, semen, and hairs.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.96-96
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• 2003

Nek2 (NIMA-related protein) is a mammalian cell cycle-regulated kinase that involves in chromosome condensation and centrosome regulation and NuMA (nuclear mitotic apparatus protein) is involved in spindle assembly during a cell cycle. The cellular distribution and organization of the centrosomal components is completely unknown during fertilization and embryonic development. We examined distribution of two well-known centrosomal proteins, Nek2 and NuMA in mouse gametes and embryos to get an insight in the reorganization of centrosomal proteins during germ cell development and early fertilization. Spermatogenic cells, gametes, and embryos were analyzed with anti-Nek2 or -NuMA antibodies by immunological assay, RT-PCR, and overexpression through gene transfection. Mitotically or meiotically active spermatogenic cells were intensively stained with these antibodies in both centrosomes and cytoplasm, whereas the oocytes showed different staining patterns depending on the meiotic stages. During maturation, GV, GVBD, and MI stage were clearly stained with NuMA antibody in the nucleus or cytoplasm at MII. Also, Nek2 was detectable in cytoplasm as scattered spots or chromosome associated at MII. In early developmental embryo, NuMA was detected in nucleus of each blastomere, while Nek2 was detected in cytoplasm. In contrast to previously reported results, Nek2 and NuMA were detected in both decondensing head, and the centriole of demembranated and decondensed sperm or whole body of trypsin-treated sperm for Nek2. During meiotic progress in oocytes, transcripts levels were the highest in MI stage and then downregulated in MII. Also, it shows dramatically change in early developmental embryos, firstly, it was increased until 4 cell stage and reduced in 8 cell stage, and finally, transcript levels were upregulated until blastoscyst. This finding suggests that cnetrosomal component may play an important role in reorganizing of functional centrosome during fertilization process and subsequent development.