• The Korean Journal of Zoology
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• v.33 no.1
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• pp.45-52
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• 1990

In order to determine whether maturation inhibiting activity(MIA) in the cytoplasm of growing follicular oocytes would suppress the cleavage of the embryonal cells, the growing oocytes were fused with the 2 or 4 cell blastomeres and then examined for the nuclear phase of the fused giant cells 24 hr after culture. A significant number of the giant cells(60%) composed of growing mouse oocyte and 2 cell mouse blastomere(1/2) in interphase has contained 2 nuclei 24 hr after culture and most of the giant cells (90%) composed of the growing oocyte and 4 cell blastomere(1/4) also contained 2 nuclei after culture. The unfused blastomeres or the isolated blastomeres cultured without fusion treatment cleaved one cell cycle under the same culture condition. In contrast, the nucleus of the growing oocytes was disintegrated and the chromosome condensed when fused with 2 cell blastomere in mitosis. The growing rat oocytes also suppressed the nuclear disintegration of the mouse embryonal cells during culture. The data presented here showed that MIA in the growing mammalian oocyte inhibited the cleavage of the embryonal cells in interphase stage, but not in milosis stage.

• Korean Journal of Veterinary Research
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• v.63 no.1
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• pp.3.1-3.9
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• 2023

The efficiency of somatic cell nuclear transfer (NT) in pigs is low and requires enhancement. We identified the most efficient method for zona pellucida (ZP) removal and blastomere aggregation in pigs and investigated whether the aggregation of NT and parthenogenetic activation (PA) of blastomeres could reduce embryonic apoptosis and improve the quality of NT-derived embryos by investigating. Embryonic developmental competence after ZP removal using acid Tyrode's solution or protease (pronase E). The embryonic developmental potential of NT-derived blastomeres was also investigated using well-of-the-well or phytohemagglutinin-L. We analyzed apoptosis in aggregate-derived blastocysts. The aggregation rate of protease-treated embryos was lower than that of Tyrode's solution-treated embryos (69.2% vs. 88.3%). No significant difference was observed between phytohemagglutinin-L and well-of-the-well (35.7%-38.5%). However, 2P1N showed a higher number of blastocysts compared to 3N (73.8% vs. 24.3%) and an increased blastocyst diameter compared to the control and 1P2N (274 ㎛ vs. 230-234 ㎛). In blastomeres aggregated using phytohemagglutinin-L, the apoptotic cell ratio was significantly higher in 1P2N and 3N than in 3P (5.91%-6.46% vs. 2.94%, respectively). Our results indicate that aggregation of one NT embryo with two PA embryos improved the rate of blastocysts with increased blastocyst diameter.

• Current Research on Agriculture and Life Sciences
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• v.9
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• pp.29-36
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• 1991

The study was carried out to investigate the viability and the offspring production rate of single blastomeres and biopsied 4-cell mouse embryos and, also to examine the efficiency of biopsy. The results obtained are summerised as follows: 1. The separated blastomeres from 4-cell embryos and intact 4-cell embryos which were cultured in Medium 2 were developed to trophoblastic vesicle and blastocyst embryo by 82.6% and 89.5%. respectively. 2. The biopsied embryos from 4-cell embryos and intact 4-cell embryos which were cultured in Medium 2 were developed to blastocyst embryo by 83.3% and 90.4%, respectively. 3. The biospied blastomeres and the separated blastomeres from 4-cell embryos which were cultured in Medium 2 were developed to trophoblastic vesicle by 80.8% and 83.3%, respectively. 4. The biopsied embryos from 4-cell embryos and intact 4-cell embryos were transferred to recipients, and the offspring rate was 36% and 48.6%, respectively.

• Clinical and Experimental Reproductive Medicine
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• v.18 no.1
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• pp.35-40
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• 1991

To find out the suitable method for blastomeres fusion of mouse 2-cell embryo using electric stimuli, these studies were carried out with various voltages (1.0 KV, 1.2 KV, 1.5 KV, 1.7 KV and 2.0KV), pulse duration times($50{\mu}\;sec$, $75/{\mu}\;sec$, $100{\mu}\;sec$) and different fusion solutions. In addition, the fused embryos were cultured for 72-80hr to observe their subsequent development. These results were summarized as follows: 1. The proportion of the fused embryos were 50.8%(34/67), 60.7%(34/56), 70.6%(48/68), 66.7% (48/72) and 85.3% (58/68) after stimuli of 1.0KV, 1.2KV, 1.5KV, 1.7KV and 2.0KV for $100{\mu}\;sec$ with 2 times, and the electric stimulation at 2.0KV(85.3%) was the most effective voltage on the blastomere fusion. 2. For in vitro development, blastocysts of the fused embryos were cultured for 72-80hrs in $M_{16}$ medium. The group(52.1%) treated with 1.5KV for $100{\mu}\;sec$ with 2 times showd higher development rates than those any other group. However, these results were not corresponded to those of the rates of blastomere fusion. 3. There were no significant differences among the rates of blastomeres fusion to 50(70.6%), 75(71.9%), and 100(78.0%) ${\mu}sec$ stimulation at 1.5KV with two times. However, the development rates of the fused embryo in vitro were 52.1%(25/48), 28.3%(13/46) and 9.4%(3/32) at the above conditions, and the development rates of fused embryo increased as the pulse duration times increased. 4. The rates of the blastomeres fusion were 38.9% (28/72) or 70.6% (48/68) in electrolyte (PBS) or non-electrolyte(0.3M mannitol) solution. The development rates of the fused embryo were 32.1% (9/28) or 52.1%(25/48) in the above fusion solutions, and non-electrolyte-treated group showed higher development rates of embryo than that of electrolyte-treated group.

• Korean Journal of Animal Reproduction
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• v.23 no.3
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• pp.271-278
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• 1999

This study was conducted to investigate the cytogenetic properties, in vitro development, and their relationship in the bovine reconstituted embryos following cell cycle-controlled nuclear transfer. Sixteen-cell stage embryos were treated by nocodazole, and after release from nocodazole treatment, their blastomeres were separated and allowed to subsequent cleavage. Blastomeres within 1.5 h post cleavage(hpc) and at 3hpc were transferred to enucleated oocytes at MII-phase or S-phase. Donor nuclei transferred into M II-phase recipients underwent various nuclear remodeling, such as extrusion of a polar body(PB)-like structure, premature chromosome condensation(PCC) and chromatin modifications. These nuclear remodeling patterns varied by the time post cleavage of donor blastomeres. Developmental rate to the blastocyst stage differed with time post cleavage of donor blastomeres and existence of a PB-like structure. Whereas do-nor nuclei transferred into S-phase oocytes did not undergo PCC and other major modifications, and their developmental potentials less depended on the nuclei types. This result confirms that the nuclear remodeling type differs with donor and recipient cell cycle stage, which affect the development of reconstituted bovine embryos.

• Korean Journal of Agricultural Science
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• v.38 no.2
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• pp.269-275
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• 2011

The objective of this study was to develop a rapid and reliable PCR method for sexing of morula or blastocyst stage bovine embryo. BOV97M and bovine 1.715 satellite DNA sequences were selected for amplification of male and bovine specific DNA, respectively. But the unbalanced number of copies of these two repetitive sequences required some modification of PCR method. Karyotyping of blastomeres were carried for the confirmation of sex determination in bovine embryos. The coincidence rate of sex between biopsied-single blastomere and matched blastocyst was 80.0%. When in vivo- and in vitro- derived embryos were compared, 61.8% and 56.7% were male in in vitro- and in vivo-derived embryos, respectively. In vivo-derived embryos showed better hatching rate than in vitro-derived embryos following biopsy of blastomeres. In conclusion, rapid and effective PCR could be applied to sexing of bovine preimplantation embryos using single blastomere. The sensitivity of this assay may eliminate the need for biopsy of more than one nucleated blastomere and reduce trauma to the embryos derived from biopsy procedure.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.1
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• pp.15-21
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• 1999

The aim of the present study was to determine the effect of paternal sex chromosome on early development of buffalo embryos fertilized and cultured in vitro. Embryos were produced in vitro from abattoir derived buffalo oocytes. The cleaved embryos were cocultured with buffalo oviductal epithelial cells and evaluated on day 7 under the phase contrast microscope to classify development. The embryos which reached the morula/blastocyst stage were fast developing, the embryos which were at 16-32 cell stage were medium developing and the embryos below 16 cell stage were slow developing. The embryos which showed some fragmentation in the blastomeres or degenerated blastomeres, were degenerating. Sex of emberyos (n=159) was determined using PCR for amplification of a male specific BRY. 1 (301 bp) and a buffalo specific satellite DNA (216 bp) fragments. The results thus obtained show that 1) X and Y chromosome bearing sperms fertilize oocytes to give almost equal numbers of cleaved XX and XY embryos, 2) male embryos develop faster than female embryos to reach advanced stage and 3) degeneration of buffalo embryos is not linked with the paternal sex chromosome. We suggest that faster development of males is due to differential processing of X and Y chromosome within the zygote for its activation and / or differential expression of genes on paternal sex chromosome sex chromosome during development of buffalo embryos fertilized and cultured in vitro which may be attributed to a combination of genetic and environmental factors.

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.153-161
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• 1998

This study was conducted to examine the viability of nuclear transfer bovine embryos following embryo transfer. Donor embryos were treated with nocodazole to arrest their cell-cycle-stage at mitotic(M) phase. After releasing from nocodazole blastomeres were separated and transferred into the enucleated oocytes(BC), or cultured in medium with aphidicolin. Freshly cleaved blastomeres within 1.5h after cleavage(AC) and non-cleaved ones up to 3h after releasing from nocodazole(NC) were transferred into the enucleated oocytes. Blastocysts derived from nuclear transfer were transferred to Day 7~8 recipient cows. Some blastocysts were vitrified and thawed before embryo transfer. Developmental rates to the blastocyst stage were higher in AC(18.1%, P<0.05) than BC(8.6%) and NC(5.1%). Blastocyst development slightly enhanced with aphidicolin(1~2$\mu\textrm{g}$/ml) treatment(16.9~22.6%) compared to non treated control(11.1%). Survival rate fo vitrified nuclear transfer embryos after thawing was 75%(24/32). Twnety-three vitrified nuclear transfer embryos and 3 fresh ones were transferred to 23 recipients, 6 heads were pregnant and 1 male calf(24 kg) was born from a recipient cow recevied one vitrifiedthawed nuclear transfer embryo at 277 days after embryo transfer. This result suggests that the nuclear transfer embryos can developed to term after vitrification andembryo transfer.

• Development and Reproduction
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• v.21 no.4
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• pp.467-473
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• 2017

Ascidian embryos have become an important model for embryological studies, offering a simple example for mechanisms of cytoplasmic components segregation. It is a well-known example that the asymmetric segregation of mitochondria into muscle lineage cells occurs during ascidian embryogenesis. However, it is still unclear which signaling pathway is involved in this process. To obtain molecular markers for studying mechanisms involved in the asymmetric distribution of mitochondria, we have produced monoclonal antibodies, Mito-1, Mito-2 and Mito-3, that specifically recognize mitochondria-rich cytoplasm in cells of the ascidian Halocynthia roretzi embryos. These antibodies stained cytoplasm like reticular structure in epidermis cells, except for nuclei, at the early tailbud stage. Similar pattern was observed in vital staining of mitochondria with DiOC2, a fluorescent probe of mitochondria. Immunostaining with these antibodies showed that mitochondria are evenly distributed in the animal hemisphere blastomeres at cleavage stages, whereas not in the vegetal hemisphere blastomeres. Mitochondria were transferred to the presumptive muscle and nerve cord lineage cells of the marginal zone in the vegetal hemisphere more than to the presumptive mesenchyme, notochord and endoderm lineage of the central zone. Therefore, it is suggested that these antibodies will be useful markers for studying mechanisms involved in the polarized distribution of mitochondria during ascidian embryogenesis.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.3
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• pp.119-124
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• 2019

Objective: It is widely accepted that aging decreases women's fertility capacity. The aim of this study was to assess correlations between maternal age and the morphokinetic parameters and cleavage pattern of embryos. Methods: The morphokinetics of embryos derived from women < 30, 30-35, 36-40, and > 40 years of age were compared retrospectively in terms of time of second polar body extrusion, time of pronuclei appearance, time of pronuclei fading, and time of two to eight discrete cells (t2-t8). Furthermore, abnormal cleavage patterns such as uneven blastomeres at the two-cell stage, cell fusion (Fu), and trichotomous mitoses (TM) were assessed. Results: Only t5 occurred later in women aged 36-40 and > 40 years when compared with those aged < 30 and 30-35 years (p< 0.001). Other morphokinetic timing parameters, as well the presence of uneven blastomeres, were comparable between the groups (p> 0.05). However, Fu and TM were more common in women aged > 40 years than in younger women (p< 0.001). Conclusion: Maternal age was correlated with the cleavage pattern of embryos. Therefore, evaluating embryo morphokinetics may contribute to optimal embryo selection, thereby increasing fertility in patients with advanced maternal age.