• Asian-Australasian Journal of Animal Sciences
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• v.20 no.6
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• pp.866-871
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• 2007

Testis-specific protein (TSPY) is a Y-specific gene, with up to 200 copy numbers in bulls. In order to make bovine embryo sexing under farm condition more feasible, the possibility of using a non-electrophoretic method to detect the TSPY gene for sexing bovine early embryos was examined. Primers were designed to amplify a portion of the TSPY gene and a common gene as an internal control primer. PCR optimization was carried out using a DNA template from bovine whole blood. Furthermore, embryo samples were diagnosed by this method and the sexing results were contrasted with those of the Loop-Mediated Isothermal Amplification (LAMP) method. The results showed that TSPY was as reliable a sexing method as LAMP. Forty-three morula and blastocyst embryos collected from superovulated donor dairy cattle were sexed by this method, and twenty-one embryos judged to be female embryos were transferred non-surgically to recipients 6 to 8 days after natural estrus. Out of 21 recipients, 9 were pregnant (42.86%) and all delivered female calves. The results showed that the sex predicted by this protocol was 100% accurate. In conclusion, the TSPY gene was a good male specific marker and indicated that a non-electrophoretic method was feasible and accurate to detect the TSPY gene for sexing preimplantation bovine embryos.

• Journal of radiological science and technology
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• v.18 no.2
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• pp.75-86
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• 1995

Embryos and fetuses are more sensitive to various environmental agents than adults of children biological effects following the exposure, such as intrauterin, malformation, have intimate conception with the prenatal exposure. There have been many studies on radiation and other agent. However, imformation about the ultrasound effects is limited. It is very important to study the effect of ultrasound with these kinds of fatera in consideration of ultrasound protection and safty. In this study, embryonic and fefal effects of ICR mouse embryos irradiated on 24, 48, 12 and 192 hpc of preimplantation and organogenesis period at the intensity of $0.5{\sim}3\;W/cm^2$ were investigated. Many type of external malformation observed in mouse irradiated on 72 hpc and 192 hpc. However, the embryos irradiated on 24 hpc and 48 hpc, at witch embryos had less then 6 cells and were pre-compaction stage, had no sensitivity for external malformation. The threshold doses of external malformation in mouse irradiated on 72 hpc and 192 hpc, at which embryos were consisted of $16{\sim}32$ cells and neural formation stage, were $1\;W/cm^2$ and $0.5\;W/cm^2$.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.3
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• pp.199-207
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• 2001

Objective: The present study was undertaken to examine the effects of magnesium ion in the culture medium on the development of mouse fertilized oocytes either before or after pronuclear formation, and to investigate whether the effect of magnesium ion is related with the redistributional change of mitochondria. Methods : Fertilized oocytes obtained from the oviducts of mice at 15 hr after hCG injection before pronuclear formation (pre-PN) or 21 hr after hCG injection after pronuclear formation (post-PN) were used. The embryos were cultured for 3 days with basic T6 medium-magnesium free and various concentrations of magnesium ion, 0.0, 0.5, 1.0, 2.0, 4.0 or 8.0 mM, respectively. After culture, the developmental stages of embryos and the number of nuclei were evaluated. To observe the effects of magnesium ion on the mitochondrial distribution, fertilized oocytes were collected at 21 hr after hCG injection and cultured for 6 hr with various concentration of magnesium ion. As a control, fertilized oocytes with pronuclei at 27 hr after hCG injection were used. Results: The concentration of magnesium ion to accelerate the in vitro development of mouse fertilized oocytes appeared to be at 2.0 mM for the pre-PN and the post-PN stage embryos. In the mitochondrial redistribution patterns, the embryos cultured in 2.0 mM concentration of magnesium ion showed the highest percentage (22.6%) of distinct perinuclear clustering pattern comparing to other experimental group. Conclusion: The effect of magnesium ion may be related to the cytoplasmic redistribution of mitochondria. This relationship seems to connect the developmental competence of preimplantation mouse embryos in vitro. These results can suggest that higher concentration of magnesium ion (2.0 mM) than those of conventional culture medium ($0.2{\sim}1.2\;mM$) is more suitable for in vitro culture of preimplantation mouse embryos.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.1
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• pp.77-84
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• 2003

Objective : The aim of this study was to evaluate the influence of three different media on preimplatation embryo development and the expression of Bcl-2, Mcl-1, Bax, and Bok in mouse. Materials and Methods: Two-cell embryos were retrieved from ICR female mice (4 weeks old) at 48 hr after hCG injection and cultured in Ham's F-10, HTF, and G1.2 media. The developmental rate of 2-cell embryos was evaluated from 24 hr to 72 hr after culture. RT-PCR was performed for the detection of Bcl-2, Mcl-1, Bax, and Bok gene expression. Results: The rates of morula and blastocyst in HTF and G1.2 media (88%, 98.1%) were significantly higher than those in Ham's F-10 media (39.6%) at 48 hr. Likewise, the rates of hatching and hatched blastocyst in HTF and G1.2 media (21.9%, 52.9%) were higher than those in Ham's F-10 media (3.5%) at 72 hr. Bcl-2 and Bax mRNAs were highly detected in embryos cultured in Ham's F-10 when compared in embryos cultured in HTF and G1.2. In contrast, the expression of Mcl-1 and Bok was not significantly different. Conclusion: These results show that HTF and G1.2 culture media increase the rate of blastocyst formation and stimulate Bcl-2 and Bax gene expression in mouse preimplantation embryos.

• Development and Reproduction
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• v.5 no.2
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• pp.101-106
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• 2001

Present study was aimed to verify the role of insulin and TNF-$\alpha$ in development of preimplantation embryos. Mouse morula were cultured for 40 hr in the presence or absence of insulin(400 ng/ml) and TNF-$\alpha$ (50 ng/ml). The morphological development, cell number of blastomeres per blastocyst, and mitogen activated protein kinase(MAPK) activity were examined. The developmental rate and cell number per embryo were the highest in insulin treatment group and the lowest in TNF-$\alpha$ treatment group. There was no significant difference in developmental rate between control and insulin plus TNF-$\alpha$ group. Taken together, it suggested that TNF-$\alpha$ impaired embryonic development and that insulin rescued developmental impairment imposed by TNF-$\alpha$. In blastocysts, insulin treatment significantly increased MAPK activity. TNF-$\alpha$ decreased the MAPK activity in a concentration-dependent manner. In the TNF-$\alpha$(50 ng/ml) -primed embryos, activation of MAPK by insulin was attenuated. In conclusion, these results suggest that there was a cross talk between insulin and TNF-$\alpha$ by means of activation of MAPK in preimplantation embryos and that insulin might rescue damage of embryos exposed to TNF-$\alpha$.

• Clinical and Experimental Reproductive Medicine
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• v.40 no.4
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• pp.163-168
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• 2013

Objective: Preimplantation genetic diagnosis (PGD) is an assisted reproductive technique for couples carrying genetic risks. Charcot-Marie-Tooth (CMT) disease is the most common hereditary neuropathy, with a prevalence rate of 1/2,500. In this study, we report on our experience with PGD cycles performed for CMT types 1A and 2F. Methods: Before clinical PGD, we assessed the amplification rate and allele drop-out (ADO) rate of multiplex fluorescent polymerase chain reaction (PCR) followed by fragment analysis or sequencing using single lymphocytes. We performed six cycles of PGD for CMT1A and one cycle for CMT2F. Results: Two duplex and two triplex protocols were developed according to the available markers for each CMT1A couple. Depending on the PCR protocols, the amplification rates and ADO rates ranged from 90.0% to 98.3% and 0.0% to 11.1%, respectively. For CMT2F, the amplification rates and ADO rates were 93.3% and 4.8%, respectively. In case of CMT1A, 60 out of 63 embryos (95.2%) were diagnosed and 13 out of 21 unaffected embryos were transferred in five cycles. Two pregnancies were achieved and three babies were delivered without any complications. In the case of CMT2F, a total of eight embryos were analyzed and diagnosed. Seven embryos were diagnosed as unaffected and four embryos were transferred, resulting in a twin pregnancy. Two healthy babies were delivered. Conclusion: This is the first report of successful pregnancy and delivery after specific PGD for CMT disease in Korea. Our PGD procedure could provide healthy babies to couples with a high risk of transmitting genetic diseases.

• Development and Reproduction
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• v.3 no.1
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• pp.59-67
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• 1999

To investigate the role of interleukin-l$\beta$ (IL-1$\beta$) in the embryonic development, in vivo and in vitro expression patterns of IL-1$\beta$ gene in the preimplantation mouse embryos were examined by RT-PCR, and the effects of explanted mouse ovi-duct and uterine epithelial cells on the expression of IL-1$\beta$ gene in the pleimplantation mouse embryos were examined by co-culture. IL-1$\beta$ mRNA was detected in the embryos from 4-cell stage to blastocyst stage in vivo and from morula stage to hatching blastocyst stage in vitro. This transcript was not detected from the GV stage to late 2-cell stage in vivo, and not at the 4-cell and 8-cell stages in vitro. For the co-culture of late 2-cell embryos with the explanted mouse oviduct and uterine epithelial cells, oviducts and uterine epithelial cells were isolated at 48 hour alter the hCG injection. The explanted oviduct and uterine epithelial cells in co-culture groups facilitated the IL-1$\beta$ gene expression of the mouse embryos in comparison with the control. Taken together these results suggest that the presence of IL-1$\beta$ plays an important role in preimplantation embryonic development. In addition, the up-regulation of IL-1$\beta$ gene expression by the explanted oviduct and uterine epithelial cells demonstrates that embryonic expression of IL-l$\beta$ gene may be regulated by the interaction with oviductal and uterine factor (s).

• Development and Reproduction
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• v.2 no.2
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• pp.157-163
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• 1998

Preimplantation embryos of the rat was examined by the morphological changes in the cortical granule envelope (CGE), blastomere surface, and zona pellucida (ZP) of embryo after cortical reaction. The ultrastructural characteristics and CGE of embryos were observed with the scanning electron microscope and fluorescence microscope. In the ultrastructural characteristic of embryo surface, surface microvilli were shortened and the CGE-like structure existed above microvilli in eight-cell embryo. Rough spongy surface and decreased network numbers were key characters of embryonic ZP compared to unfertilized oocyte. The CGE formed by cortical reaction existed in perivitelline space during embryo development but it was thin and locally distributed ill contrast to fertilized oocyte. The present results indicate that cortical reaction forms cortical granule envelope in perivitelline space and causes not only zona hardening, but also ultrastructural changes in ZP and cell membrane of preimplantation embryos.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.102-102
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• 2002

Heparin-bindin epidermal growth factor (HB-EGF) is one of the EGF family to be expressed at the time of implantation in the mouse uterus. Although HB-EGF has been shown to stimulate the development of embryo and uterus in the mouse, its correlation between cell adhesion molecules remains undefined. Integrin $\alpha$$_{ν}$$\beta$$_3$, one of the cell adhesion molecules, is an important mediator of cell-substratum and cell-cell adhesion in implantation. In the present studies, we investigated the effects of HB-EGF on the embryonic development, initiation of implantation and expression of integrin $\alpha$$_{ν}$$\beta$$_3$ in in vitro culture, blocking of HB-EGF, RT-PCR and immunofluores cence analysis. The results showed that HB-EGF significantly improved the developmental rate of hatched embryos (24.1%, p<0.01) and outgrowth embryos (42.5%, p<0.01). On the other hand, this growth factor showed no offset before the hatching embryonic stage. Analysis of RT-PCR showed that HB-EGF upregulated the expression level of integrina $\alpha$$_{ν}$$\beta$$_3$ subunit genes on the preimplantation embryo and outgrowth of blastocyst (120hr and 144hr after hCG injection). Immunofluorescence analysis showed that the integrin $\alpha$$_{ν}$$\beta$$_3$ subunits localized at the pericellular borders and cell-cell contact areas. Increase in fluorescence intensity was observed in the HB-EGF treated embryos. Intrauterine injection of an anti-HB-EGF antiserum at day 3 significantly decreased the number of implantation sites (14.4, p<0.01) and significantly increased the number of recovered embryos(6.4, p<0.05) at day 5. From these results, it imply that HB-EGF improve the embryo development and accelerated the expression of integrin $\alpha$$_{ν}$$\beta$$_3$ in the preimplantation mouse embryos.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.81-81
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• 2003

DNA methylation is a covalent modification of DNA that can modulate gene expression and is now recognized as a major component of the epigenome. During evolution, the dinucleotide CpG has been progressively eliminated from the genome of higher eukaryotes and is present at only 5% to 10% of its predicted frequency. Approxymately 80% of the remaining CpG sites contain methylated cytosines in most vertebrates and they are distributed in a pattern that is unique in each tissue and is inversely correlated with gene expression. The pattern of methylation is faithfully maintained during cell division by the enzyme Dnmt1, the maintenance DNA methyltransferase, which catalyzes the transfer of a methyl group from S-adenosyl-methionine to the 5'-position of the cytosine ring. We have been identified bovine Dnmt1 cDNA full-length recently (AY173048) Little is known on the functions of Dnmt1 in bovine preimplantation embryos. Thus, we analyzed the specific pattern of Dnmt1 in in vitro derived/nuclear transfer bovine and in vivo derived mouse embryos to monitor the epigenetic reprogramming process. We investigated these process by using indirect immunofluresence with an antibody to Dnmt1. According to other studies, Dnmt1 accumulates in nuclei of early growing oocytes but is sequestered in the cytoplasm of mature oocytes. In 2-cell and 4-cell embryos, Dnmt1 is cytoplasmic, but at the 8-cell stage, it is present only in the nucleus. By the blastocyst stage, Dnmt1o is again found only in the cytoplasm. Thus, nuclear localization of Dnmt1o in preimplantation embryos is limited to the 8-cell stages After implantation, Dnmt1 is localized in the nucleus in mouse. However, we have found different patterns of Dnmt1 nuclear localization. Though we used the common antibody, immune-localization data revealed that Dnmt1 antibody have been detected at the nucleus in 1-cell to blastocyst embryos. Therefore, maybe we think that the functions of Dnmt1 between bovine and mice are different. In order to Identify the mechanisms that regulate DNA methylation in bovine preimplantation embryo, we have plans on using bovine oocyte and somatic specific Dnmt1 antibodies.