• Reproductive and Developmental Biology
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• v.32 no.1
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• pp.33-38
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• 2008

DNA methylation is involved in tissue-specific gene control and essential for normal embryo development Octamer-binding transcription factor 4 (Oct-4) is one of the most important transcription factors for early differentiation. This study was performed whether the bovine Oct-4 is tissue specific or developmental dependent epigenetic mark, we investigated transcripts and the methylation status of CpGs of 5'-promoter region of Oct-4 in bovine preimplantation embryos. Oct-4 transcripts were highly detected in morula and blastocyst, while they were present low levels in sperm and 2- to 8-cell stage embryos. These results suggest that de novo expression of Oct-4 initiates at morula stage of embryogenesis. Here we determined that there is a tissue-dependent differentially methylated region (T-DMR) in the 5'-promoter region of Oct-4. The methylation status of the Oct-4 T-DMR was distinctively different in the oocyte from that in the sperm and adult somatic tissues and changed from zygote to blastocyst stage, suggesting that active methylation and demethylation occur during preimplantation development. Based on these results, the 5'-promoter region of Oct-4 gene is target for DNA methylation and the methylation status changes variously during embryonic development in bovine.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.235-243
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• 1998

In this study, we investigated the effects of three kinds of culture medium (Charles and Rosenkrans; CRlaa, Tyrode's; TALP, synthetic oviduct fluid: SOF), insulin transferrin + selenium complex (ITS), macromolecules(polyvinyl alcohol: PVA, fetalb-ovine serum: FBS) and NaCl on the development of early bovine embryos. In experiment 1, there were no differences in embryo development among three kinds of embryo culture medium (CR $l_{aa}$ , TALP, SOF). In experiment 2, BSA, FBS and PVA were added each in TALP as macromolecule sources. The developmental rates of embryos in BSA or FBS added TALP were significantly higher than in PVA added one (p〈0.01), but there was no difference between BSA and FBS added groups. In experiment 3, bovine embryos were cultured in TALP with the following supplements: BSA alone(1, 3 or 8 mg/ml, each) or BSA(1, 3 or 8 mg/ml, each)+ITS (10$\mu\textrm{g}$/m1 insulin, 5 $\mu\textrm{g}$/ml transferrin, 5 ng/ml selenium). In higher concentration of BSA and ITS supplemented groups, the developmental rates over compacted morula were higher than others, but there was a significant effect of ITS only in 1 mg/ml of BSA added group (p〈0.05). In experiment 4, the effect of reduced concentration of NaCl was evaluated. The developmental rate over compacted morula in the medium containing 90 mM of NaCl was higher than in 114 mM group (p〈0.05). In conclusion, BSA could be used as a macromolecule source in bovine embryo culture, and ITS, as a serum substitute, could be used for improving of embryonic development. Also, reduction of NaCl concentration from 114 mM to 90 mM may improve the development of bovine embryos.bryos.

• Korean Journal of Environmental Biology
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• v.32 no.3
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• pp.225-233
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• 2014

In this study, we investigated the changes in the physiological and histological traits of a sea squirt (Halocynthia roretzi) with the emergence of the soft tunic syndrome induced by the water temperature control (6, 9, 12, 15, 18, 21, 24 and $27^{\circ}C$). It was observed that the induction rate of the soft tunic syndrome was highest at $15^{\circ}C$, but lowest at $24^{\circ}C$. Based on the tunic color condition and contraction strength, the whole process were classified into 4 stages as S0, S1, S2 and S3. Interestingly, there were significant differences in oxygen consumption and filtration rate were observed during S0-S3. The most distinctive aspects were change of blood cell composition at stage S3, whereas multi-vacuole cell ratio was decreased by 1/2 and morula cell ratio expanded about 10 times during S0-S3. Further, change of organ structure started following the syndrome such as degeneration of epithelial cells, microfilaments, increment in hemocytes and damage in muscle fiber have been detected in tunic, siphon, branchial sac, body wall musculature and pyloric gland. Briefly, our study results indicated that the normal physiological functions of the sea squirt can be affected due to the soft tunic syndrome induced by water temperature.

• Development and Reproduction
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• v.5 no.1
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• pp.17-21
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• 2001

This study was conducted to investigate the expression pattern of Trypsin-like enzyme and the effect of a trypsin inhibitor(benzimidine) on hatching process during in-vitro culture of mouse preimplantation embryos. The Trypsin-like enzyme was identified by rhodamine-conjugated Trypsin substrate probe. The expression of trypsin-like enzyme was firstly detected at the late morula stage, and the enzyme was uniformly localized in the trophectoderm of late blastocysts. Especially, intense fluorescence was observed in the blebbing area of hatching blastocysts. Bisbenzamidine, contained in culture media, did not alter embryonic development from 4-cell stage to the expanded blastocyst but decrease the hatching rate in ImM concentration (15.8% vs 89.7%, p<0.02). In the treatment of bisbenzimidine (5mM) for 12 hours according to the embryonic stage of mouse, the hatching rate of control (83.0%) and treatment in late blastocysts (8.7%) were significantly (p<0.01) different. From these results, we suggested that the hatching enzyme having trypsin-like activity was localized from the late morula stage, and the hatching process by this enzyme was activated in the late blastocyst stage of mouse embryos.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.109-113
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• 1998

Objective: To ananlyze the direct effect of nitric oxide (NO), generated from sodium prusside (SNP) on the embryo developments in reproductive process. Design: Ova from mouse were treated to allow fertilization in in vitro culture. And the samples of fertilized ova were alloted into five alliqutos. Each alliquot was cultured in media treated with either concentration at 0 (n=92), $25{\mu}M$ (n=84), $50{\mu}M$ (n=80), $100{\mu}M$ (n=77), $500{\mu}M$ (n=54) of SNP. Main Outcome Measure: Rates of embryonal cell cleavages, viability and cell morphology were assessed during in vitro fertilization and culture. Results: As analyse the cell cleavage at 24 hours after in vitro culture of fertilised egg in variuos NO concentration, all of egg cells of each alliquot were developed to $2\sim4$ cell stage. But the alliquot of egg cells treated with $50{\mu}M$, which were totally degenerated. And also all embryonal cells of each alliquot were developed to 8 cell stage and morula stage on culture continuosly. And the embryonal cells of each alliquot were analysed at 24 and 48 hours following the in vitro culture. The rates of cell fragmentation and fusion were $4.2{\pm}3.4%$ in control group which is not treated with NO, while experimental groups was high, as rated $23.4{\pm}6.2%$ in $25{\mu}M$, $28.2{\pm}5.7%$ in $50{\mu}M$ and $32.1{\pm}6.4%$ in $100{\mu}M$ concentration of NO. Accordingly the rate of abnormal morphology of embryonal cell in control was lower significantly than that in each alliquot of experimental groups (p<0.05). And the degenerated rates of embryonal cells were 0% in control, $17.8{\pm}6.7%$ in $25{\mu}M$, $23.6{\pm}4.7%$ in $50{\mu}M$ and $26.8{\pm}11.2%$ in $100{\mu}M$ at 8 cells and morula on culture of 48 and 72 hours. On the examination of embryonal cells developed to blastocyst through in vitro culture, the rates of degenerated cells were $16.8{\pm}7.2%$ in control, $37.5{\pm}6.2%$ in $25{\mu}M$, $73.4{\pm}4.6%$ in $50{\mu}M$, 100% in $100{\mu}M$. Conclusion: This results suggeted that the NO in any concentrations is harmful on embryos in view of morphology as well as viability of cell, and the toxicity of NO on embryo is stronger at condition in higher concentration of NO.

• Korean Journal of Veterinary Research
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• v.32 no.1
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• pp.143-151
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• 1992

Sixty Four fresh and 142 frozen embryos of dairy cattle were transferred to synchronized dairy, beef or Korean Native Cattle nonsurgically at National Animal Breeding Institute from 1985 to 1990. The results obtained were as follows ; 1. The pregnancy rate of fresh embryos(39.1%) was higher than that of frozen embryos(32.4%) and average pregnancy rate was 34.5%. 2. The pregnancy rate of grade 1 embryos was higher than that of grade 2 embryos for both fresh(41.3% vs 33.3%) and frozen embryos(35.4% vs 25.6%). 3. The pregnancy rate according to development stage of fresh embryos was increased with maturity as 29.2%, 33.3%, 50.0% and 54.5% for morula, early blastocyst, blastocyst and expanded blastocyst, respectively. For frozen embryos, the pregnancy rate of blastocyst(44.4%) was higher than those of morula(31.3%) and early blastocyst(28.0%). 4. The pregnancy rate according go recipient-donor synchrony for fresh embryos was higher when the recipients exhibited estrus 1 day earlier than the donors(43.8%) than when the recipients exhibited estrus 1 day later than the donors(38.1%) or when the recipients and donors exhibited estrus at the same time(37.0%). For forzen embryos, the pregnancy rate was decreased when the recipients and donors exhibited estrus at the same time(37.9%), when the recipients exhibited estrus 1 day later than the donors(32.0%) and when the recipients exhibited estrus 1 day earlier than the donors(23.5%), in sequence. 5. The pregnancy rate of heifers was higher than that of cows for both fresh(50.5% vs 37.9%) and frozen embryos(39.7% vs 25.7%). 6. The pregnancy rate according to recipient breed for fresh embryos was higher in dairy cattle(42.1%) and beef cattle(40.%) than in Korean Native Cattle(33.3%). For frozen embryos, the pregnancy rate was decreased beef cattle(39.1%), dairy cattle(30.3%) and Korean Native Cattle(14.3%), in sequence. 7. The pregnancy rate according to equilibrium steps of glycerol and freezing rate was higher when transferred after 3-steps equilibrium and freezing by the rate of $0.3^{\circ}C$/min from $-6^{\circ}C$ to $-35^{\circ}C$ and $0.1^{\circ}C$/min to $-38^{\circ}C$(39.4%) than when transferred after 6-steps equilibrium and freezing by the rate of $0.5^{\circ}C/min$ from $-6^{\circ}C$ to $-30^{\circ}C$(30.3%).

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.77-86
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• 1998

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free T6 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at l20hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUT1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture ($93.8{\pm}3.1$, n=35) is significantly higher than that of control and glucose-free group ($76.6{\pm}3.8$, n=35 and $68.2{\pm}4.3$, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.

• Korean Journal of Veterinary Research
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• v.36 no.4
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• pp.919-927
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• 1996

In the last few years, methods for in vitro culture of early embryo stages from oocytes matured and fertilized in vitro using suitable cell culture systems have been established. But the factors affecting pregnancy rates following transfer of bovine embryos produced in vitro were not evaluated enough. So this study was performed to investigate the effects of quality and stage of embryos, parity and Corpus Luteum quality of recipients on pregnancy rates following non-surgical transfer of bovine embryos produced in vitro. Oocytes aspirated from small antral follicles of ovaries obtained at a local slaughter house were matured, fertilized with frozen-thawed semen and co-cultured for 6-7 days by utilizing co-culture system with bovine oviduct epithelial cell in vitro. After co-culture, embryos were transfered to recipients on day 7 (estrus=day 0). Recipients were monitored by ultrasonic scanning method or observation for estrus and rectal palpation after 50 days from transfer. The results of this study are follows. 1. Of the 70 recipients, 70%(49 of 70) had not showed estrus sign between day 0 and day 50, but 22.9%(16 of 70) was diagnosed not pregnant. Therefore the overall pregnancy rate of this study was 47.1%(33 of 70). 2. The pregnancy rate of recipients transfered with excellent(66.7%) and good(54.5%) embryos were higher than that of recipients transfered with fair embryos(15.8%) (p<0.05). 3. The pregnancy rate of recipients transfered with morula, compacted morula, blastocyst and expanded blastocysts were 46.2, 55.0, 62.5 and 50.0%, respectively. 4. The pregnancy rates of recipients transfered to heifer and cow were 54.5 and 55.2%, respectively. 5. The pregnancy rates of recipients with CL score I, II(66.7, 63.6%) were higher than those of recipients with CL score III (10%), (p<0.05). Success of transfer of embryos produced in vitro depends on many variables. The important factors identified in this study were the quality of embryos and the CL score of recipient animals after non-surgical transfer of embryos matured, fertilized and cultured in vitro.

• 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.