• 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.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.275-289
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• 2002

The objective of this study was to develop an in vitro assessment of sperm fertilizing capacity of bulls and investigate the factors influencing sperm function and characteristics of frozen-thawed bovine spermatozoa. in vitro fertilization (IVF), the evaluation of motility and normal morphology, HOST (hypoosmotic swelling test), Ca-ionophore induced acrosome reaction, luminol and lucigenin-dependent chemiluminescence for the measurement of reactive oxygen species (ROS), the measurement of malondialdehyde formation for the analysis of lipid peroxidation (LPO), and the evaluation of DNA fragmentation using the method of 747-mediated nick end labelling (TUNEL) by flow cytometry were performed in frozen-thawed bovine spermatozoa. Correlations between the rates of fertilization, blastocyst formation after IVF and the values of respective assays were investigated. 1. IVF rate and blastocyst formation rate averaged 64.4% and 34.3% for spermatozoa from high -fertility bull group and averaged 18.5% and 6.2% for spermatozoa from low-fertility bull group, respectively. There were significantly different between two bull groups. Sperm motility and percentage acrosome reaction averaged 79.0% and 66.2% for spermatozoa from high-fertility bull group and averaged 40.7% and 22.9% for spermatozoa from low-fertility bull group, respectivitely. There were not different between two bull groups. 2. Luminol depenent chemiluminescence, LPO and DNA fragementation averaged 6.4, 2.0 nmol and 2.6% from spermatozoa from high-fertility bull group and averaged 6.5, 3.1 nmol and 7.4% for spermatozoa from low-fertility bull group, respectively. There were significantly different between two bull groups. There was no significant difference in lucigenin dependent chemiluminescence between two bull groups. 3. Fertilization rate was positively correlated with motility and the rate of Ca-ionophore induced acrosome reaction, but negatively correlated with the frequency of luminol-dependent chemiluminescence, the rate of LPO, and the percentage of sperm with DNA fragmentation. There was no correlation between fertilization rate and the percentage of swollen spermatozoa, normal morphology, and the frequency of lucigenin-dependent chemiluminescence. 4. Blastocyst formation rate was positively correlated with the rate of Ca-ionophore induced acrosome reaction, but negatively correlated with the frequency of luminol-dependent chemiluminescence, the rate of LPO, and the percentage of sperm with DNA fragmentation. There was no correlation between blastocyst formation rate and motility, the percentage of swollen spermatozoa, normal morphology, and the frequency of lucigenin-dependent chemiluminescence. In conclusion, these data suggest that ROS significantly impact semen quality. The assays of this study may provide a basis fur improving in vitro assessment of sperm fertilizing capacity.

• Korean Journal of Animal Reproduction
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• v.15 no.1
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• pp.7-13
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• 1991

This study was carried out to investigate the effects of pH of sperm-washing solution, sperm-washing frequency and individual bull on concentration of hydrogen ion in sperm-washed solution and sperm acrosome reaction. The results obtained were as follow. 1. When bovine sperm was washed at 4 times with SHP solution and incubated, the difference of light absorbance between sperm-washing and sperm-washed solution(${\Delta}$Ao-${\Delta}$At) was higher in 2nd sperm-washed solution than that in the other washed solutions. 2. When sperm was thrice washed with SHP solutions of pH 5.99, 6.38, 6.78, 7.10, 7.40, 7.69, 8.15, 8.45, and 8.83, ${\Delta}$Ao-${\Delta}$At was significantly increased at pH7.69 to 8.83, and ${\Delta}$Ao-${\Delta}$At in 1st sperm washed solution was significantly higher than that in 2nd and 3rd sperm washed solution. 3. When sperm of Holstein, KNC and Hereford was thrice washed with SHP solutions of pH 5.99, 6.38, 6.78, 7.10, 7.40, 7.69, 8.15, 8.45, and 8.83, Holstein showed higher ${\Delta}$Ao-${\Delta}$At of sperm Washed solution than KNC and Hereford, and ${\Delta}$Ao-${\Delta}$At in sperm washed solution was significantly increased at 7.69 for Holstein and at 8.15 for KNC and Hereford, respectively. 4. When sperm was thrice with SHP solution of pH 6.8, 7.1 and 7.4, and then incubated in mTALP of pH 7.4 for 15 minutes, in 1st and 2nd sperm washing ${\Delta}$Ao-${\Delta}$At of sperm washed solution was significantly higher at pH 7.1 and 7.4 than at pH 6.8, and the sperm acrosome reaction of pH 6.8, 7.1 and 7.4 was 49.1, 68.8 and 72.9%, respectively. The sperm acrosome reaction of pH 7.1 and 7.4 was higher than that of pH 6.8.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.33-38
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• 2000

This study was undertaken in an effort to select the sire bull in Hanwoo through in vitro fertilization of proven bull semen. It was used for in vitro fertilization that of the 20 proven bull semen with follicular oocytes derived from slaughterhouse ovaries of Hanwoo. The stage of maturation on the time course of bovine cumulus-enclosed oocytes incubated for 24 hours was found the highest(96.4%) than hose of other maturationi time. In vitro fertilization rate of bovine oocytes with proven bull sperm showed from 61.5 to 88.9%. Polyspermy of in vitro fertilized oocytes according to proven bulls were the highest KP 491(61.5%) nothing but KP 486, KP 491 and KP 497.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.134-134
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• 2003

Bovine embryos produced by in vitro maturation, feretilization and development was examined for presevation and transfer. The fertilization medium used BO medium with 5 mM/$m\ell$ caffeine and 10$\mu\textrm{g}$/$m\ell$ heparin and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to 1$\times$$10^{6}$ cells/$m\ell$ motile sperm during fertilization in vitro. At 8~10 hrs after insemination, the oocytes were transferred into CR1aa medium and cultured for 7 days. Embryos were preserved by vitrification method for transfer. When the embryos of early, blastocyst and expanded blastocyst stages were frozen-thawed, the proportions of embryos with normal morphology 83.6, 88.1 and 85.2%. (중략)

• 대한생식의학회:학술대회논문집
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• 1997.11a
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• pp.81.2-82
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• 1997

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2000.11a
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• pp.64-65
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• 2000

• Journal of Embryo Transfer
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• v.17 no.3
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• pp.187-193
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• 2002

This study was conducted to determine the effect of extract of Cervi pantotrichum cornu on human sperm motility, Four different types of media were prepared such as plain Ham's F-10 medium(control medium), control medium containing 0.3% bovine serum albumin(BSA)(medium A), control medium containing the extract of Cervi pantotrichum cornu aqua-acupuncture medium(medium B) and medium B containing 0.3% BSA(medium C). Human semen were washed and divided into 4 fractions and sperm were cultured in those medium for up to 72 hours at 37$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$ in air. A total twenty eight semen samples including 14 normozoospermia and 14 asthenospermia were used for this study. In normozoospermia group, motility of control medium and medium A, B and C were 4.1%, 1.3%, 64.5%, and 77.1%, respectively after 24 hours of incubation, and were 0.0%, 0.0%. 8.8% and 44.9%, respectively after 48 hours of incubation. In asthenospermia group, motility of control medium and medium A, B and C were 2.0%, 2.2%, 58.3% and 85.1%, respectively after 24 hours of incubation, and decreased to 0.0%, 0.2%, 5.8% and 29.6%, respectively after 48 hours of incubation. In both groups, highest sperm motility was observed in medium C group when compared with other media. Furthermore motile sperm were found in medium C after 72 hours of incubation while no motile sperm was observed in the other media. Therefore it could be concluded that the extract of Cervi pantotrichum rornu affects on the human sperm motility.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.7
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• pp.969-976
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• 2009

Use of oocytes from prepubertal animals for in vitro embryo production holds potential application for reducing generation intervals and increasing genetic progress through embryo transfer. The objective of these studies was to compare the effect of three sperm pretreatments (prior to in vitro fertilization) and seven embryo culture protocols on fertilization rate and (or) subsequent development of in vitro fertilized embryos derived from oocytes harvested from ovaries of 1-6 month old prepubertal Boer goats in China. Cleavage rates were highest for embryos fertilized with heparin-treated versus calcium ionophore- or caffeine-treated sperm. Similar rates of blastocyst development were observed using heparin- and ionophore-treated sperm, which were higher than obtained with caffeine-treated sperm. No differences in cleavage or blastocyst rates were observed following embryo culture in basal medias (synthetic oviductal fluid (SOF), Charles Rosenkrans 1 (CR1) or tissue culture medium-199 (TCM-199)) containing 10% fetal bovine serum (FBS). Cumulus or oviductal cell co-culture did not enhance cleavage or blastocyst rates relative to culture in SOF+10% FBS. Replacement of FBS in SOF medium with 0.3% BSA increased cleavage rates, but did not increase rates of blastocyst development. Sequential culture in SOF+0.3% BSA followed by SOF+10% FBS increased blastocyst yield versus continuous culture in SOF+10% FBS and tended to increase blastocyst yield versus continuous culture in SOF+0.3% BSA. These results demonstrate a pronounced effects of sperm pretreatments and in vitro embryo culture systems on rates of blastocyst development and provide a potential protocol (sperm pretreatment with heparin and sequential embryo culture in SOF+0.3% BSA followed by SOF+10% FBS) for generation of the significant numbers of in vitro produced blastocysts from oocytes of prepubertal Boer goats necessary for application of embryo transfer in rural regions of China for distribution of Boer goat genetics.

• Journal of Embryo Transfer
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• v.10 no.2
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• pp.115-120
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• 1995

This study was carried out to investigate On in vitro fertilization, survival rate and developmental rate of rapidly frozen bovine immature oocytes. Immature oocytes cultured for 1, 12, 24, 48 hours in 20% FCS + TCM-199 medium and thereafter rapidly freezing-thawed oocytes inseminated with capacitated sperm. The immature oocytes following dehydration by 1.5M DMSO + 2.0M glycerol + 0.25M sucrose + TCM 199 media + 20% FGS were directly plunged into liquid nitrogen and thawes in 3$0^{\circ}C$ water. Rapid freezing embryos co-cultured in 20% FCS + TCM-199 media containing hormones(21U/mL PMSG, 21U /mL hGG and 1 $\mu$g /mL 17$\beta$-estradiol) and cumulus cells(1 x 105-6 cells). Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The in vitro maturation and fertilization rate of immature bovine oocytes on in vitro maturation period(1, 12, 24, 48 hrs) before rapid freezing4hawed were 57.1%, 45.7%, 37.1%, 25.7% and 40.0%, 31.4%, 20.0%, 11.4%, respectively. 2. The survival rate of immature bovine oocytes on in vitro maturation period(1, 12, 24, 48 hrs) before rapid freezing-thawed were 33.3%, 26.7%, 20.0%, and 10.0%, respectively. The survival rate of rapid freezing4hawed immature oocytes was significantly lower than that of non-freezing oocytes. 3. The survival rate of rapid freezing4hawed excellent and good bovine embryos co-cultured in 20% FCS + TCM-199 media containing hormones(PMSG, hCG, 17$\beta$-estradiol) and cumulus cells 4 to 5 hrs and 20 to 24 hrs were 35.0%, 15.0% and 25.0%, 15.0% and 40.0%, 20.0% and 30.0%, 15.0%, respectively. The survival rate of embryos co-cultured in TCM-199 media containing hormones and cumulus cells was significantly higher than that of non co-culture.