• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.63-69
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• 2001

This study was carried out to examine the effects of nitric oxide compounds (hemoglobin and L-NAME) on the development of porcine in vitro maturation (IVM) and in vitro fertilization (IVF) oocytes. Cumulus cell free embryos derived from porcine IVM/IVF oocytes were cultured in NCSU23 medium containing 1~5 $\mu\textrm{g}$/$m\ell$ hemoglobin added to 44 and 96hrs in culture times, and in NCSU23 medium containing 0, 10, 50 or 100mM L-NAME. The developmental rates beyond morulae stage in 0, 1 and 5 $\mu\textrm{g}$/$m\ell$ hemoglobin groups add to 44hrs in vitro culture times were 52.4%, 57.6% and 57.4%, respectively. The addition of hemoglobin groups made it slightly higher than the control group. The proportion of embryos developed to morulae and blastocysts in 1 $\mu\textrm{g}$/$m\ell$ hemoglobin add to 96hrs after in vitro culture (70.8%) was a little higher than those of 0 and 5 $\mu\textrm{g}$/$m\ell$ hemoglobin (66.2% and 62.8%). There was no significant difference in all groups (P〉0.05). The developmental rates beyond morulae stage in 0, 10, 50 and 100mM of L-NAME groups add to 96hrs after in vitro culture were 65.2%, 73.5%, 70.1% and 53.3%, respectively 10mM and 50mM L-NAME groups were significantly higher than in 0 and 100mM of L-NAME groups (P＜0.05). In conclusions, these results indicate that L-NAME (10mM, 50mM) can increase the proportion of embryos that develop into morulae and blastocysts but hemoglobin did not affect.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.56-56
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• 2003

Beta-catenin is very important in early development including involvement in cell adhesion, cell signaling, and developmental fate specification. Cell-cell interaction is an important process during mammalian embryonic development. In preimplantation embryos, embryonic compaction is the process of increased cellular flattening and adhesion of junctional complexes and results in a polarized distribution. Beta-catenin is associated with embryonic compaction in mammals. Here, we examined the relationship between beta-catenin expression and compaction in porcine embryos derived from in vitro fertilization. First of all, we investigated beta-catenin expression in each embryonic developmental stage and also focused on expression pattern according to full, partial and non-compaction at morula stage. We used the immunocyto-chemical method in this research. To confirm compaction affects on the embryonic development, we compared between compaction and developmental rates to the blastocyst. The result showed that compaction and non-compaction rates were 14.6% and 63.8% at 4 days after IVF, respectively The developmental rates to the blastocyst and their total cell number were 50.9% vs 36.4% and 41.4$\pm$11.5 vs 26.8$\pm$12.7 in compaction and non-compaction groups. Although no difference was detected in the ratio of ICM to total cells between two groups, total cell number of the blastocysts in compaction group was superior to that of the blastocysts in non-compaction group (P<0.05). Expression of beta-catenin appeared in the boundary of membrane surface between blastomeres in 2- and 4-cell stage, and observed irregular pattern from 8-cell to blastocyst stage. We also investigated beta-catenin expression pattern according to the degree of compaction in the 3 groups; full, partial (>50%) and non-compaction. The expression signal in fully compacted embryos was stronger than those of partial and non-compacted embryos. Especially, beta-catenin expression appeared various patterns in morula stage suggesting the aberrant distribution of beta-catenin is affected by compaction patterns. Our results suggest that abnormal beta-catenin expression was affected by embryo quality and further development in porcine embryos in vitro.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.711-717
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• 2007

In this study, two cell types from porcine females, namely fetal fibroblasts (pFFs) and adult ear fibroblasts (pAEFs) and two passages (3-4 and 7-8) were investigated by evaluating the development rate, blastocyst cell number and the incidence of apoptosis. No significant differences were observed in the cleavage rates of cloned and IVF embryos. The blastocyst rates between the embryos cloned with pFFs ($15.1{\pm}3.2$) and pAEFs ($10.4{\pm}2.6$) did not differ significantly but was significantly (p<0.05) lower in pAEFs than that in IVF ($22.5{\pm}4.5$) embryos. Total cell number in pFFs ($28.4{\pm}4.3$) and pAEFs cloned blastocysts ($24.2{\pm}5.1$) was significantly (p<0.05) lesser than IVF control ($35.4{\pm}3.2$). Apoptosis rates between cloned blastocysts differed significantly (p<0.05) and were significantly (p<0.05) higher than IVF embryos. The blastocyst rates between the cloned embryos cloned with different cell passages did not differ significantly but in embryos cloned with 7-8 cell passage was significantly (p<0.05) lower than the IVF control. Apoptosis signals were detected in IVF and cloned embryos as early as day 3 and the rates of apoptosis increased concurrently with the embryo development. In conclusion, high apoptosis during in vitro preimplantation development resulted in low development rate and total cell number of cloned embryos. Moreover, based on the apoptotic incidence in cloned blastocysts, fetal fibroblasts are more suitable for production of cloned embryos in porcine.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.12
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• pp.1820-1826
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• 2007

In this study, we conducted various experiments in order to develop enhanced cultural conditions for in vitro-produced porcine embryos. All embryos were produced by in vitro maturation (IVM) and fertilization (IVF) of immature oocytes from abattoir-derived ovaries. In experiment 1, we cultured IVF embryos in 4 different groups, namely, 0% bovine serum albumin (BSA), 3% BSA, 0.05% Polyvinyl alcohol (PVA), and 0.5% Polyvinylpyrrolidone (PVP) added to the basal fluid cultural medium, Porcine zygote medium 3 (PZM-3). The rates of embryo development were higher in the group where the PZM-3 media had been supplemented with 3% BSA than the other groups. While not statistically significant, the percent of blastocysts and hatched blastocytes were 6.9% and 25.0% in the 3% BSA group vs. 1.2-6.4% and 0-16.7% in the other groups, respectively. In experiment 2, we added 10% fetal bovine serum (FBS) to PZM-3 on day 0 of culture and observed the development rate of blastocysts per day of culture from days 0 to 5. The development rate of blastocysts was higher at 15.6% on day 4 than on any other day, and was significantly higher than on day 0 or day 1 (p<0.05). The development rate of hatched blastocysts was 26.7% on day 4, and was higher than on any other day. In experiment 3, we cultured IVF embryos with different fluid culture media, grouped as 1) PZM-3+0.3% BSA (day0-day7); 2) PZM-3+0.3% BSA${\rightarrow}$day-4) PZM-3+10% FBS; 3) PZM-3+0.3% BSA${\rightarrow}$PZM-3+0.3% BSA+(day-4) FBS 10%; and 4) PZM-3+0.3% BSA+10% FBS (day0-day7). The development rates of blastocysts and hatched blastocysts were 21.5% and 53.1% in group 3, respectively, which was significantly higher than group 4 with respect to blastocyst development (5.2%, p<0.05) but not hatched blastocysts (14.3%). The total cell number (TCN) of blastocysts in group 3 was higher at $37.8{\pm}16.1$ than the other groups at $16.8{\pm}4.4$ - $30.1{\pm}10.9$; however, this was not significantly different. The results of this study showed that PZM-3 containing 0.3% BSA and supplemented with FBS during the later stage of culture on day 4 resulted in better TCNs and an increased rate of hatched blastocysts.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.323-331
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• 1996

Predetermination of sex in mammalian species has many aspects of application including the prenatal diagnoses of genetic disorders in humans and sex-selected breeding programs in the animal industry. Embryos sexing can be carried out using the polymerase chain reaction (PCR) to amplify specific sequences present in the sex chromosomes, or by fluorescent in situ hybridization (FISH) of specific probes to the X and Y chromosomes. A 3.3 kb porcine male-specific DNA fragment (pEM39) was cloned previously in our laboratory. In this study, FISH and PCR methods were employed to examine if the pEM39 can be used a sex-specific DNA probes Porcine ovaries were obtained from a local slaughter house and oocytes collected. All oocytes were subjected to in vitro maturation followed by 1n vitro fertilization. Parthenogenetically activated embryos were served as a negative control. Embryonic samples were collected at the 2-cell stages and PCR was performed to analyze DNA. Among 10 embryos examined, four embryos were identified as males and six were females. The cloned male-specific DNA fragment showed male-specificity for the cells in the liver tissue and the porcine early embryos by FISH. It was also demonstrated that the cloned male-specific DNA is localized on the hetero chromatic region of the long arm in the Y chrom-osome (Yq) as shown by the FISH and karyotyping. The results suggest that the cloned male-specific DNA fragment may be useful for predetermination of sex with a few embryonic cells. The porcine male-specific sequence can be a reliable index for embryo sexing by PCR.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.225-230
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• 2009

Sex-sorting of sperm is an assisted reproductive technology (ART) used by the livestock industry for the mass production of animals of a desired sex. The standard method for sorting sperm is the detection of DNA content differences between X and Y chromosome-bearing sperm by flow cytometry. However, this method has variable efficiency and therefore requires verification by a second method. We have developed a sex determination method based on quantitative real-time polymerase chain reaction (qPCR) of the porcine amelogenin (AMEL) gene. The AMEL gene is present on both the X and the Y chromosome, but the length and sequence of its noncoding regions differ between the X and Y chromosomes. By measuring the threshold cycle (Ct) of qPCR, we were able to calculate the relative frequency of X chromosome. Two sets of AMEL primers were used in these studies. One set (AME) targeted AMEL gene sequences present in both X and Y chromosome, but produced PCR products of different lengths for each chromosome. The other set (AXR) bound to AMEL gene sequences present on the X chromosome but absent esholthe Y-chromosome. Relative product levels were calculated by normalizing the AXR fluorescence to the AME fluorescence. The AMEL method accurately predicted the sex ratios of boar sperm, demonstrating that it has potential value as a sex determination method.

• Journal of Embryo Transfer
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• v.26 no.1
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• pp.79-84
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• 2011

This study was performed to identify the differentially methylated region (DMR) and to examine the mRNA expression of the imprinted H19 gene in day 35 of SCNT pig fetuses. The fetus and placenta at day 35 of gestation fetuses after natural mating (Control) or of cloned pig by somatic cell nuclear transfer (SCNT) were isolated from a uterus. To investigate the mRNA expression and methylation patterns of H19 gene, tissues from fetal liver and placenta including endometrial and extraembryonic tissues were collected. The mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. Bisulfite analyses demonstrated that the differentially methylated region (DMR) was located between -1694 bp to -1338 bp upstream from translation start site of the H19 gene. H19 DMR (-1694 bp to -1338 bp) exhibits a normal mono allelic methylation pattern, and heavily methylated in sperm, but not in oocyte. In contrast to these finding, the analysis of the endometrium and/or extraembryonic tissues from SCNT embryos revealed a complex methylation pattern. The DNA methylation status of DMR Region In porcine H19 gene upstream was hypo methylated in SCNT tissues but hypermethylated in control tissues. Furthermore, the mRNA expression of H19 gene in liver, endometrium, and extraembryonic tissues was significantly higher in SCNT than those of control (p<0.05). These results suggest that the aberrant mRNA expression and the abnormal methylation pattern of imprinted H19 gene might be closely related to the inadequate fetal development of a cloned fetus, contributing to the low efficiency of genomic reprogramming.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.129-137
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• 2018

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig ($GalT^{-MCP/-MCP}$). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain $GalT^{-MCP/-MCP}/CD39/CD73$ pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from $GalT^{-MCP/-MCP}$ pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate $GalT^{-MCP/-MCP}/CD39/CD73$ pig expressing CD39 and CD73 at endothelial cells.

• Journal of Embryo Transfer
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• v.17 no.3
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• pp.179-185
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• 2002

This study was performed to investigate the effect of different cryoprotectants and superoxide dismutase(SOD) on viability of frozen-thawed oocytes by vitrification method in the pig. The proportions of oocytes matured to metaphase-I stage were higher in medium with ethylene glycol and DMSO(19.9%) than in medium with glycerol and DMSO(6.5%). When the oocytes were exposed in medium containing ethylene glycol, oocyte matured to prophase-I were not observed. On the other hand. significant differences were not observed between in medium with and without SOD( 1 unit/$m\ell$) during IVM of vitrified and thawed immature oocytes. However, the maturation rate from metaphase-I to metaphase-II were higher in medium with that than without SOD. The penetration rates after IVF of oocytes frozen-thawed were also higher in medium with that than without SOD. These results indicate that frozen-thawed oocytes treated with ethylene glycol and DMSO was more protective against freezing effect and that addition of 1 unit/$m\ell$ SOD in medium fur prevent of lipid peroxidation may play a positive role in improving of viability of frozen-thawed oocytes.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.287-294
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• 2011

Autophagy is a process of intracellular bulk protein degradation, in which the accumulated proteins and cytoplasmic organelles are degraded. It plays important roles in cellular homeostasis, apoptosis, and development, but its role during early embryo development remains contentious. Therefore, in the present study, we investigated the effects of 3-methyladenine (3-MA) on early embryonic development in pigs, we also investigated several indicators of developmental potential, including mitochondrial distribution, genes expressions (autophagy-, apoptosis- related genes), apoptosis and ER-stress, which are affected by 3-MA. After in vitro maturation and fertilization, presumptive pig embryos were cultured in PZM-3 medium supplemented with 3-MA for 2 days at $39^{\circ}C$ 5% $CO_2$ in air. Developmental competence to the blastocyst stage in the presence of 3-MA was gradually decreased according to increasing concentration. Thus, all further experiments were performed using 2 mM 3-MA. Blastocysts that developed in the 3-MA treated group decreased LC3-II intensity and expressions of autophagy related genes than those of the untreated control, resulting in down-regulates the autophagy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 3-MA treated group compared with control ($6.0{\pm}1.0$ vs $3.3{\pm}0.6$, p<0.05). Also, the expression of the pro-apoptotic gene Bax increased in 3-MA treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. Mito Tracker Green FM staining showed that blastocysts derived from the 3-MA treated group had lower mitochondrial integrity than that of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. Then, the expression of the spliced form of pXBP-1 product (pXBP-1s) increased in 3-MA treated group, resulting increase of ER-stress. Taken together, these results indicate that inhibition of autophagy by 3-MA is closely associated with apoptosis and ER-stress during preimplantation periods of porcine embryos.