• Journal of Embryo Transfer
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• v.25 no.2
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• pp.127-131
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• 2010

The objective of this study was to determine the mRNA expression patterns of several putative imprinted genes in in vivo and in vitro fertilized, parthenogenetic, and cloned porcine preimplantation embryos. Both maternally (Dlk1, IGF2, Peg1/Mest and Ndn) and paternally (IGF2r, H19 and Xist) imprinted genes were selected. We have used reverse transcription polymerase chain reaction (RT-PCR) to investigate gene expression patterns in the porcine embryos. IGF2 transcripts were detected in the most of embryos. In nuclear transfer (NT), Peg1/MEST transcripts showed fluctuating pattern. Dlk1 was only expressed partially from the morula and blastocyst stage of NT embryos. Ndn gene expression was started somewhat early for in vivo embryos. However, the expressions of maternally imprinted genes were similar in all types of blastocysts (NT, in vivo and in vitro fertilized, and parthenogenetic embryos). The IGF2R gene expression level was somewhat irregular and varied among samples. However, for the majority samples of all types of embryos, IGF2R expression was diminished after one- to two-cell stages and reappeared at the morulae or blastocyst stage embryos. H19 gene was only expressed early in parthenogenetic and in vivo embryos. For NT embryos, H19 was only expressed in blastocysts. Xist expression was detected in all blastocysts with the earliest being in vivo 8-cell stage embryos and the last one being NT blastocysts. These putative imprinted genes appeared to have stage specific expression patterns with a fluctuating pattern for some genes (Peg/Mest, IGF2r, H19). These results suggest that stage specific presence of imprinted genes can affect the embryo implantation and fetal development.

• Journal of Veterinary Clinics
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• v.24 no.3
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• pp.295-299
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• 2007

The detrimental effects of gene transfection on embryo development and the molecular mechanism behind the differential expression of genes related to early embryo development were assessed in the production of transgenic cow embryos through somatic cell nuclear transfer (NT). Parthenogenetic, IVF, and transgenic NT embryos derived from ${\alpha}_1$-antitrypsin transfected ear fibroblast cells was produced. To investigate the molecular mechanism behind lower developmental competence of transgenic NT embryos, the differential mRNA expression of three genes ($IFN-{\tau}$, Oct4, Fgf4) in the 3 types of embryo (Parthenogenetic, IVF, transgenic NT) was examined. RNA was extracted from ten blastocysts derived from 3 types of embryos and reverse-transcripted for synthesis of the first cDNA. The quantification of 3 gene transcripts ($IFN-{\tau}$, Oct4, and Fgf4) was carried out in three replicate by quantitative real-time reverse transcriptase PCR. Expression level of $IFN-{\tau}$ mRNA was significantly higher in transgenic NT embryos than parthenogenetic and IVF embryos (P<0.05). However, expression level of Oct4 and Fgf4 of transgenic NT embryos was significantly lower than IVF embryos (P<0.05). Altered levels of these three mRNA transcripts may explain some of the embryonic/fetal/neonatal abnormalities observed in offspring from transgenic NT embryos.

• Reproductive and Developmental Biology
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• v.31 no.4
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• pp.273-278
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• 2007

This study was conducted to examine the protein kinase inhibitors, 6-dimethylaminopurine (DMAP) and cycloheximide (CHXM) on the development and chromosome constitution of porcine parthenogenetic embryos. In vitro matured oocytes were activated by electric stimuli (ES) or a combination of ES with culture in 2 mM DMAP or $10{\mu}g/ml$ CHXM for 4 hr. Activated oocytes were cultured in PZM-3 for 6 days. Some 1-cell embryos and blastocysts were fixed by air dry method to analyze the chromosome constitutions and/or total cell number. Blastocyst development of DMAP-treated group (26.7%) was significantly higher (p<0.05) than those of CHXM-treated and ES control groups. Ploidy in 1-cell stage embryos was not different among groups (77.3 to 81.0%), however, proportion of diploid chromosome constitutions was high in DMAP-treated group (61.9%, p<0.05). In the blastocyst stage, proportion of diploid chromosome plates was significantly high in DMAP-treated group (64.2%, p<0.05), and proportion of abnormal chromosome plates was higher in CHXM-treated group (36.6%, p<0.05) than DMAP-treated group (28.3%,). Proportion of embryos with abnormal chromosome constitutions was slightly increased by DMAP (40.0%) and CHXM (42.1%) treatment due to the increasing of mixoploid (47.4 and 52.0%). The present study shows that the DMAP treatment increase the development of porcine parthenotes. However, parthenogenetic activation by ES or combined treatment with ES and DMAP or CHXM detrimentally affects the chromosome constitutions of porcine parthenotes during early embryonic development, leads to increased abnormal ploidy in the blastocyst stage.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.5
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• pp.617-620
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• 2004

The present study compares development of rabbit embryos generated using different oocyte activation protocols and reconstructed with embryonic or cumulus cells as nuclear donor. In vivo matured oocytes were collected from New Zealand White rabbits at 16 h after ovulation treatment and were activated at18 h of post-ovulation treatment. The following schemes of oocytes activation were tested: 1) single electric pulse (EP, 3.2 kV/cm, 3${\times}$20 $\mu$s, 0.3 M mannitol)+5 min culture in the presence of 5 mM Ionomycin, 2) single electric pulse (EP, 3.2 kV/cm, (${\times}$20 $\mu$s, 0.3 M mannitol)+1 h culture in the presence of 2 mM 6-DMAP, and 3) three electric pulses 30 min apart. Cleavage rate, percentage of expanded and hatched blastocysts as well as total cell number of blastomeres of parthenogenetic embryos were significantly higher using either EP+6-DMAP or 3${\times}$EP schemes, comparing with EP+Ionomycin. Development rate up to hatched blastocyst stage of cloned rabbit embryos using the EP+6-DMAP for activation of nuclei were 19% for embryonic cell nuclei and 36% for cumulus cell nuclei. The best activation protocol optimalized in this study was the combined treatment "P+6-DMAP" which may be potentially used for nuclear transfer protocol.

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.105-108
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• 2009

This study was designed to investigate the effect of kinetin on in vitro development of parthenogenetic porcine oocytes exposed to demecolcine prior to activation. In vitro matured metaphase II stage oocytes were incubated in 0 or 2 ${\mu}$g/ml demecolcine supplemented defined culture medium for 3 h and the oocytes were activated electrically. The parthenogenetic porcine embryos were then cultured in 0 or 200 ${\mu}$M kinetin supplemented defined culture medium for 7 days. Regardless of demecolcine treatment, kinetin supplementation increased blastocyst rates significantly (7.0% versus 12.1% and 4.9% versus 8.5%; Control versus Kinetin and Demecolcine versus Kinetin + Demecolcine, respectively, p<0.05). Demecolcine treatment before activation tended to decrease blastocyst rates regardless of kinetin supplementation although it is not statistically significant. Total cell numbers in the blastocysts also tended to be elevated in embryos when supplemented with kinetin, however only the result between Kinetin and Demecolcine groups is statistically significant (37.6 ${\times}$ 7.2 versus 28.1 ${\times}$ 9.5, respectively, p<0.05). In conclusion, the present report shows that kinetin enhances developmental competence of parthenogenetic porcine embryo regardless of demecolcine pre-treatment before parthenogenetic activation when they were developed in defined culture condition.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.83-83
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• 2002

This study was to compare the characteristics of parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Mouse oocytes were recovered from superovulated 4wks hybrid F1 (C57BL/6xCBA/N) female mice. The oocytes were treated with 7% ethanol for 5 min and 5 ㎍/㎖ cytochalasin-B for 4 h. For IVF, the oocytes were inseminated with epididymal sperm of hybrid Fl male mice (1×10/sup 6//㎖). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count in blastocysts was carried out differential labelling using propidium iodide (red) and bisbenzimide(blue). (omitted)

• International Journal of Oral Biology
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• v.33 no.3
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• pp.91-95
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• 2008

Embryonic stem cells have a pluripotency and a potential to differentiate to all type of cells. In our previous study, we have shown that embryonic stem cells (ESCs) lines can be generated from murine parthenogenetic embryos. This parthenogenetic ESCs line can be a useful stem cell source for tissue repair and regeneration. The defect in full-term development of parthenogenetic ESCs line enables researchers to avoid the ethical concerns related with ESCs research. In this study, we presented the results demonstrating that parthenogenetic ESCs can be induced into osteogenic cells by supplementing culture media with ascorbic acid and $\beta$-glycerophosphate. These cells showed morphologies of osteogenic cells and it was proven by Von Kossa staining and Alizarin Red staining. Expression of marker genes for osteogenic cells (osteopontin, osteonectin, alkaline phosphatase, osteocalcin, bone-sialoprotein, collagen type1, and Cbfa1) also confirmed osteogenic potential of these cells. These results demonstrate that osteogenic cells can be generated from parthenogenetic ESCs in vitro.

• Journal of Embryo Transfer
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• v.32 no.1
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• pp.25-31
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• 2017

This study was conducted to investigate the effect of activation method on the endoplasmic reticulum (ER) stress induction, apoptosis and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by four activation methods; 1) electric stimulus (ES) (E), 2) $ES+10{\mu}M$ Ca-ionophore (A23187) treatment (EC), 3) ES+2 mM 6-dimethylaminopurine (6-DMAP) treatment (ED), or 4) ES+A23187 and 6-DMAP treatments (ECD). Parthenogenetic embryos were sampled to analyze x-box binding protein 1 (Xbp1) mRNA, ER stress-associated genes and apoptosis genes at 3 h after ES and the 1-cell and blastocyst stages. In the EC group, the band intensity of spliced Xbp1 (Xbp1s) mRNA was higher than those of the other groups at the 3 h and 1-cell stage, and higher than that of the E group at the blastocyst stage. Four ER stress-associated genes were expressed at the highest level in the EC group and weakly expressed in the ED group at 3 h after activation. However, most of the genes were highly expressed at the 1-cell and blastocyst stages with some variation in the EC and ECD groups. Expression of Bcl-2-associated X protein (Bax) and caspase-3 mRNA was significantly higher in the EC group than in the other groups at all development stages. The developmental rates to the blastocyst stage were higher in the ED and ECD groups than in the E and EC groups. These results suggest that the intracellular ER stress of parthenogenetic porcine embryos is affected by the activation method and subsequently lead to the apoptosis of embryos.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.31-36
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• 2008

We attempted to control the maturation promoting factors (MPF) activity and nuclear remodeling of somatic cell nuclear transfer (NT) bovine embryos. Bovine ear skin fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h and activated. The nuclear remodeling type of the reconstituted embryos was evaluated 1.5 h after activation. MPF activity was assessed in enucleated and chemical treated oocytes before the injection of a donor cell. Effect of chemicals on the embryonic development was evaluated with parthenogenetic embryos. MPF activity increased significantly by caffeine treatment, but decreased by vanadate treatment (p<0.05). Caffeine or vanadate had no deleterious effect on the parthenogenetic embryo development. In caffeine treated group, premature chromosome condensation (PCC) was occurred in 72.2% of NT embryos (p<0.05). In contrast, vanadate induced the formation of a pronucleus-like structure (PN) in a high frequency (68.9%, p<0.05) without PCC (NPCC). Blastocyst development of NT embryos increased by treating with caffeine (30.3%), whereas decreased by treating with vanadate (11.4%) compared to control (22.1%, p<0.05). The results indicate that caffeine or vanadate can control of MPF activity and remodeling type of NT embryos, resulting in the increased or decreased in vitro development.

• Development and Reproduction
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• v.22 no.4
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• pp.403-403
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• 2018