• Reproductive and Developmental Biology
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• v.37 no.1
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• pp.51-55
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• 2013

The present study was conducted to examine the effect of antioxidant treatment during parthenogenetic activation procedure on the reactive oxygen species (ROS) levels and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by a combination of electric stimulus and 2 mM 6-dimethylaminopurine (6-DAMP) before in vitro culture. During the activation period, oocytes were treated with $50{\mu}M$ ${\beta}$-mercaptoethanol (${\beta}$-ME), $100{\mu}M$ L-ascorbic acid (Vit. C) or $100{\mu}M$ L-glutathione (GSH). To examine the ROS level, porcine parthenogenetic embryos were stained in $10{\mu}M$ dichlorohydrofluorescein diacetate ($H_2DCFDA$) dye 20 h after culture, examined under a fluorescence microscope, and the fluorescence intensity (pixels) were analyzed in each embryo. The parthenogenetic embryos were cultured for 6 days to evaluate the in vitro development. The apoptosis was measured by TUNEL assay. The $H_2O_2$ levels of parthenogenetic embryos were significantly lower in antioxidant treatment groups ($26.9{\pm}1.6{\sim}29.1{\pm}1.3$ pixels/embryo, p<0.05) compared to control ($33.2{\pm}1.7$ pixels/embryo). The development rate to the blastocyst stage was increased in antioxidant treatment groups (32.0~32.5%) compared to control (26.9%, p<0.05), although, there was no difference in apoptosis among groups. The result suggests that antioxidant treatment during parthenogenetic activation procedure can inhibit the ROS generation and enhance the in vitro development of porcine parthenogenetic embryos.

• Reproductive and Developmental Biology
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• v.33 no.1
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• pp.13-18
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• 2009

The aim of this study was to examine the microtubule distributions of somatic cell nuclear transfer (SCNT) and parthenogenetic porcine embryos. Porcine SCNT embryos were produced by fusion of serum-starved fetal fibroblast cells with enucleated oocytes. Reconstituted and mature oocytes were activated by electric pulses combined with 6-dimethlyaminopurine treatment. SCNT and parthenogenetic embryos were cultured in vitro for 6 days. Microtubule assembly of embryos was examined by confocal microscopy 1 hr and 20 hr after fusion or activation, respectively. The proportions of embryos developed to the blastocyst stage were 25.7% and 30.4% in SCNT and parthenogenetic embryos, respectively. The frequency of embryos showing $\beta$-tubulins was 81.8% in parthenogenetic embryos, whereas 31.3% in SCNT embryos 1 hr after activation or fusion. The frequency of the embryos underwent normal mitotic phase was low in SCNT embryos (40.6%) compared to that of parthenogenetic ones (59.7%) 20 hr after fusion or activation (p<0.05). The rate of SCNT embryos with an abnormal mitosis pattern is about twice compared to that of parthenogenetic ones. The spindle assembly and its distribution of SCNT embryos in the first mitotic phase were not different from those of parthenogenetic ones. The result shows that although microtubule distribution of porcine SCNT embryos shortly after fusion is different from parthenogenetic embryos, and the frequency of abnormal mitosis 20 hr after fusion or activation is slightly increased in SCNT embryos, microtubule distributions at the first mitotic phase are similar in both SCNT and parthenogenetic embryos.

• Korean Journal of Veterinary Research
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• v.32 no.4
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• pp.663-669
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• 1992

This experiment was carried out to find out the best condition for the parthenogenetic activation of mouse eggs by treating ethanol and hyaluronidase. For the parthenogenetic activation of eggs with ethanol, cumulus cell enclosed or denuded eggs were treated with 7% ethanol in D-PBS for 5, 7 or 9 minutes. For the activation of eggs with hyaluronidase, the eggs with cumulus masses were released into D-PBS with 100 unit hyaluronidase and treated for 10, 12 or 13 minutes. All of the treated eggs were incubated in BMOC-3 solution for 5 hours at $37^{\circ}C$ at an atmosphere of 5% $CO_2$ in air. The types of parthenogenetic eggs were morphologically classified into haploid, diploid, immediate cleavage eggs under an inverted microscope. The results obtained in this experiment were summarized as follows ; 1. High activation rate(99%) had been achieved by treating the eggs with 7% ethanol for 7 minutes. 2. With 100 IU hyaluronidase, high activation rate (94%) had been achieved by treating for 12 minutes. 3. The most frequent type of parthenogenetic eggs activated with ethanol or hyaluronidase was haploid (p<0.05). 4. The eggs collected from 18 to 22 hours post HCG injection showed higher activation rate than the eggs collected at 16 hours post HCG injection. 5. No significant difference (p>0.05) in activation rate was shown in strain of mouse and in presence of cumulus cells.

• Development and Reproduction
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• v.22 no.3
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• pp.235-244
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• 2018

We investigate the effect of endoplasmic reticulum (ER) stress inhibitor treatment during parthenogenetic activation of oocytes on the ER stress generation, apoptosis, and in vitro development of parthenogenetic porcine embryos. Porcine in vitro matured oocytes were activated by 1) electric stimulus (E) or 2) $E+10{\mu}M$ Ca-ionophore (A23187) treatment (EC). Oocytes were then treated by ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxychloic acid (TUDCA, $100{\mu}M$) for 3 h prior to in vitro culture. Parthenogenetic embryos were sampled to analyze ER stress and apoptosis at the 1-cell and blastocyst stages. The x-box binding protein 1 (Xbp1) mRNA and ER stress-associated genes were analyzed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. At the 1-cell stage, although no difference was observed in Xbp1 splicing among treatments, BiP transcription level in the E group was significantly reduced by salubrinal treatment, and GRP94 and ATF4 transcription levels in EC group were significantly reduced by all treatments (p<0.05) compared to control. In the EC group, both apoptotic genes were reduced by ER stress inhibitor treatments compared to control (p<0.05) except Caspase-3 gene by TUDCA treatment. These results suggest that the treatment of ER stress inhibitor during parthenogenetic activation can reduce ER stress, and thereby reduce apoptosis and promote in vitro development of porcine parthenogenetic embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.10
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• pp.1510-1516
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• 2007

These experiments were carried out to optimize the parameters of electrical activation, methods of parthenogenetic activation and embryo culture in vitro and meanwhile to isolate embryonic stem cells-like (ESCs) derived from porcine parthenogenetic blastocysts (pPBs). These results showed that, as the electric field strength increased from 1.0 to 2.7 kV/cm, the cleavage rate of parthenogenetic embryos increased gradually but the rate of oocyte lysis was significantly increased when using 2.7 kV/cm field strength. The rate of cleavage in 2.2 and 2.7 kV/cm groups was significantly increased in comparison with that of the 1.0 kV/cm group. A voltage field strength of 2.2 kV/cm DC was used to investigate blastocyst development following activation with a single pulse of 30 or $60-{\mu}sec$ pulse duration. The optimum pulse duration was 30-${\mu}sec$, with a blastocyst rate of 20.7%. Multiple pulses were inferior to a single pulse for blastocyst yield (8.0% vs. 29.9) (p<0.05). For porcine oocyte parthenogenetic activation methods, the rates of cleavage (79.0% vs. 59.8%) and blastocysts (19.4% vs. 3.4%) were significantly increased in electrical activation in contrast to chemical activation with ionomycin/6-DMAP (p<0.05). Rates of cleavage and blastocyst formation in NCSU-23 and PZM-3 embryo media were higher than those of G1.3/G2.3 serial culture media, but there was no significant difference among the three groups. The total cell number of blastocysts in PZM-3 embryo culture media containing $5{\mu}g/ml$ insulin was significantly higher than that of the control (no insulin) ($44.3{\pm}9.1$ vs. $33.9{\pm}11.7$). For isolation of PESCs-like, the rates of porcine blastocysts attached to feeder layers and ICM colony formation in Method B (nude embryo culture) were better than those in Method A (intact embryo culture).

• Korean Journal of Animal Reproduction
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• v.18 no.3
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• pp.183-189
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• 1994

We examined early development in loach(Misgurnus mizolepis) embryos with parthenogenetic agents well-known in mammals. Female loach was superovulated with an intraperitoneal injection of 15 IU human chorionic gonadotrophin (hCG) per gram body weight. After 13 h of hCG injection, the oocytes were obtained from the abdomen. The oocytes were activated with 10% ethanol in tap water or fish Ringer's solution for 5, 10 and 15 minutes(eTW5, 10, 15 and eFRS5, 10, 15), respectively. The activation rates were 29% and 10% in eFRS10 and eFRS15, 5% and 6% in eTW10 and eTW15 by judging the cleaved blastomeres. Whereas, no parthenogenetic embryo was produced by tap water or fish Ringer's solution alone. The activation rate with the fish Ringer's solution was higher than that of tap water. No embryonic development was observed by calcium ionophore, A23187, at concentrations of 10, 20, 40 and 100$\mu$M when treated for 1, 2.5 and 5 minutes, respectively. The activation agents did not cause early development as in mammalian eggs. Therefore, the results suggest that fresh water fish may have a different egg activation pathway from that of mammals.

• 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.

• Journal of Embryo Transfer
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• v.26 no.2
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• pp.123-128
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• 2011

In vitro maturation and activation of oocytes are primary steps towards biotechnological manipulation in embryology. The objectives of the present study were to determine the oocyte recovery rate per ovary, in vitro maturation rates of oocytes and rates of parthenogenetically activation of matured oocytes in Black Bengal goats. All visible follicles were aspirated to recover follicular fluid from individual ovaries (number of ovaries = 456). The immature cumulus oocyte complexes (COCs; n = 1289) were cultured in tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal bovine serum (FBS) for 27 hours at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The matured oocytes (n = 248) were activated with 5 ${\mu}M$ ionomycin for 5 minutes followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) for 4 hours. After activation, oocytes were cultured for another 14 hours in TCM-199 supplemented with bovine serum albumin (BSA) at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The pronucleus formation in activated oocytes was determined by staining with 1% orcein (whole mount technique). Matured oocytes (n = 176) without activation stimuli were used as control. The mean number of oocytes recovered per ovary was $3.5{\pm}0.5$. The proportion of oocytes matured in vitro, confirmed by the presence of first polar body, was $42.1{\pm}4.7%$. Parthenogenetic activation, evidenced by formation of pronucleus, occurred in $37.2{\pm}15.8%$ of matured oocytes. No pronucleus formation was observed in control oocytes. In conclusion, a combination of ionomycin and 6-DMAP induces activation in one third of Black Bengal goats' oocytes.

• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.117-120
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• 2005

This study was conducted to establish the optimal temperature condition before oocyte activation in B6m F1 mouse. In experiment 1, two embryo culture media (CZB vs KSOM) were evaluated for the development of activated mouse oocytes. Parthenogenetic embryos cultured in KSOM showed better blastocyst development than ones cultured in CZB $(56.2\%\;vs\;81.0\%\;p<0.01)$. Two-hour of pre-incubation before activation significantly reduced the number of hatched blastocysts in KSOM $(22.0\%\;versus\;8.8\%\;p<0.05)$. In experiment 2, recovered oocytes were pre-incubated at different temperature conditions before activation. The experimental groups were divided by 5 as follows. Group A: pre-incubation for 120 min at $37^{\circ}C$, Group B: pre-incubation at $37^{\circ}C$ for 90 min then at $25^{\circ}C$ for 30 min, Group C: pre-incubation at $37^{\circ}C$ for 60 min then at $25^{\circ}C$for 60 min, Group D: pre-incubation at $37^{\circ}C$ for 30 min then at $25^{\circ}C$ for 90 min, and Group E: pre-incubation at $25^{\circ}C$ for 120 min before activation. Group A $(67.6\%)$ and B $(66.7\%)$ showed better development to the blastocyst stage than other groups $(Group\;C:\;50.0\%\;Group \;D:\;49.2\%\;Group\;E:\;33.3\%,\;p<0.05)$. The present study indicates that the temperature before activation affects the development of B6D2 F1 mouse parthenogenetic oocytes and exposure to room temperature should be limited to 30-min when the oocytes are left in HEPES-buffered medium for micromanipulation.

• Reproductive and Developmental Biology
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• v.37 no.2
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• pp.57-64
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• 2013

Developmental potential of cloned embryos is related closely to epigenetic modification of somatic cell genome. The present study was to investigate the effects of applying histone deacetylation inhibitor, trichostatin A (TSA) to activated porcine embryos on subsequent development of porcine parthenogenetic and nuclear transfer embryos. Electrically activated oocytes were treated with 5 nM TSA for different exposure times (0, 1, 2 and 4 hr) and then the activated embryos were cultured for 7 days. The reconstructed embryos were treated with different concentrations of 0, 5, 10 and 25 nM TSA for 1 hr. Also 5 nM TSA was tested with different exposure times of 0, 0.5, 1, 2 and 4 hr. And fetal fibroblast cells were treated with 50 nM TSA for 1, 2 or 4 hr and with 5 nM TSA for 1 hr. Cumulus-free oocytes were enucleated and reconstructed by TSA-treated donor cells and electrically fused and cultured for 6 days. In parthenogenetic activation experiments, 5 nM TSA treatment for 1 hr significantly improved the percentage of blastocyst developmental rates than the other groups. Total cell number of blastocysts in 1 hr group was significantly higher than other groups or control. Similarly, blastocyst developmental rates of porcine NT embryos following 5 nM TSA treatment for 1 hr were highest. And the reconstructed embryos from donor cells treated by 50 nM TSA for 1 hr improved the percentage of blastocyst developmental rates than the control group. In conclusion, TSA treatment could improve the subsequent blastocyst development of porcine parthenogenetic and nuclear transfer embryos.