• Reproductive and Developmental Biology
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• 제38권3호
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• pp.93-98
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• 2014

The objective of this study was to investigate the changes of oxidative stress and antioxidant enzyme during in vitro development with washing culture oil in porcine embryos. During the culture, the four types of culture oil such as paraffin oil with or without washing and mineral oil with or without washing were examined. The oil was washed with PZM-3 during 7 days and collected oil only. The embryos were stained with CellTracker$^{TM}$ Red, DCFDA and Hoechst 33342 to confirm the effects of the oil. As a results, Cleavage rates and total cell number were no difference among the four oil groups. However, ${\geq}16$ cell embryos were significantly different in fore type oil treatment and blastocyst rate was significantly higher washing paraffin treatment than in other group(p<0.05). Also, the expression of free radical were lower in washing paraffin oil than in other groups (p<0.05). On the other hand, the expression of glutathione were not significant different among paraffin oil with or without washing and mineral oil with or without washing, however washing paraffin oil and washing mineral groups were higher than other treatment groups. In conclusion, the washing oil was expected with positive effects on in vitro development in porcine embryos.

• 한국수정란이식학회지
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• 제32권4호
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• pp.275-285
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• 2017

Lysophosphatidic acid (LPA) is an important signaling molecule. Here, the effect and mechanism of LPA on the preimplantation development of porcine embryos during in vitro culture (IVC) was examined. Porcine embryos were cultured in porcine zygote medium (PZM-3) supplemented with $30{\mu}M$ LPA during different days. There was a significantly higher cleavage rate in Day 1-7 and significantly higher total cell number of blastocysts in Day 1-3 and Day 4-7. It was also found that messenger RNA (mRNA) expression level of PCNA, BCL2 and BAX in blastocysts obtained from D1-7 group were significantly higher and BCL2/BAX mRNA ratio in D1-3 group was significantly lower than control group but Day 4-7 and Day 1-7 groups were comparable with control group. Treatment with $20{\mu}M$ PLC inhibitor significantly decreased the embryo cleavage rate and blastocyst formation rate. Moreover, LPA as an activator of PLCs, enhanced the $30{\mu}M$ LPA + $20{\mu}M$ U73122 group embryo cleavage rate which similar with control group. In conclusion, the results suggest that treatment with LPA during IVC improves the porcine early embryo cleavage by activation of PLC signaling pathway and regulate the mRNA expression that contribute to total cell number of blastocysts during blastocyst formation.

• 한국수정란이식학회지
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• 제30권1호
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• pp.23-31
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• 2015

Catalpol, an iridoid glucoside, isolated from the root of Rehmannia glutinosa Libosch. It possesses a broad range of biological and pharmacological activity including anti-tumor, anti-inflammation and anti-oxidant by acting as a free radical scavenger. Therefore, in this study, the effects of catalpol on blastocyst development, expression levels of reactive oxygen species (ROS) and apoptotic index were investigated in porcine embryos. After in vitro maturation and fertilization, porcine embryos were cultured for 6 days in porcine zygote medium 3 (PZM-3) supplemented with catalpol (0, 100, 200 and $400{\mu}M$, respectively). Blastocyst development not significantly improved in the catalpol treated group when compared with control group. Otherwise, the intracelluar levels of ROS were decreased and the numbers of apoptotic nuclei were reduced in the catalpol ($100{\mu}M$) treated porcine blastocysts (P<0.05). On the other hand, blastocyst development was significantly improved in the catalpol ($100{\mu}M$) treated group when compared with the untreated catalpol group under $H_2O_2$ ($200{\mu}M$) induced oxidative stress (P<0.05). Otherwise, the intracellular levels of ROS in catalpol ($100{\mu}M$) treated group were significantly decreased in the untreated catalpol group under $H_2O_2$ ($200{\mu}M$) induced oxidative stress (P<0.05). Furthermore, the total cell numbers of blastocysts were significantly increased (P<0.05) in the catalpol ($100{\mu}M$) treated group under $H_2O_2$ ($200{\mu}M$) induced oxidative stress, whereas numbers of apoptoic nuclei were significantly reduced (P<0.05). In conclusion, our results indicate that treatment of catalpol may have important implications for improving developmental competence and preimplantation quality of porcine embryos through its anti-oxidant and anti-apoptotic effect.

• 농업과학연구
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• 제33권1호
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• pp.35-41
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• 2006

이종간의 핵이식은 확보가 쉬운 난자를 이용함으로서 용이한 수핵란의 확보와 윤리적인 문제등을 피할 수 있으므로 매우 유용한 방법이다. 본 연구에서는 이러한 이종간 핵이식의 조건을 규명하고자 유산양 태아섬유아세포를 이용하여 돼지의 난자에 이종간 핵이식을 시도하였다. 돼지와 산양의 난소에서 난포란을 채취하여 각각 NCSU-23, TCM-199에 호르몬을 첨가한 성숙배양액에 배양하여 $38^{\circ}C$, 5% $CO_2$의 배양기에서 48시간 동안 체외성숙 시켜 수핵난자를 준비하였다. 공여세포는 유산양의 태아섬유아세포는 DMEM배양액에서 배양한 후 0.25% Trypsin-EDTA 용액으로 처리하여 single-cell로 분리하여 사용하였다. 돼지와 산양의 성숙란에 공여세포를 핵치환하여 0.3 M mannitol fusion medium에 넣어 각각 DC 1.2 kV/cm $30{\mu}sec$과 DC 2.39 kV/cm $15{\mu}sec$의 조건으로 BTX를 이용 2회의 전기충격으로 융합 활성화하였다. 활성화된 돼지와 산양 핵이식란은 각각 PZM-3와 mSOF 배양액에 7일간 배양하면서 할구분열율과 배발달율을 관찰하여 동종간 핵이식란과 비교하였다. 비교결과 이종간 핵이식란의 경우 분열률이 58.9%, 배반포기로의 발달률이 5.4%로 돼지 동종간 핵이식란의 분열율이 67.4%, 배반포기로의 배발달율은 13.6% 보다 낮게 나타났고, 또한 산양의 동종간 핵이식란배아의 분열율 81%, 상실배와 배반포기로의 발달률이 54.7% 보다 낮게 나타났다. 이러한 결과로 이종간의 이식은 많은 잇점에도 불구하고 아직은 낮은 배발달률을 보이고 있어 이에 대한 핵이식 조건 및 체외배양 조건 등 많은 부분에서의 추가적인 연구가 필요한 것으로 사료된다.

• Reproductive and Developmental Biology
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• 제35권3호
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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.

• 한국수정란이식학회지
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• 제32권3호
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• pp.139-146
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• 2017

The objective of this study was to determine the effect of fructose that was supplemented to a chemically defined in vitro maturation (IVM) medium on oocyte maturation and embryonic development after parthenogenesis in pigs. The base medium for in vitro maturation (IVM) was porcine zygote medium (PZM) that was supplemented with 0.05% (w/v) polyvinyl alcohol (PVA) or 10% (v/v) porcine follicular fluid (pFF). In the first experiment, when immature pig oocytes were matured in a chemically defined medium that was supplemented with 5.5 mM glucose or with 1.5, 3.0 and 5.5 mM fructose, 3.0 mM fructose resulted in a higher nuclear maturation (91.5%) than 1.5 and 5.5 mM fructose (81.9 and 81.9%, respectively) but showed a similar result with 5.5 mM glucose (94.2%). However, there was no significant differences among groups in the embryo cleavage (89.4-92.4%), blastocyst formation (37.5-41.1%), and mean cell number of blastocyst (30.8-34.2 cells). Fructose at the concentration of 3.0 mM (1.08 pixels/oocyte) resulted in a higher intra-oocyte glutathione (GSH) content than 1.5 and 5.5 mM fructose (1.00 and 0.87 pixels/oocytes, respectively) while the cumulus cell expansion was not influenced. In the second experiment, effect of individual and combined supplementation of a chemically defined maturation medium with 5.5 mM glucose or 3.0 mM fructose was examined. No significant effect was found in the nuclear maturation (86.3-92.6%). Embryo cleavage was significantly increased by the combined supplementation with glucose and fructose (95.2%) compared to that with 3.0 mM fructose only (85.7%) while blastocyst formation (37.3-42.8%) and embryonic cell number (33.3-34.1 cells) were not altered. Effect of supplementation of pFF-containing medium with glucose and fructose + glucose was examined in the third experiment. No significant effect by the supplementation with glucose and fructose or glucose alone was observed in the nuclear maturation of oocytes (90.7-94.1%) and blastocyst formation (51.0-56.5%). Our results demonstrate that 3.0 mM fructose was comparable to 5.5 mM glucose in supporting in vitro oocyte maturation and embryonic development after parthenogenesis and could be used as an alternative energy source to glucose for in vitro maturation of pig oocytes.

• 한국수정란이식학회지
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• 제33권3호
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• pp.119-127
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• 2018

The purpose of this study is to examined the electrofusion and activation conditions for the production of porcine somatic cell nuclear transfer (SCNT) embryos. In this study, immature oocytes were cultured in TCM-199 with and without hormones for 22 hours. Skin fibroblasts cells of porcine were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion was performed with two different pulses that each one pulse (DC) of 1.1 kV/cm or 1.5 kV/cm for $30{\mu}sec$. After fusion subsequent activation were divided into three groups; non-treatment (control) and treatment with 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B for 4 hours. Transferred embryos were cultured in PZM-3 (Porcine Zygote Medium-3) in $5%\;CO_2$ and 95% air at $39^{\circ}C$ for 7 day. Apoptosis-related genes (Caspase-3, BCL-2, mTOR, and MMP-2) were analyzed by immunofluorescence staining. There was no significant difference between two different electrofusion stimuli in the cleavage rate; $64.9{\pm}4.8%$ in 1.1 kV/cm and $62.7{\pm}4.0%$ in 1.5 kV/cm. However, blastocyst formation rate (%) was significantly different among three different activation groups (no treatment, 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B) combined with electrofusion of 1.1 kV/cm. The blastocyst formation rate was $12.6{\pm}2.5$, $20.0{\pm}5.0$, and $34.9{\pm}4.3%$ in control, 2 mM 6-DMAP, and $7.5{\mu}g/ml$ cytochalasin B, respectively. Immunofluorescence data showed that expression levels of caspase-3 in SCNT embryos undeveloped to blastocyst stage were higher than those in the blastocyst stage embryos. Expression levels of Bcl-2 in blastocyst stage embryos were higher than those in the arrested SCNT embryos. These results showed that the combination of an electric pulse (1.1 kV/cm for $30{\mu}sec$) and $7.5{\mu}g/ml$ cytochalasin B treatment was effective for production of the porcine SCNT embryos.

• Reproductive and Developmental Biology
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• 제36권3호
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• pp.167-172
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• 2012

The key regulators of apoptosis are the interacting protein of the Bcl-2 family. Bcl-2, an important member of this family, blocks cytochrome C release by sequestering pro-apoptotic BH3-only proteins such as Bid, Bad, Bax and Bim. The pro-survival family members (Bcl-2, Bcl-XL, Bcl-W) are critical for cell survival, since loss of any of them causes cell death in certain cell type. However, its role during early porcine embryonic development is not sufficient. In this study, we traced the effects of Bcl-2 inhibitor, ABT-737, on early porcine embryonic development. We also investigated several indicators of developmental potential, including gene expression (apoptosis-related genes) and apoptosis, which are affected by ABT-737. Porcine embryos were cultured in the PZM-3 medium with or without ABT-737 for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without ABT-737 ($14.7{\pm}3.0$ vs $30.3{\pm}4.8%$, p<0.05). TUNEL assay showed that the number of containing fragmented DNA at the blastocyst stage increased in the ABT-737 treated group compared with control (4.7 vs 3.7, p<0.05). The mRNA expression of the pro-apoptotic gene Bax increased in ABT-737 treated group (p<0.05), whereas expressions of the anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-XL, Bcl-W) decreased (p<0.05). Also, expressions of the ER stress indicator genes (GRP78, XBP-1 and sXBP-1) increased in ABT-737 treated group (p<0.05). In conclusion, Bcl-2 is closely associated with of apoptosis- and ER stress-related genes expressions and developmental potential in pig embryos.

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.85-91
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• 2009

In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups of oocytes were cultured in TCM 199 supplemented with 10% FBS, 10% pFF or 5% FBS + 5% pFF, respectively. After 44 h maturation, oocytes with the first polar body were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}sec$. Also, matured oocytes of four groups were reconstructed and fused. Reconstructed embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. The oocytes matured in the medium supplemented with FBS or/and pFF showed significantly higher maturation rates (64.0 vs. 73.9 to 85.2%). In PA embryos, cleavage rates (89.7 vs. 77.1 to 86.6%) and blastocysts rates (30.0 vs. 16.2 to 26.2%) were significantly higher in pFF group (p<0.05). In NT embryos, there was no difference among treatments in cleavage rate, but the blastocyst rates (28.5 vs. 15.5 to 24.6%) were significantly higher in pFF group (p<0.05). The apoptosis rate was significantly higher (p<0.05) in the control than other groups (10.8 vs. 4.9 to 8.2% for PA, 3.1 vs. 0.5 to 1.3% for NT). In order to select the comparable oocyte to in vivo oocytes, each group of oocytes was stained with Brilliant cresyl blue (BCB) after 42h maturation. The matured oocytes were separated according to color of cytoplasm; stained group (BCB+) and unstained group (BCB-). The oocytes matured in the presence of FBS or/and pFF showed significantly higher staining rates (70.3 to 72.7 vs. 35.1%) (p<0.05). To verify the fact that the supplementation of FBS or/and pFF can increase the maturation rates, cdc2 kinase activity, the catalytic subunit of MPF, was determined. The cdc2 kinase activity of the oocytes matured in the medium supplemented with FBS or/and pFF was significantly higher than control group (6.7 to 9.3 vs. 3.8). In conclusion, the supplementation of FBS or/and pFF can support in vitro maturation rate of porcine oocytes through the increment of cdc2 kinase activity level in the cytoplasm.

• 한국수정란이식학회지
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• 제29권2호
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• pp.141-148
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• 2014

Quercetin and genistein, plentifully present in fruits and vegetables, are flavonoid family members that have antioxidative function and plant-derived phytoestrogen activity. The antioxidative effects of quercetin and genistein on boar sperm characteristics and in vitro development of IVF embryo were investigated. The sperm motility was increased by addition of genistein $50{\mu}M$ for 6 hr incubation compared to control (p<0.05). The sperm viability was increased by addition of quercetin 1 and $50{\mu}M$ and genestein 1 and $50{\mu}M$ for 3 hr incubation. In addition, the sperm viability seemed to be increased dose-dependantly by addition of quercetin or genistein 1 and $50{\mu}M$, respectively (p<0.05). The membrane integrities were not increased by quercetin or genistein treatments for 3 hr or 6 hr incubation period except for quercetin $1{\mu}M$ for 3 hr incubation. In mitochondrial activities, addition of quercetin $50{\mu}M$ for 6 hr incubation increased mitochondrial activity but decreased at $100{\mu}M$ concentration compared with control (p<0.05). When porcine IVF embryos were cultured in PZM-3 medium supplemented with low concentrations of quercetin ($1{\sim}10{\mu}M$), the developmental rates to morula and blastocyst increased but significantly decreased at high concentrations of quercetin ($25{\sim}50{\mu}M$). The highest developmental rate to blastocysts among all concentrations of quercetin was shown at quercetin $10{\mu}M$ (p<0.05). The developmental rates to morula or blastocysts at low ($0.01{\sim}1{\mu}M$) and high ($5{\sim}10{\mu}M$) concentrations of genistein were not significantly different among all treatment group and genistein did not affect on IVF embryo development. These results suggest that quercetin and genistein seem to have positive effects at certain concentrations on sperm characteristics such as motility, viability and mitochondrial activity. In addition, low concentrations of quercetin (1, 5 and $10{\mu}M$) in this experiment, seem to have beneficial effect on porcine IVF embryo development but genistein did not affect on it at all given concentrations ($0.01{\sim}10{\mu}M$).