• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.253-256
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• 2004

Porcine fibroblasts were transferred into enucleated bovine oocytes for the interspecies nuclear transfer (NT). After NT, the embryos were cultured in three different culture systems. The media used for the experiment were CR1aa and NCSU23. The culture systems used for the experiment were: 1. Culture in CR1aa for 7 days (CR). 2. Culture in CR1aa for 2 days and subsequently in NCSU23 for 5 days (CR-NC). 3. Culture in NCSU23 for 7 days (NC). Bovine (intraspecies) NT group was used as a control. The oocytes in bovine NT group were treated the same as interspecies NT embryos except using bovine fibroblasts as nuclear donors. Regardless of their nuclear origin (interspecies vs bovine), the embryos in CR (68.4% vs 77.2%) and CR-NC (67.8% vs 70.5%) showed better developmental competence to the 2-cell stage (p<0.05) than those in NC (41.0% vs 10.0%). Bovine NT embryos in CR-NC did not develop over the 4-cell stage after the medium replacement, while interspecies NT embryos in CR-NC continued to develop and could reach over the 8-cell stage (12.2%). Blastocysts were only found in bovine NT group (17.4%), but no blastocyst was found in interspecies NT group. This study suggests that the development of interspecies NT embryos mostly depends on their recipient cytoplasm during the culture in vitro.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.395-403
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• 1998

In this study we determined fertilization processes and developmental ability of porcine oocytes following injection of round spermatid in the presence and absence of artificial activation. Electrical stimulation at 2 h before spermatid injection significantly increased the incidence of normal fertilization as compared to those following injection without stimulation or with stimulation immediately after injection. The incidences of two pronuclear formation and apposition were not different in oocytes between following intracytoplasmic spermatid and spermatid nucleus injection. Indirect immunocyto chemistry and laser scanning confocal microscopy study revealed that microtubuels were organized from the oocyte cortex following round spermatid injection, and this seemed to move both male and female pronuclei into the oocyte center. At 6 days following injection blastocoele formation was seen in the eggs following round spermatid (25%) and round spermatid nucleus injection (27%). However, none of oocytes developed to the blastocyst stage at 6 days following sham injection. The average cell numbers of blastocysts at 8 days following injection of spermatid and spermatid nucleus were 87 to 99. These results suggested that either round spermatid or it's nudeus can be used to produce viable embryos by injection into unfertilized oocytes in the pig.

• Reproductive and Developmental Biology
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• v.36 no.3
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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.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.129-135
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• 2006

This study was conducted to investigate the effect of in vitro maturation (IVM) duration of porcine follicular oocytes on maturation rate, polyspermic rate, and subsequent embryo development. The nuclear maturation rates of oocytes matured for 36, 38, 40, 42 and 44 hr were similar between 68.0, 78.0, 79.5, 73.8 and 81.8% respectively. There was no significant difference in the rates of polyspermy after in vitro feritilization (IVF). The cleavage rate in the group of 36 hr was significantly higher in than that of 40, 44 hr (p<0.05) but not to 38 and 42 hr. The development rate to blastocyst stage was significantly higher in the group of 38 hr (23.1%) than that in the group of 44 hr (15.6%) (p<0.05) but not to 36, 40 and 42 hr. These results suggest that the aged oocytes for 44 hr is not required for the production of bias to cysts derived from porcine IVF embryos.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.33-45
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• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bel-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MIT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG ($2{\mu}M$) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.235-243
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• 2002

This study was designed to examine the ability of the bovine (MII) oocytes cytoplasm to support several mitotic cell cycles under the direction of differentiated somatic cell nuclei of bovine, porcine, mouse and human. Bovine GV oocytes were matured in TCM-199 supplemented with 10% FBS. At 20h after IVM, recipient oocytes were stained with 5 $\mu\textrm{g}$/$m\ell$ Hoechst and their 1st polar body (PB) and MII plate were removed by enucleation micropipette under UV filter. Ear skin samples were obtained by biopsy from an adult bovine, porcine, mouse and human and cultured in 10% FBS added DMEM. Individual fibroblast was anlaysed chromosome number to confirm the specificity of species. Nuclear transferred (NT) units were produced by electrofusion of enucleated bovine oocytes with individual fibroblast. The reconstructed embryos were activated in 5 $\mu$M ionomycin for 5 min followed by 1.9 mM 6-dimethylaminopurine (DMAP) in CR1aa for 3 h. And cleaved NT embryos were cultured in CR1aa medium containing 10% FBS on monolayer of bovine cumulus cell for 8 days. Also NT embryo of 4~8 cell stage was analysed chromosome number to confirm the origin of nuclear transferred somatic cell. The rates of fusion between bovine recipient oocytes and bovine, porcine, mouse and human somatic cells were 70.2%, 70.2%, 72.4% and 63.0%, respectively. Also, their cleavage rates were 60.6%, 63.7%, 54.1% and 62.7%, respectively, there were no differences among them. in vitro development rates into morula and blastocyst were 17.5% and 4.3% in NT embryos from bovine and human fibroblasts, respectively. But NT embryos from porcine and mouse fibroblasts were blocked at 16~32-cell stage. The chromosome number in NT embryos from individual fibroblast was the same as chromosome number of individual species. These results show that bovine MII oocytes cytoplasm has the ability to support several mitotic cell cycles directed by newly introduced nuclear DNA.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.339-347
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• 2001

Sperm-mediated DNA transfer has a potential to markedly simplify techniques for the generation of transgenic animals. The exogenous DNA transfer by intracytoplasmic sperm injection (ICSI) procedure has been recently introduced in the production of transgenic animals. In this study, the developmental competence and tile expression rates of transgene were investigated after injection of spermatozoon or sperm head with enhanced green fluorescent protein (EGFP) gene into the mature porcine oocytes. The porcine oocytes were injected with intact sperm, membrane-disrupted sperm or sperm head. After injection. embryos were cultured in NCSU23 medium up to the blastocyst stage, and the developmental competence and expression rates were studied. The developmental rate (67.0%) of sperm injection group was higher than that (59.7%) of sperm head injection group, and the rates of EGFP expression were also significantly different between sperm injection and sperm head injection groups (42.1 vs 20.0%) (F＜0.05). In the porcine oocytes injected with sperm treated with different methods of membrane disruption, the removal of sperm membrane did not alter the developmental competence of embryos. The rate of blastocysts at 7 days after injection with intact and membrane disrupted sperm were 15.0 and 14.2%, respectively. The EGFP expression rates, 38.4% in embryos injected with frozen-thawed sperm was higher than that, 22.4% of embryos injected with the Triton X-100 treated sperm. Prior to injection, sperm were cultured in different EGFP gene concentrations from 0.Ol to 1ng/u${mu}ell$. However, no significant difference in developmental rates of embryos among different concentrations of EGFP gene were observed. The highest expression rate of EGFP gene, 37.4% was obtained from the embryos injected with spermatozoa treated with 0.1 ng/${mu}ell$ EGFP gene. These results suggested that exogenous DNA could be attached to the membrane disrupted sperm, and that these sperm could be used as a vector carrying foreign DNA into embryos.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.169-175
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• 2006

The objective of this study was carried out to examine the polar body extrusion of in vitro matured porcine follicular oocytes as a non-invasive marker of oocyte quality to know the developmental competence in advance. The porcine oocytes matured for 48 hours were examined the polar body extrusion and some parts were stained. The examined oocytes were matured for additional $16{\sim}18$ hours and activated with 7% ethanol and cultured in $5{\mu}g/ml$ cytochalasin B for 5 hours for diploid formation. The treated oocytes were washed and cultured for 7 days. The polar body extrusion and degeneration rates were varied with $9.9{\sim}52.4%$ and $21.4{\sim}61.8%$ by repetition. The polar body extruded oocytes were shown the polar body chromosome and metaphase II plate by staining. However the non-extruded oocytes were shown expanded nucleus with 39.1%, premature chromosome condensation with 19.6%, metaphase I plate with 10.9 %, metaphase II with 13%, condensed chromatin with 6.5%, and absent nuclear material with 8.7%. The oocytes that were not examined for the polar body extrusion were cleaved 45.0%, and developed to blastocyst stage with 11.3%. In examined oocytes for polar body extrusion,. the polar body extruded oocytes were cleaved 94.2% and developed with 42.5%. This result suggests that discarding of the degenerating oocytes and oocytes that not extruded polar body will be effective for experiments of culturing effect in porcine embryos and embryo biotechnology.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.153-163
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• 2002

In vitro matured porcine oocytes have very small volume of perivitellinspace (PVS). In these respect, the effect of sucrose and polybrene on the efficiency of gene transfer was investigated. As a gene (hGH) transfer vehicle, vesicular stomatitis virus glycoprotein pseudotyped retroviral vector (VSV-G) was used. Sucrose treatment has no detrimental effect on the rates of cleavage and resulted in the enlargement of PVS for the efficient introduction of retroviral vector stocks. Introduction rates of retrovirus in 0.5, 1, 2, 3 % sucrose treatment group were higher than that of the non-treatment group (39.3, 43.3, 35.7, 40.7 % vs. 8.3 %), respectively. In addition, we observed that sucrose pretreatment during injection procedure significantly reduce the frequency of polyspermy. In general, polybrene is a polycation essential for retrovirus transduction. The groups with the addition of 0.5, 5, 50$\mu\textrm{g}$/$m\ell$ polybrene exhibited a significant effect on gene transfer compared to that of the non-addition group (56.5, 50.0, 57.1 % vs. 34.6 %), respectively But, when the oocytes were co-injected with retrovirus and 50$\mu\textrm{g}$/$m\ell$ polybrene, the rates of cleavage and blastocyst development were 43.3 and 4.6%, respectively. This rates were lower than those of the non-addition group (70.0 and 17.3 %). In conclusion, sucrose pretreatment have increased efficiency of retroviral mediated gene transfer in porcine oocytes with no damage on in vitro fertilization and embryo development. In addition, sucrose pretreatment was beneficial in polyspermy inhibition. Presence of polybrene during microinjection showed a beneficial effect on the gene transfer in porcine oocytes, in low concentration. And these results will provide an useful tool for production of transgenic pigs by retroviral mediated gene transfer.

• Journal of Embryo Transfer
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• v.22 no.2
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• pp.121-126
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• 2007

The objective of this study was carried out to examine the selection effects of in vitro matured porcine follicular oocytes with polar body extrusion and early cleavage as non-invasive marker to know the developmental competence in advance. The porcine oocytes matured for 48 h were examined the polar body extrusion. The examined oocytes were matured for additional $16{\sim}18h$ and activated with 7% ethanol and cultured in $5{\mu}g/ml$ cytochalasin B for 5 h for diploid formation. The treated oocytes were cultured and examined the cleavage after 48 h and continued culturing for 5 days. The oocytes of 21.9% were discarded in morphological selection and 32.1% oocytes were discarded by failure of first polar body extrusion. The selected oocytes were matured and activated and then after 48 h the cleavage rates were examined. In morphologically selected oocytes, 15.8% oocytes were not cleaved and 52.6% oocytes were normally cleaved and 31.6% oocytes were hyper-cleaved over 8-cell stage. However in the first polar body extruded oocytes, 7.1% oocytes were not cleaved and 73.1% oocytes were normally cleaved and 19.8% oocytes were hyper-cleaved. The morphologically selected embryos that not cleavage-selected were developed in 16.7% up to blastocyst and the morphologically selected and cleavage-selected embryos were developed in 31.7%. The polar body extruded oocytes that were not carried out cleavage selection were developed in 39.0% and the polar body extruded and cleavage-selected embryos were developed 49.0%. The first cleavage timing was examined with 12 h interval after activation. In $0{\sim}12,\;12{\sim}24,\;24{\sim}36,\;and\;36{\sim}48h$ intervals, 4.1%, 68.6%, 19.1%, and 2.3% oocytes were cleaved and 5.9% oocytes were not cleaved until 48 after activation. The cleaved oocytes in each interval were cultured and developed upto blastocyst with 0, 39.1, 9.5, and 0%, respectively. This results suggests that polar body extruded and cleaved at $12{\sim}36h$ embryo has higher developmental potential than the others.