• 한국가축번식학회지
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• 제26권1호
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• pp.79-85
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• 2002

본 연구에서는 전기자극 후 cycloheximide의 적정농도와 배양시간이 체외성숙된 돼지난자의 활성화에 미치는 영향을 조사하고자 실험을 수행하였다. 42~44시간 동안 체외성숙 된 난자는 0.1%의 hyaluronidase에서 pipetting으로 난구세포를 제거한 후, 1.2 KV/cm의 전압으로 전기자극만 하거나, 전기자극 후 cycloheximide 1, 5 및 10 $\mu\textrm{g}$/$m\ell$의 농도에서 각각 3, 5 및 7시간 동안 배양하는 구로 나누어 활성화를 유도하여 다음과 같은 결과를 얻었다. 1. 난자의 분할율 cycloheximide 10 $\mu\textrm{g}$/$m\ell$ 처리군이 86.8%로 1 $\mu\textrm{g}$/$m\ell$ 처리군의 74.4%보다 유의적(P＜0.05)으로 높았다. 배반포기 발달율은 10 $\mu\textrm{g}$/$m\ell$ 처리구보다 5 $\mu\textrm{g}$/$m\ell$ 처리구에서 유의적(P＜0.05)으로 높았다. 2. 전기자극 후 cycloheximide에서 배양시간의 비교에 있어서 5시간 처리한 돼지난자의 분할율(86.6%)이 3시간 동안 처리한 난자보다 유의적(P＜0.05)으로 높게 나타났다. 상실배의 발달율은 5시간과 7시간 동안 처리구(26.7%와 16.4%)에서 3시간 처리구(14.5%)보다 유의적(P＜0.05)으로 높은 발달율을 나타났다. 3. 전기자극을 단독처리하거나 전기 자극 후 cyclohiximide를 복합처리한 돼지난자를 8일 동안 배양하였다. 전기자극 후 cycioheximide를 복합처리한 난자의 분할율(80.1%) 및 배반포기 발달율(11.6%)은 전기자극 단독처리한 난자의 분할율(77.2%) 및 배반포기 발달율(6.6%)과는 유의적인 차이가 없었다. 4. 체외성숙된 돼지난자를 전기자극 단독 또는 전기자극 후 cycloheximide 복합처리한 후 체외 발달한 배반포기 수정란의 핵 수는 각각 18.67$\pm$5.53개와 20.71$\pm$6.16개로써 유의적인 차이가 없었다. 이상의 결과를 요약하면, 전기자극을 단독처리 하거나 전기 자극 후 cyclohiximide를 복합처리한 돼지난자 발생율은 유의적인 차이가 없었으나, 전기자극 후 cycloheximide를 복합처리 할 경우에는 5 $\mu\textrm{g}$/$m\ell$의 농도로 5시간 동안 배양하는 것이 효과적으로 돼지난자의 activation을 유기할 수 있을 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제22권3호
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• pp.328-335
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• 2009

This study was undertaken to investigate the influence of exposure and removal of four different cryoprotectants (CPAs) on the ATP content of cumulus cell-enclosed (COs) and cumulus cell-denuded (DOs) immature porcine oocytes. The in vitro nuclear maturation of the COs, exposed to and removed from the CPAs was also assessed. Both COs and DOs were exposed to 1.5 M concentrations of each CPA (ethylene glycol (EG); propylene glycol (PG); dimethyl-sulfoxide (DMSO); and glycerol (G)) for durations of 5, 15, and 30 minutes at room temperature ($23.5{\pm}1.5^{\circ}C$), and immersed in physiological saline supplemented with 20% (v/v) fetal bovine serum for 5 minutes ($39^{\circ}C$) to remove each CPA. Before, during and after exposure to each CPA, the ATP content of both the COs and the DOs was measured. After removal from each CPA an aliquot of the COs was matured for 44${\pm}$2 h, and their nuclear maturation rates were measured up to the metaphase stage of the second meiotic division (the M-II stage). The maturation rates up to the M-II stage were not significantly different between all the groups that were exposed to each CPA for 5 minutes. For 15 and 30 minute exposures, the maturation rates of the COs exposed to PG, DMSO and EG were almost the same as those of the control groups; however, the rates of G group exposed for 15 and 30 minutes were significantly lower (p<0.05) than the control group. These groups were also found to have a decrease in the ATP content of COs and DOs during and after exposure for the same periods (p<0.05). In addition, although the ATP contents of the COs after exposure to EG for any period were the same as the controls, the ATP content of the DOs exposed to EG for any period were significantly lower (p<0.05) than those of the controls. When the ATP content of the COs and DOs of each CPA were compared, the DOs exposed to PG were found to have a significantly greater level (p<0.05) than DOs exposed to G for any duration. In addition, the ATP content of DOs exposed to PG for 30 min and removal was also higher (p<0.05) than when exposed to DMSO for the same period. These findings indicate that PG may be a useful CPA for the cryopreservation of immature porcine oocytes.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.69-69
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• 2002

Transgenic animals production tools have been valuable for research and purpose. The current methods of gene transfer, microinjection and nuclear transfer, which are widely used in transgenic animal production, but all most methods has only had limited success in production of larger species. Here, we report the possibility of a sperm-mediated gene transfer method in porcine embryos. Oocytes were collected from ovaries harvested at a local slaughterhouse were matured in 500${mu}ell$ drops of TCM-199 under mineral oil at 38.5$^{\circ}C$ in a humidified atmosphere of 5%CO2 in air. After 42-43h of in vitro maturation oocytes were denuded. for sperm injection into the cytoplasm of the porcine oocytes, sperm suspension in NIM medium are subjected extraction with TritonX-100 before mixing with a green fluorescent gene (GFP). Sperm with Tritonx-100 were prepared by adding TritonX-100 to a final volume of 0.05% in the sperm suspension and mixing by trituration for 60s before two wishes in NIM medium at 2$^{\circ}C$. A(ter wishing, sperm were mixed with TritonX-100 at $25^{\circ}C$ followed by washes at 2$^{\circ}C$. Sperm were resuspended in ice cold NIM to a final volume of 400${mu}ell$ and 2-20ng/${mu}ell$ DNA were triturated on ice for 60s. All microinjection was performed in HEPES-buffered CZB medium at room temperature within 2h. After culture in NCSU-23 for 72h, percent of porcine embryos transfected GFP gene are 20.7%(6/29) in 20ng/${mu}ell$ sperm-DNA mixed group and other groups were 3.7 %(2/54)and 4.7%(3/67). These data suggests that sperm-mediated gene transfer method should be used to the production tool of transgenic pig efficiently.

• 한국가축번식학회지
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• 제23권1호
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• pp.53-61
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• 1999

본 연구는 생쥐 preantral follicle 의 체외성장 및 성숙에 있어서 gonadotrophins 인 FSH 와 LH 의 효과를 조사하기 위하여 실시하였으며, 그 결과는 다음과 같다. 1. FSH 첨가군들은 대조군에 비하여 유의하게 높은 생존율과 성숙율을 나타냈으며, 100$m\ell$D/$m\ell$ 의 FSH 농도가 preantral follicle 의 체외배양에 적정한 농도인 것으로 나타났다. 2. HMG 첨가군은 FSH 첨가군보다, 통계적 유의차는 인정되지 않았지만, 높은 생존율과 성숙율을 나타냈다. 3. FSH 와 LH 의 첨가비율이 100$m\ell$U/$m\ell$ 대 10$m\ell$D/$m\ell$(10:1) 에서 가장 높은 생존율과 성숙율을 나타냈다. 4. FSH 혹은 HMG 첨가시, 정상적인 oestradiol 과 progesterone 분비양상을 나타냈으며, HMG 첨가군에서 유의하게 높은 농도의 oestradiol 과 progesterone 을 분비하였다. 이상의 결과를 종합해 볼 때, gonadotrophins 은 preantral follicle 의 체외성장 및 성숙뿐만 아니라 steroidogenesis 에서 중요한 역할을 수행한다는 것을 확인할 수 있었다.

• 한국동물생명공학회지
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• 제34권3호
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• pp.212-221
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• 2019

Sestrin-2 (SESN2) as a stress-metabolic protein is known for its anti-oxidative effects as a downstream factor of PERK pathways in mammalian cells. However, the expression patterns of SESN2 in conjunction with the UPR signaling against to ER stress on porcine oocyte maturation in vitro, have not been reported. Therefore, we confirmed the expression pattern of SESN2 protein, for which to examine the relationship between PERK signaling and SESN2 in porcine oocyte during IVM. We investigated the SESN2 expression patterns using Western blot analysis in denuded oocytes (DOs), cumulus cells (CCs), and cumulus-oocyte complexes (COCs) at 22 and 44 h of IVM. As expected, the SESN2 protein level significantly increased (p < 0.01) in porcine COCs during 44 h of IVM. We investigated the meiotic maturation after applying ER stress inhibitor in various concentration (50, 100 and 200 μM) of tauroursodeoxycholic acid (TUDCA). We confirmed significant increase (p < 0.05) of meiotic maturation rate in TUDCA 200 μM treated COCs for 44 h of IVM. Finally, we confirmed the protein level of SESN2 and meiotic maturation via regulating ER-stress by only tunicamycin (Tm), only TUDCA, and Tm + TUDCA treatment in porcine COCs. As a result, treatment of the TUDCA following Tm pre-treatment reduced SESN2 protein level in porcine COCs. In addition, SESN2 protein level significantly reduced in only TUDCA treated porcine COCs. Our results suggest that the SESN2 expression is related to the stress mediator response to ER stress through the PERK signaling pathways in porcine oocyte maturation.

• 한국수정란이식학회지
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• 제20권2호
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• pp.79-87
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• 2005

한우 및 젖소에서 체외 수정란을 생산하여 수란 우에 이식하기 위해 도축장에서 난소를 채취하여 난자를 회수한 후 체외 성숙, 체외 수정, 체외 배양과정을 거쳐 수정란을 생산하였다. 체외 수정시 난자는 난구세포 부착 정도에 따라 난할율을 조사하였고, 한우와 젖소에서 체외수정 후 배반포율을 조사하였다. 신선 및 동결 수정란을 수란우에 각각 이식하여 산자 분만에 따른 수태율을 조사하였다. 1. 한우 난소 2,021개에서 20,387개의 난자를 회수하여 체외 수정 후 난할된 난자수는 13,541개로서 $66.4\%$의 난할율을 나타내었고, 젖소는 난소 144개에서 1,784개의 난자를 회수하여 체외 수정 후 1,113개의 난자가 난할되어 $62.4\%$의 난할율을 나타내었다. 2. 한우와 젖소에서 난구세포가 온전히 둘러싸인 난자의 율은 각각 45.0, $62.0\%$이었고 난할율은 각각 81.0, $72.0\%$이었다. 난구세포가 일부 분리된 난자의 난할율은 한우, 젖소 각각 63.5, $51.3\%$이었다. 난구세포가 완전 분리된 난자의 난할율은 한우, 젖소 각각 41.0, $41.7\%$이었다. 3. 한우와 젖소 체외수정에서 난자가 배반포로 발달된 율은 전체 난자수에 비교하여 각각 27.0, $23.0\%$이었고, 난할된 난자수에 비교하여 각각 40.6, $36.9\%$이었다. 4. 신선 수정란을 이식한 결과, 한우에서는 278두에 이식하여 159두가 산자를 분만하여 $57.2\%$, 젖소에서는 15두에 이식하여 8두가 분만함으로써 $53.3\%$의 임신율을 나타내었다. 5. 동결 수정란을 이식한 결과, 한우에서는 44두에 이식하여 18두가 산자를 분만하여 $40.9\%$, 젖소에서는 11두에 이식하여 4두가 분만함으로써 $36.4\%$의 임신율을 나타내었다.

• Asian-Australasian Journal of Animal Sciences
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• 제20권3호
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• pp.334-339
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• 2007

Reactive oxygen species (ROS) generate during electrical activation of oocytes which has detrimental effects on embryo survival when overwhelmed. The present study was designed to investigate the ability of L-ascorbic acid, a novel water soluble antioxidant, to reduce the ROS level in developing embryos and their subsequent effects on embryo development in vitro. The compact cumulus oocyte complexes (COCs) were cultured in tissue culture medium (TCM)-199 supplemented with 10 ng/ml epidermal growth factor, 4 IU/ml pregnant mare serum gonadotropin (PMSG), and human chorionic gonadotropin (hCG) and 10% (v/v) porcine follicular fluid (pFF) for 44 h. After maturation culture, the denuded oocytes were activated with a single DC pulse of 2.0 kV/cm in 0.3 M mannitol solution containing 0.5 mM of HEPES, 0.1 mM of $CaCl_2$ and 0.1 mM of $MgCl_2$ for $30{\mu}s$ using a BTX Electro-cell Manipulator. The activated oocytes were cultured in modified North Carolina State University-23 (mNSCU-23) medium for 168 h. The level of $H_2O_2$ in each embryo was measured by the dichlorohydrofluorescein diacetate (DCHFDA) method at 48 h after activation. The blastocyst formation rate was significantly higher when culture medium was supplemented with 50 and $100{\mu}M$ L-ascorbic acid (31.2 and 38.7%, respectively) compared to non-supplemented (16.1%) group. Accordingly, significantly more cells in blastocyst were found for 50 and $100{\mu}M$ L-ascorbic acid (50.0 and 56.4, respectively) compared to 0 and $200{\mu}M$ L-ascorbic acid (36.5 and 39.8, respectively). L-ascorbic acid reduces the $H_2O_2$ level in developing embryos in a dose-dependant manner. The $H_2O_2$ level (pixels/ embryos) was 191.5, 141.0, 124.0 and 163.3 for 0, 50, 100 and $200{\mu}M$ L-ascorbic acid, respectively. So, we recommend to supplement 50 or $100{\mu}M$ L-ascorbic acid in porcine in vitro culture medium.

• 한국수정란이식학회지
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• 제32권1호
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• pp.17-24
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• 2017

Ganglioside GD1a is specifically formed by the addition of sialic acid to ganglioside GM1a by ST3 ${\beta}$-galactoside ${\alpha}$-2,3-sialyltransferase 2 (ST3GAL2). Above all, GD1a are known to be related with the functional regulation of several growth factor receptors, including activation and dimerization of epidermal growth factor receptor (EGFR) in tumor cells. The activity of EGF and EGFR is known to be a very important factor for meiotic and cytoplasmic maturation during in vitro maturation (IVM) of mammalian oocytes. However, the role of gangliosides GD1a for EGFR-related signaling pathways in porcine oocyte is not yet clearly understood. Here, we investigated that the effect of ST3GAL2 as synthesizing enzyme GD1a for EGFR activation and phosphorylation during meiotic maturation. To investigate the expression of ST3GAL2 according to the EGF treatment (0, 10 and 50 ng/ml), we observed the patterns of ST3GAL2 genes expression by immunofluorescence staining in denuded oocyte (DO) and cumulus cell-oocyte-complex (COC) during IVM process (22 and 44 h), respectively. Expression levels of ST3GAL2 significantly decreased (p<0.01) in an EGF concentration (10 and 50 ng/ml) dependent manner. And fluorescence expression of ST3GAL2 increased (p<0.01) in the matured COCs for 44 h. Under high EGF concentration (50 ng/ml), ST3GAL2 protein levels was decreased (p<0.01), and their shown opposite expression pattern of phosphorylation-EGFR in COCs of 44 h. Phosphorylation of EGFR significantly increased (p<0.01) in matured COCs treated with GD1a for 44 h. In addition, ST3GAL2 protein levels significantly decreased (p<0.01) in GD1a ($10{\mu}M$) treated COCs without reference to EGF pre-treatment. These results suggest that treatment of exogenous ganglioside GD1a may play an important role such as EGF in EGFR-related activation and phosphorylation in porcine oocyte maturation of in vitro.

• 한국수정란이식학회지
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• 제31권2호
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• pp.117-121
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• 2016

Although piglets have been delivered by embryo transfer (ET) with in vitro produced (IVP) embryos and blastocysts, a success rate has still remained lower level. Unlike mouse, human, and bovine, it is difficult to a production of piglets by in vitro fertilization (IVF) because of an inappropriate in vitro culture (IVC) system in pig. Therefore, the present study was conducted to investigate whether minimized exposure time in IVC can improve the pregnancy and delivery rates of piglets. Immediately after IVM, the oocytes were denuded and co-incubated with freshly ejaculated boar semen for 3.5 to 4 hours at $38.5^{\circ}C$ under 5% $CO_2$ in air. To avoid long-term exposure to in vitro state, we emitted IVC step after IVF. After that the presumptive zygotes were transferred into both oviducts of the surrogate on the same day or 1 day after the onset of estrus. Pregnancy was diagnosed on day 28 after ET and then was checked regularly every month by ultrasound examination. The 3 out of 4 surrogates were determined as pregnant (75%) and a total of 5 piglets (2 females and 3 males) were delivered at $118.3{\pm}2.5$ days of pregnancy period. In conclusion, a short-term exposure time may be an important factor in the production of IVP-derived piglets. It can be apply to the in vitro production system of transgenic pig by IVF, cloning, and pronuclear microinjection methods.