• 한국발생생물학회지:발생과생식
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• 제22권3호
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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.

• Reproductive and Developmental Biology
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• 제31권4호
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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.

• Reproductive and Developmental Biology
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• 제37권1호
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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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• 제35권2호
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• pp.191-195
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• 2011

The present study was conducted to examine the reactive oxygen species (ROS) generation levels in porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by the combination of electric stimulus and 6-DMAP before in vitro culture. Porcine oocytes and parthenogenetic embryos were stained in 10 ${\mu}M$ dichlorohydrofluorescein diacetate (DCF) or 10 ${\mu}M$ hydroxyphenyl fluorescein (HPF) dye each for 30 min at $39^{\circ}C$. The fluorescent emissions from the samples were recoded as JPEG file and the intensity of fluorescence in oocytes and embryos were analyzed. $H_2O_2$ and $^{\cdot}OH$ radical levels of porcine oocytes were reduced immediately after electric stimulation. However, $H_2O_2$ and $^{\cdot}OH$ radical levels of parthenogenetic embryos were increased with time elapsed after electric stimulation from 0 h to 3 h and after DMAP culture. During in vitro culture, $H_2O_2$ and $^{\cdot}OH$ radical levels were gradually increased from the one-cell stage to the two- and four-cell stages. The result of the present study suggests that the ROS was not increased by electric pulse in porcine embryos. Rather than it seems to be associated with the stage of development and the culture condition.

• 대한의용생체공학회:의공학회지
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• 제45권1호
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• pp.26-36
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• 2024

Functional brain imaging techniques have been used to diagnose psychiatric disorders such as dementia, depression, and autism. Recently, these techniques have also been actively used to study hearing loss. The present study reviewed the application of the functional brain imaging techniques in auditory research, especially those focusing on hearing loss, over the past decade. EEG, fMRI, fNIRS, MEG, and PET have been utilized in auditory research, and the number of research studies using these techniques has been increasing. In particular, fMRI and EEG were the most frequently used technique in auditory research. EEG studies mostly used event-related designs to analyze the direct relationship between stimulus and the related response, and in fMRI studies, resting-state functional connectivity and block designs were utilized to analyze alterations in brain functionality in hearing-related areas. In terms of age, while studies involving children mainly focused on congenital and pre- and post-lingual hearing loss to analyze developmental characteristics with and without hearing loss, those involving adults focused on age-related hearing loss to investigate changes in the characteristics of the brain based on the presence of hearing loss and the use of a hearing device. Overall, ranging from EEG to PET, various functional brain imaging techniques have been used in auditory research, but it is difficult to perform a comprehensive analysis due to the lack of consistency in experimental designs, analysis methods, and participant characteristics. Thus, it is necessary to develop standardized research protocols to obtain high-quality clinical and research evidence.

• 한국가축번식학회지
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• 제24권4호
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• pp.407-417
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• 2000

본 연구는 태아 섬유아세포를 이용한 소 핵치환 난자의 융합에 있어서 여러 조건의 전기자극 처리 후 그에 따른 발달율을 조사하였다. 임선 45 일령 태아로부터 섬유아세포를 분리하여 3~4 차례 계대배양후, 이들 단일 섬유아 세포들을 미세조작기를 이용하여 제핵된 난자의 위란강 내에 주입하였다. 본 실험에서는 첫째로, 다른 전기자극 조건의 적용에 따른 융합율 및 발달율을 비교 조사하였다. 180 V/mm 의 조건하에서 다른 조건의 전기자극 시간 (15, 30, 45 $\mu$see)을 처리하였을 때, 15 (45.5%, 120/264)나 30 $\mu$see (43.9%, 106/241) 처리구가 45$\mu$see (23.2%, 58/250) 처리구 보다 높은 융합율을 나타내었다 (P＞0.05). 그러나, 융합된 난자의 발달율에 있어서는 처리구 간에 차이가 나타나지 않았다. 다음 실험으로, 핵치환 난자의 체외발달에 있어서 15 $\mu$see 의 전기자극 시간의 조건하에서 다른 전장 (1.5, 1.8, 2.1 kV/cm)에 따른 효과를 조사하였다. 핵치환 난자의 융합이나 발달율에 전장에 따른 차이는 나타나지 않았다 (P＞0.05). 마지막으로, 체세포 핵치환과 체외수정에 의한 수정란의 체외 발달율을 비교 조사하였다. 태아 섬유아세포로 재구성된 수정란의 배반포로의 발달율은 27.4% (75/274) 로 나타났으며, 이는 체외수정에 의해 발달한 배반포의 비율(24.5%, 58/237)과 유사하다는 것을 알 수 있었다. 그러나, 핵치환에 의해 유도된 배반포의 평균 세포 수는 체외수정에 의해 유도된 배반포 보다 낮게 나타났다. 결론적으로, 본 실험에서는 체세포로 핵치환된 소 난자의 전기적 융합에 대한 조건 (1.8 kV/cm, 15 $\mu$see)을 확립하였다. 또한, 본 실험에서 나타난 결과들은 체세포로 재구성된 소 난자들이 비록 체외수정에 의해 생산된 배반포보다는 적은 세포수를 보여주었지만, 체외에서 정상적으로 배반포 단계까지 발달할 수 있다는 것을 제시하고 있다.

• Clinical and Experimental Reproductive Medicine
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• 제19권2호
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• pp.133-141
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• 1992

We examined effects of co-culture with human oviduct epithelial cells (HOEC) on the development of mouse and human embryos from early embryonic· stage to late morula or blastocyst stage (LM or B). In human, embryos were transferred and pregnancy rate was investigated. The HOEC, collected from surgically removed fallopian tube, were cultured in medium-199 supplemented with 20 % fetal cord serum (FCS). The HOEC were characterized by using immunocytochemical staining with anticytokeratin antibody and then used for cultures of mouse and human embryos. Results obtained from co-culture system were as follows. Development rate of mouse embryos was improved by co-culture system at late developmental stage (p<0.025). Human supernumerary embryos remained after transfer, unsuitable for freezing because of their poor quality, were co-cultured for 72hrs. Co-culture (78.79%) or conditioned medium (78.26%) system improved the developmemt rate, significantly, in comparision with control (11.11%)(p<0.00l). Co-cultured (85.71%) human zygotes for 24hrs showed the better development rate in comparision with control (50.00%) (p<0.01). When we transferred embryos cultured with the HOEC to patients, we obtained one pregnancy. Co-cultured human zygotes for 24hrs showed the better quality and viability for the replacement in comparision with control (p<0.01). In addition, improved pregnancy rate was obtained. Our results suggest that the co-culture system can rescue early degenerating embryos by improving early development and yield a resonable number of blastocyst for the appropriate replacement. The effect provided by cultured HOEC is not species specific for the development of embryos and it can be used to overcome in vitro blocks for the development. And also the co-culture system offers the possibility to freeze embryos at blastocyst stage which is more sucessful stage for the freezing. The HOEC monolayer may provide some stimulus via specific factor, which is unknown, to the development of embryos. Our results showed that the co-culture system with HOEC can be an alternative to conventional culture system.

• 한국발생생물학회지:발생과생식
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• 제4권2호
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• pp.237-242
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• 2000

흰쥐 시상하부에서 합성ㆍ분비되어 뇌하수체 전엽에서의 growth hormone (GH) 분비를 촉진하는 growth hormone releasing hormone (GHRH)이 시상하부 이외 조직들 (extrahypothalamic tissues)인 태반, 생식소, 그리고 뇌하수체 전엽에서도 발현됨이 보고되었다. 본 연구는 흰쥐 뇌하수체 전엽에서 발현되는 GHRH의 기능을 조사하기 위해 i)세포 배양을 시행하면서 GHRH의 세포내 함량, 분비 그리고 세포분획법 (cell-fractionation)을 사용하여 분리한 뇌하수체 세포 유형별로 GHRH 함량을 방사면역측정법으로 조사하였고, ii)체외배양 중인 뇌하수체 전엽세포의 증식에 미치는 GHRH의 효과를 측정하기 위해 [$^3$H] thymidine incorporation assay를, 그리고 iii) GHRH의 세포분열 촉진 효과와 세포내 c-fos 유전자 발현과의 상관관계를 조사하기 위해 northern blot analysis를 시행하였다. GHRH 방사면역측정법을 시행한 결과 상당량의 GHRH-like 분자들이 흰쥐 뇌하수체 전엽내에 존재하고, 체외 세포배양시 분비됨을 관찰하였다. 세포분획을 사용한 실험에서 GHRH 함량은 gonadotrope, somatotrope, lactotrope 그리고 thyrotrope 순으로 나타났다. 이 러한 결과는 흰쥐 뇌하수체 전엽에서 생성된 GHRH가 국부적인 조절인자, 특히 상이한 유형의 세포들 간의 상호조절 (cross-talk)을 통해 뇌하수체 전엽에서의 세포분열과 분화, 그리고 기능조절에 관여할 가능성을 보여주었다. GHRH는 체외 배양중인 뇌하수체 전엽세포의 [$^3$H] thymidine incorporation을 농도의존적으로 증가시켰으며, 이러한 GHRH의 세포분열 촉진 효과는 예상대로 세포내 oncogene 활성 의 증가를 통해 일어나는 것임을 c-fos northrn blot으로 확인하였다. 결론적으로, 본 연구는 흰쥐 뇌하수체 전엽에서 합성되는 GHRH가 paracrine 또는 autocrine 기작으로 GH의 분비 촉진 이외에도 세포분열의 조절함을 시사하는 것이다.

• 한국수정란이식학회지
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• 제32권1호
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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.