Earticle

본 연구는 활성화처리 방법 및 배양 조건이 돼지 단위발생란의 체외발달 및 apoptosis에 미치는 영향을 알아보기 위해 실시되었다. 도축장 유래 난소로부터 채취된 미성숙 난자를 42~44시간 동안 성숙배양한 후 사용하였다. Apoptosis는 TUNEL 방법을 사용하여 조사하였다. 실험 1에서는 성숙배양된 난자들을 electric pulse(1.2 kV/cm for 30μsec 2회, E), E + 6-dimethylaminopurine(6-DMAP) 또는 E + cycloheximide(CH) 방법으로 활성화 처리하여 PZM-3를 이용하여 5% CO2, 38.5℃에서 배양하였다. 실험 2에서는 전기자극을 이용하여 활성화처리된 난자들을 각각 PZM-3 또는 NCSU-23 배양액 내에서 배양하였다. 각 배양액 내의 난자들은 각각 20% O2 조건으로 나뉘어 배양하였다. E + 6-DMAP(36.5%) 또는 E + CH 구(32.5%)에서 E 구(27.7%)보다 유의적으로 높은 배반포 형성율을 보였다(P<0.05). 처리별 apoptosis 발생율은 각각 5.3%(E), 7.7%(6-DMAP) 및 7.1%(CH)였다. 실험 2에서는 PZM-3 구의 배반포 형성율이 NCSU-23 구에 비하여 산소분압조건과 관계없이 다소 높았다(28.2{sim}29.7% vs. 22.6~24.4%). PZM-3 및 20% O2 조건하에서 유의적으로 낮은 apoptosis 발생 비율을 나타냈다(9.2%, P<0.05). 그러므로 돼지 단위발생란을 chemical agent를 이용한 추가 활성화처리 후 PZM-3, 20% O2, 조건으로 배양하면 더 나은 배반포 발생율을 얻을 수 있다고 생각된다.

This study investigated apoptosis and in vitro development of parthenogenetic preimplantation porcine embryos. In vitro matured oocytes for 42~44h were used. Apoptotic cell death was analyzed by using a terminal deoxynucleatidyl transferase mediated deoxyuridine 5-triphosphate nick-end tabling (TUNEL) assay. In experiment 1, oocytes were activated with two electric pulses (CH) of 1.2 kV/cm for 30μsec (E), E + 6-dimethylaminopurine (6-DMAP) or E + cycloheximide (CH) and cultured in PZM-3 under 5% CO2 in air at 38.5℃. In experiment 2, oocytes were activated by E and cultured in PZM-3 or NCSU-23 under a gas atmosphere of 20% O2 (5% CO2, in air) or 5% O2 (5% CO2, 5% O2 90% N2) at 38.5℃. Oocytes activated with E+6-DMAP or E+CH showed higher blastocyst rates (36.3% and 32.5%) compared to E alone (27.7%). The frequency of apoptosis according to treatments were 5.3%, 7.7% and 7.1% respectively. Oocytes activated with E alone showed lower (P<0.05) frequency of apoptosis compared to other groups. In experiment 2, parthenotes cultured in PZM-3 showed slightly higher blastocyte rates (28.2% and 29.7%) compared to NCSU-23 (22.6% and 24.4%) regardless of atmosphere. Blastocysts generated in PZM-3 showed lower (P<0.05) apoptosis rate under 20% O2 (9.2% vs 16.9%), whereas those in NCSU-23 had slightly lower apoptosis rate under 5% O2 (14.0% vs 18.4%). This result represents that activation method and culture condition could affect the frequency of apoptosis as well as in vitro developmental rate.

본 연구는 다자이 오사무(太宰治)의 작품에서 지속적으로 등장하는 자기소멸의 방식을 고찰한다. 이를 위해 자기소멸의 은유화 방식을 아폽토시스(apoptosis)의 문학적 상상력에 의해 규명하고자 한다. 중독의 제 상황과 캐릭터의 카오스적 삶의 태도는 중독과 뒤틀림의 감각으로 발현된다. 고통을 적극적으로 드러내고 몸으로 저항하는 방식, 이러한 뒤틀림의 감각은 사회체제에 저항하는 작품 속 캐릭터를 통해 반증되고 있다. 이렇듯 억압 기제를 폭로하고 폐쇄적 존재 구조를 거부한 행위는 아폽토시스라는 자기소멸의 행위로 작품 속에서 은유화되고 있다. 다자이의 작품은 자기파괴적 양상이 두드러진다. 무화(無化)의 방식화를 통해 자신의 존재를 증명해보이는 이러한 아폽토시스 행위는 지배 담론을 거스르고 전복시키는 저항의 시도이자 존재를 현현하기 위한 자기파괴로 표현되고 있다. 다시 말해 자신을 둘러싼 억압의 경계와 질서에 균열을 일으키려는 그의 시도는 현실과 다른 삶에 대한 열망이라고 볼 수 있다.

In this study, I intend to examine the method of self-destruction consistently shown in the literature of Osamu Dazai. The metaphor of self-destruction is to be investigated throughout the literary imagination of that of apoptosis. In Dazai's works, various situations of addiction as well as the character's chaotic attitude toward life are expressed with a twist: a way of actively revealing pain and resisting with one's own body. Such a sense of a twist is proved against the characters that resist the social system. The act of exposing the mechanism of repression and rejecting the structure of a closed existence is metaphorized in the work as that of an act of self-destruction, an ‘apoptosis’. What stands out in Dazai's work is the self-destructive aspect. The act of apoptosis, which proves one's existence through the method of annihilation, is expressed as a resistance attempt to defy and overthrow the dominant discourse as well as that of self-destruction; to manifest one's existence. Therefore the imagination of the apoptosis creates a crack in the order of reality and pushes boundaries which suggest the possibility of a life that is completely different from the real one.

Parthenogenetic embryo without contribution of paternal genome would be terminated during early gestation period in pigs. Therefore, the present study was designed to analyze the characteristics of parthenogenetic fetuses to identify the possible causes for fetal degeneration during early gestation period. In brief, the pig parthenogenetic fetuses were produced by embryo transfer of parthenogenetic embryos into surrogates. Then, the pig parthenogenetic fetuses were recovered at day 26 and 35 of gestation and conducted to analyze histological characteristics. In results, the parthenogenetic pig fetuses were generated and recovered at day 26 and 35 successfully. The size of parthenogenetic fetuses (n=15, 1.5 cm) recovered at day 26 of gestation were significantly smaller than normal fetuses (n=18, 2.0 cm) while the weights were comparable (0.36 vs. 0.51 g). The size and weight of parthenogenetic fetuses (n=9, 1.8 cm, 0.47 g) recovered at day 35 of gestation were significantly lower than normal fetuses (n=3, 3.67 cm, 3.71 g). The paraffin-embedded sections from parthenogenetic fetus at day 26 showed normal formation of major organs while parthenogenetic fetus at day 35 showed terminated and degenerated formation of major organs. Additionally, the apoptotic cell number of parthenogenetic fetuses (4.4) recovered at day 35 of gestation were significantly (p<0.05) higher than normal fetuses (2.0) while the result of day 26 of gestation showed comparable (1.8 vs. 2.0). Conclusively, our results can suggest that increased apoptosis in fetal tissues also leads to abnormal development of parthenogenetic pig fetus resulting delayed development until day 26 and then termination and degeneration from day 36.

The objective of this study was to examine the effects of high concentrations of glucose on porcine parthenotes developing in vitro. Addition of 55 mM glucose to the culture medium of embryos at the four-cell-stage significantly inhibited blastocyst formation, resulting in fewer cells in blastocyst-stage embryos and increased levels of apoptosis and autophagy compared to control. Quantitative reverse transcriptase (RT) PCR analysis revealed that the expression of pro-apoptotic genes (Caspase 3, Bax and Bak) and autophagy genes (Atg6 and Atg8/Lc3) were increased significantly by the addition of 55 mM glucose to the culture medium compared to control. MitoTracker Green fluorescence revealed a decrease in the overall mitochondrial mass compared to control. However, the addition of 55 mM glucose had no effect on mRNA expression of the nuclear DNA-encoded mitochondrial-related genes, cytochrome oxidase (Cox) 5a, Cox5b and Cox6b1. These results suggest that hyperglycemia reduced the mitochondrial content of porcine embryos developing in vitro and that this may hinder embryonic development to the blastocyst stage and embryo quality by increasing apoptosis and autophagy in these embryos.

Mammalian testes are maintained at a lower temperature than the abdominal region for healthy spermatogenesis. Germ cell apoptosis has been described in heat-damaged testes by cryptorchidism, but the mechanism is not yet fully understood. To elucidate the cause of germ-cell death by cryptorchidism, we surgically induced cryptorchidism in dog testes and performed histological and molecular analyses. Histological data showed that the seminiferous tubules of cryptorchid testes and epididymis contained significantly fewer germ cells. Total RNA sequencing was performed to screen overexpressed genes in cryptorchid dog testes. Clusterin RNA was significantly higher (approximately 12.8-fold) in cryptorchid testes than in normal testes. In addition, cleaved caspase-3 and -8 were detected at higher levels in cryptorchid dog testes. Real time RT-PCR and western blotting analysis showed that the expression of clusterin was higher in cryptorchid dog testes. Furthermore, clusterin was detected in extracellular regions of cryptorchid dog testes during 4 weeks after surgery. Thus, we demonstrated germ-cell specific apoptosis and expression of clusterin in extracellular regions of cryptorchid dog testes. This result will facilitate further study of spermatogenesis and the specific mechanisms by which cryptorchidism causes male infertility.

This study was conducted to investigate the endoplasmic reticulum (ER) stress and subsequent apoptosis of porcine somatic cell nuclear transfer (SCNT) embryos induced during SCNT procedures, which compared with IVF embryos. Porcine SCNT embryos were made by micromanipulation and electrofusion/activation. Control porcine embryos were prepared by in vitro fertilization (IVF). Porcine SCNT and IVF embryos were sampled at 3 h-, and 20 h-post SCNT or IVF, and at the blastocyst stage for mRNA extraction. The x-box binding protein 1 (Xbp1) mRNA, the key transcription factor in the ER stress condition was confirmed by RT-PCR analysis. The expressions of ER stress-associated genes, the C/EBP homologous protein (CHOP), binding protein (BiP), activating transcription factor 4 (ATF4), and glucose-regulated protein 94 (GRP94) were confirmed by RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. Prior to SCNT, the splicing of Xbp1 mRNA and increased expressions of ER stress-associated genes were confirmed in the somatic cells treated with tunicamycin (TM), an ER stress inducer. Expression of spliced Xbp1 (Xbp1s) mRNA in the SCNT embryos did not differ with that of IVF embryos, regardless of the embryonic stage. Expression of ER stress-associated genes in SCNT embryos was, however, significantly increased in the all embryonic stages compared to IVF embryos (p<0.05). Apoptotic gene expression was slightly high in the SCNT embryos, but not different from that of IVF embryos in each group. The result of this study indicates that excessive ER stress can be induced by SCNT procedures. Although, ER stress-derived apoptosis was not identified in this study, more research studies are needed on the relationship between SCNT and ER stress-derived apoptosis.

Schizophrenia is a severe mental disorder characterized by abnormal mental functioning and disruptive behaviors. Abnormal expression of N-methyl-D-aspartate (NMDA) receptor, one of the glutamate receptor subtypes, has also been suggested to contribute to the symptoms of schizophrenia. The effect of treadmill exercise on schizophrenia-in-duced apoptosis in relation with NMDA receptor has not been evaluat-ed. In the present study, we investigated the effect of treadmill exercise on neuronal apoptosis in the hippocampus using MK-801-induced schizophrenic mice. MK -801 was intraperitoneally injected once a day for 2 weeks. The mice in the exercise groups were forced to run on a treadmill exercise for 60 min, once a day for 2 weeks. In the present re-sults, repeated injection of the NMDA receptor antagonist MK-801 re-duced expression of NMDA receptor in hippocampal CA2-3 regions. MK-801 injection increased casapse-3 expression and enhanced cyto-chrome c release in the hippocampus. The ratio of Bax to Bcl-2 was high-er in the MK-801- induced schizophrenia mice than the normal mice. In contrast, treadmill exercise enhanced NMDA receptor expression, suppressed caspae-3 activation and cytochrome c release, and inhibited the ratio of Bax to Bcl-2. Based on present finding, we concluded that NMDA receptor hypofunctioning induced neuronal apoptosis in MK-801-induced schizophrenic mice. Treadmill exercise suppressed neuro-nal apoptosis through enhancing NMDA receptor expression in schizo-phrenic mice.

There are diverse methods of cryopreservation of mammalian embryos with variable degrees of success. Although cryopreservation technique of mammalian embryos has been advanced, freezing stress affect to cellular event such as apoptosis and autophage in embryos. The objective of the study is to investigate the affection of to survival, development, live offspring, apoptosis and autophagy on embryo. Mouse embryos were vitrified and thawed using normal straw and modified cut standard straw (M-CSS), then in vitro cultured until blastocyst stage and transferred to recipient. Recovery rates (100 vs 99.2%), survival rates (99.2 vs 78.6%), developmental rates (18.4 vs 10.7%), total cell numbers (45 vs 37), preganacy rates (34.5 vs 25%) and offspring numbers (10.1 vs 4.9 %) of M-CSS group are significantly higher than those of normal straw vitrified group. Also, rate of apoptosis in blastocysts developed using M-CSS (1.9%) was significantly lower than using normal straw vitrification (2.7%). Apoptosis-related gene, caspase 3, was expressed at the highest level in blastocysts derived from normal straw group. However, no differences of autophagy related gene, Atg6 and expression of LC3 between normal straw and M-CSS groups were observed. In conclusion, the standard vitrification procedure induces mitochondrial apoptosis in zygotes in an autophagy-independent manner, whereas the novel M-CSS procedure may improve embryo vitrification.

Doxorubicin, a widely used chemotherapeutic agent, were found rapidly undergo morphological and biochemical changes via discrete effector signaling pathways consistent with the occurrence of apoptosis of oocyte. In this report, we elucidated the molecular requirements for actions of this drug in early embryos. Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues have recently been shown in female oocyte cells. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Cleaved PARP (cPARP) may be considered a marker of apoptosis. Doxorubicin inhibited the early embryo development, but the treatment could still reach the BL (blastocyst) stage that suggested that involved in DNA synthesis and repaired progress. Herein, the higher expression of PARP family shown especially in 2, 4 cell stagy. There was evidence of expression of Caspase3 and Bcl2l1 during embryogenesis (2 cell, 4 cell, morula and BL stage), suggesting that modulations of apoptosis-related genes and PARP were cause by DXR. Furthermore, the effect of doxorubicin on early embryo development was assessed different stage rates, and apoptosis index also conformed doxorubicin modulate embryo development by regulating apoptosis-related genes and PARP family genes. In conclusion, Doxorubicin blocked pre-implantation development in early mouse embryos by altering apoptosis-related gene expression and inactivating DNA repair by Parp.

Doxorubicin, a widely used chemotherapeutic agent, were found rapidly undergo morphological and biochemical changes via discrete effector signaling pathways consistent with the occurrence of apoptosis of oocyte, and a little known is actions of this drug in early embryos. Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, also plays the important role during the apoptosis of cell. The cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Cleaved PARP (cPARP) can be a marker of apoptosis.Doxorubicin inhibited the early embryo development, but the treatment could still reach the BL (blastocyst) stagethat suggested that involved in DNA synthesis and repaired progress. Herein, the higher expression of PARP family shown especially in 2, 4 cell stagy. There was evidence of expression of Caspase3 and Bcl2l1 during embryogenesis (2 cell, 4 cell, morula and BL stage), suggesting that modulationsof apoptosis-related genes and PARP were cause by DXR. Furthermore, the effect of doxorubicin on early embryo development was assessed different stage rates, and apoptosis index also conformed doxorubicin modulate embryo development by regulating apoptosis- related genes and PARP family genes. In conclusion, Doxorubicin blocked pre- implantation development in early mouse embryos by altering apoptosis-related gene expression and inactivating DNA repair by Parp.