Dibutyryl Cyclic AMP로 처리된 생쥐난자의 수정능에 관한 연구

강해묵,이영기,조완규 한국통합생물학회 1988 동물학회지 Vol.31 No.1

The present study was carried out to examine the fertilizability of the mouse oocytes pre-ex-posed to dbcAMP which is a well-known inhibitor of the oocyte maturation. The oocytes once cultured in the dbcMP-containing medium for a certain length of, time were cultivated in the dbcMp-free medium to induced the maturation, then mixed with sperms, and observed following culture for 24 hours. The fertilization rate of cocytes was judged by the index of the number of 2-cell embryo developed 24hr following insemination. The fertilization rate of the oocyte previously incubated with dbcAMP (100 g/ml) for 2, 4, 8 16 hours was 32.3, 14.5, 4.7 and 8.8%, respectively, while that of the control was 53.3% indicating that the fertilizability was decreased as a function of time exposed to dbcAMP. The pretreatment of dbcMP, however, didn't affect the process of sperm penetration to egg. In addition, there is no prominent changes in the morphological architecture of fertielized eggs which has been exposed to dbcAMP as revealed by electron microscopic observation. Consequendy, it can be concluded that the mouse cocytes once inhibited their maturation by dbcMP may retain, in some extent, the fertilizability, although most of the fertilized egg may not proceed to further development because of the failure of pronucleus formation. dbcAMP에 의해 성숙이 억제된 생쥐난자의 수정능을 조사하기 위해 본 실험을 행하였다. dbcAMP로 일시 성숙이 억제되었던 난자를 배양액내에서 정자와 섞고 24시간 배양한 후 발생한 2세포기의 배아형성, 정자의 관입, 전액형성을 조사하여 2세포기로 배아발생이 진척된 것을 수정율의 기준으로 정하였다. dbcAMP를 처리하지 않은 난자는 약 53.3%의 수정율을 보였으며,dbcAMP가 함유되 배양액에서 배양한 후 기본 배양액에서 성숙시킨 난자들의 수정율은 dbcAMP의 처리시간에 비례하여 낮으나, 정자의 관입은 정상적으로 일어났다. 전자현미경적 관찰에 의하면 dbcAMP 처리는 난자의 미세구조에 어떠한 변화도 야기하지 않았다. 따라서 dbcAMP를 사전처리하여 성숙을 억제한 난자라할지라도 어느 정도의 수정능력을 보유하고 있다고 사료된다.

Artificial control maturation of porcine oocyte by dibutyryl cyclicAMP

Zhao, Ming-Hui,Jin, Yong-Xun,Lee, Seul-Ki,Kim, Nam-Hyung,Cui, Xiang-Shun TaylorFrancis 2014 Animal cells and systems Vol.18 No.1

<P>In this study, we investigated the effects of various durations of dibutyryl cyclic AMP (dbcAMP) treatment on the in vitro maturation (IVM) and subsequent development of parthenogenetically activated embryos. Immature porcine oocytes were cultured with or without 1 mM dbcAMP during the first 20, 28, or 36 h of culture, and then incubated for an additional 24 h without dbcAMP. The expression of <I>Wee1B, Myt</I>, and <I>Cdc25B</I> and the level of maturation promoting factor (MPF) in metaphase II oocytes were analyzed by real-time PCR (qRT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. The distribution of actin microfilaments in oocytes was also assessed. Subsequently, apoptotic cells in blastocysts from each group were visualized by transferase-mediated dUTP nick-end labeling staining. Results showed that oocytes extruded the first polar body between 12 and 18 h after being released from dbcAMP. MPF activity in oocytes at 28 + 24 h and 36 + 24 h after dbcAMP treatment was higher than that in the control group. Significantly more blastocysts were present among embryos in 28 + 24 h (54.28% vs. 39.11%, <I>P</I> < 0.05) and 36 + 24 h (47.24% vs. 32.94%, <I>P</I> < 0.05) groups than among embryos cultured in the absence of dbcAMP. However, the number of total and apoptotic cells was not significantly different between groups. The distribution of actin microfilaments was abnormal in oocytes cultured for 60 h without dbcAMP. In addition, the expression of <I>Wee1B, Myt</I>, and <I>Cdc25B</I> was higher in the control group at 44 h than in the dbcAMP group, but there were no differences in expression at the other time points. In conclusion, dbcAMP treatment delays oocyte maturation and maintains oocyte quality.</P>

생쥐 난자의 체외 성숙에 미치는 Melatonin의 영향

안희진,배인하,Ahn, Hee-Jin,Bae, In-Ha 대한생식의학회 2004 Clinical and Experimental Reproductive Medicine Vol.31 No.3

Objective: Melatonin, which is secreted by pineal gland play an important role in the regulation of ovarian function via seasonal rhythm and sleep in most mammals. It also has a role in the protection of cells by removing toxic oxygen free radicals brought about by metabolism. In the present study, effects of melatonin on the mouse oocyte maturation were examined using two different culture conditions provided with 5% or 21% oxygen concentration. Material and Method: Immature mouse oocytes were obtained from the ovarian follicles of $3{\sim}4$ weeks old ICR strain mice intraperitoneally injected with 5 I.U. PMSG 44 hour before. Under stereomicroscope, morphologically healthy oocytes with distinct germinal vesicle (GV) were liberated from the graafian follicles and collected using mouth-controlled micropipette. They were then cultured for 17 hour at $37^{circ}C$, 5% $CO_2$ and 21% $O_2$ (95% air) or 5% $CO_2$, 5% $O_2$ and 90% $N_2$. New modified Hank's balanced salt solution (New MHBS) was used as a culture medium throughout the experiments. Effects of melatonin were examined at a concentration of $0.0001{\mu}M$, $0.01{\mu}M$ or $1.0{\mu}M$. For the prevention of spontaneous maturation of immature oocytes during culture, dibutyryl cyclic AMP (dbcAMP) and/or hypoxanthine were included in the medium. Results: Under 21% oxygen condition, oocytes cultured in the presence of $0.01{\mu}M$ melatonin showed a significantly higher maturation rates, in terms of germinal vesicle breakdown (95.0% vs 89.0%) and polar body formation (88.1% vs 75.4%), compared to those cultured with $0.0001{\mu}M$ or $1.0{\mu}M$ melatonin. However, no difference was observed in oocytes cultured under 5% oxygen whether they were treated with melatonin or not. In the presence of $0.01{\mu}M$ melatonin, oocytes either cultured under 21% or 5% oxygen exhibited no difference in the polar body formation (85.6% vs 86.7%). However, in the absence of melatonin, oocytes cultured under 21% oxygen exhibited lower polar body formation (74.7%). When oocytes were cultured in the presence of dbcAMP alone or with varying concentrations of melatonin, those treated with both compounds always showed better maturation, i.e., germinal vesicle breakdown and polar body formation, compared to those cultured with dbcAMP alone. At the same concentration of melatonin, however, oocytes exposed to 21% oxygen showed poor maturation than those to 5% oxygen. Similar results were obtained from the experiments using hypoxanthine instead of dbcAMP. Conclusion: Based upon these results, it is suggested that melatonin could enhance the meiotic maturation of mouse oocytes under 21% oxygen concentration, and release oocytes from the meiotic arrest by dbcAMP or hypoxanthine regardless of the concentration of oxygen, probably via the removal of oxygen free radicals.

DBcAMP와 retinoic acid를 이용한 마우스 배아줄기의 평활근세포 분화

박성수 ( Sung Soo Park ),강주원 ( Ju Won Kang ) 한국가축위생학회 2008 韓國家畜衛生學會誌 Vol.31 No.4

The differentiation of mouse embryonic stem(ES) cell into smooth muscle cells(SMC) may play a major role in cardiovascular development and under pathophysiological conditions. Therefore, in the present study, we have examined the differentiation of ES cells and its related gene expression. SMC differentiation was indicated by cellular morphology and time-dependent induction of dibutyryl adenosine 3,5-cyclic monophosphate(DBcAMP)and retinoic acid(RA) on smooth muscle α-actin(SMαA), smooth muscle myosin heavy chain(SMMHC) gene expression. The control was undifferentiated ES cells(protein expressions represent 50-60kDaOct-4). The results of this study show that morphology of embryoid body and confirmation of SMαA expression by immunocytochemistry. Moreover, SMMHC and desmin expression was significantly increased by time dependent manner(5, 7, 15 days), in contrast to SMαA expression was slightly decreased on 15days. In conclusion, DBcAMP and RA stimulate mouse ES cells differentiation into SMC and enhanced SMαA, SMMHC and desmin expression.

Comparative Analysis of Dibutyric cAMP and Butyric Acid on the Differentiation of Human Eosinophilic Leukemia EoL-1 Cells

The Korean Association of Immunologists 2015 Immune Network Vol.15 No.6

<P>Purification of enough numbers of circulating eosinophils is difficult because eosinophils account for less than 5% peripheral blood leukocytes. Human eosinophilic leukemia EoL-1 cells have been considered an <I>in vitro</I> source of eosinophils as they can differentiate into mature eosinophil-like cells when incubated with dibutyryl cAMP (dbcAMP) or butyric acid. In this study, the viability and phenotypic maturation of EoL-1 cells stimulated by either dbcAMP or butyric acid were comparatively analyzed. After treatment with 100 µM dbcAMP or 0.5 µM butyric acid, EoL-1 cells showed morphological signs of differentiation, although the number of nonviable EoL-1 cells was significantly increased following butyric acid treatment. Stimulation of EoL-1 cells with 0.5 µM butyric acid more effectively induced the expression of mature eosinophil markers than stimulation with dbcAMP. These results suggest that treatment of EoL-1 cells with 0.5 µM butyric acid for limited duration could be an effective strategy for inducing their differentiation. Considering that expression of CCR3 was not sufficient in EoL-1 cells stimulated with 0.5 µM butyric acid, treatment of the chemically stimulated EoL-1 cells with cytokines, which primarily support eosinophil maturation, would help to obtain differentiated EoL-1 cells with greater functional maturity.</P>

Comparative Analysis of Dibutyric cAMP and Butyric Acid on the Differentiation of Human Eosinophilic Leukemia EoL-1 Cells

정윤재 대한면역학회 2015 Immune Network Vol.15 No.6

Purification of enough numbers of circulating eosinophils is difficult because eosinophils account for less than 5% peripheral blood leukocytes. Human eosinophilic leukemia EoL-1 cells have been considered an in vitro source of eosinophils as they can differentiate into mature eosinophillike cells when incubated with dibutyryl cAMP (dbcAMP) or butyric acid. In this study, the viability and phenotypic maturation of EoL-1 cells stimulated by either dbcAMP or butyric acid were comparatively analyzed. After treatment with 100 μM dbcAMP or 0.5 μM butyric acid, EoL-1 cells showed morphological signs of differentiation, although the number of nonviable EoL-1 cells was significantly increased following butyric acid treatment. Stimulation of EoL-1 cells with 0.5 μM butyric acid more effectively induced the expression of mature eosinophil markers than stimulation with dbcAMP. These results suggest that treatment of EoL-1 cells with 0.5 μM butyric acid for limited duration could be an effective strategy for inducing their differentiation. Considering that expression of CCR3 was not sufficient in EoL-1 cells stimulated with 0.5 μM butyric acid, treatment of the chemically stimulated EoL-1 cells with cytokines, which primarily support eosinophil maturation, would help to obtain differentiated EoL-1 cells with greater functional maturity.

Cyclic AMP analog와 PGE2가 마우스 조골세포의 활성에 미치는 영향

김형룡,김장숙 원광대학교 생체재료·매식연구소 1996 원광생체재료·매식 Vol.5 No.2

To maintain its functional integrity, bone is continuously remodelled by a process involving resorption by osteoclasts and bone formation by osteoblasts. In order to respond to changes in the physical environment or to trauma with the relevant action, this process is strictly regulated by locally synthesized or systemic fators. Prostaglandin E_2(PGE_2) is perhaps one of the best studied of these factors, having been known to affect bone cell function for several decades. PGE_2 has both anabolic and catabolic activities. Excess levels of PGE_2 have been implicated in a number of pathological states associated with bone loss such as a number of chronic inflammatory conditions such as periodontal disease and rheumatoid arthritis. PGE_2 and other arachidonic acid metabolites have been shown to be potent stimulators of osteoclastic bone resorption in organ culture. The anabolic effects of PGE_2 were first noticed when an increase in periosteal woven bone formation was seen after the infusion of PGE_2 into infants in order to prevent closure of the ductus arteriosus. The cellular basis for the catabolic actions of PGE_2 has been well characterized. PGE_2 increase osteoclast recruitment in bone marrow cell cultures. PGE_2 also has a direct action on osteoclast serving to inhibit activity and can also indirectly activate osteoclast via other cells in the vicinity, presumably osteoblast. The cellular mechanisms for the anabolic actions of PGE_2 are not nearly so well understood. The purpose of this paper is to study the anabolic actions of PGE_2 on osteoblastic clone MC3T3E1 cells. The effects of DBcAMP and PGE_2 on the induction of alkaline phosphatase(ALP) was investigated in osteoblastic clone MC3T3E1 cells cultured in medium containing 0.4% fetal bovine serum. DBcAMP and PGE_2 stimulated ALP activity and MTT assay in the cells in a dose-dependent manner at concentrations of 10-500ng/ml. Cycloheximide, protein synthesis inhibitor, inhibited the stimulative effect of PGE_2 and DBcAMP on ALP activity in the cells. PGE_2 also increased the intracellular cAMP content in a dose-dependent fashion with a maximal effect at 500ng/ml. Our present results suggest that PGE_2 stimulate the differentiation of osteoblasts and are involved in bone formation in vivo, as well as in bone resorption.

Cyclic AMP analog와 PGE₂가 마우스 조골세포의 활성에 미치는 영향

김형룡 대한골대사학회 1996 대한골대사학회지 Vol.3 No.1

Testosterone과 dibutyryl cyclic AMP가 거세한 흰쥐 부정소의 $\beta$ -glucosidase와 몇가지 glycosidase 활성에 미치는 영향 및 부정소 상피세포의 여러 유형에 관한 연구

최임순,정경순 한국통합생물학회 1989 동물학회지 Vol.32 No.3

$\beta$-glucosidase, $\beta$-glucuronidase, N-acetyl-$\beta$-glucosaminidase의 성적 성숙과의 연관서을 조사하기 위하여 흰쥐의 복강내로 testosterone과 dibutryjry cyclic AMP 를 투여하여 위이 효소들의 활성도를 측정하였다. 그 결과 겨세한 실험군에서$\beta$-glucosidase와 N-acetyl-$\beta$-glucosaminidase이 활성도도 거세후 7일째에는 유의성있는 감소효과를 나타내었다. testosterone을 7일간 계속 투여한 경우에는 세 효소의 활성도가 모두 유의성있게 증가하였고 dbcAMP 투여군의 경우는 거세 14일째되는 실험군과 비슷하거나 감소하는 경향이 있었다. 미세구조를 관찰한 결과 부정소 상피세포의 유형은 크게 주세포와 기저세포로 나눌 수 있었으며 주세포는 일반적인 원주상피의 형태를 나타냈으며 소낭을 많이 포함하고 있는 narrow cell이 존재하였다. 특히 부정소미에는 다른 상피세포에 비해 전자밀도가 낮은 light cell이 존재했고 기저세포는 부정소 부위마다 비슷한 형태를 가지는 것으로 나타났으며 상피세포 사이에는 이동능력을 가진 halo cell이 존재했다. The activities of $\beta$-glucosidase, $\beta$-glucuronidase and N-acetyl-$\beta$-glucosaminidase were measured to investigate the relationships of them to sexual maturity. Peritoneal injections of testosterone and dibutyryl cAMP to rats were carried out. As a result, the activities of $\beta$-glucosidase and N-acetyl-$\beta$-glucosaminidase were significantly decreased from the third day and that of P -glucurondiase on the seventh day in the castrated groups. In addition, ihe activities of these three enzymes were significantly increased in the testosterone treated groups for 7 days. In case of dbcAMP injection, the activities of these three enzymes were similar to those of castrated groups or had a tendency to be decreased. On electron microscopic examination, principal cells, basal cells and narrow cells were observed in all regions of epididymis. Principal cells were general forms of columnar epithelial cells. Narrow cells had a number of small vesicles and light cells showed low electron density in comparison to other epithelial cells in cauda epididymis. Halo cells were migrating leucocytes btween epithelial cells.

Comparative Analysis of Dibutyric cAMP and Butyric Acid on the Differentiation of Human Eosinophilic Leukemia EoL-1 Cells

YunJae Jung 대한면역학회 2015 Immune Network Vol.15 No.6