• Clinical and Experimental Reproductive Medicine
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• 제26권3호
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• pp.363-368
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• 1999

연구목적: 본 연구는 동해방지제인 EFS40을 이용한 초자화동결이 생쥐 미성숙란의 cytoskeleton과 염색체의 성상에 미치는 영향을 indirect immunocytochemistry방법과 염색체 분석법으로 알아보고자 실시하였다. 연구재료 및 방법: 본 실험은 생쥐 미성숙란을 EFS40 (40% ethylene glycol, 18% ficou과 0.5 M sucrose가 들어있는 M2배양액)으로 초자화 동결하여 융해한 후 16시간동안 체외 성숙을 유도하여, 제 1극체가 나타난 성숙된 난자를 기준으로 동해제노출군 또는 대조군과 비교 조사하였다. 결과: 초자화동결된 미성숙란의 응해 후 생존율과 체외성숙율은 90.3%과 64.7%로써, 동해제노출군 (86.7%, 69.2%)과 대조군 (100%, 58.3%)에 유사하였다. 초자화동결이 미성숙란의 microtubule과 microfilament에 미치는 영향을 조사하였던 바, 동결군의 microtubule과 micro-filament의 정상적인 형성율 (93.9%, 100.0%)은 동해 제노출군 (94.4%, 100.0%)과 대조군 (100.0%, 100.0%)의 성적과 유사하게 나타났다. 또한, 초자화동결군에서 정상적인 염색체수를 가진 난자의 비율도 65.8%로써, 대조군(79.6%)과 노출군 (69.0%)의 결과와 유의한 차이가 없었다. 결론: 생쥐 미성숙란을 EFS40에 노출하고 동결하는 것이 미성숙란의 cytoskeleton과 염색체성상에 영향을 미치지 않으며, 본 연구에서 사용된 EFS40을 이용한 초자화동결법은 생쥐 미성숙란 동결에 적합하다는 것을 알 수 있었다.

• 한국가축번식학회지
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• 제23권1호
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• pp.85-92
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• 1999

본 연구는 동결동결보호제의 종류와 배발달단계가 생쥐의 OPP vitrification 동결방법에 미치는 영향을 알아보고자 실시하였다. 동결속도, 동결보호제 및 배발달단계는 vitrification 방법에 따른 수정란의 생존성에 영향을 미칠 수 있다. 본 연구에 사용된 동결보존액은 40% (v/v) ethylene glycol, 18% (w/v) Ficoll, 0.3 M sucrose와 5% FCS가 첨가된 D-PBS (EFS) 및 16.5% ethylene glycol, 16.5% dimethyl sulfoxide, 0.5 M sucrose 와 5% D-PBS (EDS)을 이용하였다. 배반포기배는 hCG 처리후 90시간째에 자궁으로부터 채취하여 실험 1에 이용하였고, 실험 2와 3에서는 zygote 를 hCG 처리후 18시간에 난관에서 채취하여 mHTF 배양액에 5% $CO_2$, 37$^{\circ}C$ 조건하에 배양하면서 2-, 4-, 8-cell, compacted morula, 또는 blastocyst를 이용하였다. 실험 1에서 배반포기배의 적당한 동결보존액을 결정하기 위하여 EFS 또는 EDS로 OPP vitrification 을 실시하였다. 재확장배반포기에 의한 생존율은 대조군과 EDS 처리군 (100, 100%) 이 EFS 군 (95.0%) 보다 유의적 (P＜0.05)으로 높게 나타났으나, 부화배반포기에서는 EFS 군 (90.0%) 이 대조군 (100%) 및 EDS 군 (95.0%) 보다 유의적으로 낮은 발달율을 보였다. 실험 2에서는 zygote, 2-, 4-, 8-cell, 상실배 빛 배반포기 등의 초기배에서도 OPP vitrification 동결방법이 적당한지를 판단하기 위하여 실시하였다. Zygote (70.0%) 는 동결융해 후 배발달율이 2, 4, 8, 상실배 및 배반포기배에 비하여 유의적으로 낮은 발달율을 보였다 (89.7, 90.0, 92.8, 97.6 및 97.5%) (P＜0.05). 또한 동결융해란의 할구수에서는 대조군 및 배반포기배 (35.7$\pm$2.98 및 39.6$\pm$2.81)에서 zygote, 2-, 4- 8-cell, 상실배 (29.8$\pm$3.21, 31.3$\pm$3.83, 29.3$\pm$3.58, 28.9$\pm$3.21 및 30.8$\pm$2.93) 보다 유의적으로 높게 나타났다 (P＜0.05) 실험 3에서는 zygote의 VS1 에 노출시간에 따른 생존율을 조사한 결과 융해후 2-cell (91.6, 88.5 및 88.9%) 및 배반포기 (83.3, 74.3 및 69.4%) 까지 배발달율은 1,2 및 3분간의 노출시간에 따른 유의적인 차이를 보이지 않았다. 또한 융해후 노출시간에 따른 할구수에서도 유의적인 차이를 보이지 않았다 (36.4$\pm$4.76, 32.4$\pm$4.67 및 27.6$\pm$4.52). 이상의 결과에서 OPP vitrification 방법은 EFS 또는 EDS 동결보존액에 따른 유의적인 차이 없이 이용될 수 있는 것으로 판단된다. 배발달단계에 따른 생존율은 zygote 의 초기배는 2, 4, 8, 상실배 및 배반포기보다 유의적으로 저조한 생존율을 보였다. Zygote의 VS1에 노출시간에 따른 생존율도 1 분간의 노풀시간에서 높은 배발달율을 보였다. OPP vitrification 동결보존방법으로 생쥐수정란의 동결보존에 유용하게 이용가능한 것으로 판단된다.

• Clinical and Experimental Reproductive Medicine
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• 제25권1호
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• pp.87-92
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• 1998

본 연구는 생쥐 미수정란을 초자화 동결하였을 때, 체내/외 발달율에 미치는 영향을 연구하고자 실시하였다. 생쥐 미수정란은 30, 35, 40% ethylene glycol, 18% ficoll과 0.5 M sucrose가 함유된 M2 배양액으로 구성 된 EFS30, 35, 40을 이 용하여 초자화 동결되었다. 노출 또는 초자화 동결-융해 후, 형태학적으로 정상적인 미수정란은 $1-2\times10^6/ml$ 농도의 정자로 체외수정되었고, 수정 율과 체내/외 발달율 그리고 배 반포의 세포수 (inner cell mass 와 tropectoderm cell)가 조사되었다. 본 실험에서 얻어진 결과는 다음과 같다. 35%의 ethylene glycol이 함유된 EFS35에서 EFS30과 EFS40보다 높은 분할율을 나타냈다. 초자화 동결-융해 후 체외수정된 미수정란의 2-세포기까지의 발달을 (51.1%)은 동결없이 초자화 동결액에 노출만된 군 (60.0%)과 대조군 (68.2%)에 비해 유의하게 감소하였다 (p<0.05). 그러나 이들 처리군에 있어서 난할된 난자로 부터의 배반포기 배까지의 발달율에는 유의한 차가 없었다. (75.0, 73.3 과 80.0%). 또한 초자화 동결된 군의 배반포기배 세포수 $(92.5{\pm}2.9)$도 노출군 $(98.5{\pm}5.3)$, 대조군 $(100.9{\pm}4.8)$과 유사하였다. 초자화 동결-융해 하여 얻어진 배반포기배를 가임신 생쥐에 이식하였을 때 체내발달율인 산자발달율 (50.7%)과 착상율 (80.0%)도, 대조군 (58.2, 78.2%)과 유사하였다. 이러한 결과로 보아, 생쥐 미수정란은 ethylene glycol를 기본으로 한 EFS35라는 초자화 동결액을 이용하여 동결보존될 수 있음을 알 수 있었다.

• The Korean Journal of Physiology and Pharmacology
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• 제2권2호
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• pp.217-223
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• 1998

Oxygen-derived free radicals have been implicated in many important functions in the biological system. Electrical field stimulation (EFS) causes arterial relaxation in animal models. We found that EFS applied to neither muscle nor nerve but to Krebs solution caused a relaxation of rat aorta that had been contracted with phenylephrine. In the present study, therefore, we investigated the characteristics of this EIRF (electrolysis-induced relaxing factor) using rat isolated aorta. Results indicated that EIRF acts irrespective of the presence of endothelium. EIRF shows positive Griess reaction and is diffusible and quite stable. EIRF-induced relaxation was stronger on PE-contracted aorta than on KCl-contracted one, and inhibited by the pretreatment with methylene blue. Zaprinast, a cGMP-specific phosphodiesterase inhibitor, potentiated the EIRF-induced relaxation. $N^G-nitro-L-arginine$, NO synthase inhibitor, did not inhibit the EIRF-induced relaxation. Deferroxamine, but not ascorbic acid, DMSO potentiated the EIRF-induced relaxation. These results indicate that electrolysis of Krebs solution produces a factor that relaxes vascular smooth muscle via cGMP-mediated mechanism.

• Clinical and Experimental Reproductive Medicine
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• 제29권1호
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• pp.13-20
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• 2002

Objective: This study was carried out to establish the effectiveness of the vitrification method and the optimal cryoprotectants in the cryopreservation of human embryonic stem cells (ESC). Materials and Methods: Human ESC clumps established at Seoul National University Hospital (SNUhES 1) were cryopreserved with the vitrification method using the EM grid. EDS and EFS40 were used as vitrification solutions. Results: Between the EDS and EFS40 groups, there was no significant difference in the recovery rate after cryopreservation of human ESC. The formation rates of ESC colonies in the vitrified groups were significantly lower than those in the control ESC group (p<0.05, p<0.05). In addition, the formation rate of ESC colonies in the EDS group was significantly higher than that in the EFS40 group (p<0.05). The ESC colonies in the vitrified groups were significantly smaller after culture duration of 2 and 4 days, respectively, compared with the control ESC group (p<0.1, p<0.05). However, these effects could be reduced to nonsignificant level by the additional culture of ESC colonies. The vitrified human ESC retained the properties of pluripotent cells, including the expression of cell surface. markers for the undifferentiated cells such as alkaline phosphatase and SSEA-4 (stage-specific embryonic antigen-4), and the expression of transcription factor Oct-4 (octamer-binding transcription factor-4), and the normal karyotype. Conclusion: The vitrification method using the EM grid and EDS solution was confirmed to be very effective for the cryopreservation of human ESC.

• The Korean Journal of Physiology
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• 제26권1호
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• pp.45-54
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• 1992

Effects of pH, $PCO_2$, and adenosine on the vascular contractility were investigated in the pig coronary arteries. The helical strips of isolated coronary arteries were immersed in the HEPES or $HCO_3^-/CO_2$-buffered Tyrode's solution equilibrated with 100% $O_2\;or\;95%\;O_2-5%\;CO_2\;at\;35^{\circ}C$. The contraction was recorded isometrically using a force transducer. The amplitudes of contraction induced by ACh, high $K^+$, and electrical Held stimulation (EFS) were decreased by elevating extracellular pH (pHo) and were increased by lowering pHo. A shift from $0%\;CO_2\;to\;5%\;CO_2$ at constant pHo (pH 7.4) reduced the contractions induced by ACh, high $K^+$, EFS. However the contraction induced by 100mM $K^+$ was less influenced by the change of pHo or $CO_2$. The contraction induced by ACh in $Ca^{2+}$free Tyrode's solution as well as the contraction developed by the addition of extracellular of $Ca^{2+}$ were decreased by lowering pHo and were increased by elevating pHo. High $K^+$ (25mM) induced contraction at pH 6.8 was not returned to the level of the contraction at pH 7.4 by the elevation of extracellular. calcium $[Ca^{2+}]_o$. Adenosine-induced relaxation was more significant with 5% $CO_2$ than 0% $CO_2$ in the high $K^+$-induced contraction and was more significant with low pHo than high pHo in the contraction induced by EFS. From the above results, it is suggested that $H^+$ and $CO_2$ inhibit $Ca^{2+}$ influx as well as $Ca^{2+}$ release from intracellular $Ca^{2+}$ storage sites and enhance the relaxing effect of adenosine in the pig coronary artery.

• The Korean Journal of Physiology
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• 제30권2호
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• pp.219-229
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• 1996

In order to investigate intra-pancreatic cholinergic roles on insulin action in exocrine secretion, the pancreas was isolated from rats and continuously perfused with modified Krebs-Henseleit solution. Intra-arterial infusion of insulin (100 nM) or cholecystokinin (CCK, 14 pM) alone resulted in stimulation of the volume flow and amylase output. Also insulin potentiated the action of CCK in the exocrine secretion. Tetrodotoxin and atropine completely abolished the potentiating action of insulin and CCK as well as the action of insulin alone, but did not change the action of CCK alone. In order to see an effect of intra-pancreatic neural activation on the insulin action, electrical field stimulation (EFS) with parameters of 20 V, 2 msec and 8 Hz was applied to the isolated pancreas for 10 min under 2.5 or 18 mM glucose background. The EFS voltage-dependently elevated the flow rate and amylase output, and potentiated exocrine secretion in 18 mM glucose infusion compared with 2.5 mM glucose. The potentiating effects of EFS and 18 mM glucose were not observed in the streptozotocin-treated pancreas although it was perfused with 18 mM glucose. However, it was restored when the diabetic pancreas was perfused with porcine insulin(100 nM). Tetrodotoxin and atropine inhibited the pancreatic secretion induced by EFS with the background of 18 mM glucose. The results of present investigation indicate that the intra-pancreatic cholinergic tone exerts a stimulatory influence on the action of insulin in pancreatic exocrine secretion of rats.

• 대한약리학회지
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• 제19권2호
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• pp.1-8
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• 1983

Prostaglandin(PG) is ubiquitously distributed in most mammalian tissue and their actions are complicated. Especially in autonomic nervous system, there are evidences indicating that PGs act as neuromodulators i.e., PGs, which are released in the vicinity of autonomic neuroeffector junctions, influence the release and the response of the neurotransmitter. Present study was undertaken to elucidate the interrelationship between $PGF_{2\alpha}$ and adrenergic ${\alpha}_2-receptor$ function in electrical field stimulation induced contractile response of vas deferens in rat. Male rat, weighing 150{\sim}200\;g, was sacrificed and vas deferens was obtained. The isolated vas deferens strip was placed between two platinum electrodes in temperature controlled $(37^{\circ}C)$ muscle chamber containing Tyrode's solution and the electrical field stimulation(EFS) induced contraction was recorded with Grass Polygraph(Model 7) via force displacement transducer (FT .03, Grass). The results are summarized as follows: 1) Electrical field stimulation for 1sec( 1 msec, 40 cps) induced contraction of vas deferens was completely blocked by tetrodotoxin. 2) Bretylium caused marked inhibition of the EFS-induced contraction, hut tyramine and cocaine augmented the contraction. 3) EFS-induced contraction was inhibited or little affected in distal portion of vas deferens by norepinephrine or methoxamine, but the contraction was rather augmented by the ${\alpha}-agonists$ in proximal portion. 4) Clonidine inhibited the EFS-induced contraction proportionally to the concentration in distal portion, which was blocked by yohimbine pretreatment, but in the presence of $PGF_{2\alpha}$ the blockade by yohimbine was reversed. 5) Indomethacin pretreatment reduced the effect of clonidine, but addition of $PGF_{2\alpha}$ after washing-out the indomethacin caused the contraction to the control level. From these results it is suggested that PG synthesis is a necessary step and the PG itself has a permissive role in ${\alpha}_2-adrenoceptor$ action in rat vas deferens.

• 대한수의학회지
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• 제36권3호
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• pp.599-605
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• 1996

This study was designed to examine the mechanism of penile erection in adult bull by analyzing the responses of bovine proximal retractor penile muscle strips(BRP) to electtical field stimulation(EFS), exogenous nitric oxide(NO), NO synthesis precursor(L-arginine), NO synthase inhibitors(L-NAME, L-NMMA), guanylate cyclase inhibitor(methylene blue) and nonspecific potassium channel blocker(tetraethylammonium, TEA) treatments. Isometric tension of BRP was measured using physiograph. Results were summarized as follows: 1. EFS of nonadrenergic noncholinrgic(NANC) nerve in BRP produced frequency-dependent inhibitory responses to the contraction induced by co-treatment of epinephrine, guanethidine and atropine. The inhibitory responses to EFS were blocked by tetrodotoxin(TTX, $1{\mu}M$). 2. Treatment of L-NAME ($10,\;20{\mu}M$) inhibited the relaxation to EFS whereas L-NMMA ($100{\mu}M$) had no effect. 3. Treatment of NO($20,\;40{\mu}M$; as an acidified solution of $NaNO_2$) induced concentration-dependent relaxation whereas preincubation of TTX($1{\mu}M$) and L-NAME($20{\mu}M$) had no effect on the relaxation response. 4. L-arginine treatment(10mM) blocked the inhibitory effect of L-NAME($20{\mu}M$). 5. Pretreatment of methylene blue($40{\mu}M$) reduced the NANC-induced relaxation of BRP. 6. Tetraethylammonium(TEA, 80mM) reduced NANC relaxation. These results suggest that NO may act as a NANC neurotransmitter in BRP and the effects might be mediated by cGMP and potassium channel.

• 한국수정란이식학회지
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• 제13권1호
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• pp.1-9
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• 1998

In order to improve the cryopreservation by vitrification or slow freezing of nuclear transplant rabbit embryos, the effects of factors affecting embryo cryopreservation such as cryoprotectants, equilibration, cooling rate and post-thaw dilution on post-thaw survial and development were determined using intact embryos of morular stage. And the post-thaw development of nuclear transplanted embryos cryopreserved under the optimal conditions examined was compared between vitrification and slow freezing. The cryoprotectant solution used was ethyleneglycol-ficoll-sucrose (EFS) or ethyleneglycol-poly-vinylpyrrolidone-galactose- I (EPG- I ) for vitrification, and EPG- II for slow freezing. To examine the viability of frozen-thawed embryos, the nuclear transplanted embryos were co-cultured in TCM-199 plus 10% FBS with bovine oviduct epithelial cells(BOEC) for 24 hrs and the intact morulae were co-cultured with BOEC for 5 days and 3 days to hatching blastocyst stage in 39 ˚C 5% $CO_2$ incubator. The results obtained were as follows: Following vitrification with EFS, the post-thaw development of rabbit morulae to hatching blastocyst was significantly(P<0.05) higher in compacted stage(82.4%) than in early morular stage(60.0%). The post-thaw development of compacted morulae to hatching blastocyst was similarly high in vitrification with EFS(82.4%), EPG- I (85.0%) and in slow freezing with EPG- II (83.3%). Following vitrification with EPG- I, the post-thaw development of intact rabbit morulae to hatching blastocyst was similar as 78.0% and 85.0% in 1-step and 2-step post-thaw dilution, respectively. The post-thaw development of nuclear transplanted rabbit embryos of compacted morulae stage to hatching blastocyst was similarly 43.6% and 40.0% in vitrification with EPG- Iand slow freezing with EPG- II, respectively. These results indicated that the rabbit nuclear transplant and intact embryos of morulae stage could be well cryopreserved with either vitrification or slow freezing procedure.