참개구리와 옴개구리 여포의 프로제스테론 생성과 난자의 성숙

권혁방,김지열,고선근 한국통합생물학회 1990 동물학회지 Vol.33 No.2

참구개구와 옴개구리의 여포를 생체외 배양하면서 progesterone (P$_4$)생성과 난자의 성숙 및 cyclic(cAMP)의 조절작용을 조사하였다.참개구리의 여포에 뇌하수체 추출물(frog pituitary homogenate.FPH)을 처리하면 농도에 의존하여 여포의 P$_4$생성이 증가하였으며 난자의 성숙(핵붕괴)이 일어났다. 이들 여포들을 배양하면서 3시간 간격으로 호르몬이 여포에 축적된 양, 배양액에 분비된 양 및 난자의 성숙율을 조사한 결과 FPH처리군에서 P$_4$는 3-6시간에 최고치 (여포내,약 400pg/여포;분비량,약 800pg/여포)를 나타내었으며 난자의 핵붕괴는 9-12시간에 일어났다. 상기 여포들과 같은 조건으로 배양하면서 forskolin과 3-isobutyl-1-methylxanthine(IBMX)을 배양액에 처리하여 간접적으로 여포내 cAMP의 농도를 높여주면 FPH와 유사한 양사으로 호르몬의 생성을 촉진하였다. 그러나 난자의 성숙은 전혀 일어나지 않았다. 옴개구리의 여포를 배양하면서 FPH를 처리하였을 때는 아무 처리를 하지않은 대조군과 비교하여 거의 P$_4$의 생성을 촉진하지 않았으며 난자의 성숙도 유도하지 못했다, 그러나 이들에게 forskolin과 IBMX를 처리하면 P$_4$의 생성을 현저하게 촉진하여 다량의 P$_4$가 여포와 (약 800pg/여포) 배양액에 (1700pg/여포)축적되었다. Progesterone production and oocyte maturation in ovarian follicles of Rana nigromaculata and Rana rugosa were investigated. Addition of frog pituitary homogenate (FPH) to the in utiro cultured follicles of R. nigromaculata stimulated a marked increase in the accumulation and secretion of progesterone (P$_4$) by the follicles and induced their oocyte maturation (germinal vesicle breakdown, GVBD) in a dose dependent manner. The FPH (0.1 pituitary equivalent/2 ml)-inducted P4 peak appeared in 3-6 hours and followed by the oocyte GVBD in 9-12 hours after the hormone stimulation. lncreae of intrafollicular cAMP levels with forskolin (an adenylatecyclase stimulator) and 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) mimic the FPH action in the stimulation of P$_4$ production but not in the induction of oocyte maturation. The in uitro cultured follicies of R. rugosa behaved very differently from other amphibian follicles. Addition of FPH-(0. 1 pit. equivl2 ml) to the culture medium neither stimulated P$_4$ production by the follicles nor induced the oocyte GVBD. However, treatment of the follicles with forskolin and IBMX drastically stimulated both the intrafollicular accumulation (800 pg/follicle) and secretion (1700 pg/follicle) of P$_4$ by the follicles during culture period. Thus, the data suggest that the follicles are ready to respond to cAMP increase but not to the FPH stimulation in terms of P$_4$ production.

북방산 개구리(Rana dybowskii)의포의 프로젝트론 생서에 대한 cAMP의 조절작용

권혁방,안연섭,김지열,윤용달 한국통합생물학회 1988 동물학회지 Vol.31 No.3

The pattern of progesterone production and secretion of frog(R. dybowskii) follicles was investigated in follicle culture in vitro. Involvement of cAMP in the regulation of the steroid production by the follicles was also investigated by manipulating endogeneous cAMP level with forskolin and/or 3-isoburyl- 1 - methylxanthine(IBMX). Endogeneous follicular progesterone level increased rapidly in one hour of culture by treatment of frog pituitary homogenate(FPH) and reached peak level at 2 hours or later. But the absolute amount of progesterone produced (60-300 pg/follicle) or the peak time of the honnone level was different between individual animals. Basal level of progesterone in untreated sister follicles was very low (around 10 pg/follicle) and nearly undetectable in most cases regardless of culture lime. Secretion level of progesterone by the follicles obtained by measunng the honnone in the culture media was just the reflection of the intrafollicular level. Exogeneously added forskolin, an adenylate cyclase stimulator, and/or IBMX, a phosphodiesterase inhibitor, could mimic FPH action in terms of progesterone production and secretion. Thus, it seems clear that FPH regulates progesterone production via cAMP system in the follicle cells. 북방산개구리의 여포를 인공배양하면서 여포의 progesterone(p$_4$) 의 생성양상과 cAMP의 조절작용을 조사하여 보았다. 배양중인 여포에 뇌하수체 추출물(frog pituitary homogenate,FPH)을 처리하였을 때 배양 한시간 부터 여포내 p$_4$의 양이 급격히 증가하였다. 그러나 생성된 p$_4$의 최대양이나(약 60-300pg/follicle),peak를 이루는 시간이(2시간 이후)개체에 따라 차이가 있었다. FPH의 처리를 받지않은 대조군에서는 배양기간에 관계없이 여포내에서 대략 10pg/follicle정도의 p$_4$가 측정되었으나 예외인 개체도 있었다. 배양액내로 분비된 p$_4$의 양은 여포내에 생성된p$_4$양의 약 60%정도 이었다. 배양액에 adenlate cyclass의 촉진제인 forskolin이나phosphodiesterase의 저해제인 3-isoburyl-1- methylxanthine(IBMX)을 동시에 혹은 따로 처리하면 p$_4$의 생성과 분비가 역시 증가하며 모든 양상이 FPH의 자극에 의한 것과 거의 같았다. 따라서 개구리여포의 스테로이드생성은 cAMP를 통하여 조절된다는 것과 여포세포내에는 cAMP의 생성과 분해에 관계하는 효소들이 있다는 것을 알았다.

돼지 난구세포의 분산조절에 관한 연구 : cAMP와 Calcium의 작용

권혁방,이원교,김남중,김문규,나철호 한국통합생물학회 1987 동물학회지 Vol.30 No.2

It is the purpose to investigate the physiological toxity and mutagenicity by heavy metal compounds such as potasium dichromate, sodium arsenite and selenium dioxide on the development of Drosophila melanogaster. These chemical compounds were highly toxic at every developmental stage of Drosophila. The relative values of fecundity, viability and developmental time obtained on food media containing 5 different levels of potasium dichromate (500, 600, 700, 800, and 900 ppm), also 5 different levels of sodium arsenite and selenium dioxide (10, 15, 20, 25 and 30 ppm). Resulting in the relative values of fecundity and viability generally decreased, on the contrary developmental time generally reduced as the concentration of chemicals in the food increased. A significant reduction in the fecundity was observed between 800 and 900 ppm (p<0. 01) on the potasium dichromate media, between 15 and 20 ppm (p<0. 05) on the sodium arsenite media, between 20 and 25 ppm (p<0. 02) on the selenium dioxide media. A significant reduction in the viability was observed between 700 and 800 ppm (p<0. 01) on the potasium dioxide media, between 25 and 30 ppm (p<0. 01) on the sodium arsenite media, between 20 and 25 ppm (p<0. 01) on the selenium dioxide media. The developmental time was significantly reduced between 800 and 900 ppm (p<0. 001) on the potasium dioxide media, between 15 and 20 ppm (p<0. 001) on the sodium arsenite media, and between 25 and 30 ppm (p<0. 01) on the selenium dioxide media.

우수 대학연구소 탐방-전남대학교 호르몬연구센터

권혁방,Gwon, Hyeok-Bang 한국대학교육협의회 2005 大學敎育 Vol.133 No.-

배양중인 개구리 여포의 cAMP 흡수와 분해

권혁방,나철호,안련섭,김경진 한국통합생물학회 1991 동물학회지 Vol.34 No.2

양서류 여포를 배양할 때 외부에서 cAMP를 첨가하면 호르몬에 의한 난자의 성숙이 억제된다는 많은 보고가 있었다. 그러나 실제 외부의 cAMP가 여포내로 들어간다는 보고는 아직 없다. 본 연구에서는 배양액내의 cAMP가 여포내로 침투해 들어가는 현상과 들어간 cAMP의 분해과정을 radioimmunoassay로 조사하였다. 개구리 여포를 배양하면서 배양액에 난자의 성숙을 억제하는 농도의cAMP(2.5 mM)를 첨가한 후 일정시간 간격으로 여포내 축척된 cAMP의 농도를 조사한 결과 2시간에서 이미 기본수준(여포당 약 3 p mole)의 수십배로 증가하였다.(여포당 90 p mole). cAMP를 포함한 배양액에서 6시간 배양 후 보통 배양액으로 옮겨 배양하면서 일정 시간마다 여포내 cAMP의 농도를 측정한 결과 6시간 내에 cAMP농도가 여포당 160 p mole에서 약 10 p mole로 급격히 낮아졌다. 그러나 18시간 후에도 기본 수준으로까지 내려가지는 않았다. 이러한 cAMP의 감소과정이 progesterone이나 isobuty methylxanthine (IBMX)에 크게 영향을 받지 않았다. 배양중인 여포를 forskolin(9 u m)으로 자극했을 때에는 기본 수준의 약 3배정도로 cAMP의 농도가 증가하였다. 본 결과는 배양액내의 cAMP가 여포내로 투과해 들어가고 이들은 곧 여포에 의해 분해된다는 것을 시사하고 있다. We previously showed that transient exposure of Rana dybowskii follicles to exogenous cAMP in vitro could induce meiotic maturation. The present experiments were carried out to acertain whether the exogenous cAMP penetrate into the follicles. Isolated follicles were precultured in the medium containing cAMP (2.5 mM) for 6 hours and then cultured further in plain medium for 18 hours. The change of intrafollicular cAMP levels during the culture period were examined by utilizing cAMP radioimmunoassay (RIA). The intrafollicular levels of cAMP increased about thirty times of the basal level (about 3 p mole/follicle) in two hours and reached a peak in six hours (170 p mole/follicle) during the preculture period. However, when the follicles were transferred to plain medium, the levels decreased markedly in six hours to very low levels (about 10 p mole/ follicle), and kept the same levels thereafter. But the levels did not decrease to the basal levels. The increase and decrease of the intrafollicular cAMP was not affected by the presence of isobutyl methylxanthine (IBMX) or progesterone. The data suggest that exogenous cAMP pene-trate into the follicles and the cAMP accumulated by the follicles are degraded very rapidly.

한국산개구리(북장산개구리와 참개구리) 난자의 생체외 배양에 의한 성숙유도에 관하여

권혁방,조장현,최충길 한국통합생물학회 1988 동물학회지 Vol.31 No.2

전라남도 일대에서 서식하는 북방상개구리(R. dybowskii)와 참개구리(R. nigromaculara)를 채집하는 생체외배양에 의한 여포난자의 성숙을 유도하였다. 북방산개구리의 여포난자는 배양액(amphibian Ringer's soluion AR)에 첨가한 progesterone, 0.1 $\mu$g/2 ml에 의해 난자의 성숙(핵붕괴)이 유도 되었으며 참개구리의 난자는 1 $\mu$g/2 ml (frog pituitary homogenate FPH)을 얻어서 그 효과를 조사해본 결과 북방산개구리에서는 0.01 pituitary equivalent/2 ml에서, 참개구리는 0.1 pit equiv./2 ml에서부터 여포난자의 성숙이 일어났다. 난자의 성숙에 요하는 시간은 두 개구리에서 모두 9-15시간이었으며 호르몬에 대한 반응성, 성숙기간 등은 개구리 재료로 가장 많이 사용되는 법개구리(R. pipiens)와 거의 일치하였다. 특이하게 2개월 이후에 사용한 북방산개구리의 여포난자는 호르몬의 도움없이도 성숙이 일어났으며 성숙기간도 3시간으로 매우 빨라졌다. 난소조각을 배양했을 때 자발적으로 성숙을 일으키는 여포들은 자발적인 배란까지도 일으키는 것을 발견하였다. Korean frogs (R. dybowskii and R. nigromaculara) were collected from chonnam area and their oocyte maturation was induced by using in ultro follicle culture system. Follicles were isolated from the frog ovary and cultured for 24 hr in (amphibian Ringer's soluion AR) at 22 C in the presence or absence of hormones. Follicular cocytes of R. dybowskii were induced to mature (germinal vesicle breakdown, GVBD) by the presence of progesterone, 0.1 $\mu$g/2 ml and that of R. nigromaculata by 1 $\mu$g/2 ml of progesterone. Follicles of the frogs were also responded to (frog pituitary homogenate FPH) in terms of their cocyte maturation. Follicular cocytes of R. dybowskii were induced to mature by FPH at concentration of 0.01 pituitary equivalent/2 ml and that of R.nigromaculata at 0.1 pit equiv./2 ml. The culture time required for the maturation of bath frog follicles was 915 hr. The responsiveness of the follicles of korean frogs to hormones (progesterone or FPH) was nearly the same as that of R. pipiens which are most commonly used amphibians. Particularly, follicular cocytes of R. dybowskii used from February matured spontaneously without stimulation of hormones during in vitro culture. Furthermore, those cocytes were spontaneous- ly ovulted when the ovarian fragments were cultured in a flask.

단백질분해효소들의 양서류 난자에 대한 성숙유도와 억제작용에 관하여

권혁방,고선근,박현정 한국통합생물학회 1990 동물학회지 Vol.33 No.1

양서류의 여포난자를 생체외에서 배양하면서 호르몬을 처리하면 난자의 성숙(핵붕괴)을 일으킨다. 본 연구는 북방산개구리의 여포난자를 배양하면서 난자내 단백질 분해효소들의 활성변화를 유도하여 이것이 난자의 성숙에 어떠한 영향을 미치는가를 조사하였다. chymotrypsin의 저해제로 알려진 N$\alpha$ -tosyl-L-phenylalanine-chloromethyl-ketone(TPCK)을 배양액에 처리하면 비교적 낮은 농도(0.001-1 $\mu$M)에서는 호르몬의 도움없이도 난자의 성숙을 유도하나 높은 농도 (100 $\mu$M)에서는 호르몬에 의한 난자의 성숙까지도 억제하는 이중적인 효과를 나타내었다. Trypsin의 저해제인 N$\alpha$ -tosyl-L-phenylalanine-chloromethyl-ketone(TPCK)은 성숙유도능력이 없을 뿐 아니라 progesterone에 의한 난자의 성숙을 억제하였다. Trypsin을 직접 처리했을 때에는 농도에 의존하여(0.001-1$\mu$g/2ml) 호르몬의 도움없이도 난자의 성숙을 유도함을 발견하였다. TLCK나 TPCK의 억제효과는 성숙 초기에만 나타났다. 본 결과는 양서류 난자의 성숙조절 과정에 몇종의 단백질 분해효소들이 참여한다는 것을 시사해주고 있다. Fully grown amphibian oocytes undergo their maturation (germinal vesicle breakdown, GVBD) during in vitro follicle culture when they are stimulated with frog pituitary homogenate (FPH) or progesterone. Present experiments were designed to determine whether proteolytic enzymes are involved in the regulation of the matunation process. Treatment of a $\alpha$ -chymoiyypsin inhibitor, N a -tosyl-L-phenylalanine-chloromethyl-ketone(TP) to the oocytes exhibited a biphasic phenomenon, the induction of the maturation without added hormone at relatively low doses (0.001-1 $\mu$M) and inhibition of the hormone induced oocyte maturation at a high dose (100 $\mu$M). Treatment of a trypsin inhibitor, N a -tosyl-L-lysine-chloromethyl ketone(TLCK) to the oocytes did not induce the maturation, but rather suppressed the hormone induced oocyte maturation in a high dose(100 $\mu$ M). Treatment of exogenous iyypsin to the oocyte induced their maturation without added hormone in a dose dependent manner (0.001-1 $\mu$ M). The data presented here indicate that some proteolytic enzymes play a role in the regulation of the maturation(meiotic arrest or reinitiation) in amphibians.

생쥐 난자-난구 복합체의 성숙과 분산에 관한 연구 : 세포내 cAMP의 조절

권혁방,고선근,임욱빈 한국통합생물학회 1987 동물학회지 Vol.30 No.1

생쥐 난자-난구 복합체를 인공배양하면서 adenylate cyclase의 촉진제인 forskilin과 phosphodiesterase의 저해제인 3-isobutyl-1-methylxanthine(IBMX)을 배양액에 첨가하여 이들이 난구세포의 분산과 난자의 성숙(핵붕괴)에 미치는 효과를 관찰한 결과 다음과 같았다. 1. Forskilin은 0.001$\mu$M에서 부터 난구세포의 분산을 유도하기 시작하여 (36%) 0.1-10$\mu$M 구간에서 최대의 분산율을 나타내었고 (80-90%) 100$\mu$M에서는 그 효과가 줄어들었다(60%). 이때 난자의 핵 붕괴는 10$\mu$M까지 정상으로 일어나다 (75-80%) 100$\mu$M에서 부분적으로 억제되기 시작하였다(40%). 2. IMBX는 0.01$\mu$M에서 부터 난구세포의 분산을 유도하기 시작하여 (30%) 1-1,000$\mu$M의 전 구간에서 최대의 분산율(81-89%)을 나타내었다. 난자들은 10$\mu$M의 농도까지 정상적으로 핵 붕괴를 일으키었으나(90% 이상) 100$\mu$M 이상에서 급격히 억제되었다(14%). 3. 난구세포의 분산을 유도하는데 필요한 최소의 자극기간을 조사해 본 결과 HCG는 2분, FSH와 forskilin은 15-30분, IBMX는 2시간이었다. 위 결과로 부터 생쥐 난구세포에서 cAMP의 농도를 높이는데 adenylate cyclase 와 phosphodiesterase의 두 효소가 모두 중요한 기여를 하며 난구세포는 단 기간에 생긴 cAMP의 peak로서 분산이 유도될 수 있으나 난자의 성숙억제 과정에서는 지속적인 cAMP의 존재가 필요하다는 것을 알았다. Cyclic AMP (cAMP) was known to play a key role in the regulation of cumulus expansion and oocyte maturation of mammalian cumulus-oocyte complexes (COC's) in vivo and in vitro. The present experiments were conducted to know how intracellular level of cAMP in these cells is controlled. Intracellular cAMP level was modulated by culturing mouse CGC's with an adenylate cyclase stimulator, forskolin, a phosphodiesterase inhibitor, 3-isobutyl-1-methyixanthine (IBMX), human chorionic gonadotrophin (HCG), or follicle stimulating hormone (FSH). The rate of cumulus expansion and germinal vesicle break-down (GVBD) was checked after culture and used as a biological end point. Forskolin in the medium began to stimulate the expansion of the complexes at 1 nM and induced maximum expansion (80~90%) at 0 1~10 $\mu$M. The expansion rate was reduced to 60% when forskolin concentration was increased to 100 $\mu$M. Oocyte GVBD occurred normally (75~82%) in the presence of 10 $\mu$M of forskolin, but partial suppression was appeared at 100 pM of the drug (40%). IBMX also stimulated the expansion from the concentration of 0.01 pM and induced full expansion (81~89%) between the concentration of 1-1000 $\mu$M. Meiotic resumption was occurred normally under 10 $\mu$M of IBMX, but suppressed drastically from the concentration of 100 $\mu$M. The minimum exposing time to hormone or drugs required to trigger cumulus expansion was two minutes with HCG, 15~30 minutes with FSH and fors kolin, and two hours with IBMX. The data presented here seemed to imply that intracellular cAMP level in cumulus cells is regulated by both adenylate cyclase and phosphodiesterase and cumulus expansion is induced by a peak of cAMP while meiotic arrest is maintained by continuous presence of cAMP.

생쥐 초기배아에 미치는 스테로이드 홀-몬의 영향

권혁방,정순오 최신의학사 1976 最新醫學 Vol.19 No.2

The experiments were undertaken in order to study the effect of various steroid hormones on germinal vesicle break-down of mouse oocytes and to determine if there was a different sensitivity of various aged preimplantation mouse embryos to progesterone. Follicular oocytes were obtained from ovaries of A-strain female mouse and early embryos were flushed from their fallopian tubes after an appropriate time of mating. The incubation medium was a modified Krebs-Ringer bicarbonate sol. supplemented with 0.4% bovine serum alumin (BSA). A microtube culture method (Cho, 1974) for the oocytes and embryos were adopted to avoid contamination of steroid hormones which are soluble in oil. Ten to twenty oocytes or embryos were placed in the culture medium in microtube and cultured at 37°C, gassed with 5% CO2 in air humidified in an incubator. After culture, the oocytes or embryos were observed directly through the microtube under an inverted phase microscope. The results from the experiments were as follows. 1. The activity of the steroid hormones on germinal vesicle break-kown of mouse oocytes was dependent upon specificity of the hormones. 2. Oocyte was very resistant to progesterone in germinal vesicle break-down whereas fertilized ova was very sensitive to that by blocking the development of the embryos, from 1-cell to 2-cell stage. 3. When 1-cell, 2-cell, 8-16 cell embryos were cultured for 24 hours in medium containing progesterone, 8-16 cell embryos were more resistant to progesterone than the others in cleavage inhibition. 4. When the embryos were exposed to progesterone long, they became more sensitive to the hormone.

Effects of Organotin Compounds on Follicular Steroidogenesis in Frogs

권혁방,김승창,김안나,이성호,안련섭 한국발생생물학회 2009 발생과 생식 Vol.13 No.3

Some organotin compounds such as butyltins and phenyltins are known to induce impo-sex in various marine animals and are considered to be endocrine disruptors. In this study, the effect of organotins on follicular steroidogenesis in amphibians was examined using ovarian follicles of Rana dybowskii and Rana catesbeiana. Isolated follicles were cultured for 6 or 18 h in the presence and absence of frog pituitary homogenate (FPH) or various steroid precursors, and the levels of product steroids in the culture media were measured by radioimmunoassay. Among the butyltin compounds, tributyltin (TBT) strongly and dose-dependently inhibited the FPH-induced synthesis of pregnenolone (P5) and progesterone (P4) by the follicles. TBT also strongly suppressed the conversion of cholesterol to P5 and partially suppressed the conversion of P5 to P4. A high concentration of dibutyltin (DBT) also inhibited steroidogenesis by the follicles while monobutyltin and tetrabutyltin had negligible effects. The toxic effect of TBT or DBT was irreversible and a short time of exposure (30 min) was enough to suppress steroidogenesis. All the phenyltin compounds significantly inhibited FPH-induced P5 synthesis by the follicles. The effective dose of 50% inhibition by diphenyltin was 0.04 μM and those of monophenyltin and triphenyltin were 0.24 ㎛ and 0.3 μM, respectively. However, none of the phenyltin compounds significantly suppressed the conversion of P4 to 17α-hydroxyprogesterone (17α-OHP) (by 17α-hydroxylase), 17α-OHP to androstenedione (AD) (by C17-20 lyase), or AD to testosterone by the follicles. Taken together, the data show that among the steroidogenic enzymes, p450scc in the follicles is the most sensitive to organotin compounds and that an amphibian follicle culture system can be a useful screening model for endocrine disruptors.