大韓不妊學會誌 : 第 33 卷第 1 號 2006 Kor. J. Fertil. Steril., Vol. 33, No. 1, 2006, 3 한양대학교자연과학연구소, 한양대학교자연과학대학생명과학과 1 김상수 이창주 윤현태 1 윤용달 1, * Expression of Inhibin in the Whole-body γ-irradiated Mouse Ovary Sang Soo Kim, Chang Joo Lee, Hyun-Tae Yoon 1, Yong-Dal Yoon 1,* The Research Institute for Natural Sciences, Hanyang University, Seoul 133-791, Korea, 1Laboratory of Toxicogemonics, Department of Life Science, Hanyang University, Seoul 133-791, Korea Objective: The purposes of the present study were to investigate the effect of γ-radiation on the expression of inhibin-α proteins and genes for inhibin α, βa, and βb in the ovary. Methods: Immature mice were whole-body γ-irradiated with 25% of a lethal dose. At time 0, 3, 6, 12, and 24 hours after the irradiation,the ovaries were collected and used for immunohistochemistry for inhibin-α, and RT_PCR for inhibin-α, βa, and βb. Results: The expression of the immunoreactive inhibins-α was maintained at 12 hours post-irradiation and reduced thereafter. The expression of inhibin-α mrna was significantly increased with the time after the irradiation. However there were no significant changes in the expression of βa and βb mrnas. Conclusion: It might be thought that inhibin acts as one of the regulatory factors in the γ-radiation-induced follicular atresia in mice Key Words: Mouse ovary, Follicle, Inhibin, Radiation 난포의폐쇄는성숙단계이전의원시난포및성장 성숙하고있는여러다양한단계에서의난포가퇴화하여소멸하는현상을의미한다. 난포가퇴화하게되면난포내스테로이드호르몬의생합성율이변하며, 1,2 형태학적으로과립층세포의핵이응축되고증식율이감소하면서, 난포막세포층이비대해지고결국기저막이붕괴하게되는데, 최근연구결과에의하면, 난포의퇴화는과립층세포의세포자멸사에의하여매개된다고보고되고있다. 3~5 세포자멸사는조직내정상적인세포의항상성유지를위해면역학적반응이유발되지않는생리학적과정으로서, 6,7 세포외부신호와특정환경에반응하여세포 가능동적으로사멸하는것을뜻한다. 난포의폐쇄기전을연구하기위해인위적으로폐쇄를유발하는방법중에는이온화방사선특히 X선 8 과감마선조사 9~12 가포함된다. 생쥐에서방사선에의해난포의퇴화가유발하며, 이러한퇴화의유발효과는성장을개시한 1차난포에비해원시난포에서더빠른것으로알려져있다. 11,13 또한방사선의조사선량에따라난자의수가감소하고, 14 난자내염색체이상도더욱증가하며, 15,16 난포의퇴화를가속화시키게되는것으로알려져있다. 9 뇌하수체에서 FSH의생합성과분비를선택적으로억제하는것으로알려져있는분자량 32~34 kd 주관책임자 : 윤용달, 우 ) 133-791 서울특별시성동구행당동산 17, 한양대학교자연과학대학생명과학과 Tel: (02) 2220-0955, Fax: (02) 2294-0955, e-mail: ydyoon@hanyang.ac.kr * 본연구는한국과학재단선도기초과학연구실연구비 (M02-2004-000-20849-0) 지원에수행되었음. - 35 -
의당단백질인인히빈은 TGF-β 가계에속하며 α와 βa 혹은 βb 소단위가인히빈 A와인히빈 B를형성한다. 17~20 인히빈 B가 FSH의자극에의해발달중인난포의과립층세포에서분비되는주된산물이며, 인히빈 A는우성난포및황체에서분비된다. 21 인히빈은생식소를포함한여러유형의세포에대한성장, 증식, 그리고분화를자극하는기능을갖고있다. 22,23 또한, 발달중인난포로부터분비되는인히빈은난포의계속적인발달뿐아니라난포내스테로이드호르몬의생합성을조절하며, 난소및부신피질에서종양억제인자로도작용한다. 23~25 혈청내인히빈은폐경이후에측정되지않을정도로감소되지만점액종양 (mucinous carcinoma) 이나과립세포암종 (granulosa cell tumor) 의경우계속적으로분비되어혈청내진단표지로이용된다. 26,27 현재방사요법을받은환자의경우난소의기능부전이유발되는것으로보고되었지만, 28 난소내인히빈의발현변화에있어감마선과관련된연구는많지않다. 따라서, 본연구는미성숙생쥐에감마선을전신조사한후시간에따라난소내인히빈의발현변화를유전자및단백질수준에서측정하였다. 연구대상및방법 1. 실험동물본실험에서는 3주령의 ICR 품종의생쥐를사용하였으며먹이와물을상시먹을수있는조건으로 1주일이상적응시킨후 ( 명 / 암 = 12/12 시간 ) 실험에사용하였다. 2. 감마선조사이온화방사선의조사는기보고된방법에따라한국원자력연구소의감마선발사장치 (Panoramic Irradiator, Atomic Energy of Canada Ltd.) 에서감마선 ( 60 Co, Source strength: approximately 150 TBq, Panoramic Irradiator, Aomic Energy of Cannada Ltd) 을 LD 25 (6.42 Gy) 의조사량으로 1시간동안전신조사하였다. 9,11 방사선조사후 0, 3, 6, 12, 24시간에난소를적출하여본실험에사용하였으며실험군당생쥐는 5마리로하였다. 3. 면역조직화학염색및관찰생쥐난소는 4% paraformaldehyde에 12시간고정하였으며, 파라핀을침투시켜포매한후 5 µm의두께로박절하였다. 통상적인헤마톡실린-에오신 (hematoxylin-eosin) 염색을실시하였으며, 인히빈단백질에대한면역조직화학염색을실시하였다. 파라핀을제거한후재-수화시킨절편에서인히빈에대한면역조직화학염색을실시하였다. 조직절편은 37 에서 30분간 0.1% trypsin이첨가된 CaCl 2 용액에담근후다시 30분간 2 N HCl과반응시켰다. 즉시조직절편은 0.1% 붕사 (borax) 용액에담가반응을종결시켰으며, 흐르는물로수세하였다. 절편을 10 mm 구연산완충용액 (ph 6.0) 에 5분간 2번담근후 5분간마이크로파오븐 (750 W) 을이용 3 회가열하였다. 가열이끝난절편은실온에서 15분간방치한후, Tris-완충식염수 (TBS; 0.05 M Tris- HCl + 0.15 M NaCl, ph 7.6) 으로수세하였다. 비특이적결합을방지하기위해우선 3% H 2 O 2 가들어있는메탄올용액에 15분간담가내인성과산화효소의활성을억제시켰으며, 5% 정상염소혈청에 10분간반응시켜비특이적결합부위를차단시킨후인히빈에대한면역조직화학염색을실시하였다. 즉, 절편을 TBS로세척한후 1차항체 (1:100) 가들어있는습윤기에넣고 4 에서 17시간동안반응시켰다. 인히빈에대한항체는 Oxform Bio-Innovation Ltd. (Oxfordshire, UK) 사의제품을사용하였다. 조직절편은바이오틴 (biotin) 이부착된항-생쥐 IgG (1: 200) 와 20분간반응시켰다. 마지막으로, 절편은 TBS 로세척한후 streptoavidin-abcomplex-hrp (1:100) 용액에 10분간반응시켰다. 염색은 DAB (3,3'-diaminobenzidine tetrahydrochloride, 0.5 mg/ml), 0.01 M imidazole, 그리고 0.3% H 2 O 2 가포함된 TBS에서시행하였다. 대조염색으로는 1차항체를배제한후동일과정으로진행하였다. 그후절편을흐르는물에수세하였으며메이어헤마톡실린 (Mayer's hematoxylin) 으로대조염색을실시한후카나다발삼 (Canada balsam) 으로봉입하였다. 난소의조직절편중가장큰절편을선정기보고된방법에따라, 10 광학현미경 (Zeiss, Jena, Germany) 하에서원시난포 (primordial follicle), 1차난포 (primary follicle), 강소 - 36 -
형성전난포 (preantral follicle), 강소형성난포 (antral follicle) 를확인하여관찰하였다. 4. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 인히빈소단위유전자의발현변화를알아보기위해전체 RNA 1 µg을 5 역전사효소완충용액, dntp (2.5 µm), 올리고 dt 15 프라미머 (primer), RNase 저해제, AMV 역전사효소가포함된용액으로 70분동안 42 에서반응시킨후, 95 에서 5분간효소를불활성화시켰다 (Takara, PCR Thermal Cycler 480). 중합효소연쇄반응 (polymerase chain reaction; PCR) 은동일한양의합성된 cdna에 MgCl 2 완충용액, 10 PCR 완충용액, dntp (2.5 mm), 10 pm의각프라이머들을이용하여 Taq DNA 중합효소용액속에서중합반응을수행하였다. 최초 94 에서 1분간변 성후, 94 에서 1분, 각각의프라이머에따른붙임 (annealing) 온도에서 1분, 72 에서 2분간을한주기로하여 30회반복하여증폭하였고, 마지막으로 72 에서 5분간신장반응을수행하였다 (Bio-rad, icycler TM Thermal Cycler). 반응후 5 µg의 PCR 산물을 6x의 40% 글리세롤내 0.25% 브로모페놀블루 (bromophenol blue) 용액에넣은후, 1% 아가로스젤에서전기영동하였다. 전기영동이끝난젤은 Vilber Lourmat 사의 Bio-Print로스캔한후 Bio-Profil Bio-1D Windows Application V10.02로분석하였다. 본실험에사용된프라이머서열은 Table 1에요약하였다. 5. 통계학적분석각실험군간의결과에대한유의성검정은분산분석 (ANOVA) 및 Student's t test를사용하였고, p Table 1. The primer sequences and the sizes of amplification products of β-actin, Inhibin-α, Inhibin-βA and Inhibin-βB gene used in the present study Genes Sequences Product size (bp 1 ) Cycles β-actin 2 Forward 5'-AGC CAT GTA CGT AGC CAT CC-3' Reverse 5'-CTC TCA GCT GTG GTG GTG AA-3' 228 Inhibin-α Forward 5'-GTC TGC CTC GAA GAC ATG CC-3' Reverse 5'-CTG TAC CAA GGA CAC AGG CA-3' Inhibin-βA Forward 5'-AAG AGA CCC GAT GTC ACC CA-3' Reverse 5'-CTC TCC CCC TTT AAG CCC AT-3' Inhibin-βB Forward 5'-TGG GCA GAG TGA GGC TGT TC-3' Reverse 5'-TTT CGC CTG TGT GGG TCA AC-3' 1 bp, base pair, 2 The primer sequences were adopted from Albano et al. (1993) 29 341 395 325 25 Table 2. Immunoreactivity of inhibin-α according to follicle status the control and γ-irradiated mouse ovary Normal - 37 - Atretic Nucleus Granulosa Theca Nucleus Granulosa Theca Primordial - - - - - - Primary - -/+ - - - - Preantral - +++ - - +/- - Early antral - +++ - - +/- - Normal and atretic follicles were identified according to Kim and Lee 10. -, not expressed; -/+, not always expressed; +/-, weakly and sometimes not expressed; ++, moderately expressed; +++, intensively expressed
Figure 1. Immunohistochemistry for inhibin-α proteins in the γ-irradiated immature mouse. Follicle status was identified with hematoxylin and eosin stains according to Kim and Lee (2000). The ovaries were collected before (A), and 0 (B), 3 (C), 6 (D), 12 (E), and 24 (F) hours after the irradiation. Original magnification, 100. Bars 200 µm. An, antrum; AF, antral follicle; N, nucleus; O, oocyte; SF, small follicle; POF, primordial follicle; PAF, preantral follicle. 값이 0.05보다작은경우를유의한것으로간주하였다. 결과 1. 면역조직화학적관찰인히빈단백질의난소내발현을면역조직화학적 으로조사한결과를 Figure 1에나타내었다. 감마선을조사하지않은대조군의경우난소내인히빈단백질은과립층세포에서발현되며난포의크기가클수록그리고정상난포가퇴화난포에비해더많이발현함을알수있다. 그러나, 1차난포및원시난포에서는인히빈단백질이거의발현되지않았다 (Table 2). 감마선조사후시간에따라인히빈단백 - 38 -
Figure 2. Electrophoretogram and its densitogram of RT-PCR products for inhibin-α mrna in the control and γ-irradiated mouse ovaries. The data was expressed as the mean ± SD of three independent experiments. The expression of inhibin-α mrnas was significantly increased at 6, 12 and 24 hours post irradiation. a, p<0.05 significantly higher than 0 hours post irradiation. b, p<0.01 significantly higher than normal control. Figure 3. Electrophoretogram and its densitogram of RT-PCR products for inhibin-βa mrna in the control and γ-irradiated mouse ovaries. The data was expressed as the mean ± SD of three independent experiments. The ovarian expression of inhibin-βa mrnas was not changed according to the time after the irradiation. - 39 -
Figure 4. Electrophoretogram and its densitogram of RT-PCR products for inhibin-βb mrna in the control and γ-irradiated mouse ovaries. The data was expressed as the mean ± SD of three independent experiments. The ovarian expression of inhibin-βb mrnas was not changed according to the time after the irradiation. 질의발현은감소되었으며 24시간에서는약하게발현됨을알수있었다. 2. RT-PCR 감마선을조사하기전 (control), 감마선조사직후 (0시간), 조사후 3, 6, 12, 그리고 24시간후에 RT-PCR 로확인한난소내인히빈-α 소단위유전자의발현변화를조사하였다. Figure 2에나타낸바와같이감마선조사후 0, 3, 6, 12, 24시간에서인히빈-α 유전자는각각 2.1, 2.9, 4.0, 4.8, 4.5배증가하였다. 또한감마선조사후 3, 6, 12, 24시간에서의발현양은조사직후 (0시간) 의발현양에비해 1.4, 1.9, 2.3. 2.2배가증가하였다. 그러나, 각각 Figure 3과 4에나타낸바와같이, 인히빈 βa와 βb 유전자의발현은감마선조사후시간에따라변화를보이지않았다. 고찰본연구는이온화방사선인감마선이전신조사된미성숙생쥐를이용시간에따라난소내인히빈 유전자와단백질의발현변화를조사하고자시행되었다. 포유류에서대부분의난포가배란되지못하고폐쇄되는정확한기전은현재까지도정확하게규명되지못한실정에있다. 본실험에서감마선은난포의인위적폐쇄를유발하고자조사되었는데방사선은난포의성장단계에방사능에따라그영향이다르다고알려져있다. 11,13,14 생쥐의생식세포에대한방사선민감도는연령에따라증가하며난자내염색체의이상역시증가한다. 15,16 더욱이이온화방사선은세포자멸사를유발하며, 30 난소의생리학적활성에영향을미친다. 31 특히소아암으로인해방사요법을받은환자의경우난소의기능부전이유발되는것으로보고되었다. 28 FSH 분비를조절하는폴리펩티드로서모두난포액내에존재하는것으로확인된것은인히빈, 액티빈, 폴리스타틴이있다. 인히빈은난포내과립층세포에서합성되는이형이량체의폴리펩티드로서, 32 생식소및뇌를포함하는여러조직에분포하며특히난소및시상하부기능에관여한다. 33 인히빈은 α와 βa 혹은 α와 βb가결합하여각각인히빈 A와 - 40 -
인히빈 B를구성한다. 성장을개시한전동난포에서인히빈-B가주로분비되며작은동난포에서는인히빈-B와 -A 모두가분비된다. 34 또한난포기전반기에 FSH 및 LH에의해자극된모든동난포에서인히빈 A가분비되며난포기후기에는우성난포로부터분비되는것으로알려져있다. 35 인히빈-α 단백질에대한면역조직화학염색을실시한본실험의결과인히빈 -α는전동난포와동난포의과립층세포에서분비되며감마선조사후시간에따라감소되는것을확인할수있었다. 또한, 인히빈-βA와 -βb 유전자의발현은별다른변화를보이지않은반면, 인히빈-α 유전자는감마선조사후시간에따라유의하게증가하였다. 감마선조사후 12시간까지는면역반응성인히빈-α 단백질의과립층세포내발현양이대조군에비해별다른감소를보이지않았지만, 24시간에서인히빈 -α 단백질의발현양이감소된것은감마선에의해대부분의난포가퇴화된때문으로생각된다. 그러나, 감마선조사후시간에따른인히빈-α 유전자의발현양은대조군이나 0시간군에비해유의하게높은것으로나타났다. 이는감마선에의해인히빈-α 유전자의발현이 12시간까지증가하지만 24시간에서단백질로의발현이감소되는때문으로생각된다. 한편혈청내인히빈의농도증가는점액종양 (mucinous carcinoma) 이나과립막세포종양 (granulosa cell tumor) 의내진단표지로이용되고있다. 26,27 특히과립세포암종에서인히빈 α, βa, βb 소단위에특이적인면역조직화학염색이관찰되었으며, 36,37 혈청내인히빈의양적인증가가확인되어 31 화학치료후의예후판정에대한지표로서이용되고있다. 38,39 또한, 인히빈은난포막세포에서의안드로젠생합성을직접적으로자극하고, 40 FSH를선택적으로저해하므로혈중인히빈의농도가높은일부환자에서 LH/FSH 비율이증가한다. 이들환자의경우, 혈중인히빈 B의농도는난소기능부전의정도와연관을갖는다. 41,42 본연구에서인히빈-α 유전자가감마선조사후시간에따라증가된것은흥미로운결과이다. 따라서감마선에의한난포의퇴화에인히빈이생리학적및생화학적역할을할것이라는가능성을배제 할수없다. 참고문헌 1. Hirshfield AN, Midgley AR Jr. Morphometric analysis of follicular development in the rat. Biol Reprod 1978; 19: 597-605. 2. Braw RH, Tsafriri A. Effect of PMSG on follicular atresia in the immature rat ovary. J Reprod Fertil 1980; 59: 267-72. 3. Hughes FM Jr, Gorospe WC. Biochemical identification of apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia. Endocrinology 1991; 129: 2415-22. 4. Gougeon A. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev 1996; 17: 121-55. 5. Johnson AL, Bridgham JT, Witty JP, Tilly JL. Susceptibility of avian ovarian granulosa cells to apoptosis is dependent upon stage of follicle development and is related to endogenous levels of bcl-xlong gene expression. Endocrinology 1996; 137: 2059-66. 6. Kerr JF, Winterford CM, Harmon BV. Apoptosis. Its significance in cancer and cancer therapy. Cancer 1994; 73: 2013-26. 7. Kaipia A, Hsueh AJ. Regulation of ovarian follicle atresia. Annu Rev Physiol 1997; 59: 349-63. 8. Jarrell J, YoungLai EV, McMahon A, Barr R, O'Con nell G, Belbeck L. Effects of ionizing radiation and pretreatment with [D-Leu6,des-Gly10] luteinizing hormone-releasing hormone ethylamide on developing rat ovarian follicles. Cancer Res 1987; 47: 5005-8. 9. Kim JK, Lee CJ, Song KW, Do BR, Yoon YD. Gamma-radiation accelerates ovarian follicular atresia in immature mice. In Vivo 1999; 13: 21-4. 10. Kim JK, Lee CJ. Effect of exogenous melatonin on the ovarian follicles in gamma-irradiated mouse. Mutat Res 2000; 449: 33-9. 11. Lee CJ, Park HH, Do BR, Yoon Y, Kim JK. Natural - 41 -
and radiation-induced degeneration of primordial and primary follicles in mouse ovary. Anim Reprod Sci 2000; 59: 109-17. 12. Lee CJ, Yoon YD. Gamma-radiation-induced follicular degeneration in the prepubertal mouse ovary. Mutat Res 2005; 578: 247-55. 13. Ataya K, Pydyn E, Ramahi-Ataya A, Orton CG. Is radiation-induced ovarian failure in rhesus monkeys preventable by luteinizing hormone-releasing hormone agonists?: Preliminary observations. J Clin Endocrinol Metab 1995; 80: 790-5. 14. Jacquet P, Vankerkom J, Lambiet-Collier M. The female guinea pig, a useful model for the genetic hazard of radiation in man; preliminary results on germ cell radiosensitivity in foetal, neonatal and adult animals. Int J Radiat Biol 1994; 65: 357-67. 15. Ronnback C. The age dependence of radiation sensitivity of the gonads of female mice. Acta Oncol 1988; 27: 399-405. 16. Tease C, Fisher G. The influence of maternal age on radiation-induced chromosome aberrations in mouse oocytes. Mutat Res 1991; 262: 57-62. 17. De Jong FH. Inhibin. Physiol Rev 1988; 68: 555-607. 18. Kingsley DM. The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev 1994; 8: 133-46. 19. Burger HG, Farnworth PG, Findlay JK, Gurusinghe CJ, Healy DL, Mamers P, Mason A, Robertson DM. Aspects of current and future inhibin research. Reprod Fertil Dev 1995; 7: 997-1002. 20. Vale W, Rivier C, Hsueh A, Campen C, Meunier H, Bicsak T, et al. Chemical and biological characterization of the inhibin family of protein hormones. Recent Prog Horm Res 1988; 44: 1-34 21. Illingworth PJ, Groome NP, Duncan WC, Grant V, Tovanabutra S, Baird DT, McNeilly AS. Measurement of circulating inhibin forms during the establishment of pregnancy. J Clin Endocrinol Metab 1996; 81: 1471-5. 22. Mather JP, Woodruff TK, Krummen LA. Paracrine regulation of reproductive function by inhibin and activin. Proc Soc Exp Biol Med 1992; 201: 1-5. 23. Findlay JK. An update on the roles of inhibin, activin, and follistatin as local regulators of folliculogenesis. Biol Reprod 1993; 48: 15-23. 24. Matzuk MM, Finegold MJ, Su JG, Hsueh AJ, Bradley A. Alpha-inhibin is a tumour-suppressor gene with gonadal specificity in mice. Nature 1992; 360: 313-9. 25. Matzuk MM, Kumar TR, Shou W, Coerver KA, Lau AL, Behringer RR, Finegold MJ. Transgenic models to study the roles of inhibins and activins in reproduction, oncogenesis, and development. Recent Prog Horm Res 1996; 51: 123-54. 26. Woodruff TK, Besecke LM, Groome N, Draper LB, Schwartz NB, Weiss J. Inhibin A and inhibin B are inversely correlated to follicle-stimulating hormone, yet are discordant during the follicular phase of the rat estrous cycle, and inhibin A is expressed in a sexually dimorphic manner. Endocrinology 1996; 137: 5463-7. 27. Robertson DM, Stephenson T, Pruysers E, Burger HG, McCloud P, Tsigos A, et al. Inhibins/activins as diagnostic markers for ovarian cancer. Mol Cell Endocrinol 2002; 191: 97-103. 28. Kumanov P, Nandipati KC, Tomova A, Robeva R, Agarwal A. Significance of inhibin in reproductive pathophysiology and current clinical applications. Reprod Biomed Online 2005; 10: 786-812. 29. Albano RM, Groome N, Smith JC. Activins are expressed in preimplantation mouse embryos and in ES and EC cells and are regulated on their differentiation. Development 1993; 117: 711-23. 30. Larsen EC, Muller J, Schmiegelow K, Rechnitzer C, Andersen AN. Reduced ovarian function in long-term survivors of radiation- and chemotherapy-treated childhood cancer. J Clin Endocrinol Metab 2003; 88: 5307-14. 31. Hendry JH, West CM. Apoptosis and mitotic cell death: their relative contributions to normal-tissue and tumour radiation response. Int J Radiat Biol 1997; 71: 709-19. - 42 -
32. Chapman RM. Effect of cytotoxic therapy on sexuality and gonadal function. Semin Oncol 1982; 9: 84-94. 33. Schumer ST, Cannistra SA. Granulosa cell tumor of the ovary. J Clin Oncol 2003; 21: 1180-9. 34. Murata T, Takizawa T, Funaba M, Fujimura H, Murata E, Takahashi M, Torii K. Quantitative RT-PCR for inhibin/activin subunits: measurements of rat hypothalamic and ovarian inhibin/activin subunit mrnas during the estrous cycle. Endocr J 1997; 44: 35-42. 35. Welt CK, Schneyer AL. Differential regulation of inhibin B and inhibin A by follicle-stimulating hormone and local growth factors in human granulosa cells from small antral follicles. J Clin Endocrinol Metab 2001; 86: 330-6. 36. Welt CK, Adams JM, Sluss PM, Hall JE. Inhibin A and inhibin B responses to gonadotropin withdrawal depends on stage of follicle development. J Clin Endocrinol Metab 1999; 84: 2163-9. 37. McCluggage WG, Maxwell P, Sloan JM. Immunohistochemical staining of ovarian granulosa cell tumors with monoclonal antibody against inhibin. Hum Pathol 1997; 28: 1034-8. 38. Ala-Fossi SL, Aine R, Punnonen R, Maenpaa J. Is potential to produce inhibins related to prognosis in ovarian granulosa cell tumors? Eur J Gynaecol Oncol 2000; 21: 187-9. 39. Boggess JF, Soules MR, Goff BA, Greer BE, Cain JM, Tamimi HK. Serum inhibin and disease status in women with ovarian granulosa cell tumors. Gynecol Oncol 1997; 64: 64-9. 40. Stuart GC, Dawson LM. Update on granulosa cell tumours of the ovary. Curr Opin Obstet Gynecol 2003; 15: 33-7. 41. Hillier SG, Yong EL, Illingworth PJ, Baird DT, Schwall RH, Mason AJ. Effect of recombinant activin on androgen synthesis in cultured human thecal cells. J Clin Endocrinol Metab 1991; 72: 1206-11. 42. Hughesdon PE. Morphology and morphogenesis of the Stein-Leventhal ovary and of so-called "hyperthecosis". Obstet Gynecol Surv 1982; 37: 59-77. 43. Pache TD, Chadha S, Gooren LJ, Hop WC, Jaarsma KW, Dommerholt HB, Fauser BC. Ovarian morphology in long-term androgen-treated female to male transsexuals. A human model for the study of polycystic ovarian syndrome? Histopathology 1991; 19: 445-52. = 국문초록 = 목적 : 본연구의목적은난소내인히빈-α 단백질발현과인히빈 α, βa, 그리고 βb 유전자의발현에감마선이미치는효과를연구하는것이다. 연구방법 : 미성숙생쥐에감마선을치사량의 25% 효과로전신조사하였다. 감마선조사후, 0, 3, 6, 12, 24시간이지나서난소를적출하였다. 적출한난소를이용하여, 인히빈 α에대한면역조직화학염색과인히빈 α, βa, 그리고 βb에대한 RT-PCR 을수행하였다. 결과 : 인히빈 α 면역양성반응성은 12시간방사선조사후까지유지가되었고, 이후에감소하였다. 인히빈 α mrna의발현은방사선처리후에유의하게증가하였다. 그러나, 인히빈 βa 와 βb의 mrna의발현은유의한변화가나타나지않았다. 결론 : 인히빈은감마선조사로유도된생쥐난포폐쇄에조절적요소로작용하는것으로사료된다. 중심단어 : 생쥐난소, 난포, 인히빈, 방사선 - 43 -