한국발생생물학회지:발생과생식 (Development and Reproduction)

  • 제13권4호
  • /
  • Pages.353-362
  • /
  • 2009
  • /
  • 2465-9525(pISSN)
  • /
  • 2465-9541(eISSN)
한국발생생물학회 (The Korean Society of Developmental Biology)
권호 표지

생식샘자극호르몬분비호르몬이 사람 과립-황체화 세포의 스테로이드 생성과 세포자연사에 미치는 영향

Effects of Gonadotropin Releasing Hormone on Steroidogenesis and Apoptosis of Human Granulosa-Lutein Cells

  • 이효진 (서울여자대학교 생명공학과) ;
  • 양현원 (서울여자대학교 생명공학과)
  • 발행 : 2009.12.31
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

GnRH는 국부적으로 난소에서 합성되며, 난소내 과립 및 황체세포에 직접적으로 작용하여 난소의 기능을 조절하는 것으로 알려져 있으며, 특히, GnRH는 난소내 과립-황체화 세포의 세포자연사를 유도하는 것으로 보고하고 있다. 그러나 GnRH에 의한 세포자연사가 FSH에 의해 회복될 수 있는지는 명확히 밝혀져 있지 않다. 따라서 본 실험에서 난자 채취시 획득한 사람 과립-황체화 세포를 배양한 후 5, 50, 100 ng/$m\ell$ GnRH와 1 IU/$m\ell$ FSH를 처리하고 세포의 세포자연사 여부와 분비된 progesterone$(P_4)$과 estradiol$(E_2)$ 양의 변화를 조사하였다. DNA 분절화 분석과 TUNEL 방법으로 세포자연사를 평가한 결과, GnRH는 농도 의존적으로 과립-황체화 세포의 세포자연사를 증가시켰고, 특히 100 ng/$m\ell$ GnRH을 처리한 군에서 유의한 차이를 보이며 세포자연사 비율이 증가하였다. 또한 GnRH에 의한 세포자연사의 증가는 FSH에 의해 억제되는 것을 확인할 수 있었다. 화학발광면역 측정법을 이용하여 배양내 $P_4$와 $E_2$의 양을 측정한 결과, GnRH을 처리한 후 $E_2$의 양은 변화가 없었던 반면 $P_4$의 양은 감소하였다. 이러한 GnRH의 $P_4$ 합성 억제 효과는 세포자연사 결과 마찬가지로 FSH에 의해 회복되는 것을 확인할 수 있었다. 이상의 결과는 체외수정 및 배아이식 시술시 사용되고 있는 GnRH 작용제가 난소의 기능을 억제시킬 수 있을 것으로 보이나, 다량으로 투여되는 FSH에 의해 회복될 수 있음을 보여주고 있다. 이러한 실험 결과는 난소에 대한 GnRH의 생리적 기전을 이해하고 향후 새로운 과배란 유도 방법을 개발하는데 필요한 기초 자료로 사용될 수 있을 것으로 사료된다.

GnRH and its receptor are known to express locally in the ovary and to regulate the ovarian function by affecting on granulosa and lutein cells. It has been reported that GnRH directly causes apoptosis in the granulosa and lutein cells of the ovary. However, whether the apoptosis of the cells by GnRH is recovered by FSH as an anti-apoptotic factor is not yet known. In this study, we evaluated the apoptosis and the production of progesterone $(P_4)$ and estradiol $(E_2)$ after treatment with 5, 50, and 100 ng/$m\ell$ GnRH and 1 IU/ml FSH in the granulosa-lutein cells that are obtained during oocyte-retrieval for IVF-ET. Results of DNA fragment analysis and TUNEL assay demonstrated that DNA fragmentation and the rate of apoptotic cells were increased in a dose-dependent manner showing a significant increase in the cells treated with 100 ng/$m\ell$ GnRH. In addition, we found that FSH suppresses the apoptosis of the cells induced by GnRH. In the results of chemiluminescence assay for $P_4$ and $E_2$, $P_4$ production was decreased by GnRH treatment, whereas $E_2$ production was not changed. We also demonstrated that FSH inhibits the suppressive effect of GnRH on $P_4$ production as the result of apoptosis. The present results suggest that GnRH agonist using in ovarian hyperstimulation protocol might induce the dysfunction of the ovary, but its function could be recovered by FSH. These results also will be expected to use as the basic data to elucidate the physiological role of GnRH and to develop new ovarian hyperstimulation protocols for IVF-ET.

키워드

참고문헌

  1. Amsterdam A, Keren-Tal I, Aharoni D, Dantes A, Land- Bracha A, Rimon E, Sasson R, Hirsh L (2003) Steroidogenesis and apoptosis in the mammalian ovary. Steroids 68:861-867. https://doi.org/10.1016/j.steroids.2003.09.003
  2. Ashkenazi A, Dixit VM (1998) Death receptors: signaling and modulation. Science 281:1305-1308. https://doi.org/10.1126/science.281.5381.1305
  3. Birnbaumer L, Shahabi N, Rivier J, Vale W (1985) Evidence for a physiological role of gonadotropin-releasing hormone (GnRH) or GnRH-like material in the ovary. Endocrinology 116:1367-1370. https://doi.org/10.1210/endo-116-4-1367
  4. Bicsak TA, Tucker EM, Cappel S, Vaughan J, Rivier J, Vale W, Hsueh AJ (1986) Hormonal regulation of granulosa cell inhibin biosynthesis. Endocrinology 119(6): 2711-2719. https://doi.org/10.1210/endo-119-6-2711
  5. Billig H, Furuta I, Hsueh AJ (1994) Gonadotropin-releasing hormone directly induces apoptotic cell death in the rat ovary: biochemical and in situ detection of deoxyribonucleic acid fragmentation in granulosa cells. Endocrinology 134:245-252. https://doi.org/10.1210/en.134.1.245
  6. Braw RH, Tsafriri A (1980) Effect of PMSG on follicular atresia in the immature rat ovary. J Reprod Fertil 59:267-272. https://doi.org/10.1530/jrf.0.0590267
  7. Cheng CK, Leung PC (2005) Molecular biology of gonadotropinreleasing hormone (GnRH)-I, GnRH-II, and their receptors in humans. Endocr Rev 26:283-306. https://doi.org/10.1210/er.2003-0039
  8. Choi JH, Gilks CB, Auersperg N, Leung PC (2006) Immunolocalization of gonadotropin- releasing hormone (GnRH)-I, GnRH-II, and type I GnRH receptor during follicular development in the human ovary. J Clin Endocrinol Metab 91:4562-4570. https://doi.org/10.1210/jc.2006-1147
  9. Clark MR (1982) Stimulation of progesterone and prostaglandin E accumulation by luteinizing hormone-releasing hormone (LHRH) and LHRH analogs in rat granulosa cells. Endocrinology 110:146-152. https://doi.org/10.1210/endo-110-1-146
  10. Erickson GF, Magoffin DA, Dyer CA, Hofeditz C (1985) The ovarian androgen producing cells: a review of structure/function relationships. Endocr Rev 6:371-399. https://doi.org/10.1210/edrv-6-3-371
  11. Hirshfield AN (1991) Development of follicles in the mammalian ovary. Int Rev Cytol 124:43-101. https://doi.org/10.1016/S0074-7696(08)61524-7
  12. Hsueh AJ, Erickson GF (1979) Extrapituitary action of gonadotropin-releasing hormone: direct inhibition ovarian steroidogenesis. Science 204:854-855. https://doi.org/10.1126/science.375393
  13. Hsueh AJ, Wang C, Erickson GF (1980) Direct inhibitory effect of gonadotropin-releasing hormone upon folliclestimulating hormone induction of luteinizing hormone receptor and aromatase activity in rat granulosa cells. Endocrinology 106:1697-1705. https://doi.org/10.1210/endo-106-6-1697
  14. Jones PB, Conn PM, Marian J, Hsueh AJ (1980) Binding of gonadotropin-releasing hormone agonist to rat ovarian granulosa cells. Life Sci 27:2125-2132. https://doi.org/10.1016/0024-3205(80)90494-4
  15. Kang SK, Choi KC, Yang HS, Leung PC (2003) Potential role of gonadotrophin-releasing hormone (GnRH)-I and GnRH-II in the ovary and ovarian cancer. Endocr Relat Cancer 10:169-177. https://doi.org/10.1677/erc.0.0100169
  16. Kang SK, Tai CJ, Nathwani PS, Leung PC (2001) Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid in human granulosaluteal cells. Endocrinology 142:182-192. https://doi.org/10.1210/en.142.1.182
  17. Kolena J, Channing CP (1972) Stimulatory effects of LH, FSH and prostaglandins upon cyclic 3',5'-AMP levels in porcine granulosa cells. Endocrinology 90:1543-1550. https://doi.org/10.1210/endo-90-6-1543
  18. Kumar S. (1995) ICE-like proteases in apoptosis. Trends Biochem Sci 20:198-202. https://doi.org/10.1016/S0968-0004(00)89007-6
  19. Limonta P, Moretti RM, Marelli MM, Motta M (2003) The biology of gonadotropin hormone-releasing hormone: role in the control of tumor growth and progression in humans. Front Neuroendocrinol. 24:279-295. https://doi.org/10.1016/j.yfrne.2003.10.003
  20. Magoffin DA, Reynolds DS, Erickson GF (1981) Direct inhibitory effect of GnRH on androgen secretion by ovarian interstitial cells. Endocrinology 109:661-663. https://doi.org/10.1210/endo-109-2-661
  21. Markstrom E, Svensson E, Shao R, Svanberg B, Billig H (2002) Survival factors regulating ovarian apoptosis: dependence on follicle differentiation. Reproduction 123: 23-30. https://doi.org/10.1530/rep.0.1230023
  22. Matsubara H, Ikuta K, Ozaki Y, Suzuki Y, Suzuki N, Sato T, Suzumori K (2000) Gonadotropins and cytokines affect luteal function through control of apoptosis in human luteinized granulosa cells. J Clin Endocrinol Metab 85:1620-1626. https://doi.org/10.1210/jc.85.4.1620
  23. Matsumi H, Yano T, Osuga Y, Kugu K, Tang X, Xu JP (2000) Regulation of nitric oxide synthase to promote cytostasis in ovarian follicular development. Bio Reprod 63:141-146. https://doi.org/10.1095/biolreprod63.1.141
  24. Neill JD (2002) GnRH and GnRH receptor genes in the human genome. Endocrinology 143:737-743. https://doi.org/10.1210/en.143.3.737
  25. Parborell F, Pecci A, Gonzalez O, Vitale A, Tesone M (2002) Effects of a gonadotropin-releasing hormone agonist on rat ovarian follicle apoptosis: regulation by epidermal growth factor and the expression of Bcl-2-related genes. Biol Reprod 67:481-486. https://doi.org/10.1095/biolreprod67.2.481
  26. Parinaud J, Oustry P, Bussenot I, Tourre A, Perineau M, Plantavid M, Monrozies X, Reme JM, Pontonnier G (1992) Paradoxical ovarian stimulations in the use of LHRH analogs. Eur J Obstet Gynecol Reprod Biol 47: 129-133. https://doi.org/10.1016/0028-2243(92)90042-W
  27. Pellicer A, Tarin JJ, Miro F, Sampaio M, De los Santos MJ, Remohi J (1992) The use of gonadotrophin releasinghormone analogues (GnRHa), in in-vitro fertilization: some clinical and experimental investigations of a direct effect on the human ovary. Hum Reprod 1:39-47.
  28. Pieper DR, Richards JS, Marshall JC (1981) Ovarian gonadotropin releasing hormone (GnRH) receptors: characterization, distribution, and induction by GnRH. Endocrinology 108:1148-1155. https://doi.org/10.1210/endo-108-4-1148
  29. Rippel RH, Johnson ES (1976) Inhibition of HCG-induced ovarian and uterine weight augmentation in the immature rat by analogs of GnRH. Proc Soc Exp Biol Med 152:432-436.
  30. Sealfon SC, Weinstein H, Millar RP (1997) Molecular mechanisms of ligand interaction with the gonadotropinreleasing hormone receptor. Endocr Rev 18:180-205. https://doi.org/10.1210/er.18.2.180
  31. Sifer C, Blanc-Layrac G, Bringuier AF, Porcher R, Madelenat P, Feldmann G, Benifla JL (2003) Effects of a gonadotropinreleasing hormone agonist and follicle stimulating hormone on the incidence of apoptosis in human luteinized granulosa cells. Eur J Obstet Gynecol Reprod Biol 110: 43-48. https://doi.org/10.1016/S0301-2115(03)00092-7
  32. Smith KB, Fraser HM (1991) Control of progesterone and inhibin secretion during the luteal phase in the macaque. J Endocrinol 128:107-113. https://doi.org/10.1677/joe.0.1280107
  33. Sridaran R, Lee MA, Haynes L, Srivastava RK, Ghose M, Sridaran G, Smith CJ (1999) GnRH action on luteal steroidogenesis during pregnancy. Steroids 64:618-623. https://doi.org/10.1016/S0039-128X(99)00042-2
  34. Takekida S, Matsuo H, Maruo T (2003) GnRH agonist action on granulosa cells at varying follicular stages. Mol Cell Endocrinol 202:155-164. https://doi.org/10.1016/S0303-7207(03)00077-7
  35. Tilly JL, Tilly KI, Perez GI (1997) The genes of cell death and cellular susceptibility to apoptosis in the ovary: a hypothesis. Cell Death Differ 4:180-187. https://doi.org/10.1038/sj.cdd.4400238
  36. Tsafriri A, Braw RH (1984) Experimental approaches to atresia in mammals. Oxf Rev Reprod Biol 6:226-265.
  37. Tsai NM, Hsieh RH, Au HK, Shieh MJ, Huang SY, Tzeng CR (2005) Effects of gonadotrophin-releasing hormone agonists on apoptosis of granulosa cells. Ann NY Acad Sci 1042:531-537. https://doi.org/10.1196/annals.1338.065
  38. Wang J, Steele GL, Baimbridge KG, Rodway MR, Leung PC (1992) Intracellular calcium and the signaling mechanism of luteinizing hormone-releasing hormone in rat granulosa cells. Am J Obstet Gynecol 167:541-547. https://doi.org/10.1016/S0002-9378(11)91450-1
  39. Whitelaw PF, Eidne KA, Sellar R, Smyth CD, Hillier SG (1995) Gonadotropin-releasing hormone receptor messenger ribonucleic acid expression in rat ovary. Endocrinology 136:172-179. https://doi.org/10.1210/en.136.1.172
  40. Wierman ME, Rivier JE, Wang C (1989) The structure and regulation of the pituitary gonadotrophin subunit genes. Endocrinology 124:272-278. https://doi.org/10.1210/endo-124-1-272
  41. Zeleznik AJ, Ihrig LL, Bassett SG (1989) Developmental expression of $Ca^{++}$/$Mg^{++}$-dependent endonuclease activity in rat granulosa and luteal cells. Endocrinology 125: 2218-2220. https://doi.org/10.1210/endo-125-4-2218
  42. 박은주, 김평식, 염윤희, 양현원, 박원일, 강병문 (2004) GnRHagonist에 의한 인간 과립-황체화 세포의 세포사멸은 caspase-3와 -9 및 PARP의 활성화에 의해 유도됨. 대한산부인과학회지 47:1145-1153.