臺灣博碩士論文加值系統

褪黑激素(melatonin, MLT) 是一個維持體內恆定的賀爾蒙，可以調節生理時鐘。有許多研究指出MLT可當作一種抗氧化劑。另一方面，有文獻指出在脊椎動物中，MLT可藉由抑制促性腺激素釋放素(gonadotropin)的分泌而有抑制性腺的作用。血清素(serotonin, 5HT)是MLT之前驅物，由色胺酸經由一連串酵素作用轉換而來。它有調節神經元活性、提高睡眠品質、使人冷靜並使人心情愉悅等等之功能。有研究已指出MLT和5HT都有抑制睪酮分泌之效果，然而MLT 和 5HT是如何抑制萊氏細胞中睪酮合成與其抑制機制尚未明瞭，因此，本研究之目的主要是探討MLT 和 5HT對於睪酮在萊氏細胞中的生合成所扮演的角色與其中之機制。萊氏細胞從雄性大鼠睾丸中利用percoll濃度梯度之方法分離出來。在離體實驗結果發現MLT (10-9-10-6 M) 加入萊氏細胞培養液中可抑制睪酮分泌，另外，在預處理MLT (10-8-10-6 M)一小時，第二小時MLT與人類絨毛膜促性腺激素共同培養下，其睪酮分泌量顯著遭抑制。然而5HT也有相似的結果，相較之下，色胺酸(MLT與5HT之前驅物)則沒有抑制睪酮合成之效果。我們進一步調查其分子機制，從所有結果發現MLT和5HT抑制大鼠萊氏細胞中的睪酮分泌是經由cAMP路徑。

Melatonin (MLT) is an important hormone for homeostasis, which can regulate biological clock. The antioxidant of melatonin had also been demonstrated recently. Previous studies studies indicated that melatonin has anti-gonadal effect by inhibiting gonadotropin release in vertebrates. Serotonin, 5-hydroxytryptamine (5-HT), is the precursor of melatonin biochemically derived from tryptophan. It could regulate neuron activity, and has been improving sleeping quality, making people calm, and happy. It has been demonstrated that both melatonin and serotonin could inhibit testosterone (T) release. The mechanisms of MLT and 5-HT on T production by Leydig cells are still unclear. Our aim was to investigate the mechanisms of melatonin and serotonin on T release. Leydig cells were prepared from adult male rats by continuous percoll gradient method. MLT at concentration of 10-9-10-6 M inhibited basal release of T. Besides, after pre-incubation with MLT (10-8-10-6 M) for 1 h, the hCG-evoked T release was significantly inhibited by the co-incubation of MLT (10-8-10-6 M) in vitro. Although 5-HT had similar effects, in contrast, tryptophan (the precursor of melatonin and serotonin, 10-8-10-6 M) had no effects on the steroidogenesis in rat Leydig cells. We further investigated the mechanisms of the inhibition by MLT and 5-HT. These results suggested that both MLT and 5-HT inhibit T release in rat Leydig cells via cAMP-associated pathway.

中英文名詞對照及英文縮寫表 iii
藥物作用一覽表 iv
中文摘要 v
英文摘要 vi
第壹章　文獻回顧 1
一、 雄性素 1
二、 睪丸結構及功能 1
三、 睪酮之分泌細胞 2
四、 睪酮之生合成 (詳見圖一) 2
五、 萊氏細胞睪酮分泌之調控 4
六、 萊氏細胞睪酮合成之訊息傳導路徑 6
1. cAMP 訊息傳導路徑 6
2. Inositol phosphate/ calcium訊息傳導路徑 6
3. MAPK 訊息傳導路徑 7
4. cGMP 訊息傳導路徑 7
七、 雄性素之生理作用及機轉 7
八、 褪黑激素之特性 8
1. 褪黑激素之來源 8
2. 合成作用 (詳見圖二) 8
3. 生理調節 8
4. 生理功能及其臨床上之可能應用 9
九、 血清素之特性 10
1. 血清素之來源 10
3. 生理調節 10
4. 生理功能及其臨床上之可能應用 10
十、目的與假說 11
第貳章　材料與方法 13
一、實驗動物 14
二、離體實驗 14
1. 睪丸間質細胞的製備 14
2. 萊氏細胞的製備 (詳見圖三，Ⅰ~Ⅲ) 14
3. 細胞計數及存活率計算方式 15
4. 分置細胞稀釋液 16
四、睪酮之放射免疫測定法 17
1. 原理 17
2. 方法 17
七、統計分析 18
第叁章 結 果 19
一、褪黑激素對雄性大鼠萊氏細胞分泌睪酮之效應 19
二、血清素對雄性大鼠萊氏細胞分泌睪酮之效應 19
三、色胺酸對雄性大鼠萊氏細胞分泌睪酮之效應 20
四、褪黑激素抑制雄鼠萊氏細胞合成睪酮之機制 20
五、血清素抑制雄鼠萊氏細胞合成睪酮之機制 21
第肆章 討 論 22
參考文獻 24
圖與圖誌 31
附錄 55
致謝 57

Abramets, II, Komissarov, I. V., Samoilovich, I. M. and Shovtuta, V. I. [Effect of serotonin on smooth muscle]. Biull Eksp Biol Med 83: 173-175, 1977.
Alberti, C. [Melatonin: the first hormone isolated from the pineal body]. Farmaco Sci 13: 604-605, 1958.
Aldegunde, M., Duran, R., Alfonso, M., Miguez, I. and Veira, J. Effect of long-term pinealectomy on serum LH levels and on cerebral serotonin metabolism in adult male rats. Rev Esp Fisiol 43: 351-354, 1987.
Avallet, O., Vigier, M., Perrard-Sapori, M. H. and Saez, J. M. Transforming growth factor beta inhibits Leydig cell functions. Biochem Biophys Res Commun 146: 575-581, 1987.
Barchas, J. Studies on the relation of melatonin to sleep. Proc West Pharmacol Soc 11: 22, 1968.
Benahmed, M., Reventos, J. and Saez, J. M. Steroidogenesis of cultured purified pig Leydig cells: effects of lipoproteins and human chorionic gonadotropin. Endocrinology 112: 1952-1957, 1983.
Berne and Levy Physiology 6th Updated Edition, pp.764, 2010.
Bertrand, S., Faivre, B. and Sorci, G. Do carotenoid-based sexual traits signal the availability of non-pigmentary antioxidants? J Exp Biol 209: 4414-4419, 2006.
Browning, J. Y., D'Agata, R. and Grotjan, H. E., Jr. Isolation of purified rat Leydig cells using continuous Percoll gradients. Endocrinology 109: 667-669, 1981.
Brzezinski, A., Fibich, T., Cohen, M., Schenker, J. G. and Laufer, N. Effects of melatonin on progesterone production by human granulosa lutein cells in culture. Fertil Steril 58: 526-529, 1992.
Caraty, A., Locatelli, A., Delaleu, B., Spitz, I. M., Schatz, B. and Bouchard, P. Gonadotropin-releasing hormone (GnRH) agonists and GnRH antagonists do not alter endogenous GnRH secretion in short-term castrated rams. Endocrinology 127: 2523-2529, 1990.
Carter, D. S. and Goldman, B. D. Antigonadal effects of timed melatonin infusion in pinealectomized male Djungarian hamsters (Phodopus sungorus sungorus): duration is the critical parameter. Endocrinology 113: 1261-1267, 1983.
Chen, Y. D., Kraemer, F. B. and Reaven, G. M. Identification of specific high density lipoprotein-binding sites in rat testis and regulation of binding by human chorionic gonadotropin. J Biol Chem 255: 9162-9167, 1980.
Chiao, Y. C., Cho, W. L. and Wang, P. S. Inhibition of testosterone production by propylthiouracil in rat Leydig cells. Biol Reprod 67: 416-422, 2002.
Clark, B. J., Wells, J., King, S. R. and Stocco, D. M. The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem 269: 28314-28322, 1994.
Confente, F., Rendon, M., Besseau, L., Falcon, J. and Munoz-Cueto, J. A. Melatonin receptors in a pleuronectiform species, Solea senegalensis: Cloning, tissue expression, day-night and seasonal variations. Gen Comp Endocrinol 167: 202-214, 2010.
Corbier, P., Edwards, D. A. and Roffi, J. The neonatal testosterone surge: a comparative study. Arch Int Physiol Biochim Biophys 100: 127-131, 1992.
Csaba, Z., Csernus, V. and Gerendai, I. Intratesticular serotonin affects steroidogenesis in the rat testis. J Neuroendocrinol 10: 371-376, 1998.
de Launoit, Y., Zhao, H. F., Belanger, A., Labrie, F. and Simard, J. Expression of liver-specific member of the 3 beta-hydroxysteroid dehydrogenase family, an isoform possessing an almost exclusive 3-ketosteroid reductase activity. J Biol Chem 267: 4513-4517, 1992.
Dluzen, D. E. and Ramirez, V. D. In vitro progesterone modulation of amphetamine-stimulated dopamine release from the corpus striatum of ovariectomized estrogen-treated female rats: response characteristics. Brain Res 517: 117-122, 1990.
Dufau, M. L., Baukal, A. J. and Catt, K. J. Hormone-induced guanyl nucleotide binding and activation of adenylate cyclase in the Leydig cell. Proc Natl Acad Sci U S A 77: 5837-5841, 1980.
Dunkel, L., Jia, X. C., Nishimori, K., Boime, I. and Hsueh, A. J. Deglycosylated human chorionic gonadotropin (hCG) antagonizes hCG stimulation of 3',5'-cyclic adenosine monophosphate accumulation through a noncompetitive interaction with recombinant human luteinizing hormone receptors. Endocrinology 132: 763-769, 1993.
Evseev, V. A., Trekova, N. A., Vetrile, L. A., Mikovskaya, O. I., Basharova, L. A., Gruden, M. A. and Khlopushina, T. G. Neuromodulatory effects of serotonin antibodies on behavioral responses, neurotransmitter content in the central nervous system, and ligand-binding activity of central serotonin receptors in C57BL/6 and BALB/c mice. Bull Exp Biol Med 132: 630-632, 2001.
Faria, M. J., Simoes, Z. L., Lunardi, L. O. and Hartfelder, K. Apoptosis process in mouse Leydig cells during postnatal development. Microsc Microanal 9: 68-73, 2003.
Fernstrom, M. H. and Fernstrom, J. D. Brain tryptophan concentrations and serotonin synthesis remain responsive to food consumption after the ingestion of sequential meals. Am J Clin Nutr 61: 312-319, 1995.
Foresta, C., Mioni, R., Bordon, P., Gottardello, F., Nogara, A. and Rossato, M. Erythropoietin and testicular steroidogenesis: the role of second messengers. Eur J Endocrinol 132: 103-108, 1995.
Freeman, D. A. and Ascoli, M. The low-density lipoprotein pathway of cultured Leydig tumor cells. Utilization of low-density lipoprotein-derived cholesterol for steroidogenesis. Biochim Biophys Acta 754: 72-81, 1983.
Ge, R. S., Chen, G. R., Dong, Q., Akingbemi, B., Sottas, C. M., Santos, M., Sealfon, S. C., Bernard, D. J. and Hardy, M. P. Biphasic effects of postnatal exposure to diethylhexylphthalate on the timing of puberty in male rats. J Androl 28: 513-520, 2007.
Glass, J. D. and Knotts, L. K. A brain site for the antigonadal action of melatonin in the white-footed mouse (Peromyscus leucopus): involvement of the immunoreactive GnRH neuronal system. Neuroendocrinology 46: 48-55, 1987.
Guo, Z., Benten, W. P., Krucken, J. and Wunderlich, F. Nongenomic testosterone calcium signaling. Genotropic actions in androgen receptor-free macrophages. J Biol Chem 277: 29600-29607, 2002.
Hadley, M. A., Byers, S. W., Suarez-Quian, C. A., Djakiew, D. and Dym, M. In vitro models of differentiated Sertoli cell structure and function. In Vitro Cell Dev Biol 24: 550-557, 1988.
Haider, S. and Haleem, D. J. Decreases of brain serotonin following a food restriction schedule of 4 weeks in male and female rats. Med Sci Monit 6: 1061-1067, 2000.
Hardeland, R. Antioxidative protection by melatonin: multiplicity of mechanisms from radical detoxification to radical avoidance. Endocrine 27: 119-130, 2005.
Hardeland, R., Pandi-Perumal, S. R. and Cardinali, D. P. Melatonin. Int J Biochem Cell Biol 38: 313-316, 2006.
Hedger, M. P., Khatab, S., Gonzales, G. and de Kretser, D. M. Acute and short-term actions of serotonin administration on the pituitary-testicular axis in the adult rat. Reprod Fertil Dev 7: 1101-1109, 1995.
Hirakawa, T., Galet, C. and Ascoli, M. MA-10 cells transfected with the human lutropin/choriogonadotropin receptor (hLHR): a novel experimental paradigm to study the functional properties of the hLHR. Endocrinology 143: 1026-1035, 2002.
Huang, W. J., Yeh, J. Y., Kan, S. F., Chang, L. S. and Wang, P. S. Effects of hyperprolactinemia on testosterone production in rat Leydig cells. J Cell Biochem 80: 313-320, 2001.
Huhtaniemi, I. and Toppari, J. Endocrine, paracrine and autocrine regulation of testicular steroidogenesis. Adv Exp Med Biol 377: 33-54, 1995.
Iliff-Sizemore, S. A., Ortolano, G. A., Haisenleder, D. J., Dalkin, A. C., Krueger, K. A. and Marshall, J. C. Testosterone differentially modulates gonadotropin subunit messenger ribonucleic acid responses to gonadotropin-releasing hormone pulse amplitude. Endocrinology 127: 2876-2883, 1990.
Kliesch, S. [Testosterone and infertility]. Urologe A 49: 32-36, 2010.
Lachance, Y., Luu-The, V., Verreault, H., Dumont, M., Rheaume, E., Leblanc, G. and Labrie, F. Structure of the human type II 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) gene: adrenal and gonadal specificity. DNA Cell Biol 10: 701-711, 1991.
Leydig, F. Zur anatomie der mannlichen geschlechtsorgane und Analdrusen der Saugetiere. Z. Wiss. Zool. 2: 1-57, 1850.
Lin, Y. M., Poon, S. L., Choi, J. H., Lin, J. S., Leung, P. C. and Huang, B. M. Transcripts of testicular gonadotropin-releasing hormone, steroidogenic enzymes, and intratesticular testosterone levels in infertile men. Fertil Steril 90: 1761-1768, 2008.
Lo, K. C., Lei, Z., Rao Ch, V., Beck, J. and Lamb, D. J. De novo testosterone production in luteinizing hormone receptor knockout mice after transplantation of leydig stem cells. Endocrinology 145: 4011-4015, 2004.
Lorence, M. C., Murry, B. A., Trant, J. M. and Mason, J. I. Human 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase from placenta: expression in nonsteroidogenic cells of a protein that catalyzes the dehydrogenation/isomerization of C21 and C19 steroids. Endocrinology 126: 2493-2498, 1990.
Lucas, T. F., Pimenta, M. T., Pisolato, R., Lazari, M. F. and Porto, C. S. 17beta-estradiol signaling and regulation of Sertoli cell function. Spermatogenesis 1: 318-324, 2011.
Lyseng-Williamson, K. A. Melatonin prolonged release: in the treatment of insomnia in patients aged >/=55 years. Drugs Aging 29: 911-923, 2012.
Martini, L. The 5alpha-reduction of testosterone in the neuroendocrine structures. Biochemical and physiological implications. Endocr Rev 3: 1-25, 1982.
Marwood, J. F. and Stokes, G. S. Serotonin (5HT) and its antagonists: involvement in the cardiovascular system. Clin Exp Pharmacol Physiol 11: 439-455, 1984.
McFarland, K. C., Sprengel, R., Phillips, H. S., Kohler, M., Rosemblit, N., Nikolics, K., Segaloff, D. L. and Seeburg, P. H. Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science 245: 494-499, 1989.
Mediavilla, M. D., Sanchez-Barcelo, E. J., Tan, D. X., Manchester, L. and Reiter, R. J. Basic mechanisms involved in the anti-cancer effects of melatonin. Curr Med Chem 17: 4462-4481, 2010.
Nicotera, P., McConkey, D. J., Dypbukt, J. M., Jones, D. P. and Orrenius, S. Ca2+-activated mechanisms in cell killing. Drug Metab Rev 20: 193-201, 1989.
Payne, A. H. Hormonal regulation of cytochrome P450 enzymes, cholesterol side-chain cleavage and 17 alpha-hydroxylase/C17-20 lyase in Leydig cells. Biol Reprod 42: 399-404, 1990.
Payne, A. H. and Youngblood, G. L. Regulation of expression of steroidogenic enzymes in Leydig cells. Biol Reprod 52: 217-225, 1995.
Peltoketo, H., Isomaa, V. and Vihko, R. Genomic organization and DNA sequences of human 17 beta-hydroxysteroid dehydrogenase genes and flanking regions. Localization of multiple Alu sequences and putative cis-acting elements. Eur J Biochem 209: 459-466, 1992.
Ritzenthaler, T., Nighoghossian, N., Berthiller, J., Schott, A. M., Cho, T. H., Derex, L., Brun, J., Trouillas, P. and Claustrat, B. Nocturnal urine melatonin and 6-sulphatoxymelatonin excretion at the acute stage of ischaemic stroke. Journal of pineal research 46: 349-352, 2009.
Schumacher, H., Muller, D. and Mukhopadhyay, A. K. Stimulation of testosterone production by atrial natriuretic peptide in isolated mouse Leydig cells results from a promiscuous activation of cyclic AMP-dependent protein kinase by cyclic GMP. Mol Cell Endocrinol 90: 47-52, 1992.
Schwartz, J. and Cherny, R. Intercellular communication within the anterior pituitary influencing the secretion of hypophysial hormones. Endocr Rev 13: 453-475, 1992.
Selig, W. M., Bloomquist, M. A., Cohen, M. L. and Fleisch, J. H. Serotonin-induced pulmonary responses in the perfused guinea pig lung: evidence for 5HT2 receptor-mediated pulmonary vascular and airway smooth muscle constriction. Pulm Pharmacol 1: 93-99, 1988.
Simard, J., Couet, J., Durocher, F., Labrie, Y., Sanchez, R., Breton, N., Turgeon, C. and Labrie, F. Structure and tissue-specific expression of a novel member of the rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) family. The exclusive 3 beta-HSD gene expression in the skin. J Biol Chem 268: 19659-19668, 1993.
Simard, J., de Launoit, Y. and Labrie, F. Characterization of the structure-activity relationships of rat types I and II 3 beta-hydroxysteroid dehydrogenase/delta 5 -delta 4 isomerase by site-directed mutagenesis and expression in HeLa cells. J Biol Chem 266: 14842-14845, 1991.
Sirek, A. and Sirek, O. V. Serotonin: a review. Can Med Assoc J 102: 846-849, 1970.
Srinivasan, V., Pandi-Perumal, S. R., Maestroni, G. J., Esquifino, A. I., Hardeland, R. and Cardinali, D. P. Role of melatonin in neurodegenerative diseases. Neurotox Res 7: 293-318, 2005.
Steel, R. G. and Torrie, J. H. Principles and procedures of statistics: A biometrical approach. McGraw-Hill 633, 1980.
Stocco, D. M. StAR protein and the regulation of steroid hormone biosynthesis. Annu Rev Physiol 63: 193-213, 2001.
Takatsu, S., Ishiwata, T., Meeusen, R., Sarre, S. and Hasegawa, H. Serotonin release in the preoptic area and anterior hypothalamus is not involved in thermoregulation during low-intensity exercise in a warm environment. Neurosci Lett 482: 7-11, 2010.
Tian, Y. M., Zhang, G. Y. and Dai, Y. R. Melatonin rejuvenates degenerated thymus and redresses peripheral immune functions in aged mice. Immunol Lett 88: 101-104, 2003.
Tsai, C. C., Collins, S. H. and Swanger, S. J. Ovarian Sertoli-Leydig cell tumor in an amenorrheic hirsute patient. Changgeng Yi Xue Za Zhi 19: 191-195, 1996.
Valenti, S. and Giusti, M. Melatonin participates in the control of testosterone secretion from rat testis: an overview of our experience. Ann N Y Acad Sci 966: 284-289, 2002.
Vanecek, J. Cellular mechanisms of melatonin action. Physiol Rev 78: 687-721, 1998.
Walker, F. R. A critical review of the mechanism of action for the selective serotonin reuptake inhibitors: do these drugs possess anti-inflammatory properties and how relevant is this in the treatment of depression? Neuropharmacology 67: 304-317, 2013.
Wang, P. S., Tsai, S. C., Hwang, G. S., Wang, S. W., Lu, C. C., Chen, J. J., Liu, S. R., Lee, K. Y., Chien, E. J., Chien, C. H. and et al. Calcitonin inhibits testosterone and luteinizing hormone secretion through a mechanism involving an increase in cAMP production in rats. J Bone Miner Res 9: 1583-1590, 1994.
Wang, S. W., Lin, H., Hwang, J. J. and Wang, P. S. Inhibition of testosterone secretion by digitoxin in rat testicular interstitial cells. J Cell Biochem 74: 74-80, 1999.
Wu, L., Einstein, M., Geissler, W. M., Chan, H. K., Elliston, K. O. and Andersson, S. Expression cloning and characterization of human 17 beta-hydroxysteroid dehydrogenase type 2, a microsomal enzyme possessing 20 alpha-hydroxysteroid dehydrogenase activity. J Biol Chem 268: 12964-12969, 1993.
Zhao, H. F., Labrie, C., Simard, J., de Launoit, Y., Trudel, C., Martel, C., Rheaume, E., Dupont, E., Luu-The, V., Pelletier, G. and et al. Characterization of rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase cDNAs and differential tissue-specific expression of the corresponding mRNAs in steroidogenic and peripheral tissues. J Biol Chem 266: 583-593, 1991.