睡眠在人類內分泌，醣類的代謝，心血管疾病，免疫功能及精神狀態等許多的生理及心理方面，扮演著重要的角色。睡眠剝奪時對女性生殖功能會產生不利影響。我們藉由全睡眠剝奪(total sleep deprivation, TSD)的大鼠動物模式並評估血清雌二醇，皮質酮和血清素的濃度，以探究睡眠不足時對大鼠的生殖功能產生的影響。並以幼大鼠卵巢完整分離的卵泡於體外培養方式評估皮質酮和血清素對卵泡發育和類固醇生成功能的作用。TSD鼠與TSD控制鼠兩組間血清雌二醇濃度有顯著差異，且血清素和皮質酮濃度在超過2天的TSD鼠血清中顯著升高。FSH誘導了卵泡成長和卵泡細胞數量的增加，伴隨著抑制素-α(inhibin-α)和連接蛋白43(connexin 43)表現量增加的卵泡細胞分化。貝他美松(betamethasone, BET)呈現劑量依賴性（0.001-1 µg/ml）的抑制FSH對卵泡生長的促進作用，使竇前期卵泡生長減緩。此減緩作用與卵泡細胞數量的減少，及增殖細胞核抗原，抑制素-α和連接蛋白43表現被抑制相關。使用固定劑量的FSH（50 mIU/ml）和遞增劑量的血清素共同培養大型卵泡，可降低雌二醇分泌量達20％。血清素也會減弱FSH刺激的卵泡類固醇生成速控蛋白(steroidogenic acute regulatory protein, StAR)表現量，但不影響卵泡細胞增殖。這些發現顯示TSD的對生殖功能的負面影響極可能是透過過皮質酮升高抑制卵巢內卵泡的發育，同時藉由血清素抑制大鼠卵泡中StAR蛋白的表現量而降低大鼠血清雌二醇濃度。

Sleep plays an important role in many physiological and psychological aspects of human endocrinology, carbohydrate metabolism, cardiovascular diseases, immune function and mental status. Sleep deprivation (SD) adversely affects female reproductive function. In this study, we explored the effect of lack of sleep on reproductive function using rat model of total sleep deprivation (TSD), and the in vitro cell culture of intact ovarian follicles of juvenile rats. The concentrations of serum estradiol, corticosterone and serotonin in rats were systemically measured. Mechanically dissected intact rat follicles were cultured in vitro for the evaluation of follicular development and steroidogenic functions. TSD led to a significant difference in serum estradiol concentrations between the treatment and the control groups. The serum serotonin and corticosterone concentrations were significantly elevated in groups with more than 2 days of SD. FSH induced an increase in both follicle size and follicular cell number, while follicular cell differentiation was accompanied by enhanced inhibin-α and connexin 43 expression. Preantral follicular growth was dose-dependently inhibited by betamethasone (BET; 0.001–1 µg/ml). Such inhibition led to a decrease in follicular cell numbers, the suppressed expression of a proliferating cell nuclear antigen, inhibin-α, and connexin 43. Increasing doses of serotonin could reduce the estradiol production from the large follicles cultured in a fixed FSH level (50 mIU/ml) by as much as 20%. Serotonin also attenuated the expression of FSH-stimulated follicular steroidogenic acute regulatory protein (StAR) but did not affect the follicular cell proliferation. These findings supported the notion that TSD retards the follicular development by overstimulating the ovaries with elevated corticosterone. The reduced serum level of estradiol in TSD rats is likely caused by serotonin-mediated inhibition of estradiol production and the expression of StAR protein in the follicles.

論文審定書…………………………………………………………...i
誌謝…………………………………………………………………..ii
中文摘要……………………………………………………………..iii
英文摘要…………………………………………….……………… iv
第一章、前言…..………………………….……………..………....1
1.1 睡眠概論...............................................................................1
1.2 睡眠缺乏對生理功能影響的流行病學研究……......................3
1.3 睡眠缺乏與生殖功能的關聯性…………………………….......5
1.3.1 睡眠缺乏導致壓力(Stress)的產生……………...............….5
1.3.2 雌性動物生殖系統的組成與功能……………………..........6
1.3.3 下視丘-腦下垂體-腎上腺縱軸(HPA axis)活化對下視
丘-腦下垂體-性腺縱軸(HPG axis)的影響……..……........8
1.3.4 血清素對卵巢的影響………………………………............9
1.4 研究目的與實驗設計………………………………….…......10
第二章、材料與方法…………………………………………......12
2.1 睡眠剝奪動物模式的建立…………………………..…......12
2.1.1 動物照顧 ……………………………………......................12
2.1.2 腦波及肌電圖電極埋置 ……………………….......…........12
2.1.3 TSD動物模式的建立…………………….................…......13
2.2 雌二醇、皮質酮、黃體激素及血清素濃度測定………...…..14
2.3 卵泡分離與培養………………………………………............15
2.3.1 大型卵泡分離與培養……………………………................15
2.3.2 竇前期卵泡分離與培養…………………………................16
2.4 細胞存活率分析(MTS Assay)……………………...........…..17
2.5 卵泡大小的量測...……………………………………....….....17
2.6 卵巢重量與型態.………………………………………....…...18
2.7 蛋白質電泳與西方點墨法.……………………………....…...18
2.8 蛋白質印跡脫離與再度點墨……………………………........20
2.9 統計分析 ………………………………………………….......20
第三章、結果……………………….………........……..…….......22
3.1 TSD動物模式的建立…………………………….……….......22
3.2 TSD對雌性大鼠血中皮質酮、雌性素、黃體激素與血清
素的影響…..........................................................................22
3.3 糖皮質素貝他美松弱化孕馬血清激性腺素對未成熟幼鼠
卵巢的刺激性生長...…...…..………………………………...24
3.4 貝他美松抑制FSH所促進的竇前期卵泡體外生長……....….25
3.5 貝他美松降低竇前期卵泡發育標誌蛋白的表現………….....27
3.6 血清素對卵泡細胞活力與性激素分泌功能的影響……....….27
3.7 血清素活化5-HT2A受體影響大型卵泡的賀爾蒙分泌的
能力……………………………………................…………....28
第四章、討論..…………………………………....…..……………30
第五章、參考文獻……………………………….……....…….......37

1. National Institute of Mental Health. Arousal and Regulatory Systems: Workshop Proceedings, 2012. Retrieved from https://www.nimh.nih.gov/research-priorities/rdoc/arousal-and-regulatory-systems-workshop-proceedings.shtml.
2. Campbell SS, Tobler I. Animal sleep: a review of sleep duration across phylogeny. Neurosci Biobehav Rev. 1984; 8:269–300.
3. Retrieved from http://www.psychology4a.com/biological-rhytms-and-sleep.html.
4. Van Dongen HP, Vitellaro KM, Dinges DF. Individual differences in adult human sleep and wakefulness: leitmotif for a research agenda. Sleep. 2005; 28:479-96.
5. Youngstedt SD, Kripke DF. Long sleep and mortality: rationale for sleep restriction. Sleep Med Rev. 2004; 8:159-74.
6. Youngstedt SD, Goff EE, Reynolds AM, Kripke DF, Irwin MR, Bootzin RR, Khan N, Jean-Louis G. Has adult sleep duration declined over the last 50+ years? Sleep Med Rev. 2016; 28:69-85.
7. Bringmann H. Sleep-Active Neurons: Conserved Motors of Sleep. Genetics. 2018; 208:1279-89.
8. Fuller PM, Sherman D, Pedersen NP, Saper CB, Lu J. Reassessment of the structural basis of the ascending arousal system. J. Comp. Neurol. 2011; 519:933–56.
9. Saper CB, Fuller PM, Pedersen NP, Lu J, Scammell TE. Sleep state switching. Neuron. 2010; 68:1023–42.
10. Hirshkowitz M, Hirshkowitz M, Whiton K, Albert SM, Alessi C, Bruni O, et al. National sleep foundation’s sleep time duration recommendations: methodology and results summary. Sleep Health. 2015; 1:40-3
11. Liu Y, Wheaton AG, Chapman DP, Cunningham TJ, Hua L, Croft JB. Short sleep duration among workers – United States. 2010. MMWR Morb Mortal Wkly Rep. 2012; 61:281–5.
12. Liu Y., Wheaton AG, Chapman DP, Cunningham TJ, Lu H, Croft JB. Prevalence of healthy sleep duration among adults—United States. 2014. MMWR Morb. Mortal. Wkly. Rep. 2016; 65:137–41.
13. Steptoe A, Peacey V, Wardle J. Sleep duration and health in young adults. Arch Intern Med. 2006; 166:1689–92.
14. Banks S, Dinges DF. Behavioral and physiological consequences of sleep restriction. J Clin Sleep Med. 2007; 3:519-28.
15. Miller MA, Cappuccio FP. Biomarkers of cardiovascular risk in sleep deprived people. J Hum Hypertens. 2013; 27:583–88.
16. Mahoney MM. Shift work, jet lag, and female reproduction. Int J of Endocrinol. 2010; 2010:813764.
17. Axelsson G, Rylander R, Molin I. Outcome of pregnancy in relation to irregular and inconvenient work schedules. Br J Ind Med. 1989; 46:393–8.
18. McDonald AD, McDonald JC, Armstrong B, Cherry NM, Cote R, Lavoie J, et al. Fetal death and work in pregnancy. Br J Ind Med. 1988; 45:148–8.
19. Bisanti L, Olsen J, Basso O, Thonneau P, Karmaus W. Shift work and subfecundity: a European multicenter study. European study group on infertility and subfecundity. J Occup Environ Med. 1996; 38:352–8.
20. Labyak S, Lava S, Turek F, Zee P. Effects of shiftwork on sleep and menstrual function in nurses. Health Care Women Int. 2002; 23:703–14.
21. Pal L, Bevilacqua K, Zeitlian G, Shu J, Santoro N. Implications of diminished ovarian reserve (DOR) extend well beyond reproductive concerns. Menopause. 2008; 15:1086–94.
22. Lin JL, Lin YH, Chueh KH. Somatic symptoms, psychological distress and sleep disturbance among infertile women with intrauterine insemination treatment. J Clin Nurs. 2013; 23:1677–84.
23. Singer F, Zumoff B. Subnormal serum testosterone levels in male internal medicine residents. Steroids. 1992; 57:86-9.
24. Cortes-Gallegos V, Castaneda G, Alonso R, Sojo I, Crranco A, Cervntes C, and Parra a. Sleep deprivation reduces circulating androgens in healthy men. Arch Androl. 1983; 10:33-7.
25. Andersen ML, Bignotto M, Machado RB, Tufik S. Different stress modalities result in distinct steroid hormone responses by male rats. Braz J Med Biol Res. 2004; 37:791-7.
26. Andersen ML, Alvarenga TA, Guindalini C, Perry JC, Silva A, Zager A, Tufik S. Paradoxical sleep deprivation influences sexual behavior in female rats. J Sex Med. 2009; 6:2162-72.
27. Antunes IB, Andersen ML, Baracat EC, Tufik S. The effects of paradoxical sleep deprivation on estrous cycles of the female rats. Horm Behav. 2006; 49:433-40.
28. Leproult R, Cauter EV. Effect of 1 week of sleep restriction on testosterone levels in young healthy men. JAMA. 2011; 305:2173-4.
29. Leproult R, Van Cauter E. Role of sleep and sleep loss in hormonal release and metabolism. Endocr Dev. 2010; 17:11–21.
30. Meerlo P, Sgoifo A, Suchecki D. Restricted and disrupted sleep: effects on autonomic function, neuroendocrine stress systems and stress responsivity. Sleep Med Rev. 2008; 12:197–210.
31. Park SP, Lopez-Rodriguez F, Wilson CL, Maidment N, Matsumoto Y, Engel JJ. In vivo microdialysis measures of extracellular serotonin in the rat hippocampus during sleep–wakefulness. Brain Res. 1999; 833:291–6.
32. Blier P, de Montigny C. Possible serotonergic mechanisms underlying the antidepressant and anti-obsessive-compulsive disorder responses. Biol Psychiatry. 1998; 44:313-23.
33. Wu JL, Wu RS, Yang JG, Huang CC, Chen KB, Fang KH, et al. Effects of sleep deprivation on serum testosterone concentrations in the rat. Neurosci Lett. 2011; 494:124-9.
34. Pepling ME. Follicular assembly: mechanisms of action. Reproduction. 2012; 143:139-49.
35. Chowdhury I, Garcia-Barrio M, Harp D, Thomas K, Matthews R, Thompson WE. The emerging roles of prohibitins in folliculogenesis. Front Biosci (Elite Ed). 2012; 4:690-9.
36. Rodgers RJ, Irving-Rodgers HF. Formation of the ovarian follicular antrum and follicular fluid. Biol Reprod. 2010; 82:1021-9.
37. Richards JS, Pangas SA. New insights into ovarian function. Handb Exp Pharmacol. 2010; 198:3-27.
38. Adhikari D, Liu K. Molecular mechanisms underlying the activation of mammalian primordial follicles. Endocr Rev. 2009; 30:438-64.
39. Hackbart KS, Cunha PM, Meyer RK, Wiltbank MC. Effect of glucocorticoid-induced insulin resistance on follicle development and ovulation. Biol Reprod. 2013; 88:153.
40. Propst AM, Bates GW Jr. Evaluation and treatment of anovulatory and unexplained infertility. Obstet Gynecol Clin North Am. 2012; 39:507-19.
41. Urman B, Yakin K. Ovulatory disorders and infertility. J Reprod Med. 2006; 51:267-82.
42. Frautschy SA, Liptrap RM. Anovulation and plasma hormone concentrations after administration of dexamethasone during the middle of the luteal phase in sows undergoing estrous cycles. Am J Vet Res. 1988; 49:1270-5.
43. Witorsch RJ. Effects of elevated glucocorticoids on reproduction and development: relevance to endocrine disruptor screening. Crit Rev Toxicol. 2016; 46:420-36.
44. Mahajan DK, Billiar RB, Little AB. Isolation of cortisol binding globulin (CBG) from porcine follicular fluid by affinity chromatography. J Steroid Biochem. 1980; 13:67-71.
45. Andersen CY. Concentrations of free oestradiol and progesterone in human preovulatory follicular fluid. Hum Reprod. 1991; 6:359-64.
46. Michael AE, Pester LA, Curtis P, Shaw RW, Edwards CR, Cooke BA. Directinhibition of ovarian steroidogenesis by cortisol and the modulatory role of 11 beta-hydroxysteroid dehydrogenase. Clin Endocrinol (Oxf). 1993; 38:641-4.
47. Hsueh AJ, Erickson GF. Glucocorticoid inhibition of FSH-induced estrogen production in cultured rat granulosa cells. Steroids 1978; 32:639-48.
48. Yang JG, Chen WY, Li PS. Effects of glucocorticoids on maturation of pig oocytes and their subsequent fertilizing capacity in vitro. Biol Reprod. 1999; 60:929-36.
49. Cerdà J, Reich G, Wallace RA, Selman K. Serotonin inhibition of steroid-induced meiotic maturation in the teleost Fundulus heteroclitus: role of cyclic AMP and protein kinases. Mol Reprod Dev. 1998; 49:333-41.
50. Khan-Dawood FS, Dawood MY. Baboon corpus luteum: the effect of melatonin on in vitro progesterone production. Fertil Steril. 1993; 59:896-900.
51. Tanaka E, Baba N, Toshida K, Suzuki K. Serotonin stimulates steroidogenesis in rat preovulatory follicles: involvement of 5-HT2 receptor. Life Sci. 1993; 53:563-70.
52. Bódis J, Török A, Tinneberg HR, Hanf V, Hamori M, Cledon P. Influence of serotonin on progesterone and estradiol secretion of cultured human granulosa cells. Fertil Steril. 1992; 57:1008-11.
53. Graveleau C, Paust HJ, Schmidt-Grimminger D, Mukhopadhyay AK. Presence of a 5-HT7 receptor positively coupled to adenylate cyclase activation in human granulosa-lutein cells. J Clin Endocrinol and Metab. 2000; 85:1277–86.
54. Vesela J, Rehak P, Mihalik J, Czikkova S, Pokorny J, Koppel J. Expression of serotonin receptors in mouse oocytes and preimplantation embryos. Physiological Research. 2003; 52:223–8.
55. Schaeffer HJ, Sirotkin AV. Melatonin and serotonin regulate the release of insulin-like growth factor-I, oxytocin and progesterone by cultured human granulosa cells. Exp Clin Endocrinol Diabetes. 1997; 105:109-12.
56. Amireault P, Dubé F. Intracellular cAMP and calcium signaling by serotonin in mouse cumulus-oocyte complexes. Mol Pharmacol. 2005; 68:1678-87.
57. Amireault P, Dubé F. Serotonin and its antidepressant-sensitive transport in mouse cumulus-oocyte complexes and early embryos. Biol Reprod. 2005; 73:358-65.
58. Vesela J, Rehak P, Mihalik J, Czikkova S, Pokorny J, Koppel J. Expression of serotonin receptors in mouse oocytes and preimplantation embryos. Physiol Res. 2003; 52: 223–8.
59. Fertility treatment in 2014 trends and figures. Retrieved from https://www.hfea.gov.uk/media/1783/fertility-treatment-2014-trends-and-figures.pdf.
60. 吳榮良. 睡眠剝奪對大鼠血清中的睾固酮濃度之影響.長榮大學醫學研究所碩士論文 2011.
61. Andersen ML, Martins PJ. D'Almeida, Bignotto, Tufik S. Endocrinological and catecholaminergic alterations during sleep deprivation and recovery in male rats. J Sleep Res. 2005; 14: 83-90.
62. Andersen M, Tufik S. The effects of testosterone on sleep and sleep-disordered breathing in men: its bidirectional interaction with erectile function. Sleep Med Rev. 2008, 12: 365-79.
63. Axelsson J, Ingre M, Akerstedt T, Holmbäck U, Effects of acutely displaced sleep on testosterone. J Clin Endocrinol Metab. 2005; 90: 4530-5.
64. Balkin TJ, Rupp T, Picchioni D, Wesensten NJ. Sleep loss and sleepiness: current issues. Chest. 2008; 134:653-60.
65. Booth CK, Probert B, Forbes-Ewan C, Coad RA, Australian army recruits in training display symptoms of overtraining. Mil. Med. 2006; 171:1059-64.
66. Rajaratnam SM, Arendt J. Health in a 24-h society. Lancet. 2001; 358:999-1005.
67. Axel’rod J, Reisine TD. Stress hormones:their interaction and regulation. Science. 1984; 224:452-59.
68. Johnson EO, Kamilaris TC, Chrousos GP, Gold PW. Mechanisms of stress: a dynamic overview of hormonal and behavioral homeostasis. Neurosci Biobehav Rev. 1992; 16:115-30.
69. Koehl M, Battle S, Meerlo P. Sex differences in sleep: the response to sleep deprivation and restraint stress in mice. Sleep. 2006; 29:1224-31.
70. Yang CK, Wu RS, Wu CH, Lin TR, Tsai HD. Sleep deprivation enhances peripheral serotonin secretion to regulate the large follicle steroidogenesis of rats. Taiwan J Obstet Gynecol. 2015; 54:260-5.
71. Senthilvelan M, Ravindran R, Samson J, Devi RS. Serotonin turnover in discrete regions of young rat brain after 24 h REM sleep deprivation. Neurochem Res. 2006; 31:81–4.
72. Zant JC, Leenaars CH, Kostin A, Van Someren EJ, Porkka-Heiskanen T. Increases in extracellular serotonin and dopamine metabolite levels in the basal forebrain during sleep deprivation. Brain Res. 2011; 1399:40–8.
73. Machado RB, Suchecki D. Neuroendocrine and Peptidergic Regulation of Stress-Induced REM Sleep Rebound. Front Endocrinol (Lausanne). 2016; 7:163.
74. Heiser P, Dickhaus B, Opper C, Schreiber W, Clement HW, Hasse C, Hennig J, Krieg JC, Wesemann W. Platelet serotonin and interleukin-1 beta after sleep deprivation and recovery sleep in humans. J Neural Transm (Vienna). 1997; 104:1049-58.
75. Huang TJ, Shirley Li P. Dexamethasone inhibits luteinizing hormone-induced synthesis of steroidogenic acute regulatory protein in cultured rat preovulatory follicles. Biol Reprod. 2001; 64:163-70.
76. Nuti KM, Sridharan BN, Meyer RK. Reproductive biology of PMSG-primed immature female rats. Biol Reprod. 1975; 13:38–44.
77. Chen WY, Yang JG, Li PS. Effect of dexamethasone on the expression of p34(cdc2) and cyclin B1 in pig oocytes in vitro. Mol Reprod Dev. 2000; 56:74-9.
78. Tetsuka M, Milne M, Simpson GE, Hillier SG. Expression of 11beta-hydroxy steroid dehydrogenase, glucocorticoid receptor, and mineralocorticoid receptor genes in rat ovary. Biol Reprod. 1999; 60:330-5.
79. McGee EA, Raj RS. Regulators of ovarian preantral follicle development. Semin Reprod Med. 2015; 33:179-84.
80. Garvy BA, King LE, Telford WG, Morford LA, Fraker PJ. Chronic elevation of plasma corticosterone causes reductions in the number of cycling cells of the B lineage in murine bone marrow and induces apoptosis. Immunology. 1993; 80:587-92
81. Warabi S, Tachibana Y, Kumegawa M, Hakeda Y. Dexamethasone inhibits bone resorption by indirectly inducing apoptosis of the bone-resorbing osteoclasts via the action of osteoblastic cells. Cytotechnology. 2001; 35:25-34.
82. Park SY, Jeong MS, Han CW, Yu HS, Jang SB. Structural and functional insight into proliferating cell nuclear antigen. J Microbiol Biotechnol. 2016; 26:637-47.
83. Gittens JE, Mhawi AA, Lidington D, Ouellette Y, Kidder GM. Functional analysis of gap junctions in ovarian granulosa cells: distinct role for connexin43 in early stages of folliculogenesis. Am J Physiol Cell Physiol. 2003; 284:C880-7.
84. Schoonmaker JN, Erickson GF. Glucocorticoid modulation of follicle- stimulating hormone-mediated granulosa cell differentiation. Endocrinology. 1983;113: 1356-63.
85. Raymond JR, Mukhin YV, Gelasco A, Turner J, Collinsworth G, Gettys TW, et al. Multiplicity of mechanisms of serotonin receptor signal transduction. Pharmacol Ther. 2001; 92:179-212.
86. Dubé F, Amireault P. Local serotonergic signaling in mammalian follicles, oocytes and early embryos. Life Sci. 2007; 81:1627-37.
87. Bodis J, Bognar Z, Hartmann G, Torok A, Csaba IF. Measurement of noradrenaline, dopamine and serotonin contents in follicular fluid of human graafian follicles after superovulation treatment. Gynecol and Obstet Invest. 1992; 33:165–67.
88. Terranova PF, Uilenbroek JT, Saville L, Horst D, Nakamura Y. Serotonin enhances oestradiol production by hamster preovulatory follicles in vitro: effects of experimentally induced atresia. J Endocrinol. 1990; 125:433–38.
89. Suzuki K. Serotonin stimulates steroidogenesis in rat preovulatory follicles: involvement of 5-HT2 receptor. Life Sci. 1993; 53:563-70.
90. Stocco DM. A StAR search: implications in controlling steroidgenesis. Biol Reprod. 1997; 56:328–36.