雌性個體中的類固醇荷爾蒙黃體酮與雌激素主要由卵巢顆粒細胞製造。在顆粒細胞中，黃體酮由前驅物膽固醇所衍生而成，而雌激素由苯環化酶催化雄性素(源自於鞘膜細胞)而得。實驗室過去的研究發現腦下腺分泌之卵泡促素(FSH)可刺激顆粒細胞合成類固醇荷爾蒙，而卵巢內的細胞激素轉型乙型生長因子(TGFbeta1)可以更強化FSH的作用。在一般非類固醇生成細胞(non-steroidogenic cells)中，胞內膽固醇含量受恆定性調節──高量的膽固醇致使HMG-CoA reductase (膽固醇生合成之關鍵酶)以及低密度脂蛋白受體(LDLR, 用以攝入低密度脂蛋白)表現下降。而類固醇生成細胞卻額外表現了高密度脂蛋白受體SR-BI。這些瞭解引發此論文之研究動機，並分為兩個主要研究目的：第一個研究目的是調查FSH和TGF1如何調控獲取膽固醇之相關分子HMG-CoA reductase, LDLR 與SR-BI，此涵括調查這些分子之表現量以及參與類固醇生合成之活性，以及高量的固醇(sterol)如何影響這些分子的表現與功能。另外，我們近期發現了FSH和TGFbeta1透過calcineurin活化CRTC2 (一個CREB共轉錄因子)以提升黃體酮生成相關之基因表現，承接此發現，第二個研究目的為調查FSH和TGFbeta1如何透過calcineurin和CRTC2調控苯環化酶基因(Cyp19a1)表現。
實驗發現，HMG-CoA reductase抑制劑 simvastatin大幅降低了FSH±TGFbeta1所促進之黃體酮生合成，並且，FSH+TGFbeta1增加了細胞內HMG-CoA reductase的表現。此顯示細胞需要膽固醇生合成的增加以製造類固醇荷爾蒙。TGFbeta1亦促進FSH的效應而增加了脂蛋白受體SR-BI和LDLR的表現。給予細胞hHDL3和hLDL皆增加了黃體酮生合成，而SR-BI抑制劑BLT-1完全抑制了hHDL3和部分抑制hLDL的效果，證實SR-BI和LDLR確實具有獲取膽固醇的功能，並用以增加黃體酮生合成。使用具中性脂肪親和力之螢光染劑BODIPY493/503，可觀察到攝入之膽固醇亦儲存於胞內脂肪小滴。格外重要的是，LDLR與SR-BI對於固醇的調節有不同的敏感性。給予細胞脂蛋白或25-hydroxycholesterol (細胞通透之膽固醇類似物)降低了Ldlr的表現，而Scarb1 (SR-BI基因)的表現僅有在25-hydroxycholesterol和aminoglutethimide (類固醇生成抑制劑)共同處理造成固醇大量累積的狀況下才開始降低。最後，我們使用染色質免疫沉澱法證明了固醇對於Ldlr和Scarb1的影響是透過轉錄因子SREBP-2和LRH-1的作用。
我們接著探討calcineurin和CRTC2如何參與在FSH和TGF1對Cyp19a1基因轉錄之調控。首先，FSH經24小時作用後增加Cyp19a1蛋白表現，而此效應在48小時後減弱，TGFbeta1則可增加FSH作用而維持Cyp19a1高表現量。前處理calcineurin auto-inhibitory peptide (CNI)可抑制FSH與TGFbeta1共同增加之苯環化酶活性，但不影響FSH單獨處理之效應，而在Cyp19a1基因表現也可觀察到相似的調節現象。卵巢細胞的Cyp19a1 PII啟動子含有CREB和核受體LRH-1/NR5A2、SF-1/NR5A1的結合位，而Nr5a2啟動子亦含有CREB結合位。在FSH和TGF1刺激下，CRTC2結合Cyp19a1 PII啟動子和Nr5a2啟動子的程度增加了(control < FSH < FSH+TGFbeta1),，但CREB的結合卻不受影響。LRH-1和SF-1的表現在FSH與TGF1共同處理下被提升而此受CNI所抑制，FSH單獨處理則不具效應。FSH與TGFbeta1亦促使LRH-1和SF-1結合至Cyp19a1 PII啟動子。這些結果告訴我們FSH+TGFbeta1透過calcineurin活化CRTC2以增加Cyp19a1表現，而calcineurin亦可以間接透過先增加NR5A核受體再提升Cyp19a1表現量。
總結而論，我們首先發現了卵巢顆粒細胞可自行製造膽固醇，並如非類固醇生成細胞一般具有膽固醇恆定調節之機轉，而其又另用SR-BI來規避此恆定調節機轉，以攝入高量膽固醇來提供給大量黃體酮生合成所需。我們亦發現，FSH和TGFbeta1可透過calcineurin和CRTC2來活化Cyp19a1基因表現以增加雌激素生合成。而NR5A核受體在這些過程中都扮演了重要的調節角色。

Ovarian granulosa cells are the major producer of female sex steroids progesterone and estrogen. Granulosa cells synthesize progesterone from the steroid precursor cholesterol, whereas estrogen is produced through aromatase catalysis of theca cell-derived androgen. Ovarian local factor TGFbeta1 facilitates the action of pituitary hormone FSH to promote granulosa cell steroidogenesis. It is known that in non-steroidogenic cells, cellular cholesterol is under homeostatic control involving feedback inhibition of HMG-CoA reductase (key enzyme for cholesterol de novo synthesis) and LDLR (receptor for LDL uptake), while steroidogenic cells additionally express the HDL receptor SR-BI. There are two objectives in this study. The first objective was to investigate how FSH and TGFbeta1 regulate HMG-CoA reductase, LDLR, and SR-BI in granulosa cells. This includes their 1) expression and functional involvement in steroidogenesis, and 2) responsiveness to sterol challenge and identification of potential transcription regulators. We have recently identified that FSH and TGFbeta1 induce calcineurin-dependent activation of CREB coactivator CRTC2 to upregulate steroidogenic gene expression. The second objective was to investigate how calcineurin and CRTC2 are involved in FSH and TGFbeta1 stimulation of estrogen synthesis through controlling aromatase gene (Cyp19a1) expression. Primary culture of granulosa cells from ovarian antral follicles of gonadotropin-primed immature rats was used.
HMG-CoA reductase inhibitor simvastatin suppressed the basal and FSH±TGFbeta1-stimulated progesterone synthesis, and additionally, treatment with FSH+TGFbeta1 increased HMG-CoA reductase mRNA and protein level, suggesting the involvement of cholesterol de novo synthesis during steroid synthesis in granulosa cells. Moreover, TGFbeta1 potentiated FSH to upregulate SR-BI and LDLR, and both are functional in uptaking cholesterol as hHDL3 and hLDL supplementation enhanced progesterone production, and the effect of each lipoprotein was completely or partially blocked by SR-BI selective inhibitor BLT-1. Uptaken cholesterol could also be stored in lipid droplets, and this was evidenced by utilization of the fluorescent dye BODIPY 493/503 that stains for neutral lipid. Importantly, LDLR and SR-BI responded to sterol with different sensitivity. Giving cells lipoproteins or 25-hydroxycholesterol downregulated Ldlr but not Scarb1; Scarb1 was ultimately downregulated by excessive sterol accumulation under 25-hydroxycholesterol and aminoglutethimide (inhibitor of steroidogenesis) cotreatment. Finally, ChIP analysis indicated that transcription factors SREBP-2 and LRH-1 crucially mediate Ldlr and Scarb1 differential response to sterol challenge.
Next, we asked whether FSH and TGFbeta1 regulate Cyp19a1 through calcineurin and CRTC2. We first observed FSH increased aromatase protein at 24 h which subsided by 48 h, while FSH+TGFbeta1 exerted a greater and longer effect. Further, pretreatment with calcineurin inhibitor (CNI) abolished the FSH+TGFbeta1- but not FSH-upregulated aromatase activity at 48 h, and corresponding changes of Cyp19a1 mRNA was observed at 24 h. Ovary-specific Cyp19a1 PII-promoter contains crucial response elements for CREB and nuclear receptors LRH-1/NR5A2 and SF-1/NR5A1, and Nr5a2 promoter has a CREB-binding site. Chromatin immunoprecipitation analysis revealed FSH and TGFbeta1 increased CRTC2 binding to Cyp19a1 PII-promoter and Nr5a2 promoter at 24 h post-treatment (control < FSH < FSH+TGFbeta1), while CREB was constitutively bound. Simultaneously, FSH+TGFbeta1 but not FSH alone increased the expression of LRH-1 and SF-1 and their binding to Cyp19a1 PII-promoter, and expression of both was suppressed by CNI. These results implicate that calcium-dependent calcineurin importantly mediates FSH and TGFbeta1 upregulation of Cyp19a1, acting directly through CRTC2 to enhance CREB transcriptional activity, and indirectly through upregulation of NR5As.
In conclusion, this thesis study first uncovers that ovarian granulosa cells are capable of de novo synthesizing cholesterol and retain the cholesterol homeostatic control machinery like non-steroidogenic cells, while during active steroidogenesis utilize SR-BI to evade such feedback control. Secondly, calcineurin and CRTC2 are critically involved in FSH and TGFbeta1 upregulation of Cyp19a1 transcription for estrogen synthesis. Remarkably, the NR5A nuclear receptors play important regulatory roles in both events.

Contents
Abstract...................................................i
中文摘要.................................................iii
Abbreviations..............................................v
Study background...........................................1
1. The ovary..............................................2
A. Ovarian follicle development..........................2
B. Ovarian steroidogenesis...............................4
C. FSH signaling in ovarian granulosa cells..............8
D. TGFbeta action in ovarian follicle development........9
E. Calcineurin and CRTC2 regulation of CREB activity....11
F. Role of NR5A nuclear receptors in ovarian development11
G. Role of aromatase and ovarian-specific PII-promoter..13
2. Control of cholesterol utilization and implications in the ovary................................................14
A. Cholesterol uptake from lipoproteins.................14
B. Cellular cholesterol homeostasis control.............18
3. Objectives............................................19
Materials and methods 21
Part 1. Ovarian granulosa cells utilize SR-BI to evade cholesterol homeostatic control for steroid synthesis.....29
Introduction.............................................29
Results..................................................31
Discussion...............................................37
Part 2. Calcineurin and CREB coactivator CRTC2 importantly mediate FSH and TGFbeta1 collateral upregulation of Cyp19a1 and Nr5a expression in rat ovarian granulosa cells........58
Introduction.............................................58
Results..................................................59
Discussion...............................................62
Overview..................................................73
Acknowledgment............................................76
References................................................77
Appendix: publication containing parts of this work.......90

List of figures and table
Figure 1. Ovarian steroidogenesis..........................7
Figure 2. Effect of simvastatin on FSH and TGFbeta1-induced progesterone production in granulosa cells................42
Figure 3. FSH and TGFbeta1 regulation of HMG-CoA reductase mRNA and protein expression in granulosa cells............43
Figure 4. FSH and TGFbeta1 regulation of SR-BI and LDLR protein expression in granulosa cells.....................44
Figure 5. FSH and TGFbeta1 regulation of SR-BI and LDLR subcellular localization in granulosa cells...............45
Figure 6. Effect of BLT-1 on hHDL3 supplementation in FSH and TGFbeta1-induced steroidogenesis......................46
Figure 7. Effect of BLT-1 on hLDL supplementation in FSH and TGFbeta1-induced steroidogenesis......................47
Figure 8. Fluorescent staining of neutral lipids in steroidogenic granulosa cells.............................48
Figure 9. Effect of hHDL3 supplementation on FSH and TGFbeta1-induced expression of cholesterogenic genes in granulosa cells...........................................49
Figure 10. Effect of hLDL supplementation on FSH and TGFbeta1-induced expression of cholesterogenic genes in steroidogenic granulosa cells.............................50
Figure 11. Effect of 25-OHC and AMG on FSH and TGFbeta1-induced expression of cholesterogenic genes in granulosa cells.....................................................51
Figure 12. FSH and TGFbeta1 regulation of SREBP-2 in granulosa cells...........................................52
Figure 13. FSH and TGFbeta1 regulation of LRH-1 in granulosa cells...........................................53
Figure 14. FSH and TGFbeta1 regulation of SREBP-2 and LRH-1 to Scarb1 and Ldlr promoters in granulosa cells...........54
Figure 15. Effect of 25-OHC and AMG on FSH and TGFbeta1 upregulation of lipoprotein receptors and key transcription factors SREBP-2, LRH-1, SR-BI, and LDLR in granulosa cells.....................................................55
Figure 16. A proposed working model for FSH and TGFbeta1 regulation of cholesterol uptake in steroidogenic granulosa cells.....................................................56
Figure 17. Effect of calcineurin inhibitor on FSH±TGFbeta1 stimulation of aromatase in granulosa cells...............66
Figure 18. Effect of FSH and TGFbeta1 regulation of CRTC2 and CREB binding to Cyp19a1 PII-promoter..................67
Figure 19. FSH and TGFbeta1 regulation of SF-1 in granulosa cells.....................................................68
Figure 20. Effect of FSH and TGFbeta1 regulation of LRH-1 and SF-1 binding to Cyp19a1 PII-promoter..................69
Figure 21. Effect of calcineurin inhibitor (CNI) on FSH and TGFbeta1 regulation of NR5A expression....................70
Figure 22. Effect of FSH and TGFbeta1 on CRTC2 and CREB binding to Nr5a2 promoter.................................71
Figure 23. A proposed working model of FSH and TGFbeta1 regulation of Cyp19a1 expression through calcineurin and CRTC2.....................................................72
Table 1. Primer pairs used in RT-PCR and ChIP analyses....28

References
1. Edson MA, Nagaraja AK, Matzuk MM. The mammalian ovary from genesis to revelation. Endocr Rev 2009; 30:624-712.
2. Knight PG, Glister C. TGF-b superfamily members and ovarian follicle development. Reproduction 2006; 132:191-206.
3. Ke FC, Chuang LC, Lee MT, Chen YJ, Lin SW, Wang PS, Stocco DM, Hwang JJ. The modulatory role of transforming growth factor β1 and androstenedione on follicle-stimulating hormone-induced gelatinase secretion and steroidogenesis in rat granulosa cells. Biol Reprod 2004; 70:1292-1298.
4. Dodson WC, Schomberg DW. The effect of transforming growth factor-b on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells. Endocrinology 1987; 120:512-516.
5. Dorrington JH, Bendell JJ, Khan SA. Interactions between FSH, estradiol-17b and transforming growth factor-b regulate growth and differentiation in the rat gonad. J Steroid Biochem Mol Biol 1993; 44:441-447.
6. Ke F-C, Fang S-H, Lee M-T, Sheu S-Y, Lai S-Y, Chen YJ, Huang F-L, Wang PS, Stocco DM, Hwang J-J. Lindane, a gap junction blocker, suppresses FSH and transforming growth factor b1-induced connexin43 gap junction formation and steroidogenesis in rat granulosa cells. J Endocrinol 2005; 184:555-566.
7. Chen YJ, Hsiao PW, Lee MT, Mason JI, Ke FC, Hwang JJ. Interplay of PI3K and cAMP/PKA signaling, and rapamycin-hypersensitivity in TGFb1 enhancement of FSH-stimulated steroidogenesis in rat ovarian granulosa cells. J Endocrinol 2007; 192:405-419.
8. Chen YJ, Lee MT, Yao HC, Hsiao PW, Ke FC, Hwang JJ. Crucial role of estrogen receptor-alpha interaction with transcription coregulators in follicle-stimulating hormone and transforming growth factor b1 up-regulation of steroidogenesis in rat ovarian granulosa cells. Endocrinology 2008; 149:4658-4668.
9. Fang W-L, Lee M-T, Wu L-S, Chen Y-J, Mason J, Ke F-C, Hwang J-J. CREB coactivator CRTC2/TORC2 and its regulator calcineurin crucially mediate follicle-stimulating hormone and transforming growth factor β1 upregulation of steroidogenesis. J Cell Physiol 2012; 227:2430-2440.
10. Goldstein JL, DeBose-Boyd RA, Brown MS. Protein sensors for membrane sterols. Cell 2006; 124:35-46.
11. Payne AH, Hales DB. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr Rev 2004; 25:947-970.
12. Bulun SE, Sebastian S, Takayama K, Suzuki T, Sasano H, Shozu M. The human CYP19 (aromatase P450) gene: update on physiologic roles and genomic organization of promoters. J Steroid Biochem Mol Biol 2003; 86:219-224.
13. Sánchez F, Smitz J. Molecular control of oogenesis. Biochim Biophys Acta Mol Basis Dis 2012; 1822:1896-1912.
14. Richards JS. Perspective: The ovarian follicle--A perspective in 2001. Endocrinology 2001; 142:2184-2193.
15. Kidder G, Mhawi A. Gap junctions and ovarian folliculogenesis. Reproduction 2002; 123:613-620.
16. Richards JS, Russell DL, Ochsner S, Hsieh M, Doyle KH, Falender AE, Lo YK, Sharma SC. Novel signaling pathways that control ovarian follicular development, ovulation, and luteinization. Recent Prog Horm Res 2002; 57:195-220.
17. Saragüeta PE, Lanuza GM, Barañao JL. Autocrine role of transforming growth factor β1 on rat granulosa cell proliferation. Biol Reprod 2002; 66:1862-1868.
18. Juengel JL, McNatty KP. The role of proteins of the transforming growth factor-b superfamily in the intraovarian regulation of follicular development. Hum Reprod Update 2005; 11:144-161.
19. Ingman WV, Robker RL, Woittiez K, Robertson SA. Null mutation in transforming growth factor beta1 disrupts ovarian function and causes oocyte incompetence and early embryo arrest. Endocrinology 2006; 147:835-845.
20. Russell DL, Robker RL. Molecular mechanisms of ovulation: co-ordination through the cumulus complex. Hum Reprod Update 2007; 13:289-312.
21. Stocco C, Telleria C, Gibori G. The molecular control of corpus luteum formation, function, and regression. Endocr Rev 2007; 28:117-149.
22. Simard J, Ricketts M-L, Gingras S, Soucy P, Feltus FA, Melner MH. Molecular biology of the 3β-Hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase gene family. Endocr Rev 2005; 26:525-582.
23. Jamnongjit M, Hammes RS. Ovarian steroids: the good, the bad, and the signals that raise them. Cell Cycle 2006; 5:1178-1183.
24. Hunzicker-Dunn M, Maizels ET. FSH signaling pathways in immature granulosa cells that regulate target gene expression: Branching out from protein kinase A. Cell Sig 2006; 18:1351-1359.
25. Wayne CM, Fan H-Y, Cheng X, Richards JS. Follicle-stimulating hormone induces multiple signaling cascades: evidence that activation of rous sarcoma oncogene, RAS, and the epidermal growth factor receptor are critical for granulosa cell differentiation. Mol Endocrinol 2007; 21:1940-1957.
26. Mayr B, Montminy M. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol 2001; 2:599-609.
27. Riccio A, Alvania RS, Lonze BE, Ramanan N, Kim T, Huang Y, Dawson TM, Snyder SH, Ginty DD. A nitric oxide signaling pathway controls CREB-mediated gene expression in neurons. Mol Cell 2006; 21:283-294.
28. Conkright MD, Canettieri G, Screaton R, Guzman E, Miraglia L, Hogenesch JB, Montminy M. TORCs: transducers of regulated CREB activity. Mol Cell 2003; 12:413-423.
29. Altarejos JY, Montminy M. CREB and the CRTC co-activators: sensors for hormonal and metabolic signals. Nat Rev Mol Cell Biol 2011; 12:141-151.
30. Flores JA, Veldhuis JD, Leong DA. Follicle-stimulating hormone evokes an increase in intracellular free calcium ion concentrations in single ovarian (granulosa) cells. Endocrinology 1990; 127:3172-3179.
31. Carnegie JA, Tsang BK. The calcium-calmodulin system: participation in the regulation of steroidogenesis at different stages of granulosa cell differentiation. Biol Reprod 1984; 30:515-522.
32. Tsang BK, Carnegie JA. Calcium-dependent regulation of progesterone production by isolated rat granulosa cells: effects of the calcium ionophore A23187, prostaglandin E2, dl-isoproterenol and cholera toxin. Biol Reprod 1984; 30:787-794.
33. Jayes FC, Day RN, Garmey JC, Urban RJ, Zhang G, Veldhuis JD. Calcium ions positively modulate follicle-stimulating hormone- and exogenous cyclic 3',5'-adenosine monophosphate-driven transcription of the P450(scc) gene in porcine granulosa cells. Endocrinology 2000; 141:2377-2384.
34. Takemori H, Kanematsu M, Kajimura J, Hatano O, Katoh Y, Lin XZ, Min L, Yamazaki T, Doi J, Okamoto M. Dephosphorylation of TORC initiates expression of the StAR gene. Mol Cell Endocrinol 2007; 265-266:196-204.
35. Franke TF. PI3K/Akt: getting it right matters. Oncogene 2008; 27:6473-6488.
36. Schmierer B, Hill CS. TGF[beta]-SMAD signal transduction: molecular specificity and functional flexibility. Nat Rev Mol Cell Biol 2007; 8:970-982.
37. Zhang YE. Non-Smad pathways in TGF-b signaling. Cell Res 2009; 19:128-139.
38. Teerds KJ, Dorrington JH. Immunohistochemical localization of transforming growth factor-b1 and -b2 during follicular development in the adult rat ovary. Mol Cell Endocrinol 1992; 84:R7-R13.
39. Ying S-Y, Becker A, Ling N, Ueno N, Guillemin R. Inhibin and beta type transforming growth factor (TGFβ) have opposite modulating effects on the follicle stimulating hormone (FSH)-induced aromatase activity of cultured rat granulosa cells. Biochem Biophys Res Commun 1986; 136:969-975.
40. Gitay-Goren H, Kim IC, Miggans ST, Schomberg DW. Transforming growth factor beta modulates gonadotropin receptor expression in porcine and rat granulosa cells differently. Biol Reprod 1993; 48:1284-1289.
41. Inoue K, Nakamura K, Abe K, Hirakawa T, Tsuchiya M, Matsuda H, Miyamoto K, Minegishi T. Effect of transforming growth factor β on the expression of luteinizing hormone receptor in cultured rat granulosa cells. Biol Reprod 2002; 67:610-615.
42. Gong X, McGee EA. Smad3 is required for normal follicular follicle-stimulating hormone responsiveness in the mouse. Biol Reprod 2009; 81:730-738.
43. Anttonen M, Parviainen H, Kyrönlahti A, Bielinska M, Wilson DB, Ritvos O, Heikinheimo M. GATA-4 is a granulosa cell factor employed in inhibin-α activation by the TGF-β pathway. J Mol Endocrinol 2006; 36:557-568.
44. Bennett J, Wu Y-G, Gossen J, Zhou P, Stocco C. Loss of GATA-6 and GATA-4 in granulosa cells blocks folliculogenesis, ovulation, and follicle stimulating hormone receptor expression leading to female infertility. Endocrinology 2012; 153:2474-2485.
45. Li H, Rao A, Hogan PG. Interaction of calcineurin with substrates and targeting proteins. Trends Cell Biol 2011; 21:91-103.
46. Screaton RA, Conkright MD, Katoh Y, Best JL, Canettieri G, Jeffries S, Guzman E, Niessen S, Yates JR, 3rd, Takemori H, Okamoto M, Montminy M. The CREB coactivator TORC2 functions as a calcium- and cAMP-sensitive coincidence detector. Cell 2004; 119:61-74.
47. Conkright MD, Canettieri G, Screaton R, Guzman E, Miraglia L, Hogenesch JB, Montminy M. TORCs: transducers of regulated CREB activity. Mol Cell 2003; 12:413-423.
48. Uebi T, Tamura M, Horike N, Hashimoto YK, Takemori H. Phosphorylation of the CREB-specific coactivator TORC2 at Ser307 regulates its intracellular localization in COS-7 cells and in the mouse liver. Am J Physiol Endocrinol Metab 2010; 299:E413-E425.
49. Jansson D, Ng AC-H, Fu A, Depatie C, Al Azzabi M, Screaton RA. Glucose controls CREB activity in islet cells via regulated phosphorylation of TORC2. Proc Natl Acad Sci USA 2008; 105:10161-10166.
50. Fayard E, Auwerx J, Schoonjans K. LRH-1: an orphan nuclear receptor involved in development, metabolism and steroidogenesis. Trends Cell Biol 2004; 14:250-260.
51. Hoivik EA, Lewis AE, Aumo L, Bakke M. Molecular aspects of steroidogenic factor 1 (SF-1). Mol Cell Endocrinol 2010; 315:27-39.
52. Lazarus KA, Wijayakumara D, Chand AL, Simpson ER, Clyne CD. Therapeutic potential of liver receptor homolog-1 modulators. J Steroid Biochem Mol Biol 2012; 130:138-146.
53. Yazawa T, Inanoka Y, Mizutani T, Kuribayashi M, Umezawa A, Miyamoto K. Liver receptor homolog-1 regulates the transcription of steroidogenic enzymes and induces the differentiation of mesenchymal stem cells into steroidogenic cells. Endocrinology 2009; 150:3885-3893.
54. Saxena D, Escamilla-Hernandez R, Little-Ihrig L, Zeleznik AJ. Liver receptor homolog-1 and steroidogenic factor-1 have similar actions on rat granulosa cell steroidogenesis. Endocrinology 2007; 148:726-734.
55. Yazawa T, Inaoka Y, Okada R, Mizutani T, Yamazaki Y, Usami Y, Kuribayashi M, Orisaka M, Umezawa A, Miyamoto K. PPAR-γ coactivator-1α regulates progesterone production in ovarian granulosa cells with SF-1 and LRH-1. Mol Endocrinol 2010; 24:485-496.
56. Jeyasuria P, Ikeda Y, Jamin SP, Zhao L, de Rooij DG, Themmen APN, Behringer RR, Parker KL. Cell-specific knockout of steroidogenic factor 1 reveals its essential roles in gonadal function. Mol Endocrinol 2004; 18:1610-1619.
57. Duggavathi R, Volle DH, Mataki C, Antal MC, Messaddeq N, Auwerx J, Murphy BD, Schoonjans K. Liver receptor homolog 1 is essential for ovulation. Genes Dev 2008; 22:1871-1876.
58. Pelusi C, Ikeda Y, Zubair M, Parker KL. Impaired follicle development and infertility in female mice lacking steroidogenic factor 1 in ovarian granulosa cells. Biol Reprod 2008; 79:1074-1083.
59. Falender AE, Lanz R, Malenfant D, Belanger L, Richards JS. Differential expression of steroidogenic factor-1 and FTF/LRH-1 in the rodent ovary. Endocrinology 2003; 144:3598-3610.
60. Park Y, Maizels ET, Feiger ZJ, Alam H, Peters CA, Woodruff TK, Unterman TG, Lee EJ, Jameson JL, Hunzicker-Dunn M. Induction of cyclin D2 in rat granulosa cells requires FSH-dependent relief from FOXO1 repression coupled with positive signals from Smad. J Biol Chem 2005; 280:9135-9148.
61. Britt K, Findlay J. Estrogen actions in the ovary revisited. J Endocrinol 2002; 175:269-276.
62. Rosenfeld C, Wagner J, Roberts R, Lubahn D. Intraovarian actions of oestrogen. Reproduction 2001; 122:215-226.
63. Couse JF, Korach KS. Estrogen receptor null mice: what have we learned and where will they lead us? Endocr Rev 1999; 20:358-417.
64. Mendelsohn ME, Karas RH. The protective effects of estrogen on the cardiovascular system. N Engl J Med 1999; 340:1801-1811.
65. Stein DG. Brain damage, sex hormones and recovery: a new role for progesterone and estrogen? Trends Neurosci 2001; 24:386-391.
66. Yager JD, Davidson NE. Estrogen carcinogenesis in breast cancer. N Engl J Med 2006; 354:270-282.
67. Stocco C. Aromatase expression in the ovary: Hormonal and molecular regulation. Steroids 2008; 73:473-487.
68. Mendelson CR, Kamat A. Mechanisms in the regulation of aromatase in developing ovary and placenta. J Steroid Biochem Mol Biol 2007; 106:62-70.
69. Britt KL, Drummond AE, Dyson M, Wreford NG, Jones MEE, Simpson ER, Findlay JK. The ovarian phenotype of the aromatase knockout (ArKO) mouse. J Steroid Biochem Mol Biol 2001; 79:181-185.
70. Fisher CR, Graves KH, Parlow AF, Simpson ER. Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene. Proc Natl Acad Sci USA 1998; 95:6965-6970.
71. Kraemer FB. Adrenal cholesterol utilization. Mol Cell Endocrinol 2007; 265-266:42-45.
72. Hegele RA. Plasma lipoproteins: genetic influences and clinical implications. Nat Rev Genet 2009; 10:109-121.
73. Jeon H, Blacklow SC. Sturcture and physiologic function of the low-density lipoprotein receptor. Ann Rev Biochem 2005; 74:535-562.
74. Tobert JA. Lovastatin and beyond: the history of the HMG-CoA reductase inhibitors. Nat Rev Drug Discov 2003; 2:517-526.
75. Connelly MA, Williams DL. SR-BI and cholesterol uptake into steroidogenic cells. Trends Endocrinol Metab 2003; 14:467-472.
76. Connelly MA. SR-BI-mediated HDL cholesteryl ester delivery in the adrenal gland. Mol Cell Endocrinol 2009; 300:83-88.
77. Rigotti A, Trigatti BL, Penman M, Rayburn H, Herz J, Krieger M. A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism. Proc Natl Acad Sci USA 1997; 94:12610-12615.
78. Trigatti B, Rayburn H, Viñals M, Braun A, Miettinen H, Penman M, Hertz M, Schrenzel M, Amigo L, Rigotti A, Krieger M. Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology. Proc Natl Acad Sci USA 1999; 96:9322-9327.
79. Jiménez LM, Binelli M, Bertolin K, Pelletier RM, Murphy BD. Scavenger receptor-B1 and luteal function in mice. J Lipid Res 2010; 51:2362-2371.
80. Miettinen HE, Rayburn H, Krieger M. Abnormal lipoprotein metabolism and reversible female infertility in HDL receptor (SR-BI)–deficient mice. J Clin Invest 2001; 108:1717-1722.
81. Azhar S, Luo Y, Medicherla S, Reaven E. Upregulation of selective cholesteryl ester uptake pathway in mice with deletion of low-density lipoprotein receptor function. J Cell Physiol 1999; 180:190-202.
82. Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science 1996; 271:518-520.
83. Reaven E, Chen YD, Spicher M, Hwang SF, Mondon CE, Azhar S. Uptake of low density lipoproteins by rat tissues. Special emphasis on the luteinized ovary. J Clin Invest 1986; 77:1971-1984.
84. Azhar S, Nomoto A, Leers-Sucheta S, Reaven E. Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model. J Lipid Res 1998; 39:1616-1628.
85. Reaven E, Tsai L, Azhar S. Cholesterol uptake by the 'selective' pathway of ovarian granulosa cells: early intracellular events. J Lipid Res 1995; 36:1602-1617.
86. Chapman MJ. Animal lipoproteins: chemistry, structure, and comparative aspects. J Lipid Res 1980; 21:789-853.
87. Soto E, Silavin SL, Tureck RW, Strauss JF. Stimulation of progesterone synthesis in luteinized human granulosa cells by human chorionic gonadotropin and 8-bromo-adenosine 3',5'- monophosphate: The effect of low density lipoprotein. J Clin Endocrinol Metab 1984; 58:831-837.
88. Azhar S, Tsai L, Medicherla S, Chandrasekher Y, Giudice L, Reaven E. Human granulosa cells use high density lipoprotein cholesterol for steroidogenesis. J Clin Endocrinol Metab 1998; 83:983-991.
89. Illingworth DR, Kenny TA, Orwoll ES. Adrenal function in heterozygous and homozygous hypobetalipoproteinemia. J Clin Endocrinol Metab 1982; 54:27-33.
90. Illingworth DR, Lees AM, Lees RS. Adrenal cortical function in homozygous familial hypercholesterolemia. Metabolism 1983; 32:1045-1052.
91. Vergeer M, Korporaal SJA, Franssen R, Meurs I, Out R, Hovingh GK, Hoekstra M, Sierts JA, Dallinga-Thie GM, Motazacker MM, Holleboom AG, Van Berkel TJC, et al. Genetic variant of the scavenger receptor BI in humans. N Engl J Med 2011; 364:136-145.
92. Reaven E, Tsai L, Spicher M, Shilo L, Philip M, Cooper AD, Azhar S. Enhanced expression of granulosa cell low density lipoprotein receptor activity in response to in vitro culture conditions. J Cell Physiol 1994; 161:449-462.
93. Heikkilä P, Arola J, Liu J, Kahri A. ACTH regulates LDL receptor and CLA-1 mRNA in the rat adrenal cortex. Endocr Rev 1998; 24:591-593.
94. Cherian-Shaw M, Puttabyatappa M, Greason E, Rodriguez A, VandeVoort CA, Chaffin CL. Expression of scavenger receptor-BI and low-density lipoprotein receptor and differential use of lipoproteins to support early steroidogenesis in luteinizing macaque granulosa cells. Endocrinology 2009; 150:957-965.
95. Bengoechea-Alonso MT, Ericsson J. SREBP in signal transduction: cholesterol metabolism and beyond. Curr Opin Cell Biol 2007; 19:215-222.
96. Radhakrishnan A, Goldstein JL, McDonald JG, Brown MS. Switch-like control of SREBP-2 transport triggered by small changes in ER cholesterol: a delicate balance. Cell Metab 2008; 8:512-521.
97. Fitzpatrick SL, Richards JS. Identification of a cyclic adenosine 3',5'-monophosphate-response element in the rat aromatase promoter that is required for transcriptional activation in rat granulosa cells and R2C leydig cells. Mol Endocrinol 1994; 8:1309-1319.
98. Hinshelwood MM, Repa JJ, Shelton JM, Richardson JA, Mangelsdorf DJ, Mendelson CR. Expression of LRH-1 and SF-1 in the mouse ovary: localization in different cell types correlates with differing function. Mol Cell Endocrinol 2003; 207:39-45.
99. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955; 34:1345-1353.
100. Converse CA, Skinner ER. Lipoprotein analysis: a practical approach. Oxford, UK.: IRL Press; 1992: 103-110.
101. Karpe F, Hamsten A. Determination of apolipoproteins B-48 and B-100 in triglyceride-rich lipoproteins by analytical SDS-PAGE. J Lipid Res 1994; 35:1311-1317.
102. Gillies RJ, Didier N, Denton M. Determination of cell number in monolayer cultures. Anal Biochem 1986; 159:109-113.
103. Carey MF, Peterson CL, Smale ST. Chromatin immunoprecipitation (ChIP). Cold Spring Harb Protoc 2009; 2009:pdb.prot5279.
104. Chang T-Y, Chang CCY, Ohgami N, Yamauchi Y. Cholesterol sensing, trafficking, and esterification. Annu Rev Cell Dev Biol 2006; 22:129-157.
105. Hu J, Zhang Z, Shen W-J, Azhar S. Cellular cholesterol delivery, intracellular processing and utilization for biosynthesis of steroid hormones. Nutr Metab 2010; 7:47.
106. Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 1997; 89:331-340.
107. Lopez D, McLean MP. Sterol regulatory element-binding protein-1a binds to cis elements in the promoter of the rat high density lipoprotein receptor SR-BI gene. Endocrinology 1999; 140:5669-5681.
108. Miller WL, Bose HS. Early steps in steroidogenesis: intracellular cholesterol trafficking. J Lipid Res 2011; 52:2111-2135.
109. Adams CM, Reitz J, De Brabander JK, Feramisco JD, Li L, Brown MS, Goldstein JL. Cholesterol and 25-hydroxycholesterol inhibit activation of SREBPs by different mechanisms, both involving SCAP and insigs. J Biol Chem 2004; 279:52772-52780.
110. Schoonjans K, Annicotte JS, Huby T, Botrugno OA, Fayard E, Ueda Y, Chapman J, Auwerx J. Liver receptor homolog 1 controls the expression of the scavenger receptor class B type I. EMBO Rep 2002; 3:1181-1187.
111. Azhar S, Cooper A, Tsai L, Maffe W, Reaven E. Characterization of apoB, E receptor function in the luteinized ovary. J Lipid Res 1988; 29:869-882.
112. Kovacs EJ, Messingham KAN, Gregory MS. Estrogen regulation of immune responses after injury. Mol Cell Endocrinol 2002; 193:129-135.
113. Michael MD, Michael LF, Simpson ER. A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450 (CYP19) gene. Mol Cell Endocrinol 1997; 134:147-156.
114. Tsang BK, Carnegie JA. Calcium requirement in the gonadotropic regulation of rat granulosa cell progesterone production. Endocrinology 1983; 113:763-769.
115. Parakh TN, Hernandez JA, Grammer JC, Weck J, Hunzicker-Dunn M, Zeleznik AJ, Nilson JH. Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires β-catenin. Proc Natl Acad Sci USA 2006; 103:12435-12440.
116. Brown KA, McInnes KJ, Hunger NI, Oakhill JS, Steinberg GR, Simpson ER. Subcellular localization of cyclic AMP-responsive element binding protein-regulated transcription coactivator 2 provides a link between obesity and breast cancer in postmenopausal women. Cancer Res 2009; 69:5392-5399.
117. Annicotte J-S, Chavey C, Servant N, Teyssier J, Bardin A, Licznar A, Badia E, Pujol P, Vignon F, Maudelonde T, Lazennec G, Cavailles V, et al. The nuclear receptor liver receptor homolog-1 is an estrogen receptor target gene. Oncogene 2005; 24:8167-8175.
118. Bouchard MF, Taniguchi H, Viger RS. Protein kinase A-dependent synergism between GATA factors and the nuclear receptor, liver receptor homolog-1, regulates human aromatase (CYP19) PII promoter activity in breast cancer cells. Endocrinology 2005; 146:4905-4916.
119. Yeh Y-T. Molecular mechanism of follicle-stimulating hormone (FSH) and transforming growth factor (TGFb1) regulation of aromatase expression. Taipei, Taiwan: National Yang-Ming University; 2010. Thesis.
120. Velasco M, Alexander C, King J, Zhao Y, Garcia J, Rodriguez A. Association of lower plasma estradiol levels and low expression of scavenger receptor class B, type I in infertile women. Fertil Steril 2006; 85:1391-1397.