Telomerase: the expression and regulatory mechanisms in preovulatory ovarian granulosa cells

Μέγεθος: px

Εμφάνιση ξεκινά από τη σελίδα:

Download "Telomerase: the expression and regulatory mechanisms in preovulatory ovarian granulosa cells"

Ῥεβέκκα Καψής
5 χρόνια πριν
Προβολές:

1 714 Acta Physiologica Sinica, December 25, 2005, 57 (6): , (telomeric repeat amplification protocol-enzyme linked immunoadsordent assay, TRAP-ELISA) (radioimmunoassay, RIA) (estradiol, E 2 ) (progesterone, P 0 ) MTT( ) (human chorionic gonadotropin, HCG) (follicle-stimulating hormone, FSH)(dbcAMP) (verapamil) c-myb RIA verapamil FSH E 2 P 0 dbcamp HCG c-myb E 2 P 0 MTT htert FSH HCG verapamil dbcamp, Q492.5; R Telomerase: the expression and regulatory mechanisms in preovulatory ovarian granulosa cells ZHANG Jing 1, ZHENG Yue-Hui 2,, ZHENG Li-Ping 2 1 Center of Reproduction; 2 Department of Physiology, School of Medicine, Nanchang University, Nanchang , China Abstract: The objective of this study was to analyze the expression of telomerase in granulosa cells and the influential factors in telomerase expression. TRAP-ELISA (telomeric repeat amplification protocol-enzyme linked immunoadsordent assay) method was used to study the expression and control of telomerase in rat preovulatory ovary. We also used radioimmunoassay (RIA) to determine the expression of estradiol (E 2 ) and progesterone (P 0 ). Furthermore we used MTT to study the proliferation of ovarian granulosa cells. Telomerase activity was significantly enhanced by human chorionic gonadotropin (HCG), follicle-stimulating hormone (FSH), verapamil and dbcamp, and was significantly reduced by antisense-c-myb oligodeoxynucleotide (anti-c-myb ODN) treatment. RIA was used to determine the secretion of P 0 and E 2 in all these cell culture media. We found that the secretion of these two hormones was increased when verapamil and FSH were added; no change after adding HCG and dbcamp; and reduced when anti-c-myb was added. In MTT assay, we found that the antisense htert ODN significantly inhibited the proliferation of ovarian granulosa cells. These results demonstrate that telomerase activity is present in ovary antral granulosa cells and its activity is controlled by FSH, HCG, verapamil and anti-c-myb, and is directly related with the function of proliferation. Key words: preovulatory ovarian granulosa cell; telomere; telomerase; distribution Received Accepted This work was supported by the National Natural Science Foundation of China (No ). Corresponding author. Tel: ; yuehuizheng@163.com

2 : (telomere) TTAGGG [1] (telomerase) [2] [3] RNA(hTR)(TP1/TLP1) (htert) [4] htert [5] [6] [7-9] [10-12] Eisenhauer [13] 1/50 Sprague-Dawley (SD) SD 25~29 ( ) PCR-ELISA (human chorionic gonadotropin, HCG) (follicle-stimulating hormone, FSH) (verapamil) (dbcamp) Sigma c-myb (anti-c-myb ODN): 5'-GTGTCGGGGTCTC- 715 CGGGC-3' htert (anti-htert ODN) : 5'-GGAGCGCGCGGCATCGCGGG-3' W94172 W [14] SD PMSG () 50 IU/ 48 h 1.3 [15] MEM 200 (2 000 r/min 5 min) /ml 37 60~80 /min 15 min 6 (1) (2) HCG (3) FSH (4) dbcamp (5) verapamil (6) c-myb 37 60~80 /min 6 h (estradiol, E 2 ) (progesterone, P 0 ) (telomeric repeat amplification protocol-enzyme linked immunoadsordent assay, TRAP-ELISA) 1.4 TRAP [16] µl [ (mmol/l): Tris-HCl (ph 7.5) 10 MgCl 2 1 EGTA 1 PMSF 0.1 β- 50, 0.5% CHAPS 10% ] 4 1~2 h ( r/min 15 min) mrna 5 µl PCR 50 µl ( Tris Taq P 1 -TS P 2 ) 25 10~30 min min 2 : s50 30 s72 90 s min 1 RNA DNA (TTAGGG) PCR 10% 600 V 40 min 1%

3 716 Acta Physiologica Sinica, December 25, 2005, 57(6): ELISA PCR PCR (anti-dig-pod) TMB A 450nm A 690nm E 2 P 0 (radioimmunoassay, RIA) 9% 4% 1.7 MTT /ml 20 µl 37 ( 5% CO 2 ) 24 h 4 FSH htert FSH+ htert 24 h MTT 20 µl/200 µl MEM 37 4 h 150 µl 5 min ELISA 570 nm 1.8 mean±sd SPSS FSH HCG verapamil dbcamp (P<0.01) verapamil dbcamp (P<0.05); anti-c-myb ODN (P<0.05)( 1 1) 1 FSH HCG verapamil dbcamp c-myb Table 1. Effects of FSH, HCG, verapamil, dbcamp, and anti-c-myb on the telomerase activity in granulosa cells of rat by quantitative image analysis (mean±sd, n=3) Group A Control 1.420± FSH (500 ng/l) 2.521± HCG (1 IU/L) 2.465± Verapamil (1 µmol/l) 2.628± dbcamp (0.1 mmol/l) 2.201± Anti-c-myb (20 µmol/l) 1.022± P<0.05, P<0.01 vs control; P<0.05 vs preantral follicle granulosa cells. 1. Fig.1. Telomerase activity in preovulatory ovarian granulosa cells. 1, dbcamp; 2, FSH; 3, HCG; 4, 5, marker; 6, 7, anti-c-myb ODN; 8, 9, 10, verapamil. dbcamp HCG verapamil FSH (P<0.05); anti-cmyb ODN (P< 0.05) ( 2) 2 E 2 P 0 Table 2. Effects of various factors on granulosa cell-induced P 0 and E 2 in culture medium (mean±sd, n=6) Group P 0 E 2 Control ± ± FSH (500 ng/l) ± ± HCG (1 IU/L) ± ± Verapamil (1 µmol/l) ± ± dbcamp (0.1 mmol/l) ± ± Anti-c-myb (20 µmol/l) ± ± P<0.05, P<0.01 vs control. FSH anti-htert ODN MTT FSH (P<0.01); anti-htert ODN (P<0.01); FSH anti-htert ODN (P<0.01)

4 : 3 MTT Table 3. MTT of various factors on granulosa cells (mean±sd, n=8) Group MTT Control 1.062±0.328 FSH (500 ng/l) 1.562±0.198 Anti-hTERT (20 µmol/l) 0.496±0.147 FSH+anti-hTERT 1.320±0.153 P<0.01 vs control; P<0.05 vs FSH. FSH (P<0.05)( 3) 1 verapamil FSH HCG dbcamp c-myb A 3 FSH [17] HCG FSH FSH dbcamp [18] camp camp (PKA) Ca 2+ [19] [14,20,21] FSH HCG verapamil dbcamp (P<0.01) verapamil 717 dbcamp (P<0.05); anti-c-myb ODN (P<0.05) MTT FSH (P<0.01); anti-htert ODN (P<0.01); anti-htert ODN FSH FSH anti-htert ODN (P<0.01) FSH (P<0.05) 1 Lengauer C. How do tumors make ends meet. Proc Natl Acad Sci USA 2001; 98(22): Saldanha SN, Andrews LG, Tollefesbol TO. Assessment of telomere length and factors that contribute to its stability. Eur J Biochem 2003; 270 (3): Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin

5 718 Acta Physiologica Sinica, December 25, 2005, 57(6): GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE. Extension of life-span by introduction of telomerase into normal human cells. Science 1998; 279(5349): Dahse R, Fiedler W, Ernst G. Telomeres and telomerase: biological and clinical importance. Clinc Chem 1997; 43(5): Kotoula V, Hytrioglon P, Pypasopoulou A, Saxena R, Thung SN, Papadimitriou CS. Expression of human telomerase reverse transcriptase in regenerative and precancerous lesions of cirrhotic livers. Liver 2002; 22: Liu L, Blasco M, Trimarchi J, Keefe D. An essential role for functional telomeres in mouse germ cells during fertilization and early development. Dev Biol 2002; 249(1): Liu WJ ( ), Ding J. Molecular mechanisms involved in the regulation of telomerase activity. Prog Physiol Sci ( ) 2001; 32(3): (Chinese, English abstract). 8 Wang XY ( ), Zhang ZX, Xu YJ, Cheng SX, Xiong WN. Inhibition of telomerase activity and induction of apoptosis in lung cancer cell by human telomerase reverse transcriptase gene antisense oligodeoxynucleotide. Chin J Intern Med ( ) 2002; 41(3): (Chinese, English abstract). 9 Yang XZ ( ), Chen BL, Su MQ, Xin XY, Wang DY. Telomerase activity in ovarian tumors tissues and its clinical significance. Med J Nat Def Forces Northwest China ( ) 2000; 21(4): (Chinese, English abstract). 10 Levy R. Genetic regulation of preimplantation embryo survival. Int Rev Cytol 2001; 210: Betts D, Bordignon V, Hill J, Winger Q, Westhusin M, Smith L, King W. Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle. Proc Natl Acad Sci USA 2001; 98(3): Xu J, Yang X. Telomerase activity in bovine embryos during early development. Biol Reprod 2000; 63(4): Eisenhauer KM, Gerstein RM, Chiu CP, Conti M, Hsueh AJ. Telomerase activity in female and male rat germ cells undergoing meiosis and in early embryos. Biol Reprod 1997; 56(5): Zheng YH ( ), Kuang HB, Zhong ZS, Wu LF, Reng M. Expression of c-myb in various functional states of rat ovaries. Basic Med Sci Clin ( ) 2001; 21(5): (Chinese, English abstract). 15 Zheng YH ( ), Zhong ZS, Kuang HB, Fang L, Jing X. The investigation of c-fos,c-myb,c-erbb-2 on the regulation of HCG-induced progesterone production in rat granulosa cells. Chin J Appl Physiol ( ) 2000; 16 (3): (Chinese, English abstract). 16 Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW. Specific association of human telomerase activity with immortal cells and cancer. Science 1994; 266: Kinugawa C, Muratami T, Okamura K, Yajima A. Telomerase activity in normal ovaries and premature ovarian failure. Tohoku J Exp Med 2000; 190(3): Silva JR, van den Hurk R, Matos MH, Santos RR, Pessoa C, Moraes MO, Figueiredo JR. Influences of FSH and EGF on primordial follicles during in vitro culture of caprine ovarian cortical tissue. Theriogenology 2004; 61(9): Mussche S, Leybaert L, D Herde K. First and second messenger role of calcium: survival versus apoptosis in serum-free cultured granulosa explants. Ann NY Acad Sci 2000; 92(6): Zheng YH ( ), Fang L, Kuang HB, Zou GH. Effect of c-myb on the progesterone and estradiol production of rat luteal cells. Basic Med Sci Clin ( ) 1999; 19 (2): (Chinese, English abstract). 21 Zheng YH ( ), Zhong ZS, Kuang HB, Fang L, Wu QM. Effect of c-fos antisense oligodexynucleotide on the HCGinduced progesterone and estradiol production in rat luteal cells. Acta Zool Sin () 1999; 45(2): (Chinese, English abstract).

Σχετικά έγγραφα

Cellular Physiology and Biochemistry

Cellular Physiology and Biochemistry Original Paper 2016 The Author(s). 2016 Published The Author(s) by S. Karger AG, Basel Published online: November 25, 2016 www.karger.com/cpb Published by S. Karger AG, Basel 486 www.karger.com/cpb Accepted: