714 Acta Physiologica Sinica, December 25, 2005, 57 (6): 714-718 http://www.actaps.com.cn 1 2, 2 1 2 330006 (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; R335 +.5 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 330006, 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 2004-07-05 Accepted 2005-09-19 This work was supported by the National Natural Science Foundation of China (No. 30260035). Corresponding author. Tel: +86-791-6363096; E-mail: yuehuizheng@163.com
: (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) 1 1.1 SD 25~29 ( 0219602) 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 W94173 1.2 [14] SD PMSG () 50 IU/ 48 h 1.3 [15] MEM 200 (2 000 r/min 5 min) 1 10 6 /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] 2 10 5 100 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 (4 12 000 r/min 15 min) mrna 5 µl PCR 50 µl ( Tris Taq P 1 -TS P 2 ) 25 10~30 min 1 94 5 min 2 : 94 30 s50 30 s72 90 s 29 72 10 min 1 RNA DNA (TTAGGG) PCR 10% 600 V 40 min 1%
716 Acta Physiologica Sinica, December 25, 2005, 57(6): 714-718 1.5 ELISA PCR PCR (anti-dig-pod) TMB A 450nm A 690nm 0.2 1.6 E 2 P 0 (radioimmunoassay, RIA) 9% 4% 1.7 MTT 96 2 10 6 /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 SPSS11.5 2 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±0.4237 FSH (500 ng/l) 2.521±0.0295 HCG (1 IU/L) 2.465±0.1155 Verapamil (1 µmol/l) 2.628±0.1578 dbcamp (0.1 mmol/l) 2.201±0.2313 Anti-c-myb (20 µmol/l) 1.022±0.0978 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 26.3602±8.098 450.4855±117.50 FSH (500 ng/l) 81.8437±15.069 574.856±125.723 HCG (1 IU/L) 25.6524±6.022 399.289±60.1869 Verapamil (1 µmol/l) 80.508±17.706 360.381±63.946 dbcamp (0.1 mmol/l) 18.350±3.6237 476.2154±172.1996 Anti-c-myb (20 µmol/l) 8.1623±0.9391 305.018±22.691 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)
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