10199728 2004 5
AP alkaline phosphatase ATP adenosine triphosphate BCIP 5-bromo-4-chloro-3-indolyl- phosphate 5 4 3 camp cyclic adenosine monophosphate CBAVD congenital bilateral absence of the vas deferens CF cystic fibrosis CFTR cystic fibrosis transmembrane conductance regulator CUAVD congenital unilateral absence of the vas deferens DAB diaminobenzidine DPC Diphenylamine-2-carboxylate E 2 estrogen ENaC epithelial Na conductance FBS fetal bovine serum FSH follicle stimulating hormone FSK forskolin GAPDH glyceraldehydes phosphate dehydrogenase HBSS Hank s blanced salt solution Hank's LH luteinizing hormone IL-1 interleukin-1-1 IOD integrated optical density 1
mrna messenger ribonucleic acid MSD membrane-spanning domain NBD nucleotide binding domain NBT nitrobenzene thiocyanate PAF platelet activiting factor PBS phosphate buffer saline PGE 2 prostaglandings E 2 E 2 PGF 2 prostaglandings F 2 F 2 P progesterone PI pancreatic insufficiency PMSF Phenylmethylsulfonyl fluoride RT-PCR reverse transcription-polymerase chain reaction SDS sodium decdecyl sulfate SP streptavidin-peroxidase - SSC sodium chloride/sodium citrate buffer TBS tris-buffered saline Tris TEMED N,N,N,N -tetra methy lethylene diamine N,N,N,N - Tris trishydroxymethylaminomethane 2
camp (CFTR) CFTR CFTR western blotting CFTR mrna 1 CFTR mrna, 2 CFTRmRNA 3 CFTR mrna, western blotting 170KDa CFTR RT-PCR CFTR CFTRmRNA CFTR CFTRmRNA CFTRmRNA CFTR mrna CFTRmRNA CFTR CFTR CFTR CFTR forskolin( ) forskolin 3
DPC CFTR camp camp CFTR PGE 2 forskolin PGF2 CFTR [ ] camp 4
ABSTRACT BACKGROUND: As a camp-regulated Cl - channel, cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in the active secretion of electrolytes and fluid in epithelial cells. Women with CFTR gene mutations are less fertile generally assumed to cervical factors. However, there is little known about CFTR protein expression in human endometrium and its possible roles in reproduction. METHODS AND RESULTS: CFTR protein and mrna levels in human endometrium were analyzed using immunohistochemical and in situ hybridization methods respectively. Significant expression of CFTR protein was only seen in the glandular cells from late-proliferative to all secretory phases, consistent with western blot analysis. While CFTR mrna was presented only around ovulatory period with high levels. In cultured glandular cells, the productions of CFTR protein and mrna were all stimulated by estradiol and inhibited by progesterone. Forskolin-activated Cl - current in endometrial epithelial cells with a linear I/V relationship was detected by whole-cell patch clamp technique. The stimulated channel activities were suppressed by DPC, a CFTR channel blocker. Furthermore, PGE 2 -activated Cl - current also was detected. However the current magnitude induced by PGE 2 was significantly reduced than that stimulated by forskolin. CONCLUSIONS: (1) CFTR mrna and protein localized in human endometrial epithelial cells and the amounts were varied cyclically; (2) CFTR expression in cultured glandular cells was up- and down- regulated by estradiol and progesterone respectively; (3) CFTR in human endometrium functions as a camp-activated Cl - channel. It can be regulated by PGE 2 and play an important role in modulating ion composition of human uterine fluid. 5
In summary, the present work was the first to demonstrate the expression of CFTR protein and mrna was cyclic dependent in human endometrium during the menstrual period. The expression of functional CFTR in human endometrial epithelium was thought to be associated with the composition of human uterine fluid. The abundant CFTR mrna and protein around the ovulatory period may drive Na + and fluid from plasma into the uterine lumen to make optimal electrolyte composition and sufficient fluid volume for sperm migration and capacitation. In addition, continuable expression of CFTR proteins has been detected in all secretory phase. Prostaglandin E 2 released by gametes or embryos could activated CFTR channel and thereby inducing chloride ion and water across the endometrial epithelium, it is believed to benefit embryo implantation. Further research of CFTR regulation may improve our knowledge about human reproductive physiology and be helpful to facilitate embryo implantation in treatment of infertile patients by using assisted reproductive technology. [Key words] cystic fibrosis transmembrane conductance regulator; endometrium; camp- activated chloride channel; patch clamp 6
cystic fibrosis transmembrane conductance regulator CFTR camp CFTR cystic fibrosis,cf 95 98% [1,2] CF [3] CFTR CFTR CFTR [4] 1989 Riordan CF [5] 7q31 250kb 27 cdna 6129bp 1480 168kDa ATP (ATP-binding cassette) (motif), membrane-spanning domain MSD nucleotide binding domain, NBD, R- (regulatory domain,rd) R 1991 [6,7,8] CFTR camp 6-10ps, 7
- I-V, Br - Cl - >I - >F -, [9] CFTR CFTR CFTR PDZ PDZ [10,11] CFTR [12,13] CFTR 5 [14] CFTR CFTR R ATP NBD Carson 8
[15] : camp A(PKA) R ATP NBD C3 NBD1 ATP (O1) NBD2 ATP [16] NBD NBD ATP NBD ADP, NBD ATP 3 CFTR CFTR CFTR camp, camp CFTR, CFTR epithelial Na conductance, ENaC CFTR [17,18] CFTR 9
[19] 1 PKA [20] ; 2 CFTR ENaC [21] ; 3 ENaC [22], CFTR CFTR/ENaC Devidas CFTR ATP [23] ATP G CFTR [24] [25,26] [27] GTP- CFTR CFTR [28,29] CFTR CF CFTR camp [30] Ajonuma CFTR [31] CFTR CFTR camp,, \, 10
( ) 30 [32] 2000 CF 1/26 [33] CF 1/3500 [34] 1/15876 [35] 1/9 [36] CFTR CFTR 1000 (The Cystic Fibrosis Genetic Analysis Consortium; http:www.genet.sickkids.on.ca/cftr) F508, (1) F508, 10 3, 508 CF, CF 70 (2) F508,, 40 (3) 1 2 (4) 20 (5) 10 (6), intron8 5T,, [37], CFTR, CFTR NBF 9,10,11,12 19,20,21, 3,4,7 17b, NBF1 11, 5 11
( )CF DeltaF508 (1/n) >0.5%) percent Finland 25000 4 46.2 Lucotte et al. [1995]; Kere et al. [1994] Mexico 8500 15 41.6 Grebe et al. [1994] Sweden 7300 10 66.6 Lucotte et al. [1995]; Schaedel et al. [1999]; Poland 6000 11 57.1 Romeo et al. [1989] North Ireland 5350 7 56.6 Lucotte et al. [1995] Russia 4900 12 54.5 Romeo et al. [1989] Denmark 4700 6 87.5 Lucotte et al. [1995] Norway 4500 6 60.2 Lucotte et al. [1995] Netherlands 3650 9 74.2 Lucotte et al. [1995] Spain 3500 21 52.7 Lucotte et al. [1995]; Collee et al. [1998] Greece 3500 22 52.9 Lucotte et al. [1995] Germany 3300 17 71.8 Lucotte et al. [1995] United States 2835 10 68.6 Lucotte et al. [1995]; Romeo et al. [1989] Czech Republic 2833 10 70 Kosorok et al. [1996]; Romeo et al. [1989] United Kingdom 2600 5 75.3 Lucotte et al. [1995]; Romeo et al. [1989] Australia 2500 8 76.9 Lucotte et al. [1995]; Romeo et al. [1989] Italy 2438 9 50.9 Romeo et al. [1989] France 2350 12 67.7 Lucotte et al. [1995]; Gasparini et al. [1999]; Switzerland 2000 9 57.2 Romeo et al. [1989] Ireland 1800 7 70.4 Lucotte et al. [1995] 12
11 245 18.98 CFTR : CFTR [38], 5 1 CFTR,, 7% CF ; 2 CFTR, CF, F508, CF 85 ; 3 CFTR, camp,, 3% CF ; 4 ; 5, mrna CFTR,, CFTR CF : CFTR, [39], (pancreatic insufficiency, PI) [40], severe mutation mild mutation 13
CFTR pancreatic sufficiency, PS CF F508 CFTR >100mmol/L CF CF CF [41] CF CFTR CFTR ( ) CFTR 1993 Trezise CFTR [42] Tizzano CFTRmRNA [43], CFTR CFTR,cAMP Cl - [44],, [45], [46], [47], [48], [49], CFTR CFTR,, CFTR CFTR [50] CFTR 14
[43] Sertoli Leydig Sertoli Sertoli CFTR [51] CFTR CFTR Intron8 5T CF Boockfor Sertoli CFTRmRNA [52] camp CFTR ( ) CFTR 1972 Taussig CF [53] CF 1 CFTR (congenital absence of the vas deferens, CAVD) 6% 14% CAVD 18% 50% [54] congenital bilateral absence of the vas deferens, CBAVD 15