title: Expression of estrogen receptor and maintenance of hormone-responsive phenotype in bovine fetal uterine cells.
abstract: Objectives were to establish conditions for preparation, growth, and maintenance of a primary culture cell model of fetal uterine cells, and to determine whether cells maintained under those conditions would maintain their capacity to respond to estrogen stimulation in vitro. Fetal uteri (n = 19) were enzymatically dispersed and grown on Type 1 collagen in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum. Fetal-uterine cells appeared fibroblast-like and exhibited positive immunostaining for both vimentin and cytokeratin. Effects of gestational age (GA), passage number (p), and hormonal treatment on appearance of specific mRNAs were determined by RT-PCR; relative concentrations of products determined by densitometry were analyzed as the ratio of target cDNA to the GAPDH loading control. Cells expressed mRNAs for estrogen receptor (ER), TGF-beta, EGF-R, PRL-R, IL-1 alpha, and IL-6. ER mRNA was greater at 185-200 than at 100-110 d GA (P < 0.01). All specific mRNAs examined were greater in p5 cells than p2 at both 100-110 (P < 0.01) and 185-200 d GA (P < 0.02). There was no effect of estradiol on these specific mRNAs in cells from 100-110 d GA; at 185-200 d GA, there was an estradiol (1.0 nM) effect both at 6 hr (P < 0.001) and 24 hr (P < 0.02). Overall, there was an effect of 8-br-cAMP (1 mM; 6 h) on specific mRNAs in cells at both 100-110 (P < 0.001) and 185-200 d GA (P < 0.001). In p5 cells from Day 185-200 GA, there was increased cell proliferation (P < 0.001) in response to estradiol (1 nM; 24 hr). These data suggest that primary fetal uterine cells retain their age-specific and hormone-responsive phenotype under these in vitro conditions.
authors.affiliation: Department of Infectious Disease and Physiology, College of Veterinary Medicine, Oklahoma State University, Stillwater 74078-0350, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Malayer
authors.forename: V M
authors.initials: VM
authors.lastname: Woods
mesh_terms: D000818:Animals; D002417:Cattle / Q000196:embryology / Q000235:genetics*; D018929:Cell Culture Techniques / Q000379:methods / Q000662:veterinary; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis; D004958:Estradiol / Q000032:analysis / Q000494:pharmacology*; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D005865:Gestational Age; D007150:Immunohistochemistry; D007633:Keratins / Q000032:analysis; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis; D011960:Receptors, Estrogen / Q000032:analysis / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection / Q000235:genetics; D014599:Uterus / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D014746:Vimentin / Q000032:analysis
chemical_list: D012333:RNA, Messenger; D011960:Receptors, Estrogen; D014746:Vimentin; D004958:Estradiol; D007633:Keratins; D015500:Chloramphenicol O-Acetyltransferase