title: Effects of storage and passage of bovine luteal endothelial cells on endothelin-1 and prostaglandin F2alpha production.
abstract: To establish a storage system for isolated bovine luteal endothelial cells (LECs), we investigated the basal and tumor necrosis factor (TNF) alpha-stimulated production of endothelin-1 (ET-1) and prostaglandin (PG) F2alpha in unfrozen and frozen-thawed LECs until passage 10. LECs were obtained from developing corpora lutea (CL; days 5-7 of the estrous cycle) using enzymatic digestion and magnetic beads coated with lectin BS-1. The LECs were frozen at -80 C or further cultured and/or passaged until passage 10 in DMEM/Ham's F-12 supplemented with 10% calf serum. The hormonal productions of unfrozen and frozen/thawed LECs were compared through passages 2-10. When both the unfrozen and frozen/thawed cells reached confluence, the culture medium was replaced with fresh medium containing 0.1% bovine serum albumin (BSA), and the cells were incubated with TNFalpha (50 ng/ml) for 12 h. The basal productions of ET-1 and PGF2alpha by the unfrozen and frozen/thawed LECs were similar at passage 2. The basal production of PGF2alpha by LECs was not altered by passage and storage at -80 C, whereas the basal production of ET-1 decreased from passage 2 and 3 to passage 4 in the unfrozen LECs and from passage 2 to passage 3 in the frozen/thawed LECs. However, production of ET-1 by the unfrozen and frozen/thawed LECs was similar between passages 4-10 and passages 3-10, respectively. Exposure of LECs to TNFalpha increased (P<0.05) ET-1 and PGF2alpha production by the unfrozen and frozen-thawed LECs in all passages examined. Thus, LECs obtained from developing CLs and stored until passage 10 can be used for study of the physiology of LECs in vitro.
authors.affiliation: Laboratory of Reproductive Endocrinology, Graduate School of Natural Science and Technology, Okayama University, Japan.
authors.forename: Tomas J
authors.initials: TJ
authors.lastname: Acosta
authors.forename: Shin
authors.initials: S
authors.lastname: Yoshioka
authors.forename: Junichi
authors.initials: J
authors.lastname: Komiyama
authors.forename: Seung-Hyung
authors.initials: SH
authors.lastname: Lee
authors.forename: Anna T
authors.initials: AT
authors.lastname: Grazul-Bilska
authors.forename: Dariusz J
authors.initials: DJ
authors.lastname: Skarzynski
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Okuda
mesh_terms: D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D003338:Corpus Luteum / Q000166:cytology*; D015237:Dinoprost / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D019332:Endothelin-1 / Q000378:metabolism*; D005260:Female; D005615:Freezing; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D019332:Endothelin-1; D014409:Tumor Necrosis Factor-alpha; D015237:Dinoprost