Effects of Storage and Passage of Bovine Luteal Endothelial Cells on Endothelin-1 and Prostaglandin F2α Production

• Published in: Journal of Reproduction and Development Vol. 53; no. 3; pp. 473 - 480
• Main Authors: ACOSTA, Tomas J., YOSHIOKA, Shin, KOMIYAMA, Junichi, LEE, Seung-Hyung, GRAZUL-BILSKA, Anna T., SKARZYNSKI, Dariusz J., OKUDA, Kiyoshi
• Format: Journal Article
• Language: English
• Published: THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT 01.06.2007
• Subjects: Bovine; Corpus luteum; Cytokines; Endothelin-1; Endothelium; Prostaglandins
• ISSN: 0916-8818; 1348-4400
• DOI: 10.1262/jrd.18142

To establish a storage system for isolated bovine luteal endothelial cells (LECs), we investigated the basal and tumor necrosis factor (TNF) α-stimulated production of endothelin-1 (ET-1) and prostaglandin (PG) F2α 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 TNFα (50 ng/ml) for 12 h. The basal productions of ET-1 and PGF2α by the unfrozen and frozen/thawed LECs were similar at passage 2. The basal production of PGF2α 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 TNFα increased (P<0.05) ET-1 and PGF2α 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.