title: Establishment of adult mouse Sertoli cell lines by using the starvation method.
abstract: Sertoli cells were isolated from the testes of 6-week-old mice and stable Sertoli cell lines with higher proliferation rates were subcloned after starvation of primary cultured cells. After two rounds of this subcloning, 33 subcloned lines were selected on the basis of their proliferation rates. In addition, these subclones were screened according to their phagocytic activity and the characteristics of mature Sertoli cells, such as the expression of androgen receptors (ARs) and progesterone receptors, by using western blotting and immunocytochemical analysis, in addition to their morphology and proliferation rates. After the third round of subcloning, 12 subclones were selected for the final selection using RT-PCR for identification of genes specifically expressed by various testicular cells. Three clones were selected that expressed Sertoli-cell-specific genes, i.e., stem cell factor, clusterin, AR, α-inhibin, transferrin, Wilms' tumour-1, Müllerian inhibitory substance, sex-determining region Y-box 9, FSH receptor (Fshr) and occludin; however, these clones did not express globulin transcription factor 1, steroidogenic factor or androgen-binding protein. These clones also expressed growth and differentiation factors that act on germ cells, such as leukaemia inhibitory factor, transforming growth factor β1 and basic fibroblast growth factor 2, but did not express c-kit (specific for germ cells), LH receptor and 3β-hydroxyl-dehydrogenase (specific for Leydig cells). Immunocytochemical data confirmed the expression of clusterin in these clones. Furthermore, the Bromodeoxyuridine incorporation assay confirmed the proliferation activity of these clones through Fshr after treatment with FSH. These clones are considered to be valuable tools for the study of Sertoli cell-specific gene expression and function.
authors.affiliation: Department of Urology, St Marianna University School of Medicine, 2-16-1, Sugao, Kawasaki, Kanagawa 216-8511, Japan. pxx13340@nifty.ne.jp
authors.forename: Yoko
authors.initials: Y
authors.lastname: Sato
authors.forename: Kaoru
authors.initials: K
authors.lastname: Yoshida
authors.forename: Shiari
authors.initials: S
authors.lastname: Nozawa
authors.forename: Miki
authors.initials: M
authors.lastname: Yoshiike
authors.forename: Michiko
authors.initials: M
authors.lastname: Arai
authors.forename: Takeshige
authors.initials: T
authors.lastname: Otoi
authors.forename: Teruaki
authors.initials: T
authors.lastname: Iwamoto
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D033481:Chromosomes, Mammalian / Q000378:metabolism; D002999:Clone Cells; D051152:Clusterin / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D015870:Gene Expression; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010587:Phagocytosis; D012333:RNA, Messenger / Q000378:metabolism; D011944:Receptors, Androgen / Q000378:metabolism; D011980:Receptors, Progesterone / Q000378:metabolism; D012708:Sertoli Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D015415:Biomarkers; C495243:Clu protein, mouse; D051152:Clusterin; D003470:Culture Media; D012333:RNA, Messenger; D011944:Receptors, Androgen; D011980:Receptors, Progesterone