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, a-inhibin, transferrin, Wilms' tumour-1, Mü llerian 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 b1 and basic fibroblast growth factor 2, but did not express c-kit (specific for germ cells), LH receptor and 3b-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. Reproduction (2013) 145 505-516

miR-762 promotes porcine immature Sertoli cell growth via the ring finger protein 4 (RNF4) gene

“…Each Sertoli cell can support limited germ cells; therefore, the Sertoli cell number in seminiferous tubules is important for germ cell production28. Immature Sertoli cells determine the final number of mature Sertoli cells1629. Our results indicated that miR-762 promoted cell proliferation, inhibited cell apoptosis, and accelerated DNA damage repair in immature Sertoli cells, thus suggesting a potential function of miR-762 in pig spermatogenesis.…”

Functional importance of JMY expression by Sertoli cells in mediating mouse spermatogenesis

“…Primary Sertoli cell culture was carried out as described (Sato et al, 2013; Holembowski et al, 2014) with some modifications. Briefly, to obtain Sertoli cells for primary culture, testes of adult mice were decapsulated in HBSS and seminiferous tubules were dispersed in a HBSS solution containing collagenase (0.1%)/hyaluronidase (0.1%)/DNase (0.04%) for 20 min at 34°C.…”

Establishment and applications of male germ cell and Sertoli cell lines

“…1 (Blagosklonova et al 2002, Sharpe et al 2003, Hai et al 2014, whereas AR, SGP2 ( Sulfated glycoprotein 2), GATA1 and p27 kip1 can be used as mature hallmarks for these cells (Regadera et al 2001, Sharpe et al 2003, Sato et al 2013. In addition, WT1, GATA4, SOX9, VIM, SCF and GDNF serve as whole-stage markers for Sertoli cells.…”

“…In addition, this cell line has recently been utilized to elucidate the pharmacological roles and ERK/Akt signaling pathway of diosgenin and offer novel therapeutic methods to treat male infertility (Wu et al 2015). Starvation method has also been applied to generate three Sertoli cell lines, including A31C4, C22A4 and C24A4 (Sato et al 2013). Similar to TM4 cell line, these Sertoli cell lines were created without inducing ectogenic oncogenes, and thus their behavior is close to primary Sertoli cells and they can be applied to basic research on the interactions among male germ cells and Sertoli cells.…”

“…Additionally, the G5, A31C4, C22A4 and C24A4 Sertoli cell lines can secret numerous growth factors, e.g. leukemia inhibitory factor (LIF), transforming growth factor-β (TGF-β) and bFGF (Hofmann et al 2003, Sato et al 2013, which are extremely important for the survival and proliferation of SSCs. Therefore, these Sertoli cell lines might be used as an excellent feeder layer for long-term culture and expansion of SSCs.…”