Proliferation Of TM4 Cells Research Articles

Flurochloridone induces Sertoli cell apoptosis through ROS-dependent mitochondrial pathway

Flurochloridone (FLC), a selective herbicide used on a global scale, has been reported to have male reproductive toxicity which underlying mechanism is still largely unknown. The present study was conducted to determine the effects of FLC on Sertoli cell and explore its mechanism by using normal mouse Sertoli (TM4) cell line. Our data indicate that FLC suppressed proliferation of TM4 cells in a dose- and time-dependent manner. Further studies confirmed that FLC induced apoptosis in TM4 cells, accompanied by reactive oxygen species (ROS) accumulation, intracellular calcium increase, opening of mitochondrial permeability transition pore, depolarization of the mitochondrial membrane potential (MMP) and decrease of adenosine triphosphate (ATP) level. Meanwhile, changes of B-cell lymphoma-2 (Bcl-2) family proteins expression, release of cytochrome c and the activation of caspase-9 and caspase-3 were also confirmed. These results indicate that FLC induces TM4 cells apoptosis through the mitochondrial apoptotic pathway. In addition, pretreatment with ROS scavenger N-acetyl-L-cysteine (NAC), could significantly alleviate FLC-induced TM4 cells apoptosis and MMP depolarization. In conclusion, our results suggested that FLC induced TM4 cells apoptosis and it was regulated by mitochondrial dysfunction and oxidative stresses.

Dibutyl phthalate promotes juvenile Sertoli cell proliferation by decreasing the levels of the E3 ubiquitin ligase Pellino 2

BackgroundA previous study showed that dibutyl phthalate (DBP) exposure disrupted the growth of testicular Sertoli cells (SCs). In the present study, we aimed to investigate the potential mechanism by which DBP promotes juvenile SC proliferation in vivo and in vitro.MethodsTimed pregnant BALB/c mice were exposed to vehicle, or DBP (50, 250, and 500 mg/kg/day) from 12.5 days of gestation until delivery. In vitro, CCK-8 and EdU incorporation assays were performed to determine the effect of monobutyl phthalate (MBP), the active metabolite of DBP, on the proliferation of TM4 cells, which are a juvenile testicular SC cell line. Western blotting analysis, quantitative PCR (q-PCR), and flow cytometry were performed to analyse the expression of genes and proteins related to the proliferation and apoptosis of TM4 cells. Coimmunoprecipitation was used to determine the relationship between the ubiquitination of interleukin 1 receptor-associated kinase 1 (IRAK1) and the effect of MBP on promoting the proliferation of TM4 cells.ResultsIn the 50 mg/kg/day DBP-exposed male mice offspring, the number of SCs was significantly increased. Consistent with the in vivo results, in vitro experiments revealed that 0.1 mM MBP treatment promoted the proliferation of TM4 cells. Furthermore, the data showed that 0.1 mM MBP-mediated downregulation of the E3 ubiquitin ligase Pellino 2 (Peli2) increased ubiquitination of IRAK1 by K63, which activated MAPK/JNK signalling, leading to the proliferation of TM4 cells.ConclusionsPrenatal exposure to DBP led to abnormal proliferation of SCs in prepubertal mice by affecting ubiquitination of the key proliferation-related protein IRAK1 via downregulation of Peli2.

Signaling related with biphasic effects of bisphenol A (BPA) on Sertoli cell proliferation: A comparative proteomic analysis

BackgroundBiphasic effects on cell proliferation of bisphenol A (BPA) can occur at lesser or greater exposures. Sertoli cells play a pivotal role in supporting proliferation and differentiation of germ cells. The mechanisms responsible for inverse effects of great and low concentrations of BPA on Sertoli cell proliferation need further study. MethodsWe utilized proteomic study to indentify the protein expression changes of Sertoli TM4 cells treated with 10−8M and 10−5M BPA. The further mechanisms related to mitochondria, energy metabolism and oxidative stress were investigated by qRT-PCR and Western-blotting analysis. ResultsProteomic studies identified 36 proteins and two major clusters of proteins including energy metabolism and oxidative stress expressed with opposite changes in Sertoli cells treated with 10−8M and 10−5M BPA, respectively, for 24h. Exposure to 10−5M BPA resulted in greater oxidative stress and then inhibited cell proliferation, while ROS scavenger NAC effectively blocked these effects. Exposure to 10−8M BPA caused higher intercellular ATP, greater activities of mitochondria, and resulted in significant proliferation of TM4 cells, while oligomycin A, an inhibitor of ATP synthase, abolished these growth advantages. ConclusionsOur study demonstrated that micromolar BPA inhibits proliferation of Sertoli cells by elevating oxidative stress while nanomolar BPA stimulates proliferation by promoting energy metabolism. General significanceMicromolar BPA inhibits cell proliferation by elevating oxidative stress while nanomolar BPA stimulates cell proliferation by promoting energy metabolism.

Involvement of activating ERK1/2 through G protein coupled receptor 30 and estrogen receptor α/β in low doses of bisphenol A promoting growth of Sertoli TM4 cells

Sertoli cells play a pivotal role in supporting proliferation of germ cells and differentiation during spermatogenesis in mammals. Nanomolar concentrations of Bisphenol A (BPA) can significantly stimulate the proliferation of mouse immature Sertoli (TM4) cells. However, mechanisms by which BPA caused these effects were still unclear. In the present study, an inverse U-shaped curve was observed when treating TM4 cells with increasing doses of BPA: 1 to 10nM BPA significantly stimulated the proliferation of TM4 cells and increased the proportion of cells in S phase; >1μM BPA caused lesser proliferation of cells. Exposure of TM4 cells to G15 or ICI 182,780, which are specific antagonists of GPR30 and estrogen receptor α/β (ERα/β), respectively, abolished BPA-induced proliferation of cells, which suggests that both GPR30 and ERα/β were involved in the observed effects of BPA. Furthermore, exposure to BPA caused rapid (5min) activation of ERK1/2 via both GPR30 and ERα/β. Blocking the GPR30/EGFR signal transduction pathway by antagonists suppressed both phosphorylation of ERK and BPA-induced cell proliferation. BPA up-regulated mRNA and protein expression of GPR30 in a concentration-dependent manner. In summary, the results reported here indicated that activating ERK1/2 through GPR30 and ERα/β is involved in low doses of BPA that promoted growth of Sertoli TM4 cells. The GPR30/EGFR/ERK signal is the downstream transduction pathway in BPA-induced proliferation of TM4 Sertoli cells.

The Seminiferous Growth Factor Induces Proliferation of TM4 Cells in Serum-Free Medium1

The seminiferous growth factor (SGF) of the mammalian testes induces DNA synthesis and cell proliferation of Balb/c 3T3 cells (Bellvé and Feig, 1984; Rec Prog Hormone Res 40:531-567). In this study, SGF was purified 80,000- to 100,000-fold from calf testes and used to examine the growth of TM4 cells in a chemically defined medium. Cells were seeded sparsely in Dulbecco's Modified Eagles/Ham's F12 medium (1:1;v:v) (DME/F12 degrees), containing epidermal growth factor (EGF; 1 ng/ml), insulin (1; 10 micrograms/ml), and transferrin (Tr; 5 micrograms/ml) (DME/F12). After 24 h, the medium was replaced with DME/F12 degrees supplemented with SGF, EGF, 1, or Tr, in two-, three- or four-way combinations. Cell numbers were quantified after another 48 h of culture. EGF, I, and Tr, alone or in two-way combinations, were not mitogenic for TM4 cells. By contrast, SGF (1 U) alone, or with any two of these factors, stimulated TM4 cell proliferation to commensurate levels, and to twofold greater numbers than occurred with the combination of EGF, I, and Tr. Synergisms or inhibitions were not measurable. Follicle-stimulating hormone, luteinizing hormone, prolactin, acidic fibroblast growth factor, or basic fibroblast growth factor was weakly or not mitogenic for TM4 cells. The effect of SGF on cell proliferation was inhibited by 1 microM - 1 nM retinoic acid, but not by retinol or retinyl acetate. SGF was mitogenic for bovine adrenal capillary endothelial cells, an effect that was potentiated by 10 micrograms heparin/ml. Thus, SGF can induce proliferation of TM4 cells and capillary endothelial cells. The former provides a sensitive, and selective, serum-free, bioassay system for SGF activity.

Adverse effects of gossypol analogs on TM4 cell mitochondrial function

Three aqueous-soluble gossypol Schiff's bases, SP562: bis-8,8′-[(N-(2-(dimethylamino) ethyl))-iminomethylene]-1, 1′,6,6′,7,7′-hexahydroxy-5,5′-diisopropyl-3,3′-dimethyl-2,2′-binaphthalene dihydrochloride; SP563: bis-8,8′-[(N-(2-(diethylamino)ethyl))-iminomethylene]-1, 1′,6,6′,7,7′-hexahydroxy-5, 5′-diisopropyl-3,3′-dimethyl-2,2′-binaphthalene dthydrochloride; and SP564: bis-8,8′-[(N-(2-(diethylamino)propyl))-iminomethylene]-1, 1′,6,6′,7,7′-[hexahydroxy-5, 5′-dilsopropyl-3,3′-dimethyl-2,2′-binaphthalene dihydrochloride, were examined for their effects on TM4 cell mitochondrial function and proliferation. Among the three gossypol analogs, SP562 had the most severe effect on decreasing TM4 cell population that accumulated rhodamine 123 into their mitochondria. This adverse effect exerted by SP562, however, was not as strong as that caused by (±)gossypol acetic acid of the same molarity. On the other hand, SP564 had the most severe effect on proliferation of TM4 cells. This severe effect was even more so than that caused by an equimolarity of (±)gossypol acetic acid.