Effects of glial cell line-derived neurotrophic factor, fibroblast growth factor 2 and epidermal growth factor on proliferation and the expression of some genes in buffalo (Bubalus bubalis) spermatogonial cells

2013 ◽  
Vol 25 (8) ◽  
pp. 1149 ◽  
Author(s):  
Prashant H. Kadam ◽  
Sushila Kala ◽  
Himanshu Agrawal ◽  
Karn P. Singh ◽  
Manoj K. Singh ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Fibroblast Growth Factor ◽  
Cell Line ◽  
Glial Cell ◽  
Sertoli Cell ◽  
Neurotrophic Factor ◽  
Feeder Layer ◽  
Fibroblast Growth ◽  
Epidermal Growth

The present study evaluated the effects of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF) 2 and epidermal growth factor (EGF) on proliferation and the expression of some genes in spermatogonial cells. Spermatogonial cells were isolated from prepubertal buffalo testes and enriched by double enzyme treatment, filtration through 80- and 60-μm nylon mesh filters, differential plating on lectin-coated dishes and Percoll density gradient centrifugation. Cells were then cultured on a buffalo Sertoli cell feeder layer and formed colonies within 15–18 days. The colonies were found to predominantly contain undifferentiated Type A spermatogonia because they bound Dolichos biflorus agglutinin and did not express c-kit. The colonies expressed alkaline phosphatase, NANOG, octamer-binding transcription factor (OCT)-4 and tumour rejection antigen (TRA)-1–60. Cells were subcultured for 15 days, with or without growth factor supplementation. After 15 days, colony area and the relative mRNA abundance of PLZF were higher (P < 0.05) following supplementation with 40 ng mL–1 GDNF + 10 ng mL–1 EGF + 10 ng mL–1 FGF2 than with the same concentrations of GDNF alone or GDNF plus either EGF or FGF2. Expression of TAF4B was higher (P < 0.05) in the presence of FGF2, whereas the expression of THY1 was not affected by growth factor supplementation. In the Sertoli cell feeder layer, EGF and FGF2 decreased (P < 0.05), whereas GDNF increased (P < 0.05), the relative mRNA abundance of ETV5 compared with control. In conclusion, an in vitro culture system that incorporates various growth factors was developed for the short-term culture of buffalo spermatogonia.

scholarly journals SNT, a differentiation-specific target of neurotrophic factor-induced tyrosine kinase activity in neurons and PC12 cells.

1993 ◽  
Vol 13 (4) ◽  
pp. 2203-2213 ◽  
Author(s):  
S J Rabin ◽  
V Cleghon ◽  
D R Kaplan
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Fibroblast Growth Factor ◽  
Pc12 Cells ◽  
Neurotrophic Factor ◽  
Kinase Activity ◽  
Fibroblast Growth ◽  
Tyrosine Kinase Activity ◽  
Epidermal Growth

To elucidate the signal transduction mechanisms used by ligands that induce differentiation and the cessation of cell division, we utilized p13suc1-agarose, a reagent that binds p34cdc2/cdk2. By using this reagent, we identified a 78- to 90-kDa species in PC12 pheochromocytoma cells that is rapidly phosphorylated on tyrosine following treatment with the differentiation factors nerve growth factor (NGF) and fibroblast growth factor but not by the mitogens epidermal growth factor or insulin. This species, called SNT (suc-associated neurotrophic factor-induced tyrosine-phosphorylated target), was also phosphorylated on tyrosine in primary rat cortical neurons treated with the neurotrophic factors neurotrophin-3, brain-derived neurotrophic factor, and fibroblast growth factor but not in those treated with epidermal growth factor. In neuronal and fibroblast cells, where NGF can also act as a mitogen, SNT was tyrosine phosphorylated to a much greater extent during NGF-induced differentiation than during NGF-induced proliferation. SNT was phosphorylated in vitro on serine, threonine, and tyrosine in p13suc1-agarose precipitates from NGF-treated PC12 cells, indicating that this protein may be a substrate of kinase activities associated with p13suc1-p34cdc2/cdk2 complexes. In addition, SNT was associated predominantly with nuclear fractions following subcellular fractionation of NGF-treated PC12 cells. Finally, in PC12 cells, NGF-stimulated tyrosine phosphorylation of SNT was dependent on the levels of Trk tyrosine kinase activity and was constitutively induced by expression of pp60v-src. However, Ras was not required for constitutive SNT tyrosine phosphorylation, suggesting that this protein functions distally to Trk and pp60v-src but in a pathway parallel to that of Ras. SNT is the first identified specific target of differentiation factor-induced tyrosine kinase activity in neuronal cells.

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