Retinol-induced changes in the phosphorylation levels of histones and high mobility group proteins from Sertoli cells

Chromatin proteins play a role in the organization and functions of DNA. Covalent modifications of nuclear proteins modulate their interactions with DNA sequences and are probably one of the multiple factors involved in the process of switch on/off transcriptionally active regions of DNA. Histones and high mobility group proteins (HMG) are subject to many covalent modifications that may modulate their capacity to bind to DNA. We investigated the changes induced in the phosphorylation pattern of cultured Wistar rat Sertoli cell histones and high mobility group protein subfamilies exposed to 7 µM retinol for up to 48 h. In each experiment, 6 h before the end of the retinol treatment each culture flask received 370 KBq/ml [32P]-phosphate. The histone and HMGs were isolated as previously described [Moreira et al. Medical Science Research (1994) 22: 783-784]. The total protein obtained by either method was quantified and electrophoresed as described by Spiker [Analytical Biochemistry (1980) 108: 263-265]. The gels were stained with Coomassie brilliant blue R-250 and the stained bands were cut and dissolved in 0.5 ml 30 per cent H2O2 at 60oC for 12 h. The vials were chilled and 5.0 ml scintillation liquid was added. The radioactivity in each vial was determined with a liquid scintillation counter. Retinol treatment significantly changed the pattern of each subfamily of histone and high mobility group proteins.

Biomatrix effect on Sertoli cells phospholipids

In order to investigate the influence of biomatrix on Sertoli cell morphology and on the phospholipids content, these cells were isolated from tests of 15-day old Wistar rats and plated ontoplastic coated with extracellular matrix extracted froma seminiferous tubules, here denoted biomatix. When the Sertoli cells were cultured on biomatrix they did not from a monolayer until day 7 of culture, while cells plated onto plastic did so 48 h after plating. On day 5 of culture. Sertoli cells were incubated for 48 h with 5 µCi/ml 32P. There was no difference in 32P incorporation into lipids of cells plated onto biomatrix or plastic. However, there was a larger amount of phospholipid phosphate in cells plated onto biomatrix than onto plastic. When the phospholipds were analyzed by bidimensional thin-layer chromatography, no diferences were detected in their distribution; however, there was a significant decrease in the percentage of sphingomyelin in cells plated onto biomatrix when compared to plastic. These results showed that the cells cultured on biomatrix change their phospholipids content, but not their distribution. The importance of a small reduction in sphingomyelin content remains to be investigated