The low molecular weight heat shock protein plays a role in rat Sertoli cell response to a germ cell paracrine signal.

Abstract

Sertoli cells phosphorylate a protein termed GC1 (Ireland & Welsh, 1987) of 27kDa and pI of 5.6 in response to a germ cell paracrine factor. GC1 and an adjacent 27kDa phosphoprotein are synthesized in response to Cd$\sp{2+}$. The mammalian low molecular weight heat-shock protein (hsp27) has three isoforms, two of which are phosphorylated. Based on these similarities, it was hypothesized that GC1 is an isoform of Sertoli cell hsp27. In these studies, CdCl$\sb2$ and heat-shock increased two phosphorylated isoforms of hsp27 over a period of hours. Heat-shock and CdCl$\sb2$ induced incorporation of $\sp3$H-labeled amino acids into three isoforms of hsp27, indicating the appearance of the phosphorylated isoforms is due to increased synthesis. An increased proportion of the phosphoproteins was found in the low speed pellet fraction of homogenized Sertoli cells after heat-shock, with the phosphoproteins returning to the supernatant fraction by 4h. It is concluded that GC1 is an isoform of Sertoli cell hsp27. A sheep antibody to a conserved hsp27 peptide sequence was prepared. Sertoli cell protein retained on an antibody affinity column was a single band of 27kDa on SDS-PAGE. In hsp27 phosphorylation studies of Sertoli cell, rat pulmonary endothelial cell, and TR-ST cell cultures, both Sertoli cells and endothelial cells showed an increase of phosphorylated forms of hsp27 in response to heat-shock, while TR-ST cells did not. In indirect immunofluorescence studies using affinity-purified peptide antibodies on the three cell types, dispersed cytoplasmic immunolocalization of hsp27 was seen in control Sertoli cell, endothelial cell, and TR-ST cell cultures. Increased perinuclear hsp27 was seen in all three cell types after heat-shock, with dispersed cytoplasmic distributions seen again after recovery from heat-shock. On the basis of silver-stained 2-D PAGE, the relative abundance of hsp27 in the three cell types was: endothelial cells $>$ Sertoli cells $>$ TR-ST cells. It is concluded that hsp27 plays a central role in the pathway of Sertoli cell responses to paracrine signals in the seminiferous tubule.