Testis is one of the most active organs, producing male gamete cells (sperm) continuously. As well as through histological investigations, testis has been studied by biochemical methods and revealed to have characteristic components. We have previously shown that spermatogenic cells undergoing meiosis are especially rich in calmodulin, a calcium-binding modulator protein. We now wish to investigate which proteins are being targeted by calmodulin. Specifically, a calmodulin-binding assay of testis extract after non-denatured PAGE (native polyacrylamide gel electrophoresis) showed two positive bands. One of these displayed a strong affinity to calmodulin in calcium-dependent manner, the other (540kDa) band showing weak affinity, but was more in abundance. We analyzed the partial amino acid sequence of the components and performed database searches. The former band was found to be a complex of three kinds of heat shock protein (APG-1/94kDa osmotic stress protein, APG-2/94kDa ischemia-responsive protein, and testis-specific major heat-shock protein, HSP70), the latter band being HSP86. These findings indicate that heat shock proteins are prominent components of spermatogenic cells and are likely to play a role during meiotic cell division in correlation with the calcium-calmodulin signal. However, the exact function of these heat shock proteins has not been elucidated yet. The isolated, heat-shock protein complex of 350kDa was negatively stained and observed under the electron microscope. Subsequent single particle analysis of the complex revealed a ring structure of 115 Å in diameter that resembled other molecular chaperons. Immuno-electron microscopy and freeze-fracture replica are under investigation with a view to understanding the distribution of heat-shock protein complexes in spermatogenic cells in general.
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