Fluorescent ruthenium chelates have been prepared and investigated for staining proteins separated by electrophoresis. Ruthenium II tris (bathophenanthroline disulfonate) appears to be suitable for detection of proteins, with sensitivities in the nanogram range, although sensitivity does not reach the level of optimized silver staining methods. This compound can be efficiently detected either with UV tables or with commercial laser fluorescence scanners. A further advantage of this detection method is its compatibility with mass spectrometry., These factors make this method a good compromise for proteomics studies.
During spermatogenesis, the various classes of germ cells synthesize proteins necessary for their own functioning and for regulation of the Sertoli cells. However, the nature of these proteins has been little studied, especially in spermatogonia, the germ stem cells. In this study, the electrophoretic patterns of high-resolution, silver-stained, two-dimensional polyacrylamide gels of intracellular spermatogonial protein extracts were studied by computerized gel image analysis. We detected 675 individual spots, some of which we identified by mass spectrometry and database searching. We present here a first set of 53 proteins identified. They include housekeeping proteins never before detected in spermatogonia, ten proteins previously detected in the reproductive tract but not in spermatogonia, including stathmin, a protein previously shown to be involved in cell proliferation and differentiation, and one new testicular protein named translationally controlled tumor protein (TCTP), also known as a growth-related protein. Immunohistochemistry demonstrated that the two latter proteins were indeed highly expressed in spermatogonia in situ, and their possible involvement in spermatogonial division and proliferation is currently under investigation in our laboratory. We conclude that this type of experimental strategy, known as proteomics, is a very powerful way to analyze germ cell proteins comprehensively and should rapidly greatly improve our understanding of spermatogenesis.
