Proteome analysis of spermatogonia: identification of a first set of 53 proteins

Abstract

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.

Tat1, a Novel Sulfate Transporter Specifically Expressed in Human Male Germ Cells and Potentially Linked to RhoGTPase Signaling

“…Spermatogonia were prepared from testes of 9 days post-partum male Harlan Sprague-Dawley rats. Seminiferous epithelial cells were dispersed by enzyme treatment and separated by sedimentation at unit gravity as described previously (19), leading to greater than 90% pure spermatogonia. Pachytene spermatocytes and early spermatids were prepared by centrifugal elutriation with a purity greater than 90% according to a method previously described (20), with the exception that enzymatic dissociation of cells was replaced by a mechanical dispersion.…”

Testicular postgenomics: targeting the regulation of spermatogenesis

“…Proteomic technologies have also strongly impacted our knowledge of the developing spermatogonia. With the view of gaining insights into the biology of spermatogonial cells, our group established reference proteome maps of mature spermatogonia freshly isolated from rat 9-dpp testis (Guillaume et al 2000). Another study provided information about the low-copy number proteins expressed in these cells through the prefractionation of protein cell extracts on two-dimensional gels with a narrow pH range (Com et al 2003).…”

New Insights into the Rat Spermatogonial Proteome

“…Complex techniques are required to isolate and enrich them (10,11), and it is very difficult to culture them (6,12). However, several groups have identified a number of genes and proteins characterizing spermatogonia in different mammals (13,14). In the latter study (14), we combined two-dimensional electrophoresis (2-DE) 1 and mass spectrometry to investigate the protein content of spermatogonia.…”

“…However, several groups have identified a number of genes and proteins characterizing spermatogonia in different mammals (13,14). In the latter study (14), we combined two-dimensional electrophoresis (2-DE) 1 and mass spectrometry to investigate the protein content of spermatogonia. This led to the identification of 53 spermatogonial polypeptides, two of which were further studied in detail within the testis because of their possible implication in proliferation and differentiation events (translationally controlled tumor protein (15) and stathmin (16)).…”

“…We report here the identification of 156 additional polypeptides by mass spectrometry and searches of protein sequence databases. The identified proteins have been classified according to their main known/postulated functions and these data, together with those obtained in our previous study (14), will be used to create a Web-based proteomic database of the rat testis (TESTIS-2DPAGE).…”