Preparation, isolation and characterization of stage-specific spermatogenic cells for cellular and molecular analysis

Spermatogenesis constitutes a remarkable program of cell differentiation, which involves dramatic changes in cell morphology, biochemistry and gene expression(1,2). But the study of mate germ cells is complicated by the exceptional organization of the seminiferous epithelium and by the Lack of established cell Lines that are able to recapitulate any of the multiple differentiation steps of the spermatogenesis program in vitro. Cell types with differences in their sedimentation properties or cell surface markers can be isolated from the whole tissue by various methods, but these methods do not allow accurate identification of all the differentiation stages. As a consequence of strict paracrine regulation by Sertoli cells, spermatogenesis proceeds in synchronized waves along the seminiferous tubules, and every given cross-section of the tubule contains only certain cell types in a specific combination (Fig. 1). The Light absorption pattern of a seminiferous tubule, as seen under a dissection microscope, correlates with defined stages of the spermatogenic wave, which makes it possible to isolate specific stages on the basis of their transillumination properties(3-6). The accuracy of the isolation of specific stages can be improved by combining it with phase-contrast microscopy of Live cell preparations(7-10). The staging of the spermatogenic cycle is best characterized in rat and mouse(11,12). Here, we describe a method designed to identify, isolate and characterize mouse mate germ cells at specific steps of differentiation by transillumination-assisted microdissection. The distinctive morphological features of germ cells, as detected by phase-contrast microscopy, are detailed. Although the method described here generates small quantities of cells and therefore does not allow for further isolation of the different cell types comprising each differentiation stage, the applications of this method are powerful and diverse. It enables the monitoring of spermatogenic differentiation events, and in combination with biochemical analyses, it allows the characterization of the molecular mechanisms governing these processes. This method can be used for the identification of the effects of cytotoxic and environmental factors on male reproductive function, as well as for the rapid and comprehensive diagnosis and characterization of sperm cell defects attributable to infertility. In combination with gene targeting models, it enables the characterization of the critical factors involved in the cell cycle, chromatin dynamics, spermatid differentiation, stem cell biology and fertility.