The mammalian spermatogenic pathway is a complex process that involves the mitotic proliferation of spermatogonia, the meiotic division of spermatocytes, chromosomal condensation, the production of sperm-specific proteins, and the morphogenic differentiation of spermatids to mature sperm. Elucidating the molecular basis of mammalian spermatogenesis is of great importance, not only because spermatogenesis is a fundamental biological event in the organism but because understanding of this mechanism could lead to the development of new therapeutics to overcome certain genetic diseases. Numerous cytological studies have described, in detail, the dramatic morphological changes of the differentiating spermatogenic cells, however, the molecular basis for many of these steps has yet to be elucidated. What has hampered the progress of such research is the lack of suitable experimental systems. Establishing cultured cell lines of testicular cells has been attempted for many times without much success. We have studied the molecular basis for the mammalian spermatogenic pathway, making use of two unique systems; cell-free transcription reactions in nuclear extracts of animal organs and primary culture of rat testicular cells. Expression of a variety of genes is activated or inactivated as the spermatogenic pathway progresses. The isozyme for glycolytic enzyme phosphoglycerate kinase (PGK) changes in spermatogenic cells from somatic-type PGK-1 to sperm-type PGK-2. We showed that the PGK isozyme switch occurs at the mRNA level during the pachytene spermatocyte stage, suggesting that Pgk-1 transcription ceases and Pgk-2 transcription is instead induced at this stage. The mechanism of the Pgk transcription switch was then investigated by cell-free transcription and DNA transfection experiments. We found that the activation of Pgk-2 transcription in the testis is caused mainly by an Ets family transcription factor named TAP-1, and that the same protein is likely to be involved in the inactivation of the Pgk-1 gene during spermatogenesis. Silencer-like negative regulatory DNA sequences and proteins bound to them seemed to be responsible for the repression of Pgk-1 transcription in the somatic tissues. Throughout the spermatogenic pathway, spermatogenic cells remain in close contact with each other and with somatic Sertoli cells in the seminiferous tubules. Although a number of reports have suggested that Sertoli cells play an important role in spermatogenesis, little is known about the mechanism by which these cells influence spermatogenic cell differentiation.4+ f1p4