Mammalian spermatogenesis is a highly organized system that can produce largenumber of spermatozoa continuously. Spermatogenesis takes place within theseminiferous tubules that are composed of germ cells and Sertoli cells that aremorphologically and functionally unique somatic cells specialized to supportspermatogenesis. Sertoli cells directly contact with germ cells, and regulate their properlocalization and release into lumen. They also provide nutrients and growth factorsessential for the differentiation of germ cells and the stem cell maintenance. Furthermore, Sertoli cells play a pivotal role for maintaining the integrity of the seminiferous epithelium by forming tight junction. Consequently, dysfunction of Sertoli cells leads to the disruption of spermatogenesis and male infertility. Therefore, understanding of the Sertoli cell’s function is crucial to clarify the microenvironment for spermatogenesis. Spermatogenesis progresses in a spatially and temporally regulated manner, known as spermatogenic wave. During spermatogenesis, germ cells in different developmental stages form groups and synchronously differentiate. In the mouse testis, twelve germ cell groups, known as seminiferous epithelial stages I- XII, can be recognized and they are arranged in order along the tubule length showing the spatial continuity. This cyclical program in spermatogenesis is called “seminiferous epithelial cycle”. One cycle takes 8.6 days in the mouse and 12.9 days in the rat. When rat germ cells are transferred to mouse testis, spermatogenesis proceed with the cycle characteristic of rat germ cells. Therefore it is believed that the cycle length is primarily regulated by germ cells. It has been known that Sertoli cells change their gene expression patterns in accordance with the continuous alteration of the epithelial stages. Because heterogeneous gene expression in Sertoli cells was observed even in testes lacking differentiating germ cells, it is suggested that cyclic gene expression in Sertoli cells can occur independently of synchronous spermatogenic differentiation. Furthermore, in the early phase of the first round spermatogenesis when intact seminiferous epithelial cycle was not established, the wave-like expression in Sertoli cells was associated with the timing of spermatogonia differentiation. Growing evidence suggests interesting possibility that Sertoli cells support stage-specific events of spermatogenesis by creating stage-specific microenvironments. It has been suggested that retinoic acid (RA) signaling is involved in the regulation of seminiferous epithelial cycle. In vitamin A-deficient (VAD) mice, spermatogenesis was arrested at spermatogonia stage, and the injection of vitamin A (retinol) or RA into VAD mice triggers initiation of synchronized spermatogenesis in all seminiferous tubules. Moreover, it was reported that Sertoli cell-specific deletion of RARα resulted in disruption of the stage-dependent gene expression in Sertoli cells. These evidences suggest the involvement of RA signaling in the stage-dependent expression of Sertoli cells. However, it remains elusive how RA signaling controls the periodicity of Sertoli cells and whether other signaling pathway(s) is involved in the regulation. Furthermore, importance of periodicity in Sertoli cells for spermatogenesis is also unknown. In part I, to understand the periodicity of Sertoli cells, I performed comprehensive analysis of stage-dependent gene expression in Sertoli cells by using microarray, and identified 419 stage-dependent genes. In part II, I investigated implication of Notch signaling in Sertoli cells, because the list of stage-dependent genes contained Notch1 receptor. I found that the Notch ligand, Jagged2 was expressed in germ cells, and Notch1 receptor was expressed and activated in Sertoli cells in stage-dependent manner. To examine the involvement of Notch signaling in periodicity of Sertoli cells and spermatogenesis, I inactivated Notch signaling in Sertoli cells by using cre-loxP system.However, my data demonstrated that genetic ablation of Notch signaling in Sertoli cells did not affect spermatogenesis nor stage-dependent gene expression in Sertoli cells as long as I examined, suggesting that Notch signaling in Sertoli cells is dispensable for mouse spermatogenesis and the stage-dependent expression. In part III, I studied relationship between stage-dependent gene expression in Sertoli cells and RA signaling. I found that RA signaling was periodically activated in stage VII-XII seminiferous tubules, and stage-dependent genes showing peak at stage I-VI and VII-XII tended to be suppressed and activated by RA signaling, respectively. To examine the significance of the periodicity in Sertoli cells for spermatogenesis, I disrupted stage-dependent gene expression in Sertoli cells by means of lentivirus mediated suppression of RA signaling, and found it led to stage-specific defects in germ cell differentiation, delayed recovery of tight junction component and abnormal morphology of Sertoli cells. Taken together, the stage-dependent gene expression in Sertoli cells is primarily regulated by periodic activation of RA signaling, and is important for stage-specific events of spermatogenesis
