Project Summary/Abstract: The cycle of the seminiferous epithelium is a characteristic of spermatogenesis in mammals. The cycle serves to distribute the developmental stages of spermatogenesis in constant periodic intervals along the seminiferous tubules. The function of the cycle is to assure a constant source of spermatozoa over the relatively extended developmental time period. We have previously shown in the mouse testis that the cycle is initiated by pulses of retinoic acid (RA) originally generated by the Sertoli cells at stages VIII and IX of the cycle. The pulse of RA forces the undifferentiated spermatogonia to irreversibly enter the differentiation pathway. At stages VIII and IX we have shown in the adult mouse that the Sertoli cells demonstrate an increased abundance of hundreds of transcripts that allow for multiple functions such as RA synthesis, junction formation, uptake of residual bodies, spermiation, and others. In the absence of germ cells there are no cyclic activities of Sertoli cells detectable along the tubules, thus, accentuating the requirement for interactions between Sertoli and germ cells for normal spermatogenesis. We propose to examine the influence of spermatogonia at stages VIII and IX on the transcriptome of Sertoli cells and their functions during the development of complete spermatogenesis. We propose that this influence on the transcriptome and on the generation of the RA pulse is partially or wholly a result of Sertoli-germ cell interactions through the Notch signaling pathway. We will test this hypothesis by examining the influence of germ cells with Notch ligand gene deletions on the transcriptome and RA biosynthesis in normally developing Sertoli cells and in Sertoli cells with Notch receptor deletions. We will utilize a new technology designated capped small RNAseq or nTIseq to obtain information on active promoters and transcriptional start sites in the presence and absence of an intact Notch signaling pathway. We will pay special attention to transcripts regulating RA biosynthesis such as RDH10. The results of this study will clarify the interactive roles of germ and somatic cells in spermatogenesis and possibly implicate the Notch pathway in the etiology of male infertility.