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The testis, however, remains mysterious, without a clear understanding of its circadian functions. Our time-series transcriptome analysis reveals more than 1000 rhythmically expressed genes in the zebrafish and mouse testes, respectively. Canonical circadian clock genes are rhythmically expressed in Sertoli cells and regulate retinoic acid (RA) production, which is also evidenced by their co-expression with RA synthesis genes in single Sertoli cells. Genetic and pharmacological manipulations and temporal desynchronization revealed that the circadian clock-regulated RA signaling synchronizes spermatogonial differentiation via zbtb16a and promotes fertilization via izumo1 in zebrafish. 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