Time-series single-cell transcriptomics reveals pervasive daily rhythmicity and nocturnal spermatogenesis in the zebrafish testis

The vertebrate testis has long been regarded as lacking strong cellular daily rhythmicity. Here, we use time-series single-cell RNA sequencing to construct a temporal atlas of the adult zebrafish testis. We identify previously unknown cell-type-specific markers and previously unidentified testicular subtypes and reveal that Sertoli cells, spermatogonia, and spermatids exhibit robust oscillations in key circadian clock genes. In contrast, Leydig cells and spermatocytes primarily exhibit rhythmic expression of key functional genes. We show that intercellular communication networks also exhibit daily rhythmicity. Pseudotime trajectory analysis demonstrates a notable nocturnal preference for germ cell progression. Disrupting circadian input via constant light reduced germ cell numbers and impaired fertility. Genetic ablation of <i>clock1a</i> arrests spermatogonial differentiation, whereas mutation of the rhythmic spermatocyte gene <i>hmgb1a</i> substantially reduces meiotic cells. Together, our findings redefine the testis as a highly rhythmic organ in which a precisely timed, nocturnally coordinated cellular symphony drives spermatogenesis.