Digital Chromosome Banding Reveals Distinct Spatiotemporal Dynamics and Sexual Dimorphism in Meiotic Silencing

In mammals, meiotic silencing of unsynapsed chromatin (MSUC) is initiated by the DNA damage response (DDR) pathway, as marked by yH2AX. During normal male meiosis, MSUC is restricted to the unsynapsed sex chromosomes, a process known as meiotic sex chromosome inactivation (MSCI). While the initiation of MSCI has been well studied, its full silencing dynamics and underlying structural mechanism remain unclear. In contrast to MSCI, broader MSUC can occur on autosomes in response to synapsis failure,out its cell-to-cell variability obscures its quantification. To address these challenges, we introduce "digital-chromosome-banding", a single-cell-based approach that allows quantitative analysis of MSCI and MSUC atchromosomal resolution. Using this approach, we identified two distinct silencing transitions during MSCI,occurring from zygonema to early pachynema and from early to mid-pachynema. The latter step coincides withmature sex body formation and involves a gel-like diffusion barrier to enforce transcriptional repression.Applying this approach to synapsis-defective mouse models (Spol1-/, Tardbp cKO, and Nelfb cKO), weobserved divergent MSUC patterns that correlate with the severity of asynapsis Comparative analysis between sexes also uncovered notable sexual dimorphisms in meiotic silencing. Together, our data provide aquantitative framework to dissect the spatiotemporal dynamics and sexual differences of meiotic silencing.