Diurnal testosterone oscillations gate drug delivery via phase separation of ZO-1 at the blood-testis barrier in mice.

The blood-testis barrier is essential for sperm development but also blocks the delivery of non-hormonal male contraceptives and other germ-cell-targeting therapies. However, how this barrier opens and closes in a controlled manner remains poorly understood. Here we show that the barrier exhibits time-of-day-dependent changes in permeability, allowing drugs to enter selectively during the light phase while remaining sealed during the dark phase. This gating mechanism is driven by daily fluctuations in testosterone. When testosterone rises at night, it activates calcium channels in supporting cells of the testis, leading to dephosphorylation of a key scaffolding protein called zonula occludens-1. This triggers the protein to undergo liquid-liquid phase separation, forming condensates that tighten the barrier. During the day, when testosterone drops, the condensates dissolve and the barrier becomes more permeable. Manipulating this calcium pathway pharmacologically enhances the entry of a contraceptive candidate without causing permanent toxicity. A specific disordered region within zonula occludens-1 is identified as the core driver of this testosterone-responsive condensation. Our study establishes diurnal hormone oscillations as a physiological rheostat for biomolecular condensate formation, offering a chronotherapeutic strategy to optimize drug delivery across biological barriers.