Abstract
<title>Abstract</title> <p>Leydig cells support spermatogenesis primarily through testosterone (T) production. However, whether T alone is sufficient remains unresolved. Here, using Leydig cell–deficient mice with deletion of a fetal Leydig enhancer (ΔFLE), we demonstrate that T supplementation is sufficient to drive spermatogenesis up to the elongated spermatid stage in vitro. In an organ culture system, neonatal ΔFLE testes exhibited meiotic arrest under conditions of negligible T, consistent with their in vivo phenotype. T supplementation restored spermatogenic progression. However, efficiency varied markedly depending on T concentration. While 1 µM T induced elongating spermatids, it was associated with severe luminal dilation and low efficiency. In contrast, 0.01 µM T supported the highest spermatogenic efficiency, whereas 0.001 µM T was insufficient to complete meiosis. These findings establish that T is sufficient to support spermatogenesis in the absence of Leydig cells in vitro. This work also highlights a previously underappreciated concentration-dependent constraint of T for efficient progression of spermatogenesis in vitro.</p>