Leydig cells are known for their tremendous demand for cholesterol during acute de novo biosynthesis of the male hormone. Looking beyond the essential cholesterol storage and mobilization, we uncover the molecular composition of MA-10 Leydig cells and describe different metabolic systems that support steroidogenic function. Functional testing via limiting use of specific metabolite stockpiles identified crucial components and novel paths to support steroid biosynthesis. Our findings build a metabolomic framework for steroidogenesis and provide new fundamental insights into its biosynthetic regulation.

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