Knockout of hsd3b1 disrupts steroid hormone homeostasis and compromises gonadal development in zebrafish

Steroid hormones are central regulators of gonadal differentiation, endocrine homeostasis, and reproductive function, with impaired steroid biosynthesis leading to metabolic dysfunction and reproductive disease. 3β-Hydroxysteroid dehydrogenase (3β-HSD), encoded by HSD3B, catalyzes the conversion of inactive Δ⁵-3β-hydroxysteroid precursors into bioactive Δ⁴-ketosteroids, representing a pivotal enzymatic step in steroidogenesis. In this study, CRISPR/Cas9-mediated disruption of hsd3b1 in zebrafish produced pronounced endocrine and reproductive abnormalities, including aberrant gonadal development, interrenal hyperplasia, hyperpigmentation, and reduced fertility. Integrated RNA sequencing and targeted steroid hormone metabolomics further demonstrated extensive disruption of steroid hormone homeostasis in hsd3b1 mutants, with attenuated sex-specific metabolic differences between males and females. These findings establish hsd3b1 as an essential determinant of steroidogenic balance and gonadal development in zebrafish. The hsd3b1 mutant line provides a robust and tractable vertebrate model for investigating how steroidogenic disruption reshapes reproductive development and endocrine physiology.