Multi-omics integration reveals Vha68-3 as a testicular aging-specific factor that coordinates spermatid elongation through mitochondrial metabolic homeostasis.

Background: Testicular aging has profound effects on spermatogenesis, sperm function, and the spermatogenic microenvironment, contributing to reduced male fertility. However, the precise molecular mechanisms by which mitochondria influence spermiogenesis during aging still remain largely unclear.

Methods: Vha68-3 KO flies were generated using the CRISPR/Cas9 technique. Testicular phenotypes and functions were mainly observed through immunofluorescence staining and transmission electron microscopy. Multi-omics study was mainly conducted through single-cell RNA sequencing and transcriptome-metabolomics association analysis. Vha68-3 binding proteins were identified via liquid chromatography-tandem mass spectrometry. The therapeutic potential of modulating mitochondrial metabolism for testicular aging mainly relied on the dietary intake of related compounds in fruit flies.

Results: In this study, we identified Vha68-3, a testis-specific subunit of the V-type adenosine triphosphate (ATP) synthase, predominantly localized in the tails of elongated spermatids, as a key age-related regulator of male fertility and spermatid elongation in Drosophila testes. Crucially, Vha68-3 deficiency impaired mitochondrial homeostasis in elongated spermatids during testicular aging. Through a multi-omics approach, including single-cell transcriptomics, protein interaction mapping of Vha68-3, and transcriptome-metabolome integration, we identified pyruvate metabolism as a critical pathway disrupted by Vha68-3 deficiency. Moreover, dietary supplementation with pyruvate (PA), S-lactoylglutathione (SLG), and phosphoenolpyruvate (PEP) effectively alleviated mitochondrial dysfunction and testicular aging linked to Vha68-3 deficiency.

Conclusions: Our findings uncover novel mechanisms by which mitochondrial metabolism regulates spermatid elongation and propose potential therapeutic strategies to combat mitochondrial metabolic disorders in aging testes.