Adenine nucleotide translocase 2 (Ant2) is required for individualization of spermatogenesis of Drosophila melanogaster

Successful completion of spermatogenesis is crucial for the perpetuation of the species. In Drosophila, spermatid individualization, a process involving changes in mitochondrial structure and function is critical to produce functional mature sperm. Ant2, encoding a mitochondrial adenine nucleotide translocase, is highly expressed in male testes and plays a role in energy metabolism in the mitochondria. However, its molecular function remains unclear. Here, we identified an important role of Ant2 in spermatid individualization. In Ant2 knockdown testes, spermatid individualization complexes composed of F‐actin cones exhibited a diffuse distribution, and mature sperms were absent in the seminal vesicle, thus leading to male sterility. The most striking effects in Ant2‐knockdown spermatids were decrease in tubulin polyglycylation and disruption of proper mitochondria derivatives function. Excessive apoptotic cells were also observed in Ant2‐knockdown testes. To further investigate the phenotype of Ant2 knockdown in testes at the molecular level, complementary transcriptome and proteome analyses were performed. At the mRNA level, 868 differentially expressed genes were identified, of which 229 genes were upregulated and 639 were downregulated induced via Ant2 knockdown. iTRAQ‐labeling proteome analysis revealed 350 differentially expressed proteins, of which 117 proteins were upregulated and 233 were downregulated. The expression of glutathione transferase (GstD5, GstE5, GstE8, and GstD3), proteins involved in reproduction were significantly regulated at both the mRNA and protein levels. These results indicate that Ant2 is crucial for spermatid maturation by affecting mitochondrial morphogenesis. A new testis specific gene Ant2 was identified in Drosophila melanogaster. In Ant2‐knockdown males, the overall appearance of testis was almost the same with control groups. Further, needle‐shaped spermatid nuclei were observed in Ant2‐knockdown males, however, most of the spermatid nuclei were scattered, and no mature sperms were observed in seminal vesicle of Ant2‐knockdown testes. Immunofluorescence staining of the testis from Ant2 RNA interference flies showed that spermatid individualization complexes composed of F‐actin cones exhibited a diffuse distribution, and microtubule polyglycylation defect. To further investigate the phenotype of Ant2 knockdown in testes at the molecular level, complementary transcriptome and proteome analyses were performed. At the mRNA