Abstract
The adenine nucleotide translocases (Ant) facilitate the transport of ADP and ATP by an antiport mechanism across the inner mitochondrial membrane, thus playing an essential role in cellular energy metabolism. We recently identified a novel member of the Ant family in mouse, Ant4, of which gene configuration as well as amino acid homology is well conserved among mammals. The conservation of Ant4 in mammals, along with the absence of Ant4 in nonmammalian species, suggests a unique and indispensable role for this ADP/ATP carrier in mammalian development. Of interest, in contrast to its paralog Ant2, which is encoded by the X chromosome and ubiquitously expressed in somatic cells, Ant4 is encoded by an autosome and selectively expressed in testicular germ cells. Immunohistochemical examination as well as RNA expression analysis using separated spermatogenic cell types revealed that Ant4 expression was particularly high in spermatocytes. When we generated Ant4-deficient mice by targeted disruption, a significant reduction in testicular size was observed without any other distinguishable abnormalities in the mice. Histological examination as well as stage-specific gene expression analysis in adult and neonatal testes revealed a severe reduction of spermatocytes accompanied by increased apoptosis. Subsequently, the Ant4-deficient male mice were infertile. Taken together, these data elucidated the indispensable role of Ant4 in murine spermatogenesis. Considering the unique conservation and chromosomal location of the Ant family genes in mammals, the Ant4 gene may have arisen in mammalian ancestors and been conserved in mammals to serve as the sole and essential mitochondrial ADP/ATP carrier during spermatogenesis where the sex chromosome-linked Ant2 gene is inactivated.
Highlights
It has been believed that humans posses three members of the ANT family of genes: ANT1 (SLC25A4), which is expressed primarily in the heart and skeletal muscle; ANT2 (SLC25A5), which is expressed in rapidly growing cells and is inducible; and ANT3 (SLC25A6), which appears to be constitutively expressed in all tissues [14, 15]
The Autosomal Ant4 Gene Is Conserved in Mammals—The deduced amino acid sequence of Ant4 is well conserved among mammals (Table 1); a phylogram indicates that Ant4 is relatively distant from the other mammalian adenine nucleotide translocases (Ant) family peptides, Ant1, 2, and 3 (Fig. 1)
The Ant2 gene, which is ubiquitously expressed in somatic organs, is encoded by the X chromosome and the Ant3 gene, which has been identified in only a portion of mammalian species so far, including human, cow, and dog, is located on the X chromosome
Summary
Immunostaining—Testes were harvested from 6-week-old wild type or mutant male mice. All of the mice have been maintained under standard specific pathogen-free conditions, and the procedures performed on the mice were reviewed and approved by the University of Florida Institutional Animal Care and Use Committee. Deparaffinized and rehydrated 5-m tissue sections were stained with rabbit polyclonal antibodies against mouse Ant4 [5] or a cleaved Caspase-3 (Cell Signaling Technologies, Danvers, MA). The purities of recovered cell types were assessed on the basis of morphological characteristics when viewed under phase optics and were Ն85% for prospermatogonia, spermatogonia, and juvenile spermatocytes (preleptotene, leptotene plus zygotene, and juvenile pachytene) and Ն95% for adult pachytene spermatocytes and round spermatids [44]. Real time PCR was performed using the TaqMan gene expression assay (Applied Biosystems) according to the manufacturer’s instructions. The targeting construct was generated by sequential subcloning of the 5Ј homology arm, 3Ј homology arm, and diphtheria toxin gene into the pNF-SIBN vector. Following excision from pCR2.1TOPO vectors, the 5Ј homologous arm was ligated into the XhoI site, and the 3Ј homologous arm was ligated into the SalI site in the pNF-SIBN targeting vector. Chromosomal location, number of exons, and deduced amino acid identities of the Ant , and 4 genes
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