Abstract
Thioredoxins compose a growing family of proteins that participate in different cellular processes via redox-mediated reactions. We report here the cloning, developmental expression, and location of murid Sptrx-2. Mouse and rat SPTRX-2 proteins display a high homology to their human ortholog in the thioredoxin and NDP kinase domains, and the coding genes are located at syntenic positions. Northern blotting and in situ hybridization confirmed the testis-specific expression of murine Sptrx-2 mRNA, mostly in round spermatids. Immunohistochemical analysis of the 19 steps of rat spermiogenesis showed that SPTRX-2 expression becomes prominent in the cytoplasmic lobe of step 15-18 spermatids and diminishes in step 19 just before spermiation. However, in the spermatid tail, SPTRX-2 immunoreactivity increased from step 15 to 19 and was confined to the principal piece. By immunogold electron microscopy, SPTRX-2 was first detected scattered throughout the cytoplasm of the axoneme in step 14-15 spermatids, but began to be incorporated by step 16 into the fibrous sheath (FS). During steps 17-18, the labeling increased over the ribs and columns of the assembled FS. It peaked in step 19 and remained in the FS of epididymal spermatozoa. Immunoblots of isolated FS obtained from spermatozoa confirmed that SPTRX-2 is an integral component of the FS and a post-obstruction autoantigen in vasectomized rats. Our data indicate that SPTRX-2 incorporation into the FS lags well behind FS assembly, suggesting it is required during the final stages of sperm tail maturation in the testis and/or epididymis, where extensive disulfide bonding of FS proteins occurs.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF548543 and AF548544
All of the structural amino acids that are conserved in previously characterized mammalian thioredoxins are conserved in mouse and rat SPTRX-2, including those residues shown to be essential for catalysis, maintenance of three-dimensional structure, or protein-protein interactions such as Asp-26, Trp-31, Pro-75, and Gly-91
We recently reported that human SPTRX-2, a protein composed of thioredoxin and NDP kinase domains, is present only in male germ cells [9]
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF548543 and AF548544. Thioredoxins are a class of multifunctional proteins that participate in a variety of redox reactions by the reversible oxidation of the cysteine residues in their conserved active-site Cys-Gly-Pro-Cys [1]. Based on this active-site sequence, seven thioredoxin proteins in humans have been reported to date. The complexity of the fertilization process is increased by the advent of internal fertilization and by the functional adaptations of the oocyte vestments To circumvent these hurdles, the spermatozoon has acquired a highly specialized morphology comprising cytoskeletal components, which appear to have no structural counterpart in somatic cells. Nine ODFs associate with the corresponding microtubule doublets of the axoneme, whereas in the principal piece, ODF3 and ODF8 are fused to the two longitudinal columns of the FS, which are bridged by FS ribs and together surround the seven remaining ODFs [14, 18]
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