EVOLUTIONARY ASPECTS

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The striking finding that out of eight “classical” thioredoxins in the human genome half are either highly expressed in testis or spermatid-specific invites speculations about why, from an evolutionary point of view, it was necessary for these proteins to arise in the male reproductive tract. No definitive answer to this question can be given, but an overview of the biochemical mechanisms that underlie the formation and maturation of the male gamete, as well as comparative genomics, can be of some help finding plausible explanations.

A phylogenetic analysis of the eight human thioredoxins clearly positions the spermatid-thioredoxins into two clusters (Figure 8): Sptrx-2 and Txl-2 are in the same branch which indicates that could have arisen as consequence of a genomic duplication event from a common ancestor as the intron/exon organization of their respective genes (at least in the thioredoxin domain) is identical (81, 82). The acquisition of thioredoxin and NDP kinase domains in the same polypeptide is a relatively recent event in evolution as the first organisms to present such a protein are sea urchins (echinoderms) and ascidian Cyona intestinalis (tunicate) (65, 69). On the other hand, Sptrx-1 and Sptrx-3 are clustered in another branch together with Trx-1. Analysis of the genomic organization of these three genes is conclusive in determining their common origin. The Sptrx-1 open reading frame does not contain introns (38, 56) indicating that it originated as a retrotransposition of the Trx-1 gene. Earlier, Trx-1 ancestor also underwent a genomic duplication which gave rise to Sptrx-3 retaining identical genomic organization to that of human Trx-1 (Jiménez et al., unpublished results). The remaining members of the family are derived from ancestors already present in lower eukaryotes such as Drosophila melanogaster, Caenorhabditis elegans and Saccharomyces cerevisiae (19, 57, 71). Thus, the phylogenetic analysis supports a late but rapid evolution of the thioredoxin family which can be traced to the emergence of a higher complexity of the internal fertilization process and a requirement for the spermatozoa to acquire additional cytoskeletal structures.

One might wonder why tissue-specificity of thioredoxins occurs only in testis but not any other organ or tissue, including the ovary. A likely explanation might be the evolution of the fertilization process which has made it increasingly difficult for the spermatozoon to reach the oocyte, as a consequence of internal fertilization and functional adaptations of the oocyte vestments (20, 104). To overcome these difficulties, higher vertebrates evolved the spermiogenesis mechanism which is basically a metamorphosis in which a somatic-cell-like germ cell (haploid round spermatid) is converted into a highly specialized and differentiated structure (spermatozoa) in an orchestrated and complex sequence of events that involves morphological, physiological and biochemical changes (7). These changes are unique events that do not happen in any other cell and can be grouped into (a) formation of the acrosome and sperm head skeleton, (b) nuclear condensation, (c) development of the flagellum and its accessory structures and (d) reorganization/reduction of the cytoplasm and cellular organelles (22). It is therefore reasonable to speculate that spermatid-specific thioredoxins were acquired through evolution to accomplish the above mentioned changes required for spermiogenesis to take place. Supporting this hypothesis is the fact that no orthologues of any Sptrxs or Txl-2 are present in the genome of lower eukaryotes such as nematodes (C. elegans) or insects (Drosophila), phyla whose spermatozoa are much simpler. In contrast, Sptrxs are ubiquitously expressed in spermatozoa of mammals, ranging from marsupials and rodents to ungulates and primates (Figure 5). Marsupials and humans are the only studied species in which both Sptrx-1 and Sptrx-2 can be detected in the flagellum (Figure 5). The first orthologues we find in the evolutionary scale are IC1 proteins of sea urchin and the ascidian Cyona intestinalis (65, 69) which have identical domain organization to that of Sptrx-2 and are components of the dynein machinery of the sperm axoneme. Interestingly, Sptrx-2 is not associated with the axoneme but with the FS of the mammalian spermatozoa. Instead, Txl-2 with its microtubule-binding capacity is found in the sperm axoneme. Thus, it seems that the association of thioredoxin and kinase domains is required for the function of the sperm axoneme and most likely necessary to supply energy and modulate the microtubule thread-milling. The role of Sptrx-2 in the FS is still unknown. As non-mammalian orthologues have not been identified for Sptrx-1 or Sptrx-3, studies to determine the testis-specific role of these proteins in mammals are underway.