Spermatid-specific thioredoxins

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During the last few years our group has characterized a number of thioredoxins encoded by the human genome. One of the most exciting results of this quest has been the discovery of a group of thioredoxins with a tissue-specific expression in spermatids and mature spermatozoa, the reason for which they have been named Sptrx-1, Sptrx-2 and Sptrx-3, respectively (for Sperm thioredoxins; (56, 81 and Jiménez et al., unpublished results). In addition, we have recently reported another member of this family, Txl-2 (thioredoxin-like 2), a microtubule binding protein highly expressed not only in sperm manchette and axoneme, but also in other ciliated tissues (82). These novel thioredoxins were first identified by in silico search for human expressed sequence tags (EST) with the conserved active site CGPC (Figure 2).

The first identified spermatid-specific thioredoxin was human Sptrx-1, a polypeptide of 486 amino acids organized into two distinct domains: an N-terminal domain consisting of 23 repeats of a 15-residue motif and a C-terminal domain typical of thioredoxins (56). Mouse and rat Sptrx-1 orthologues have an identical structure to that of human Sptrx-1. The N-terminal repetitive domain has no homology with any other protein in the databases and is predicted to organize as a coiled-coil protein (38). Human Sptrx-1 elutes as a 400 kDa protein in gel filtration chromatography, consistent with either an oligomeric form and/or a highly asymmetrical structure. Furthermore, Sptrx-1 behaves both as reductant and oxidant in vitro and crystallization/circular dichroism experiments indicate that the N-terminal repetitive domain of Sptrx-1 is largely unstructured and labile (37, 56). By northern blot and in situ hybridization we have found that Sptrx-1 mRNA is exclusively expressed in testis, with transcripts localized to round and elongating spermatids. Consistent with the mRNA expression, Sptrx-1 protein is synthesized within elongating spermatids, localized in close association to the assembling longitudinal columns of the FS, but not the ribs that connect these two columns, during tail elongation (Figure 4 and 5) (37, 111). This particular expression pattern, to our knowledge not described for any other protein so far, strongly supports the possibility that Sptrx-1 could be a part of a nucleation center for the formation of the longitudinal columns and the transverse ribs of the FS (111). The spermiogenesis process in general, and the sperm tail formation in particular, are characterized by a progressive increase of disulfide bonding, which starts at the spermatid stage and continues during epididymal transit (9, 12, 87). Together with its unique spatial and temporal expression pattern in the tail of elongating spermatids, the dual reducing/oxidizing activity of Sptrx-1 in vitro suggests that Sptrx-1 could indeed participate in the regulation of FS assembly by supporting the formation of disulfide bonds during sperm tail morphogenesis. In addition, its reducing activity might be required to rectify incorrect disulfide pairing and generate suitable pairs between the different FS constituents (37). Finally, the reduction of the disulfide bonds in the flagella of abnormal spermatozoa could serve as a quality control signal for their marking and elimination during epididymal passage (98).

Human Sptrx-2 is a 588 amino acid protein which is also organized in two different domains, that have been characterized in more detail than those of Sptrx-1. Thus, Sptrx-2 consists of an N-terminal thioredoxin domain followed by three repeats of nucleoside diphosphate kinase (NDPk) domain, the first of which is not complete (81). Mouse and rat orthologues have also been cloned, which share similar organization (Miranda-Vizuete et al., submitted for publication). NDP-kinases (also known as nm23) constitute another well-known family of structurally and functionally conserved proteins identified across a wide range of species from bacteria to human. NDP-kinases catalyse the transfer of g-phosphates between nucleosides and deoxynucleoside di- and tri-phosphates, playing a pivotal role in maintaining a balanced pool of nucleotides. In addition to the kinase function, nm23 proteins have been implicated in cell growth, cancer progression and development (34, 48, 73). Similarly to Sptrx-1, northern blot and in situ hybridization shows that Sptrx-2 mRNA is only expressed in testis at the round and elongating spermatid stages ((81) and Miranda-Vizuete et al., submitted for publication). Although recombinant human Sptrx-2 expressed in bacteria can be easily purified, it failed to show any enzymatic activity as a thioredoxin using NADPH and thioredoxin reductase, or DTT as electron donors. Furthermore, no NDP kinase activity was detected (A. Karlsson, personal communication). By light immunocytochemistry and immunofluorescence analyses, we detected Sptrx-2 protein in the principal piece of the spermatid and spermatozoon flagellum, suggesting that Sptrx-2 is a structural component of the FS. We confirmed this observation by immunogold-electron microscopy in rat seminiferous tubules where Sptrx-2 was found in both the longitudinal columns and the ribs of the FS (Miranda-Vizuete et al., submitted for publication). A detailed developmental analysis of both Sptrx-1 and Sptrx-2 expression detected differences in their respective patterns. While Sptrx-1 expression peaks at steps 14-16 of the rat spermiogenesis cycle, Sptrx-2 is incorporated into the FS at a later stage, peaking from step 15 to 19 ((111) and Miranda-Vizuete et al., submitted for publication). Yet another important difference between these two proteins is that while Sptrx-1 appears to be required during FS assembly but not in fully differentiated mature sperm FS, Sptrx-2 remains a structural component of the mature FS and can be detected in cauda epididymal or ejaculated spermatozoa. Given this pattern, Sptrx-2 may be necessary for the post-testicular events such as epididymal sperm maturation, hyperactivation/capacitation, or even for fertilization and zygotic development. In this regard, we have shown previously that the sperm tail FS, where Sptrx-2 resides, is one of the first sperm structures degraded in the zygotic cytoplasm at fertilization. The solubilization of FS precedes the degradation of paternal mitochondria and ODF, and coincides with the early stages of male pronuclear development (97). Extensive studies have demonstrated the requirement of disulfide bond reduction for the successful processing of the sperm nucleus and sperm accessory structures during mammalian fertilization (99) and Sptrx-2 may be involved in regulating this process. Furthermore, although we could not detect any kinase activity in recombinant Sptrx-2, its well conserved NDP-kinase domain supports the idea that Sptrx-2 could be a phosphate donor for the phosphorylation of other FS proteins. Phosphorylation is the major regulatory mechanism in spermatozoa and underlies important processes during the acquisition of fertilizing capability by the spermatozoon, such as capacitation and hyperactivation (25, 60, 109).

Our continuous search for novel members of the thioredoxin family yielded a close homologue of Sptrx-2, which we later named Txl-2 (thioredoxin-like 2). Txl-2 has similar yet distinct domain organization to that of Sptrx-2, namely an N-terminal thioredoxin domain followed by one NDP kinase domain (82) instead of three as in Sptrx-2. While cloning human Txl-2 from a testis library, we found that in addition to a full-length variant coding for a 330 amino acid residues, an alternative splicing variant lacking exon number five was present, resulting in a shorter protein of 291 amino acid residues. Due to the striking similarity both in sequence and domain organization and the fact that we cloned Txl-2 from a testis cDNA library, we initially assumed Txl-2 to be a novel testis-specific protein. However, mRNA analysis indicated that indeed Txl-2 should be placed between the ubiquitous and the tissue-specific thioredoxins. When attempting to determine the size and tissue distribution of Txl-2 mRNA by northern blot analysis, we consistently failed to detect any signal despite the use of low stringency conditions, extended time of exposure or use of different probes, suggesting that Txl-2 mRNA might be expressed at very low levels. To improve the sensitivity we used real time PCR which confirmed that expression of Txl-2 mRNA in adult tissues is very low with highest levels found in testis and lung and lower levels were found in a variety of additional tissues (82). In clear parallel with Sptrx-2, we were unable to detect any thioredoxin or kinase enzymatic activity of recombinant Txl-2 expressed in bacteria. This lack of activity despite the presence of two well characterized enzymatic domains in both proteins raises the possibility that translational modifications or interaction with other proteins or cofactors might be required for their function. Specific antibodies against recombinant human Txl-2 detected Txl-2 in close association to the cilia of the lung airway epithelium and the microtubule-based spermatid manchette and axoneme (82) (Figure 6). This particular localization strongly suggested that Txl-2 was a microtubule-binding protein. To prove this point, we performed in vitro microtubule binding assays using recombinant full-length and D5Txl-2 splicing variant. The result clearly demonstrated that the D5Txl-2 variant binds microtubules with very high affinity while full-length Txl-2 binding is weak (82). This is the first report of a member of the thioredoxin family with a microtubule-binding activity. However, there are reports of other members of the NDP kinase family with such an activity (49, 72, 79). Consistent with this result, we have also identified Txl-2 in the cilia of the brain ependymal cells (Pelto-Huikko and Miranda-Vizuete, unpublished results).

The subcellular localization of Txl-2 in spermatid manchette deserves further examination. The manchette is a transient microtubule-based structure that caudally surrounds the spermatid nucleus (58). The manchette recently has been proposed to be a transient storage location for both signalling proteins involved in nucleocytoplasmatic trafficking and structural proteins that are eventually sorted to the centrosome and the developing spermatid tail (40, 41). However in this case, Txl-2 seems to associate to the spermatid manchette and axoneme simultaneously during spermatid elongation (82).

Although we still do not know the function of Txl-2, its localization in close association to microtubules of cilia and flagella suggests that Txl-2 could control microtubule stability and maintenance. Its putative disulfide reducing activity, by virtue of the thioredoxin domain, might regulate ligand interactions and microtubule assembly as it has been reported that cysteine residues in tubulin are critical for those events (13). More importantly, Chlamydomonas flagellar protein p72 and sea urchin sperm axoneme protein IC1 have been reported to have NDP kinase activity and suggested to be the suppliers of GTP for microtubule assembly (65, 70). Thus it is conceivable that Txl-2 plays this role in the formation, functioning and maintenance of mammalian axonemes.

Finally, we have very recently identified a third testis-specific member of the thioredoxin family, Sptrx-3 (Figure 2). By in situ hybridization, Sptrx-3 mRNA displays an expression pattern similar to Sptrx-1 and Sptrx-2, mainly present in round and early elongating spermatids (Jiménez et al., unpublished results). Sptrx-3 differs from the other testis/spermatid-specific proteins in that Sptrx-3 is composed of only one thioredoxin domain. In addition, multiple splicing variants have been identified. The fact that its mRNA is expressed at the spermatid level indicates that the protein is most probably required in the later steps of spermiogenesis or in the mature spermatozoa. This localization is consistent with our more recent data on immunolocalization of Sptrx-3 in the developing acrosome and Golgi (Oko et al., unpublished results). A scheme showing the localization of the different thioredoxins in spermatozoa is depicted in Figure 7. Perinuclear and Golgi-localization of Sptrx-3 during spermiogenesis is consistent with immunodetection of Sptrx3 protein in the redundant cytoplasm and nuclear vacuoles of infertile men’s semen samples (Figure 5), suggestive of incomplete spermatogenesis and rejection of cytoplasmic droplets and residual bodies.