Regular articleNm23/NDP kinases in human male germ cells: role in spermiogenesis and sperm motility?
Introduction
Flagellar and ciliary movements are powered by the ATP-fueled motor protein dynein bound to microtubule doublets. The movement is caused by the sliding/bending of the peripheral microtubules, driven by successive mechano-chemical cycles of attachment–detachment of dynein arms on adjacent microtubules [1]. The energy necessary for this sliding results from ATP hydrolysis by dynein. ATP is primarily generated by oxidative phosphorylation by mitochondria of the flagellar middle piece and by glycolysis [2], [3]. Simplistically in sea urchin, energy transport from sperm mitochondrion along the tail is due to the phosphocreatine shuttle [3], [4]. However, this mechanism is probably not sufficient to maintain endogenous ATP stores in the vicinity of dynein arms, and other enzymes, responsible for nucleotide synthesis or channeling, are probably involved. Adenylate kinase, which furnishes ATP at the expense of ADP, has been detected in sperm flagella [5]. In the 1970s, nucleotide diphosphate (NDP) kinase, which catalyzes phosphoryl exchanges between nucleoside di- and triphosphates, was detected in the flagella of sea-urchin sperm [6], [7] and Chlamydomonas [8] for which it was proposed to replenish GTP pools necessary for microtubule assembly. At that time, the molecular identity of the enzyme(s) could not be known, as the first NDP kinase primary structures had not yet been determined [9], [10]. The NDP kinases have since been identified as the Nm23 and Awd proteins [11], which are also involved in the control of tumor aggressiveness and in Drosophila development [reviewed in 12]. Numerous observations have documented involvement of NDP kinase in signal transduction, in particular through GTP-utilising processes, but the mechanisms remain obscure [reviewed in 13]. The possible role of nucleoside diphosphate (NDP) kinases in sperm functions is unknown.
Nine human Nm23/NDP kinase-homologous proteins have been identified and can be divided into two groups [reviewed in 14]. Group I comprises four ubiquitously expressed proteins sharing about 60% sequence identity; all have catalytic activity and are therefore named NDP kinases A to D. The most abundant and most extensively studied NDP kinases are isoforms A and B, which are 88% identical and can form hetero- and homohexamers in vitro and in vivo. They are mainly found in the cytosol, although a nuclear location has also been reported [15], [16]. The second group includes the less-conserved proteins Nm23-H5 [17], Nm23-H6 [18], Nm23-H7 (unpublished, Genbank accession numbers AF153191), Nm23-H8 [19], and Nm23-H9 [20]. Nm23-H5, -H7, -H8, and -H9 are abundantly if not exclusively expressed in testis. With the exception of Nm23-H6, the only member of the second group with known NDP kinase enzymatic activity, they all possess several domains. Two and three tandem repeat C-terminal NDP kinase domains are found in Nm23-H7 and -H8, respectively. The N-terminal region of Nm23-H7 is a DM10 domain of unknown function that is found three times in tandem in the Chlamydomonas p72 (or Rib72) protein [21], [22] also containing EF-hand motifs, predicted to bind calcium. This protein, which was shown to associate with flagellar microtubules [22] and possesses a human ortholog, was proposed by Patel-King et al. [21] to participate in the regulation of a flagellar NDP kinase system. Nm23-H8 and -H9 contains one thioredoxin domain preceding the NDP kinase region(s). Interestingly, Nm23-H8 is highly homologous to the axonemal dynein intermediate chain of sea urchin [23] and Ciona intestinalis [24] and was found in human sperm flagella [19].
We identified nm23-H5 by homology with nm23-H4 (NDP kinase D) in EST data banks. The deduced Nm23-H5 protein is 212 residues long and has a unique 51-amino-acid C-terminal extension homologous to dpy30 protein, which is involved in X chromosome dosage compensation in Caenorhabditis elegans [17]. Nm23-H5 gene orthologs have been found in EST rodent and Drosophila data banks. The nm23-H5 mRNA is expressed in testis, specifically in germinal cells, spermatogonia, and early spermatocytes [17].
The aim of this study was to analyse Nm23-H5 protein distribution in human germinal cells and sperm cells at different maturation stages, in parallel with the ubiquitous group I NDP kinases for which specific antibodies are available. We found that Nm23-H5 and NDP kinases A and B are differently distributed in spermatids and mature spermatozoa and could therefore be involved at various levels of sperm differentiation and function.
Section snippets
Materials
Ethylene glycol-bis(succinic acid n-hydroxy-succinimide ester) (EGS), dimethyl sulfoxide (DMSO), P1,P5-di(adenosine-5′) pentaphosphate (Ap5A), Triton X-100, bovine serum albumin (BSA), 3,3′-diaminobenzidine (DAB), paraformaldehyde (PFA), Tween 20, Dulbecco’s phosphate-buffered saline (PBS), and monoclonal anti-α-tubulin antibody (cloneB-5-1-2) were from Sigma (Saint Quentin Fallavier, France). Percoll was obtained from Amersham Biosciences (Orsay, France). Human tubal fluid (HTF) medium was
Specificity of anti-Nm23-H5 antibodies
The full-length Nm23-H5 protein fused to a hexahistidine tag at the NH2 terminus was expressed and purified as described by Munier et al. [17] and used as immunogen to raise polyclonal antibodies in rabbits. These affinity-purified antibodies specifically recognised Nm23-H5, and not the other human A, B, C, and D NDP kinases or the E. coli enzyme, as shown by ELISA (Fig. 1A) and Western blotting (Fig. 1B).
Distribution of Nm23-H5 and group I human NDP kinases in sperm
In situ hybridization experiments have previously demonstrated the presence of Nm23-H5
Discussion
Despite the known involvement of nucleotides in providing energy for flagellar movement, the precise role of NDP kinases, the enzymes responsible for the last step of their synthesis, is unclear. Many nucleoside triphosphate-requiring systems are present in sperm, including the dynein machinery, which utilizes ATP to generate motility [1], [3]. Several members of the GTPase family (heterotrimeric and Ras-related) as well as members of the ERK signaling cascade have been reported [32].
Acknowledgements
We are indebted to Professor Ioan Lascu for the gift of some of the recombinant Nm23/NDP kinases and antibodies and to Dr. M. Konrad for the gift of the plasmids used to express the recombinant human C and E. coli NDP kinases. We thank Professor P. Jouannet for his constant interest in this work. We are also indebted to Cynthia Lebon (CECOS) for supplying frozen sperm. This work was supported by funds from INSERM, Association pour la Recherche contre le Cancer and by Grant 1752 from the
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These authors contributed equally to this work.