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Research Article |
Center for Research on Reproduction and Women's Health, Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104
ABSTRACT
Proper sperm function depends on adequate ATP levels. In the mammalian flagellum, ATP is generated in the midpiece by oxidative respiration and in the principal piece by glycolysis. In locations where ATP is rapidly utilized or produced, adenylate kinases (AKs) maintain a constant adenylate energy charge by interconverting stoichiometric amounts of ATP and AMP with two ADP molecules. We previously identified adenylate kinase 1 and 2 (AK1 and AK2) by mass spectrometry as part of a mouse SDS-insoluble flagellar preparation containing the accessory structures (fibrous sheath, outer dense fibers, and mitochondrial sheath). A germ cell-specific cDNA encoding AK1 was characterized and found to contain a truncated 3' UTR and a different 5' UTR compared to the somatic Ak1 mRNA; however, it encoded an identical protein. Ak1 mRNA was upregulated during late spermiogenesis, a time when the flagellum is being assembled. AK1 was first seen in condensing spermatids and was associated with the outer microtubular doublets and outer dense fibers of sperm. This localization would allow the interconversion of ATP and ADP between the fibrous sheath where ATP is produced by glycolysis and the axonemal dynein ATPases where ATP is consumed. Ak2 mRNA was expressed at relatively low levels throughout spermatogenesis, and the protein was localized to the mitochondrial sheath in the sperm midpiece. AK1 and AK2 in the flagellar accessory structures provide a mechanism to buffer the adenylate energy charge for sperm motility.
gametogenesis, sperm, sperm motility and transport, spermatogenesis
INTRODUCTION
Mammalian sperm motility is driven by the flagellum and is dependent on the availability of an adequate and continued supply of ATP. The flagellum can be segregated into three domains, all of which contain the axoneme, a 9+2 microtubular structure. The midpiece contains the mitochondrial sheath and 9 outer dense fibers (ODFs). The principal piece represents the major portion of the tail and is comprised of the fibrous sheath and 7 ODFs. A small end piece has only the axoneme surrounded by the plasma membrane of the sperm. ATP is used by the dynein ATPases that function as the flagellar motors and in protein kinase A-mediated signal transduction pathways to regulate motility. There are a number of factors that make the generation and delivery of ATP difficult in a flagellum that reaches a length of >100 µm in some species [1]. The motile wave is propagated along the length of the flagellum, necessitating the availability of ATP to the dynein motors present throughout the tail. As mentioned above, mitochondria are present in the midpiece, which means that ATP generated by oxidative phosphorylation must be transported to other regions and/or that ATP is produced locally. Invertebrates such as sea urchins utilize a phosphocreatine shuttle system to actively move ATP down the length of the tail [2]; however, such a system either is not present or is of limited importance in mammalian sperm [3].
Mammalian sperm utilize glycolysis to generate ATP in the principal piece of the tail. A variety of glycolytic enzymes are localized to the fibrous sheath [4–7]. For example, hexokinase is present in both the longitudinal columns and transverse ribs of this structure [5]. The importance of glycolysis was elegantly demonstrated by the gene targeting of Gapdhs, the sperm-specific isoform of glyceraldehyde 3-phosphate dehydrogenase [8]. Gapdhs-null male mice have immotile sperm and consequently are infertile. Furthermore, glycolysis is required for mammalian sperm to acquire a hyperactivated form of motility [9].
We recently performed a proteomic analysis of an SDS-insoluble tail preparation containing the flagellar accessory structures—fibrous sheath, outer dense fibers, and the mitochondrial sheath—but lacking the axoneme and plasma membranes of the flagella [10]. This study identified a number of proteins involved in the generation and utilization of ATP. In addition to glycolytic enzymes, e.g., aldolase, triosephosphate isomerase, phosphoglycerate kinase, and glyceraldehyde 3-phosphate dehydrogenase, we determined that both adenylate kinase 1 (AK1) and 2 (AK2) are present in the flagellar accessory structures. Adenylate kinases (ATP:AMP phosphotransferase, EC 2.7.4.3) are present in prokaryotes and eukaryotes and participate in maintaining energy homeostasis in cells [11–14]. These enzymes buffer the adenylate energy charge by catalyzing the reaction, 2ADP 
ATP+AMP, thus producing either ADP or stoichiometric amounts of ATP and AMP, depending on the concentrations of the three nucleotides [15]. Among the members of the AK family, the major AK isoform, AK1, is found in the cytosol of tissues with high-energy demands, e.g., skeletal muscle, while the more ubiquitously expressed AK2 is localized mainly to mitochondria [16].
Although there is a considerable body of literature on the role of AK in heart and skeletal muscle tissues [17, 18], relatively little is known about this enzyme family in mammalian germ cells and sperm. Schoff et al. [19] showed that AK activity exists in bovine sperm flagella and can generate sufficient ATP to produce normal motility in digitonin-permeabilized cells treated with MgADP. We found that AK1 and AK2 are localized in different compartments of the flagellum, suggesting that these enzymes play important roles in buffering the adenylate energy charge in sperm.
MATERIALS AND METHODS
Isolation of Germ Cells and Sperm
All animal procedures were approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Male germ cells were prepared from decapsulated testes of adult mice (CD1 retired breeders, Charles River Laboratories) by sequential dissociation with collagenase and trypsin-DNase I [20, 21]. The cells were separated at unit gravity in a 2%–4% BSA gradient in an enriched Krebs-bicarbonate medium to purify pachytene spermatocytes, round spermatids, and condensing spermatids. Both the pachytene spermatocyte and round spermatid populations were at least 85% pure, while the condensing spermatid population was approximately 40%–50% pure (contaminated primarily with anuclear residual bodies and some round spermatids).
Epididymal sperm were collected by mincing the caudae epididymides and allowing the sperm to swim out in PBS. The sperm were collected by centrifugation at 800 x g for 5 min at room temperature, and SDS-resistant head and tail structures were separated [22]. Briefly, sperm were homogenized in 1% SDS, 75 mM NaCl, 24 mM EDTA, pH 6.0 (S-EDTA), layered on 1.6 M sucrose gradient in S-EDTA, and centrifuged at 5000 x g for 1 h at room temperature. The SDS-resistant tail structures were collected from the interface.
Generation of AK2 Antibody and Immunological Procedures
An affinity-purified rabbit polyclonal antibody was generated to AK2 using the peptide Ac-RSYHEEFNPPKEPMKDDIC-amide (amino acids 150–167, accession number NP_058591) (Quality Controlled Biochemicals, Hopkinton, MA).
Proteins from epididymal sperm and tail preparations were separated by 10% SDS-PAGE and transferred to Hybond ECL nitrocellulose membranes (Amersham Biosciences, Buckingham, UK). The membranes were blocked with TBST (25 mM Tris-HCl, pH 8.0; 125 mM NaCl; 0.1% Tween 20) containing 5% nonfat dry milk (NFDM) and incubated with primary antibody [rabbit anti-mouse AK1, a generous gift of Dr. Edwin Janssen (Radboud University, The Netherlands) 1:10 000 dilution in 5% NFDM in TBST; rabbit anti-mouse AK2, 1:1000 dilution in 5% NFDM in TBST]. After washing with TBST, the blots were incubated with secondary antibody [donkey anti-rabbit IgG conjugated with horseradish peroxidase (Amersham, Buckinghamshire, UK), 1:5000 dilution in 5% NFDM in TBST]. The bound enzyme was detected with the ECL kit according to the manufacturer's directions (Amersham) and exposed to film. As a control, the anti-AK1 was neutralized by adding a 10-fold molar excess of the peptide in 200 µl 5% NFDM in TBST and incubating the mixture at 4°C overnight. After centrifugation to remove any particulate material, the supernatant was used for immunoblot analysis.
Epididymal sperm were collected, attached to slides, fixed with 4% paraformaldehyde for 15 min, and permeabilized with –20°C methanol for 2 min. In some cases, sperm were treated with S-EDTA to separate heads and tails and to solubilize the membrane and axoneme prior to attaching to slides. The slides were washed with PBS, and the samples were incubated with 10% goat serum in PBS (blocking solution) for 30 min at room temperature and then with the primary antibody (either anti-AK1 antibody, 1:2500 dilution in 10% goat serum or anti-AK2 antibody, 1:50 dilution in 10% goat serum) in blocking solution for 1 h at room temperature. For a control, PBS-goat serum was substituted for the primary antibody. After washing with PBS, the samples were incubated with the secondary antibody [donkey anti-rabbit IgG linked either with Alexa Fluo-488 (for AK1) or with 568 (for AK2); Molecular Probes, Eugene, OR] (1:500 dilution in 10% goat serum) in blocking solution for 1 h at room temperature. After washing with PBS, the samples were mounted with coverslips using Fluoromount-G (Southern Biotechnology Associates, Inc, Birmingham, AL), examined using an inverted microscope (Nikon Eclipse TE 2000-u, Nikon Corp), and photographed with a CFW-1310C color digital camera (Scion Corp., Frederick, MD).
For immunoelectron microscopy, sperm and SDS-insoluble accessory structures were isolated and fixed with 4% paraformaldehyde and 0.25% glutaraldehyde; all reagents were purchased from Electron Microscopy Sciences (Fort Washington, PA). The samples were embedded in Lowicryl K4M that was then polymerized with ultraviolet light (365 nm) for 5 days. Ultrathin sections were cut and mounted on nickel grids coated with Formvar. To prevent nonspecific binding, grids were incubated with blocking buffer (PBS with 1.0% BSA and 2% normal goat serum) for 30 min at room temperature. The sections were incubated with primary antibody (anti-AK1 antibody 1:1000; anti-AK2 antibody 1:50 in blocking buffer) for 1 h at room temperature, washed in buffer, and incubated with 15 nm gold particle-labeled anti-rabbit IgG (1:10 in blocking buffer) (Ted Pella, Inc., Redding, CA). After a 1 h incubation at room temperature, the grids are rinsed with buffer followed by deionized water for 3 min, air-dried, and then examined with a FEI Tecnai G2 electron microscope. Images were collected with a Gatan Camera (Gatan, Inc., Pleasanton, CA).
Isolation of Germ Cell AK1 cDNAs
A probe corresponding to a portion of an Ak1ß cDNA (nucleotides 143–492, accession # NM_021515) was amplified by PCR. After labeling with [
32P]dCTP (3000 Ci/mmol) by random priming (Invitrogen, Piscataway, NJ), the probe was used to screen a total of 500 000 plaques from a mixed germ cell cDNA library in the HybriZAP 2.1 vector (Stratagene, La Jolla, CA). The library was plated and the plaques transferred to Hybond N+ membranes (Amersham Biosciences) that were treated with DNA denaturing solution (1.5 M NaCl, 0.5 M NaOH) and then DNA neutralizing solution (1.5 M NaCl, 0.5 M Tris-HCl, pH 8.0). Membranes were pre-hybridized in Rapid-hyb buffer (Amersham Biosciences) for 15 min at 65°C and then incubated overnight at 42°C in Rapid-hyb buffer containing the radiolabeled probe. The membranes were washed with 2 x SSPE/0.1% SDS at room temperature and then with 0.1 x SSPE/0.1% SDS at 65°C and developed by exposure to film. Positive clones were purified by two additional rounds of screening and converted to plasmids according to the manufacturer's directions. All clones were sequenced in both directions using an ABI 3100 DNA sequence analyzer and the BigDye chemistry (Applied Biosystems, Inc., Foster City, CA). The sequences were analyzed using MacVector (Accelrys, Madison, WI) and Sequencher (Gene Codes, Ann Arbor, MI) software.
Preparation of RNA and RT-PCR
RNA was prepared from germ cells, testis, and various somatic tissues using Tri Reagent (Sigma; St. Louis, MO). Reverse transcription using 1 µg mRNA was performed using SuperScript II Reverse Transcriptase according to the manufacturer's instructions (Invitrogen Corp., Carlsbad, CA). Products were amplified using Extaq DNA Polymerase (Takara Co., Toyko, Japan) and the appropriate primers (Table 1). Amplicons were cloned into the pCR2.1-TOPO (Invitrogen). Plasmid DNA was prepared and sequenced as described above.
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For quantitative RT-PCR assays, primers were designed using Primer Express 1.5 Taqman Primer Design software (Applied Biosystems) (Table 2). Products were amplified with the SYBR Green PCR Master Mix and analyzed with the ABI 7900 HT Sequence Detection system. The following PCR protocol was used: 1) denaturation (50°C for 2 min, 95°C for 10 min), 2) amplification and quantification (95°C for 15 sec, 60°C for 1 min) repeated for 40 cycles, 3) a dissociation curve program (95°C for 15 sec, 60°C for 15 sec, 95°C for 15 sec), and, 4) cooling at 4°C. Amplicons were analyzed by generating a dissociation curve and determining the threshold cycle (Ct) value for each transcript. The relative quantification of gene expression was analyzed by the 2-
CT method [23]. The mRNA corresponding to ribosomal protein S16 (accession number: BC082286) was used as a control [24].
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Two-dimensional Gel Electrophoresis and Mass Spectrometry
Two-dimensional gel electrophoresis was performed as previously described except that 7-cm Immobiline DryStrips (pH 3–10, nonlinear) were used [10]. Equipment and reagents were from Amersham Bioscience (Uppsala, Sweden). Briefly, samples (60 µl containing 100 µg protein) were mixed with 65 µl rehydration buffer (8 M urea, 2% w/v CHAPS, 0.5% IPG buffer [pH 3–10, nonlinear], 2 mg/mL DTT) and loaded in the IPGphor strip holder. The DryStrips were placed in the holder and overlaid with 
2 ml DryStrip cover fluid. Strips were hydrated at 30 V for 14 h and then focused for a total of 80 kVh at 20°C on the IPGphor IEF system. After electrophoresis, each strip was equilibrated with 5 ml equilibration buffer A (6 M urea, 100 mM Tris-HCl pH 8.8, 30% v/v glycerol, 1% w/v SDS, 1% DTT) by rocking for 15 min, and then with 5 ml equilibration buffer B (8 M urea, 100 mM Tris-HCl pH 6.8, 30% v/v glycerol, 1% w/v SDS, 2.5% iodoacetamide) for an additional 15 min. For the second dimension, the strips were placed on top of a Novex Bis-Tris 4%–12% gel (Invitrogen). After electrophoresis, proteins were stained with Colloidal Coomassie Blue for subsequent protein identification.
Two-dimensional gels were scanned with a Typhoon 9400 scanner (Amersham), and the spots analyzed with DeCyder software (Amersham) and picked manually. After digestion with trypsin, a portion of the digest was analyzed directly by MALDI-TOF for molecular weight determination and MALDI-TOF/TOF for sequence information using a Voyager 4700 proteomics analyzer mass spectrometer (Applied Biosystems). Another portion of the digest was subjected to nanoLC/Qstar-XL analysis (Applied Biosystems). The data were acquired and analyzed with Analyst QS. The protein identification and database search were performed with Mascot dll script of Analyst QS; the combined MS and MS/MS data were used for the Mascot database search. A protein score of >70 with a confidence identification of >95% was considered acceptable.
RESULTS
Our previous proteomic analysis of the SDS-insoluble flagellar accessory structures identified a number of proteins involved in the generation and utilization of ATP [10]. In particular, we identified both cytoplasmic AK1 and mitochondrial AK2 as part of these structures. Two highly homologous isoforms of somatic AK1 have been described. AK1 (accession number: AAH14802) and AK1ß (accession number: NP_067490) are 194 and 210 amino acids long, respectively, with AK1ß having an additional 16 amino acids at its amino terminal end. AK2 (accession number: NP_058591) is a 239 amino acid protein with 30% identity to AK1 and AK1ß. All three proteins contain an ATP-binding site and the AK signature domain.
We confirmed that AK1 and AK2 were present in sperm and the SDS-insoluble tail fraction by immunoblotting. A band of the predicted size for AK1 (Mr 22 000) was seen in proteins from both samples when probed with anti-AK1 (Fig. 1A); this band was absent when the immunoblot was probed with normal rabbit serum (data not shown). An abundant AK1-immunoreactive band of Mr 56 000 also was present in sperm, but not in the SDS-insoluble tail preparation, which initially suggested to us that a larger, soluble isoform of AK1 might be present in these cells. Using anti-AK2, a band of the expected size (Mr 28 000) was seen in protein extracts from sperm and an SDS-insoluble tail preparation (Fig. 1B). Preabsorption of the antibody with the peptide used as the antigen eliminated immunoreactivity (data not shown). Both AK1 and AK2 were not enriched in the SDS-insoluble tail preparation compared to levels of the proteins in intact sperm, indicating that portions of these proteins were solubilized under these extraction conditions. The most abundant axonemal protein, tubulin, is not seen in the tail preparation, indicating that our SDS-insoluble preparation contained accessory structures but not components of the axoneme [10].
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Identification of cDNAs in Germ Cells Encoding Adenylate Kinase 1
To identify cDNAs encoding AK1 in spermatogenic cells, we screened a mixed germ cell cDNA library. Two different cDNA clones, a 1961 nt long form (Ak1_v1) and a 819 nt short form (Ak1_v2), corresponding to AK1 were isolated and sequenced. Ak1_v1 was not a full-length cDNA, as it lacked the nucleotide sequence encoding the amino-terminal end of the protein and the 5' UTR; however, it was identical to the Ak1/Ak1ß cDNAs throughout the remaining open reading frame and 3' UTR. In comparison, the Ak1_v2 cDNA was a full-length transcript, as it contained an in-frame stop codon upstream of the initiator methionine. This sequence has been assigned the GenBank database accession number DQ486026. While this cDNA contained a different 5' UTR and a truncated 3' UTR when compared to Ak1_v1, the protein encoded by Ak1_v2 was identical to somatic AK1. Alignment of Ak1_v2, Ak1_v1, and various Ak1 cDNAs and ESTs to the Ak1 genomic sequence showed that Ak1_v2 shared most of its exons with Ak1_v1 (Fig. 2). However, its first exon (containing the majority of the 5' UTR) was upstream of the 5' UTR of Ak1_v1, suggesting that Ak1_v2 was transcribed from an alternative promoter. A cDNA (accession number: AK046613) was characterized from adipose tissue of a 4-day-old neonate and had the same 5' UTR as Ak1_v2; however, an amplicon corresponding to this cDNA was not present in adult adipose tissue when assayed by RT-PCR (Fig. 3). Several ESTs from testis and round spermatids were identical to regions of Ak1_v2.
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Testis-Specific Expression of Ak1_v2
To determine whether the Ak1_v2 cDNA was testis-specific, RT-PCR was performed using primers that would only amplify RNA corresponding to this transcript. An amplicon of the predicted size was detected when testicular RNA, but not RNA from a variety of somatic tissues, was used (Fig. 3A). When primers corresponding to common regions of Ak1 were utilized, amplicons were detected in known sources of Ak1 mRNA, such as heart and skeletal muscle, in addition to testis (Fig. 3B). In comparison, Ak2 mRNA was present in all tissues examined (Fig. 3C). An amplicon corresponding to ß-actin was present in all samples, confirming the integrity of the RNA (data not shown).
Consistent with the RT-PCR results, immunoblot analysis showed a restricted pattern of AK1 expression in testis and somatic tissues, i.e., a band of Mr 22 000 was present in heart, skeletal muscle, and testis (Fig. 4). In contrast, a band of Mr 28 000 corresponding to AK2 was present in nearly all tissues analyzed, although at varying levels. Higher molecular weight bands that were immunoreactive with anti-AK2 were also detected in testis and liver. Preabsorption of the antibody with the peptide used as the antigen eliminated immunoreactivity of these bands (data not shown).
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AK1 and AK2 mRNAs and Proteins are Differentially Expressed During Spermatogenesis
To examine the expression of adenylate kinases 1 and 2 during spermatogenesis, we used quantitative real-time RT-PCR and immunoblotting. Ak1 mRNA was not detected in pachytene spermatocytes (Fig. 5A). It was first detected at low levels in round spermatids, indicating that the mRNA is post-meiotically expressed. Transcript levels became much more abundant in condensing spermatids compared to round spermatids (>20-fold). Ak1 mRNA in condensing spermatids was present at 30-fold higher levels compared to Ak2 mRNA (compare Fig. 5, A and B). AK1 was only detected in condensing spermatids, demonstrating that it is post-meiotically transcribed and translated (Fig. 5C).
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In contrast, Ak2 mRNA was present at low but relatively equivalent levels in pachytene spermatocytes, round spermatids, and condensing spermatids (Fig. 5B). AK2 also was present in these cell types (Fig. 5D). Its lower level in condensing spermatids compared to the other cell types most likely reflects the loss of flagella from condensing spermatids as a consequence of the enzymatic treatment required to prepare purified spermatogenic cell populations.
In addition to AK1 and AK2, other murine adenylate kinases have been identified using the UniGene database. To determine whether additional Ak mRNAs were present in spermatogenic cells, RT-PCR was performed using mixed germ cell RNA and specific primers to Ak3–5, 7, Taf9 (homologous to human AK6) [25], and a RIKEN clone (accession number: NM_001033874) that encodes a protein with an AK signature domain and an ATP binding site. All of these transcripts were present in mixed germ cells (Fig. 6). Based on the amount of input template cDNA required for amplification, the levels of these adenylate kinase mRNAs were in the general range of Ak2 and not nearly as abundant as Ak1.
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The Mr 56 000 Sperm Protein Is Not an AK1 Isoform
An abundant AK1-immunoreactive band of Mr 56 000 was detected in sperm extracts (Fig. 1); however, we never identified a cDNA encoding a protein of this size. Quantitative RT-PCR showed that the Ak1 mRNA was expressed post-meiotically (Fig. 5A). On the other hand, the Mr 56 000 band appeared earlier during spermatogenesis, i.e., in pachytene spermatocytes, indicating it is not an AK1 isoform (Fig. 5C). To identify this immunoreactive band, we separated sperm protein extracts by 2D gel electrophoresis. Three spots corresponding to the Mr 56 000 band were cored from the gel and identified after trypsin digestion and mass spectrometry. Two spots were identified as ß-tubulin, an abundant sperm protein that has a molecular size and pI consistent with the location of the cored spot in the 2D gel (Table 3). However, our AK1 antibody did not immunoreact with purified tubulin (data not shown). The third spot was identified as ATP synthase ß-chain (EC 3.6.3.14; accession number: P56480). This protein shares homologous regions with AK [26] and has been reported to be present in rat sperm [27]. We conclude that the antibody is most likely reacting with ATP synthase ß-chain; since this protein is not seen in the SDS-flagellar protein preparation (Fig. 1), it is not part of the accessory structures.
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AK1 and AK2 are Differentially Localized in Sperm
AK1 was localized to the entire length of the sperm flagellum in a punctate pattern (Fig. 7A). Because we were concerned that the larger Mr 56 000 band recognized by anti-AK1 might confound any results seen in sperm, the localization was repeated using SDS-insoluble tail structures, which do not contain this protein (Fig. 1). A similar pattern was observed, i.e., a punctate pattern throughout the length of the sperm tail (Fig. 7C). Immunoelectron microscopy showed that AK1 was associated with both the outer dense fibers and the outer microtubular doublets of intact sperm (Fig. 7, E and F). AK1 was also retained in the ODFs of the SDS-insoluble tail structures, which lack the axonemal components (Fig. 7, G and H). The association of AK1 with the ODFs and axoneme would allow the interconversion of ATP and ADP between the fibrous sheath where ATP is produced by glycolysis and the axoneme where ATP is consumed by the dynein ATPases.
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As expected from previous studies in somatic cells that showed that AK2 is found in mitochondria, AK2 localized to the midpiece of the sperm flagellum, the site of the mitochondrial sheath (Fig. 8A). A similar localization pattern was found when SDS-insoluble tail structures were analyzed, confirming that AK2 is part of the accessory structures (data not shown). Immunoelectron microscopy showed that AK2 was present in the mitochondrial sheath in both sperm and SDS-insoluble accessory structures (Fig. 8C–F). The protein was found clustered in the inner aspect of the sheath; this clustering indicates that AK2 functions in spatially restricted microdomains. In addition, the mitochondrial localization of AK2, close to the outer dense fibers, suggests that AK1 and AK2 work in concert to maintain the adenylate energy charge in the sperm flagellum.
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DISCUSSION
A substantial and continuing supply of ATP is necessary for the various events associated with mammalian sperm function. The hydrolysis of ATP by the axonemal ATPases in the long flagellum supplies the energy required to regulate sperm movement in a coordinated manner. We, and others, have hypothesized that ATP is generated by multiple mechanisms and that this production is compartmentalized along the flagellum so that ATP can function in a localized fashion [3, 5, 8]. Because oxidative phosphorylation generates more ATPs than glycolysis per glucose equivalent, the ATP necessary for mammalian sperm motility was thought to be generated by mitochondrial respiration in the midpiece (the most proximal region of the sperm tail, where all the mitochondria are located). This ATP either would have to diffuse to other regions of the tail or be actively transported down the flagellum from the midpiece, possibly by a phosphocreatine shuttle system similar to that seen in sea urchin sperm [2]. However, the length of the mammalian flagellum probably precludes efficient diffusion [28], and the phosphocreatine system is either poorly developed or absent in these sperm [3].
Glycolysis is a major ATP generator for mammalian sperm motility. When oxidative phosphorylation in mouse sperm mitochondria is suppressed, sperm remain motile as long as glucose is present in the medium [29]. However, motility is greatly diminished when sperm are incubated in a medium without glucose. Glycolytic enzymes have been localized to the fibrous sheath in the principal piece, a more distal region of the tail to the mitochondria, indicating that glycolysis could supply localized ATP to the ATPases along the entire length of the principal piece. Male mice carrying a targeted deletion of the gene encoding the sperm-specific glyceraldehyde 3-phosphate dehydrogenase are infertile; their sperm are immotile and have very low cellular ATP levels [8]. Other studies demonstrated that glycolysis is necessary for hyperactivated motility [30]. We previously showed that glucose is necessary for the tyrosine phosphorylation of a subset of sperm proteins associated with capacitation, while uncouplers of oxidative phosphorylation do not affect this process [31].
In addition to oxidative phosphorylation and glycolysis, another way to generate cellular ATP is via adenylate kinase, where two molecules of ADP can be interconverted to one molecule of ATP and one molecule of AMP. Because AKs have an equilibrium constant approaching 1, they maintain the adenylate energy charge at a relatively constant value [15]. In locations where ATP is being used rapidly, e.g., the sperm flagellum, the reaction is in the direction of ATP production, allowing the cell to scavenge energy from ADP during peaks of net energy consumption.
Our proteomic analysis identified both AK1 and AK2 in the accessory structures surrounding the axoneme, leading us to hypothesize that these enzymes are in positions to buffer the adenylate energy charge in the flagellum [15]. AK1 and 2 are differentially localized in the sperm flagellum, with AK2 being in the mitochondrial sheath and AK1 located at the outer dense fiber-outer microtubular doublet interface. This localization is reminiscent of SPAG4, which has been postulated to act as part of the link between the ODF and axoneme [32]. SPAG4 binds ODF1 (but not ODF2), but whether AK1 binds either SPAG4 or ODF1 is not known.
There are at least seven murine genes (Ak1–7) and a RIKEN clone (accession number: NM_001033874) predicted to encode adenylate kinases. (The approved gene symbol for Ak4 is Ak3L1.) These isozymes have different expression and/or localization patterns. While we focused on AK1 and AK2 in this study because of their locations in the flagellar accessory structures, we also found mRNAs corresponding to all the known AK isoforms in mixed germ cells. This differs from recent analyses using the less sensitive Northern blot technique that did not find all of the Ak genes expressed in the testis [16]. Future experiments need to be directed towards determining whether all the different Ak transcripts are translated and if (and where) the proteins are present in sperm.
The multiplicity of AKs expressed in murine germ cells is similar to that of the flagellated protozoan parasite, Trypanosoma brucei, which expresses 7 adenylate kinase genes [33]. Of note, three of the AK proteins are present in the flagellum either in the axoneme or the extra-axonemal paraflagellar rod, a structure that is required for motility in this organism. Multiple AKs are also found in the flagellum of Chlamydomonas reinhardtii [34, 35]. Similar to mammalian sperm, Chlamydomonas lacks creatine kinase, eliminating the possibility of ATP transport by a phosphocreatine shuttle system. In addition, the finding that motility is reactivated in the presence of ADP alone suggests the presence of AKs [36]. A Chlamydomonas adenylate kinase with three AK domains is anchored by two outer dynein arm proteins, ODF5p and Oda10p [35]. Another Chlamydomonas protein, CPC1, is found in the central pair of the axoneme and contains an unusual AK domain [34]. The presence of two proteins with AK activity explains why mutation of either protein alone reduces but does not totally eliminate motility in Chlamydomonas. Homologues of Cpc1 are found in a number of mammalian organisms having motile flagella, suggesting that additional proteins with AK function are present in the axoneme [34]. However, such proteins would not be identified in our proteomic analyses, as they would be solubilized during the isolation of the accessory structures.
The disruption of the adenylate kinase gene, adk1, in Saccharomyces pombe is lethal [37]. When this gene is disrupted in Saccharomyces cerevisiae, there is a compensatory metabolism mechanism to keep the cells alive; however, they have a petite phenotype and can not grow under nonfermentative conditions [38, 39]. The ablation of the murine Ak1 gene resulted in mice which are viable, although the phenotype indicated that AK1 is critical for the maintenance of skeletal muscle energetic economy [17, 40]. In particular, the potential of myofibers to sustain nucleotide ratios is decreased, and a higher ATP turnover rate is seen. Metabolic stress amplified this reduction in energy homeostasis. Importantly, glycolysis and the guanylate and creatine kinase phosphotransfer pathways are upregulated, presumably compensating for the loss of AK1. Similar results are seen in hearts of AK1-null animals, i.e., an upregulation of alternative high-energy transfer pathways prevents gross abnormalities in the absence of metabolic stress [18].
The function of AK1 and AK2 in sperm remains unclear. AK activity was detected in bovine sperm flagellum and can generate sufficient ATP to produce motility in digitonin-permeabilized cells treated with MgADP [19]. When this enzyme activity is inhibited by P1,P5-di(adenosine 5')-pentaphosphate, an AK-specific inhibitor, motility is disrupted. Surprisingly, a fertility defect was not reported in Ak1-null male mice, although a detailed assessment of sperm function was not performed [17]. It is possible that the Ak1-null sperm compensate by other mechanisms, e.g., upregulation of other AK proteins and/or glycolysis in the fibrous sheath of the tail. The Ak2 gene has not been eliminated by targeting, although its widespread expression pattern suggests that the Ak2-null phenotype would be embryonic lethal. Future experimentation should offer insights into the role of the adenylate kinases in sperm function.
ACKNOWLEDGMENTS
We are grateful to Drs. Bé Wieringa and Edwin Janssen for generously providing the AK1 antibody. Mass spectrometry and peptide microsequencing were provided by the Proteomics Core Facility of the Geonomics Institute and the Abramson Cancer Center at the University of Pennsylvania. We thank Dr. Chao-Xing Yuan and Christine Busch of this facility for their expertise and guidance during the course of this work.
FOOTNOTES
1 Supported by NICHD HD06427 (S.B.M. and G.L.G.). The germ cell-specific AK1 sequence described in this work has been assigned the GenBank database accession number DQ486026. 
2 Correspondence: Stuart B. Moss, Center for Research on Reproduction and Women's Health, 1312 BRB II, 421 Curie Blvd., University of Pennsylvania Medical School, Philadelphia, PA 19104. FAX: 215 573 7627; smoss{at}mail.med.upenn.edu
Received: 2 May 2006.
First decision: 26 May 2006.
Accepted: 13 June 2006.
REFERENCES
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 V. Thimon, O. Koukoui, E. Calvo, and R. Sullivan Region-specific gene expression profiling along the human epididymis Mol. Hum. Reprod., October 1, 2007; 13(10): 691 - 704. [Abstract] [Full Text] [PDF]
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