BIOLOGY OF REPRODUCTION 75, 492–500 (2006)
Published online before print 21 June 2006.
Adenylate Kinases 1 and 2 Are Part of the Accessory Structures in the Mouse
Wenlei Cao, Lisa Haig-Ladewig, George L. Gerton,3and Stuart B. Moss2,3
Center for Research on Reproduction and Women’s Health, Department of Obstetrics and Gynecology, University of
Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104
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 charac-
terized and found to contain a truncated 30UTR and a different
50UTR 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 interconver-
sion 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 local-
ized 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
gametogenesis, sperm, sperm motility and transport,
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 9þ2 microtu-
bular 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 lm in some species . 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 ; however,
such a system either is not present or is of limited importance in
mammalian sperm .
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 . The importance of glycolysis was
elegantly demonstrated by the gene targeting of Gapdhs, the
sperm-specific isoform of glyceraldehyde 3-phosphate dehy-
drogenase . 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 .
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 . This study identified a number
of proteins involved in the generation and utilization of ATP.
In addition to glycolytic enzymes, e.g., aldolase, triosephos-
phate 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 phospho-
transferase, EC 188.8.131.52) 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 , ATPþAMP, thus
producing either ADP or stoichiometric amounts of ATP and
AMP, depending on the concentrations of the three nucleotides
. 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 mitochon-
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.  showed
that AK activity exists in bovine sperm flagella and can
1Supported 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.
2Correspondence: 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; e-mail: email@example.com
Received: 2 May 2006.
First decision: 26 May 2006.
Accepted: 13 June 2006.
? 2006 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
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 spermato-
cyte and round spermatid populations were at least 85% pure, while the
condensing spermatid population was approximately 40%–50% pure (contam-
inated 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 3 g for 5 min at room temperature, and SDS-resistant
head and tail structures were separated . 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 3 g for 1 h at room
temperature. The SDS-resistant tail structures were collected from the interface.
Generation of AK2 Antibody and
peptide Ac-RSYHEEFNPPKEPMKDDIC-amide (amino acids 150–167, acces-
sion 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) 1:10000 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 ll 5% NFDM in TBST and incubating the
mixture at 48C 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 ?208C 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.,
Isolation of Germ Cell AK1 cDNAs
A probe corresponding to a portion of an Ak1b cDNA (nucleotides 143–
492, accession # NM_021515) was amplified by PCR. After labeling with
[a32P]dCTP (3000 Ci/mmol) by random priming (Invitrogen, Piscataway, NJ),
the probe was used to screen a total of 500000 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 Nþ 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 658C and then incubated overnight at
428C in Rapid-hyb buffer containing the radiolabeled probe. The membranes
were washed with 23SSPE/0.1% SDS at room temperature and then with 0.1
3SSPE/0.1% SDS at 658C 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 lg
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
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 (508C for 2 min, 958C for 10 min), 2)
amplification and quantification (958C for 15 sec, 608C for 1 min) repeated for
40 cycles, 3) a dissociation curve program (958C for 15 sec, 608C for 15 sec,
958C for 15 sec), and, 4) cooling at 48C. 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-DDCTmethod . The mRNA corresponding to ribosomal protein S16
(accession number: BC082286) was used as a control .
Two-dimensional Gel Electrophoresis and
Two-dimensional gel electrophoresis was performed as previously
described except that 7-cm Immobiline DryStrips (pH 3–10, nonlinear) were
used . Equipment and reagents were from Amersham Bioscience (Uppsala,
Sweden). Briefly, samples (60 ll containing 100 lg protein) were mixed with
65 ll 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 208C 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
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