BIOLOGY OF REPRODUCTION 82, 504–515 (2010)
Published online before print 4 November 2009.
Molecular Complex of Three Testis-Specific Isozymes Associated with the Mouse
Sperm Fibrous Sheath: Hexokinase 1, Phosphofructokinase M, and Glutathione
S-Transferase mu class 51
Noriko Nakamura,3,4Chisato Mori,5and Edward M. Eddy2,4
Gamete Biology Section,4Laboratory of Reproductive and Developmental Toxicology, National Institute
of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina
Department of Bioenvironmental Medicine,5Graduate School of Medicine, Chiba University, Chuo-ku, Chiba, Japan
Mammalian sperm require ATP for motility, and most of it is
generated by glycolysis. The glycolytic enzymes segregate to the
principal piece region of the flagellum, where some are bound
tightly to a novel cytoskeletal structure defining this region, the
fibrous sheath (FS), and others are easily extracted with
detergents. One of the latter is the spermatogenic cell-specific
variant isozyme of hexokinase type 1 (HK1S), characterized by
an N-terminal 24-amino acid spermatogenic cell-specific region
(SSR). Yeast two-hybrid screens carried out using the SSR as bait
determined that HK1S is tethered to muscle-type phosphofruc-
tokinase (PFKM) in the principal piece region. This led to the
identification of four testis-specific Pfkm splice variants, one that
overlapped a variant reported previously (Pfkm_v1) and three
that were novel (Pfkm_v2, Pfkm_v3, and Pfkm_v4). They differ
from Pfkm transcripts found in somatic cells by encoding a novel
67-amino acid N-terminal extension, the testis-specific region
(TSR), producing a spermatogenic cell-specific PFKM variant
isozyme (PFKMS). An antiserum generated to the TSR demon-
strated that PFKMS is present in the principal piece and is
insoluble in 1% Triton X-100 detergent. In subsequent yeast
two-hybrid screens, the TSR was found to interact with
glutathione S-transferase mu class 5 (GSTM5), identified
previously as a spermatogenic cell-specific component of the
FS. These results demonstrated that HK1S is tethered in the
principal piece region by PFKMS, which in turn is bound tightly
to GSTM5 in the FS.
flagellum, gamete biology, gametogenesis, glycolysis,
multienzyme complex, sperm, spermatogenesis, testis
Glycolysis is the major source of ATP required for sperm
flagellar activity, capacitation, and fertilization [1–3], and the
enzymes for this process are localized in the principal piece
region of the flagellum. This places the glycolytic enzymes and
ATP production along most of the length of the flagellum in
proximity to the dynein ATPase activity generating the
flagellar beat. Some of these enzymes are known to be
products of genes expressed specifically in spermatogenic
cells, including lactate dehydrogenase C (LDHC) , phos-
phoglycerate kinase 2 (PGK2) [5, 6], glyceraldehyde 3-
phosphate dehydrogenase S (GAPDHS) , and two aldolase
A isozymes (ALDOART1 and ALDOART2) . Others are
products of variant transcripts, including spermatogenic cell-
specific hexokinase type 1 (HK1S) [9, 10] and another aldolase
A isozyme (ALDOA_V2) . Furthermore, mammalian sperm
contain atypical isozymes of phosphoglucose isomerase ,
triosephosphate isomerase , phosphoglycerate mutase ,
and enolase [14, 15], but it remains to be determined if they are
products of genes or variant transcripts expressed specifically
in spermatogenic cells.
The principal piece region accounts for about 70% of the
length of the mouse sperm flagellum and is characterized by
the presence of a fibrous sheath (FS). This novel cytoskeletal
structure serves as a mechanoelastic structure involved in
shaping the flagellar waveform , is composed of more than
20 proteins, and is a scaffold for glycolytic enzymes, signaling
pathway components, and chemoprotective proteins .
Earlier studies indicated that some sperm glycolytic enzymes
cofractionate with flagellar components , are difficult to
extract , are associated with insoluble components ,
and are present in multienzyme complexes [20, 21]. More
recent studies showed that GAPDHS [22, 23], lactate
dehydrogenase A (LDHA), aldolase A , and pyruvate
kinase (PK)  are tightly bound to the FS.
It remains to be determined how the glycolytic enzymes
segregate to the principal piece region of the flagellum and are
anchored in this region. Some of the glycolytic enzymes are
highly resistant to extraction, including GAPDHS ,
ALDOART1, ALDOA_V2 , LDHA, and PK . An
unusual feature of GAPDHS is a novel proline-rich N-terminal
105-amino acid sequence not present in GAPDH , while
ALDOART1 has a novel 55-amino acid N-terminal region, and
ALDOA_V2 has a novel 54-amino acid N-terminal region .
It has been suggested that the novel N-terminal sequences are
involved in delivering these enzymes to the principal piece and/
or attaching them to the FS [8, 23, 24, 26].
While some of the glycolytic enzymes are highly resistant to
extraction, others found in the principal piece region are readily
solubilized by detergents, including HK1S, LDHC, and PGK2
. While LDHC and PGK2 lack novel N-terminal features,
HK1S has a novel N-terminal spermatogenic cell-specific
region (SSR). In somatic cells, HK1 is tethered to porin
(voltage-dependent anion channel) in the outer mitochondrial
membrane by its N-terminal porin-binding domain [27, 28].
Replacement of the porin-binding domain by the SSR produces
1Supported by the Intramural Research Program of the NIH, National
Institute of Environmental Health Sciences.
2Correspondence: Edward M. Eddy, Gamete Biology Section, Labora-
tory of Reproductive and Developmental Toxicology, National Institute
of Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, NC 27709. FAX: 919 541 3800;
3Current address: Department of Pharmacology, Physiology and
Toxicology, Marshall University, Huntington, WV 25755.
Received: 2 August 2009.
First decision: 4 September 2009.
Accepted: 15 October 2009.
? 2010 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
Downloaded from www.biolreprod.org.
the spermatogenic cell-specific HK1S isozyme . This led us
to hypothesize that HK1S is localized to the principal piece by
associating with other sperm components or multienzyme
complexes in this region [9, 26]. This hypothesis was tested in
the present study by using the SSR as bait in a yeast two-hybrid
screen to search for HK1S-binding partners. We found that
HK1S is tethered via its SSR to a previously unidentified
spermatogenic cell-specific phosphofructokinase variant iso-
zyme (PFKMS) confined to the principal piece. A subsequent
yeast two-hybrid screen determined that PFKMS binds via its
novel 67-amino acid N-terminal testis-specific region (TSR) to
glutathione S-transferase mu class 5 (GSTM5), a highly
insoluble spermatogenic cell-specific component of the FS
identified previously by peptide sequencing . This demon-
strates that direct interactions occurring between two glycolytic
enzymes and between a glycolytic enzyme and an FS protein,
mediated by spermatogenic cell-specific peptide sequences, are
responsible for anchoring HK1S and PKFMS to the FS.
MATERIALS AND METHODS
Materials and Animals
All reagents were purchased from Sigma (St. Louis, MO [http://www.
sigma-aldrich.com]) unless indicated otherwise.
CD-1 mice used for protein experiments and immunohistochemistry were
obtained from Charles River Laboratories (Raleigh, NC [http://www.criver.
com]). All animal studies were approved by the National Institute of
Environmental Health Sciences Institutional Animal Care and Use Committee
and were performed according to U.S. Public Health Service guidelines.
Yeast Two-Hybrid Screening
Yeast two-hybrid screening was carried out as recommended by the
supplier (BD Biosciences Clontech, San Jose, CA [http://www.clontech.com]).
Yeast AH109 competent cells were prepared with the Frozen-EZ Yeast
Transformation II kit (Zymo Research, Orange, CA [http://www.zymoresearch.
com]). The cDNAs encoding the SSR of Hk1s cDNA and the TSR of Pfkm_v2
cDNA were subcloned into the yeast expression vector pKBTG7 (BD
Biosciences Clontech) to generate pKBGT7-SSR and pKBGT7-TSR, respec-
tively. Yeast AH109 cells were cotransformed with a mouse testis cDNA
library (BD Biosciences Clontech) and pKBGT7-SSR or pKBGT7-TSR using
the lithium acetate method .
Amplification of 50cDNA Ends
Rapid amplification of 50cDNA ends (50RACE) was performed using a
Marathon-ready testis cDNA library and the BD Advantage 2 PCR kit (BD
Biosciences Clontech) according to the supplier’s recommended procedure.
Adaptor primer 1 from the kit and primer (50-CTG ATG TGC TCG CCA CCG
TCC ACC A-30) were used for PCR amplification. RACE products were
ligated into the pGEM-T vector (Promega, Madison, WI [http://www.promega.
com]) for sequencing.
The SSR, GSTM5 full length (FL), and GSTM5 mutants (GSTM5-N
[amino acids 5–85] and GSTM5-C [amino acids 95–195]) were subcloned into
the bacteria expression vector FLAG-CTC (Sigma). The TSR, PFKMS-FL, and
PFKMS mutants (PFKMS-N [amino acids 16–383] and PFKMS-C [amino
acids 403–685]) were subcloned into the pGEX-4T-1 vector (GE Healthcare
Life Sciences; Piscataway, NY [http://www.gehealthcare.com]) for GST pull-
down assays. In addition, HK1S, TSR, PFKMS-FL, and GSTM5-FL were
subcloned into the pcDNA3.1(þ)-His/Myc (Invitrogen, Carlsbad, CA [http://
www.invitrogen.com]) vector for in vitro translation.
Preparation of Recombinant TSR
Recombinant protein containing the TSR was produced for use as an
immunogen. The TSR-encoding region of the Pfkm_v2 cDNA was subcloned
with pET-21b vector (EMD Chemicals, Inc., Gibbstown, NJ [http://www.
emdbiosciences.com]) and expressed in Escherichia coli BL21(DE3) (Stra-
tagene, Cedar Creek, TX [http://www.stratagene.com]), and exponential growth
was induced with 0.5 mM isopropyl-D-thiogalactoside. The recombinant protein
was purified using Ni-NTA beads (EMD Chemicals, Inc.) and the MagneHis
Protein purification system (Promega) as recommended by the suppliers.
Purification of Antiserum to TSR
Serum was collected from New Zealand white rabbits following
immunization with the recombinant TSR peptide (Covance, Denver, PA
[http://www.covance.com]). Proteins were precipitated from the serum by
saturating with ammonium sulfate at 48C for 2 h and were collected by
centrifugation at 24553 3 g for 30 min at 48C. The pellet was resuspended in
PBS and dialyzed against 10 mM Tris (pH 7.5) overnight at 48C. The dialysate
was affinity purified using recombinant TSR linked to CNBr-4B beads (GE
Healthcare Life Sciences) as recommended by the supplier. This is referred to
hereafter as TSR antiserum.
Northern Blot Analysis
Total RNA was extracted from testes of mice aged 10–30 days and from
tissues (brain, heart, kidney, liver, spleen, lung, skeletal muscle, thymus, ovary,
and epididymis) of adult mice using Trizol reagent (Invitrogen), separated by
electrophoresis on 1.0% agarose gels containing 0.66 M formaldehyde, and
transferred to Hybond N nylon membranes (GE Healthcare Life Sciences) in
103 saline-sodium citrate (SSC). The probes were labeled with [32P] using a
random prime labeling kit (Roche Applied Sciences, Indianapolis, IN [http://
roche.com]) with the 320-base pair (bp) cDNA encoding the TSR from
Pfkms_v2 as template. Hybridization was performed in Hybrisol I (Millipore
Corporation, Billerica, MA [http://www.millipore.com]) at 428C overnight. The
membranes were washed with 23 SSC containing 0.1% SDS at room
temperature for 15 min and then in 0.13SSC containing 0.1% SDS at 508C for
15 min, and they were subjected to autoradiography.
Recombinant GST-PFKMS-FL, GST-PFKMS mutants (GSTM5-N and
GSTM5-C), GST-phosphofructokinase (PFK), and GST proteins were
incubated with recombinant FLAG-tagged SSR protein, and GST pull-down
assays were carried out using the lMACS GST-tag isolation kit (Miltenyi
Biotech, Auburn, CA [http://www.miltenyibiotec.com]). Elutes were analyzed
by immunoblot analysis with antibodies to GST (RGST-45A-Z; Immunology
Consultants Laboratory, Inc., Newberg, OR [http://www.icllab.com]) and to
FLAG (F7425; Sigma). FLAG pull-down assays were performed as
recommended by the supplier (Sigma). Recombinant GST-TSR protein was
incubated with FLAG vector only protein or with recombinant FLAG-tagged
GSTM5-FL (FLAG-GSTM5-FL), the N-terminal portion of GSTM5 (FLAG-
GSTM5-N), or the C-terminal portion of GSTM5 (FLAG-GSTM5-C) protein
for 2 h at 48C. FLAG-M2-agarose (A2220; Sigma) was added to the protein
mixtures, and after incubation overnight at 48C, the agarose beads were washed
with Tris-buffered saline (TBS), mixed with 13 sample buffer, and heated at
958C for 5 min. The materials released were separated by SDS-PAGE and
immunoblotted with antibody to GST.
In Vitro Translation
Using plasmids already described, in vitro translation of the SSR of HK1S
and the TSR of PFKMS and GSTM5 was carried out in the TNT coupled
reticulocyte lysate system (Promega) according to the supplier’s instructions.
Translation was performed for 2 h at 308C in a volume of 25 ll containing 20
lCi of [35S] methionine (GE Healthcare Life Sciences). After incubation, the
labeled proteins were separated from unincorporated [35S] methionine using
MicroSpin G-25 columns (GE Healthcare Life Sciences).
Testes were homogenized in a lysis buffer containing 20 mM Tris-HCl (pH
8.0), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% SDS,
0.5% NP-40, 1% Triton X-100, and protease inhibitor cocktail (Roche Applied
Sciences). Homogenates were incubated for 30 min on ice and then centrifuged
at 158003g for 30 min, and the supernatant was used for immunoprecipitation
studies. The in vitro-translated [35S]-labeled proteins were incubated with testis
lysate for 2 h at 48C. Ten micrograms of antibody was added and incubated for 2
h at 48C, and then Immunopure immobilized protein G beads (Pierce
Biotechnology, Rockford, IL [http://piercenet.com]) were added and incubated
for 2 h at 48C on a rotating platform. The beads were washed with TBS, mixed
with 13 sample buffer, and heated for 5 min at 868C. After centrifugation at
5000 3 g for 3 min at room temperature, the supernatants were separated by
TESTIS-SPECIFIC ISOZYMES IN THE FIBROUS SHEATH
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quiescent sperm and as monomers in actively motile sperm;
and 4) the dimer to monomer conversion involved reduction of
disulfide bonds. In the present study, we report the following
findings: 1) PFK activity was lower in immotile sperm than in
motile sperm, 2) most of the PFK enzymatic activity remained
in the detergent-insoluble pellet fraction and was extracted only
partially with 6 M urea, 3) PFKMS was present in dimers in
extracts of quiescent sperm and in monomers in extracts of
actively motile sperm analyzed under nonreducing conditions,
and 4) PFKMS was present as monomers under reducing
conditions. Thus, enzymatic activity of both HK1S and
PFKMS is lower in quiescent than in actively motile sperm,
and the change in activity correlates with a dimer to monomer
conversion. However, HK1S is solubilized more easily than
PFKMS, and previous studies [24, 29] demonstrated that
GSTM5 remains present in the FS fraction after a rigorous
extraction procedure. These results indicate that a hierarchy of
binding affinities exists from low to high between HK1S and
PFKMS, between PFKMS and GSTM5, and between GSTM5
and other FS components. However, it remains to be
determined how the glycolytic enzymes are transported to the
principal piece region of the flagellum and how the others are
held in this region. While some of the enzymes have novel N-
terminal regions that might be involved in these processes (e.g.,
GAPDHS, ALDOART1, and ALDOART2), some do not (e.g.,
PGK2 and LDHC), and the mechanisms involved in the
segregation of most enzymes to this region remain to be
The authors thank Eugenia H. Goulding for valuable technical support,
Clyde Roger and Linwood Koonce for help with animals, other members
of the Gamete Biology Group for constructive suggestions, and Tracy
Clement and Oleg Alekseev for helpful comments on the manuscript.
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