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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SDS-PAGE, and the gels were immersed in Amplify (GE Healthcare Life
Sciences) for 30 min, dried, and exposed to Hyperfilm MP (GE Healthcare Life
Sciences) at ?708C.
Sample Preparation Procedures
Skeletal muscle and testes were homogenized in lysis buffer (20 mM PBS,
1% Triton X-100, 0.2 mM sodium orthovanadate, and 0.1 mM PMSF),
incubated for 30 min on ice, and centrifuged at 158003g for 30 min, and the
soluble fraction (supernatant) was collected. Sperm used in most studies were
isolated from the cauda epididymis, incubated in M2 medium (Sigma) for 5
min at room temperature, pelleted in Eppendorf tubes by centrifugation at 7500
3g for 3 min, and washed in PBS. To prepare sperm samples for SDS-PAGE,
the pellet was suspended in PBS containing 0.1% Triton X-100 (PBSTX) and
an equal volume of 23 sample buffer (4% SDS, 100 mM Tris-HCl [pH 6.8],
20% glycerol, 2% 2-mercaptoethanol, and 0.001% bromophenol blue).
For comparative solubility studies, the sperm pellet was suspended in PBS
containing 1% Triton X-100 or urea (1 M, 2 M, 3 M, or 4 M) for 2 h on ice or
in PBS containing 6 M urea for 2 h at room temperature. After centrifugation at
12334 3 g for 15 min at 48C, the supernatants were suspended in 23 sample
buffer. For sequential solubility studies, sperm were treated using a procedure
modified from one reported previously . Sperm were suspended in NP-40
lysis buffer (1% NP-40,10 mM Hepes [pH 7.5], 50 mM NaCl, 10 mM EDTA,
1% NP-40, 0.2 mM PMFS, and 10 lg/ml of aprotinin) for 1 h on ice,
centrifuged at 500 3 g for 10 min at 48C, and the supernatant was collected.
The pellet was washed with 50 mM Tris-HCl (pH 8.2), resuspended in urea
lysis buffer (6 M urea, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 6 M urea, 0.2
mM dithiothreitol [DTT], and 10 lg/ml of aprotinin) for 2 h on ice, and
centrifuged at 120003g for 20 min at 48C, and the supernatant was collected.
Pellets (6 M urea-insoluble fraction) were suspended in 13 sample buffer. For
studies of quiescent sperm, sperm were collected from the caput and cauda
epididymis directly into 1.5-ml tubes without dilution as described previously
, suspended in PBSTX, and centrifuged at 45283g for 5 min 48C, and the
supernatants and pellets were collected. For activated sperm, sperm were
collected as already described. Samples of quiescent and activated sperm from
caput and cauda epididymis were suspended in 23sample buffer containing 2%
2-mercaptoethanol (reducing conditions) or without 2-mercaptoethanol (non-
Proteins extracted from sperm and skeletal muscle were separated by SDS-
PAGE on 4%–20% gradient ready gels or 10% ready gels (Bio-Rad
Laboratories, Hercules, CA [http://www.bio-rad.com]) and were transferred
from the gels to Immobilon-P PVDF membranes (Millipore Corporation). The
membranes were immunostained with TSR antiserum and antisera to SSR ,
PFKM (100-1156; Rockland Immunochemicals, Inc., Gilbertsville, PA [http://
www.rockland-inc.com]), a-tubulin (T5168; Sigma), b-actin (A5441; Sigma),
or GSTM5 (gift of Dr. Irving Listowsky, Albert Einstein College of Medicine,
Bronx, NY)  and detected using ECL reagents (GE Healthcare Life
Sciences) as recommended by the supplier.
Testes were fixed in Bouin solution, dehydrated, and embedded in paraffin,
and 6-lm sections were prepared using standard procedures. Slides were
deparaffinized and immunostained with TSR antiserum and an Elite ABC kit
for rabbit IgG (Vector Laboratories, Burlingame, CA [http://www.vectorlabs.
Pfkm_v3, and Pfkm_v4 transcripts are de-
rived from different combinations of exons,
and their 50ends (upstream of Pfkm exon 1)
differ in length. M indicates the putative
initiation methionine. B) The genomic
structure of Pfkm, indicating the location of
the exons giving rise to the variant tran-
scripts. The rectangles show the location of
the spermatogenic cell expressed exons (-s1
to -s5) and exons (1–3) transcribed in
somatic and germ cells. The Pfkm_v1
transcript uses exon -s1, and Pfkm_v2,
Pfkm_v3, and Pfkm_v4 use exons -s2 and
-s3. In addition, Pfkm_v2 uses exon -s5, and
Pfkm_v3 uses exon -s4.
A) The Pfkm_v1, Pfkm_v2,
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com]), and the peroxidase-labeled secondary antibody was detected using 3,30-
diaminobenzidine tetrahydrocholoride (Sigma) as the chromogen.
Sperm were isolated from the cauda epididymis as already described, fixed
in 2% paraformaldehyde in PBS for 15 min at room temperature, suspended in
50 mM glycine in PBS for 30 min to block free aldehyde groups, and applied to
slides as described previously . The slides were immersed in ice-cold
methanol for 1 min to permeabilize the sperm, immunostained with TSR
antiserum and antiserum to rabbit IgG conjugated to fluorescein isothiocyanate
(MP Biomedicals, Irvine, CA [http://mpbio.com]), and observed using an
Axioplan microscope (Carl Zeiss, Thornewood, NJ [http://www.zeiss.com/
micro]), and images were recorded using a QImaging camera and software
(QImaging, Tucson, AZ [http://www.qimaging.com]).
PFK Enzymatic Assay
Sperm isolated from the cauda epididymis as already described were
permeabilized for 30 min on ice in PBS containing 1% Triton X-100, 0.2 mM
sodium orthovanadate, and 0.1 mM PMSF. The sample was centrifuged at
12000 3 g for 5 min at 48C, the supernatant was collected, and the pellet was
resuspended in PBST. Enzymatic activity was measured in the pellet and
supernatant of sperm from the cauda epididymis as described previously .
In brief, the sample (30 ll) was assayed at 258C in 1 ml of assay buffer (pH 8.2)
containing 75 mM glycelglycine, 75 mM disodium b-glycerophosphate, 3 mM
Na2-EDTA, 18 mM MgCl2, 9 mM ammonium sulfate, 10 mg/ml of NADH, 3
mM DTT, 30 mM ATP, 30 mM fructose-6-phosphate, 18 U/ml of
triosephosphate isomerase, 18 U/ml of a-glycerophosphate dehydrogenase,
and 36 U/ml of aldolase. Enzymatic activity was determined using a UV
spectrophotometer (Amersham Pharmacia Biotech, Piscataway, NJ) at 340 nm
to measure the decrease in NADH. After 3 min of preincubation, the reaction
was initiated by adding 1–3 lg of sample. The negative control included assay
buffer without fructose-6-phosphate. The protein concentration of the pellet and
supernatant was measured with Bradford reagent (Bio-Rad Laboratories).
These assay results are reported as the average of four separate experiments,
each performed in duplicate. Data are expressed as the mean 6 SD of the four
Quantitative Real-Time RT-PCR
Total RNA was extracted using Trizol reagent from testes of mice aged 10–
30 days old. The cDNA for quantitative real-time RT-PCR (qPCR) was
synthesized with reverse transcriptase (RT; Applied Biosystems, Foster City,
CA). The qPCR analyses were performed using SYBR Green PCR Master Mix
reagents (Applied Biosystems) and the ABI PRISM 7900HT Sequence
Detection System (Applied Biosystems) according to the manufacturer’s
protocols. The cDNA served as a template in a 50-ll reaction mixture and was
processed using an initial denaturation step at 958C for 10 min, followed by 45
amplification cycles (958C for 10 sec and 608C for 1 min) and one dissociation
stage cycle (958C for 15 sec, 608C for 15 sec, and 958C for 15 sec). The
reactions were carried out using primer for upstream exon -s2 (50-
and for Pfkm exon 1 (50-GAGTGGATCATGACCCAT-30and 50-GCATCTC
CACCAGAGGTCA-30). The relative steady-state transcript levels were
calculated using cycle time (Ct) values and the following equation: Relative
Quantity ¼ 2?DDCt. The expression levels were normalized using primer for
Gapdh (50-TGCACCACCAACTGCTTAG-30and 50-GATGCAGGGATGAT
GTTC-30) as an internal control for each sample. The relative ratios (folds) of
transcript levels in each sample were calculated using the Day 10 value as 1.
The qPCR reactions were performed in triplicate with each of the samples.
To confirm the presence of the Pfkm variants in mouse testis, total RNA
was extracted from testis using Trizol reagent according to manufacturer’s
instructions (Invitrogen). Total RNA (1 lg) was treated with RNase-free DNase
I and was reverse transcribed using oligo dT primers. The PCR was carried out
using forward primers in the upstream exons -s1 (50-ACCTGGCTCCTC
CCCGTGTT-30), -s2 (50-GAAGCTTCAACCTCCAACA-30), -s3 (50-GAA
GTAGCCATGCGTAGAGA-30), -s4 (50-CCATGTTGACCTTTGACACT-30),
-s5 (50-GACGTGGAGGGAGCGT-30), and a reverse primer located in exon 1
(50-GCATCTCCACCAGAGGTCA-30). The 20-ll reaction mixture was
processed using an initial denaturation step at 958C for 2 min, followed by
50 amplification cycles (958C for 30 sec, 548C for 30 sec, and 728C for 20 sec)
and one step at 728C for 7 min. Products were separated on 2% agarose gels.
HK1S Is Tethered to the FS by PFKM
The localization of HK1S mainly in the principal piece
region of the flagellum led to the hypothesis that HK1S is
tethered through its SSR to proteins associated with the FS [10,
26]. This was tested in the present study using the SSR as bait
in a yeast two-hybrid screen. Of the seven cDNA clones
isolated and characterized, one encoded a 246-bp sequence
with high identity to amino acids 321–401 in mouse muscle-
type PFK (Pfkm [GenBank accession number BC005526.1]).
The identification of PFKM as a binding partner of HK1S was
confirmed using pull-down assays (described herein).
Identification of Testis-Specific Splice Variants of Pfkm
The use of 50RACE and a mouse testis cDNA library to
further characterize the Pfkm transcript giving rise to the yeast
prey clone led to the unexpected identification of four Pfkm
variant transcripts (Pfkm_v1, Pfkm_v2, Pfkm_v3, and
Pfkm_v4). They are distinguished from Pfkm transcripts in
somatic cells by the presence of additional sequences on their
50ends that are identical to regions approximately 3.5–16
kilobase (kb) upstream of the Pfkm translation start site on
chromosome 15. These sequences are encoded by five exons
and are referred to provisionally as spermatogenic cell exons
-s1 to -s5 (Fig. 1, A and B).
The Pfkm_v1 transcript incorporates exon -s1, which
contains a Pfkm variant sequence reported previously 
(TE-PFK-M [GenBank accession number D21867]) plus an
additional 60 nucleotides on the 50end (GenBank accession
number GQ428207). All or part of the exon -s1 sequence is
present in more than 60 expressed sequence tags (ESTs), many
ubiquitous (PFKM) and testis-specific
(PFKMS) PFK isozymes. The sequences in
the dashed-line box are present in the
ubiquitous PFKM in somatic cells, and the
PFKM translation initiation codon (M) is
underlined. The sequences in the solid-line
box are present in the TSR of PFKMS. The
PFKMS initiation codons (M) are underlined
Amino acid sequences of the
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of which map to UniGene Mm.272582 containing sequences
related to Pfkm. However, we were unable to identify a likely
ATG start codon upstream of exon -s1, and a BLASTP search
failed to identify significant similarities between the hypothet-
ical 39- residue peptide sequence encoded by the exon and any
mouse proteins, suggesting that exon -s1 and thus Pfkm_v1 are
The 50ends of Pfkm_v2, Pfkm_v3, and Pfkm_v4 differed
(Fig. 1A), but all contained an identical sequence originating
from exon -s2 and exon -s3 (GenBank accession numbers
GQ428206 and GQ428205) that encodes a 67-amino acid
sequence not present in the typical PFKM isozyme. This
sequence is referred to hereafter as the TSR and is the hallmark
of the spermatogenic cell-specific PFKM isozyme, provision-
ally named PFKMS (Fig. 2). Putative ATG translation start
codons were identified for exon -s3 and exon -s4 (GenBank
accession number GQ428204) but not for exon -s5 (GenBank
accession number GQ428203), suggesting that it is not
translated. The PCR results suggested that transcript levels
for Pfkm_v1, Pfkm_v2, and Pfkm_v3 were lower than Pfkm_v4
transcript levels in adult mouse testis (Supplemental Fig. S1
available at www.biolreprod.org). Using the BLAST program
to query the National Center for Biotechnology Information
databases, we found that the sequence of exon -s2 was
contained in an EST from newborn mouse brain (CB523377)
that included exon 1 of Pfkm, but there were no significant
matches with other RNA or EST sequences. In addition, part of
exon -s5 and all of exon -s4, exon -s3, and exon -s2 were
identical to regions in a sequence from newborn mouse thymus
that was purportedly from a cDNA clone (AK138788) but
contained intronic sequences. The four Pfkm variants also
contained the Pfkm coding sequence.
Northern blot analysis was used to determine if the variants
were expressed in other tissues. When a portion of the
sequence encoding the TSR (Fig. 3A) was used to probe RNA
from 11 tissues, an approximate 4.4-kb transcript was detected
only in testis (Fig. 3B). When this probe was used for Northern
blot analysis with RNA from testes of mice aged 10–35 days,
transcripts were first seen on Postnatal Day 16 (Fig. 3C),
corresponding to the age when midpachytene spermatocytes
are present in the synchronous first wave of spermatogenesis.
Pfkm_v2 (A) and RNA from various tissues of CD-1 mice (B) or whole
testes of juvenile mice 10–35 days old (C). The Rpl7 transcript was used as
an internal control.
Northern blot analyses using a probe of the TSR (arrows) from
coimmunoprecipitation assays. A) Diagram of recombinant proteins
containing either TSR of PFKMS fused to GST (GST-TSR) or all or part of
PFKM fused to GST, including full length (GST-PFKM-FL), the N-terminal
region, and the C-terminal region (GST-PFKM-N and GST-PFKM-C). B)
GST-PFKM-FL, GST-PFKM-N, and GST-PFKM-C proteins were immobi-
lized on glutathione-sepharose resin and incubated with FLAG-tagged SSR
protein, and the recombinant proteins that interacted were eluted from the
resin. The eluted recombinant proteins were separated by SDS-PAGE, and
then Western blot analysis was performed with antibodies to FLAG (upper
panel) or GST (lower panel). C) In vitro-translated [35S]-HK1S added to
testis lysate coimmunoprecipitated with PFKMS when TSR antiserum and
protein G beads were added but not when TSR antiserum was deleted (-)
or rabbit IgG was added instead of TSR antiserum.
The SSR of HK1S interacts with PFKM in a GST pull-down and
NAKAMURA ET AL.
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