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From the * Department of Zoology, Graduate School
of Agriculture, Kyushu University, Fukuoka, Japan;
Taisho Pharmaceutical Co, Ltd, Saitama,
Japan.
| Correspondence to: Dr Hiroshi Iida, Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, Higashiku Hakozaki 6-10-1, Fukuoka 812-8581, Japan (e-mail: iidahiro{at}agr.kyushu-u.ac.jp). |
| Received for publication March 29, 2004; accepted for publication May 24, 2004. |
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Abstract |
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Key words: Spermatogenesis, spermiogenesis, spermatid, calcium-binding protein, differential display
It has been reported that a considerable number of genes are specifically expressed in haploid spermatids, most of which are transcribed after the postmeiotic stage (Fujii et al, 1999) and that transcription machinery including RNA polymerase II, TATA-binding protein, and TF IIB are known to be overexpressed in rodent spermatids (Schmidt et al, 1995). In addition to the precise regulation of stringent stage-specific gene expression, posttranscriptional control is especially important toward the end of spermatogenesis because global transcription ceases several days before the completion of spermatogenesis (Sassone-Corsi, 1997). Thus, spermiogenesis is a complicated but very interesting phenomenon in terms of morphological changes, expression of specific genes, and transcriptional/translational regulation.
To investigate the molecular mechanisms regulating the drastic morphological changes from round spermatids to spermatozoa, we used differential display in combination with cDNA cloning techniques to isolate genes that are predominantly expressed in haploid spermatids. By this approach, we have recently isolated several novel genes that are expected to be involved in differentiation of spermatogenic cells. Spergen-1 is a small protein of 154 amino acids, which is associated with mitochondria of both elongating spermatids and matured spermatozoa (Doiguchi et al, 2002b), and it might be involved in mitochondria sheath formation during spermiogenesis by working as an adhesive molecule between mitochondria (Doiguchi et al, 2002a). In addition to Spergen-1, we have recently isolated Spergen-2, encoding a 56-kDa protein localized in germ cells' nuclei (Iida et al, 2003), and Spetex-1, encoding a 63-kDa protein present in the cytoplasm of elongated spermatids (Iida et al, 2004). In the present study, we report another novel gene, designated as Spergen-3, which encodes a 75-kDa protein bearing EF-hand motifs.
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Materials and Methods |
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Differential Display
The mRNA differential display method
(Liang and Pardee, 1992;
Blanchard and Cousins, 1996)
was carried out using RNA map Kit (GenHunter, Nashville, Tenn). Briefly, total
testis RNAs were isolated from Wistar rats of age 1, 2, 3, 4, 5, 6, 7, and 8
weeks, as described previously (Iida et
al, 1999; Doiguchi et al,
2002b). RNAs were reverse-transcribed (RT) with oligo-(dT) primers
anchored to the beginning of the poly(A) tail. The resulting cDNAs were
amplified with oligo-(dT) primers and arbitrary primers. The cycling
parameters were as follows: 94°C for 30 seconds, 40°C for 2 minutes,
72°C for 30 seconds for 40 cycles. The amplified cDNAs were separated on
6% urea-polyacrylamide gels, fixed, and stained by the silver sequence system
(Promega, Madison, Wis). cDNA fragments for which expression levels were
developmentally increased were recovered directly by cutting out the gel
slices. After elution by boiling the gel slices in distilled water for 15
minutes, cDNA fragments were reamplified by using the same primers as the ones
used in the initial polymerase chain reaction (PCR) for differential display.
The cDNA fragments were then purified by electrophoresis, cloned into the pGEM
easy T-vector (Promega), and sequenced using a DNA sequencer (Applied
Biosystem, Foster City, Calif).
Complementary DNA (cDNA) cloning
To obtain the full-length cDNA encoding the rat gene, plaque hybridization
was performed by the standard method
(Sambrook et al, 1989). Rat
testis 5'-stretch plus cDNA library was obtained from CLONTECH Lab Inc.
(Palo Alto, Calif). The probe for plaque hybridization was a PCR-reamplified
340-base DNA fragment that was labeled with Digoxigenin (DIG)-dUTP by DIG High
Prime DNA Labeling Kit (Roche Molecular Biochemicals, Germany). The hybridized
probe was immunodetected by anti-DIG antibody conjugated with alkaline
phosphatase, and then recorded on x-ray films with the chemiluminescence
substrate CSPD (Roche Molecular Biochemicals). Isolated cDNA clones were
sequenced using a DNA sequencer (Applied Biosystem).
5' Rapid Amplification of cDNA End
The 5' rapid amplification of cDNA end (RACE) was performed using the
5' RACE system kit (Gibco BRL, Rockville, Md). Based on the sequence
data of a cDNA fragment isolated by cDNA library screening, first-strand cDNA
was synthesized from total RNA by SuperScript II reverse transcriptase using a
gene-specific primer (5'-GTT CTC TAC TTG CTG AGA GCT G-3'). After
addition of dCTP at the 3' end by terminal deoxynucleotidyl transferase,
the cDNA was amplified by PCR using a first nested primer (5'-CTC TAG
GGC CAC CTC TGG CAG-3') and an abridged anchor primer (5'-GGC CAC
GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3', I = deoxyinosine). The
resultant PCR products were used as template for a second PCR using the second
nested primer (5'-CCA TCA GGA CAT TCT GTT CCA C-3') and an
abridged universal amplification primer (5'-GGC CAC GCG TCG ACT AGT
AC-3'). A 5' RACE product was cloned into pGEM-T easy vector
(Promega) and sequenced using a DNA sequencer (Applied Biosystem).
Northern Blot Analysis
A Northern blot membrane loaded with 15 µg total RNA from 2-, 3-, 7-,
and 8-week-old rat testis was hybridized with the 598-base DNA that was
obtained by PCR using 1438 bp cDNA as a template
(Figure 1E). The probe was
purified by gel electrophoresis and labeled with DIG-dUTP, according to the
instruction manual of Roche Molecular Biochemicals. The primers used to
amplify the DNA fragment were 5'-GAG CTG GAG ATG GCC AGA GCT G-3'
(forward) and 5'-GTT CTC TAC TTG CTG AGA GCT G-3' (reverse).
Hybridization was performed as previously reported
(Iida et al, 2003). mRNA
hybridized with the probe was immunologically detected by anti-DIG antibody
conjugated with alkaline phosphatase, and then recorded on x-ray films with
the chemiluminescence substrate CSPD (Roche Molecular Biochemicals). Ribosomal
RNAs were visualized by staining the membrane with methylene blue.
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Reverse Transcription-Polymerase Chain Reaction
For RT-PCR analysis, cDNA strands were synthesized from 2 µg of total
RNA by using a first-strand synthesis kit (Amersham Pharmacia Biotech,
Buckinghamshire, England) with random primers. The reverse-transcribed cDNA
was used as a PCR template to synthesize a gene. The primers used to obtain
full-length genes were 5'-ATG GAA AAC AGA AAC ACC CAC AC-3'
(forward) and 5'-GTG GGT TCG GGC TGG CAC GGA G-3' (reverse). The
primers used to amplify the cDNA fragment of 598 bp were 5'-GAG CTG GAG
ATG GCC AGA GCT G-3' (forward) and 5'-GTT CTC TAC TTG CTG AGA GCT
G-3' (reverse). The PCR-amplified DNAs were cloned into pGEM-T easy
vector and sequenced using a DNA sequencer (Applied Biosystems). Primers for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 5'-TGA
AGG TCG GTG TCA ACG GAT TTG GC-3' (forward) and 5'-CAT GTA GGC CAT
GAG GTC CAC CAC-3' (reverse).
In Situ Hybridization
In situ hybridization was carried out as previously reported
(Iida et al, 2001;
Doiguchi et al, 2002b). In
brief, frozen sections of rat testes were preincubated for 30 minutes at
42°C in a hybridization buffer (20 mM Tris-HCl [pH 8.0], 0.3 M NaCl, 2 mM
EDTA, 50% formamide, 1 mg/mL Bovine Serum Albumin, 0.02% Ficoll, 0.02%
polyvinylpyrolidone, 1 mg/mL herring sperm DNA), and hybridized for 5 hours at
42°C in the hybridization buffer containing a DIG-labeled sense or
antisense RNA probe of 598 nucleotide length
(Figure 1E). After
hybridization, the sections were washed for 1 hour in 2x SSC with 50%
formamide at 42°C, incubated for 30 minutes at 37°C with RNase A (20
µg/mL), and bound cRNA was detected using anti-DIG alkaline
phosphatase-conjugated antibody (1:500 dilution, Roche Molecular
Biochemicals), and visualized with NTB-BCIP (Roche Molecular
Biochemicals).
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Results |
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In order to estimate the size of mRNA of the gene, Northern blot analysis was conducted using the PCR-amplified 598-bp cDNA fragment as a probe (Figure 1E). As shown in Figure 2, a transcript of approximately 2.5 kb was detected in 7- and 8-week-old rat testes, but not detectable in 2- and 3-week-old rat testes. The length of 1.44 kb cDNA isolated from the library was shorter than the expected size of the gene deduced from Northern blot analysis, suggesting that the 1.44-kb cDNA might be truncated at the N-terminus. Additional screening of 5 x 105 phage plaques failed to obtain cDNAs longer than 1.44 kb. We, therefore, carried out 5' RACE to obtain the missing 5' region of the gene, which yielded a 1803 nucleotide-length cDNA that contained a missing 1004 nucleotides of the gene at the 5' end (Figure 1B). The total length of the gene obtained by cDNA library screening and 5' RACE was 2442 base (Figure 1C), with an open reading frame (ORF) of 2055 bases, which was in agreement with the size of the gene predicted from Northern blot analysis. Because the gene is predominantly expressed in spermatogenic cells in rat testis (see below), it was designated as Spergen-3 (a spermatogenic-cell specific gene-3, Accession No. AB168097).
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To confirm that Spergen-3 containing a 2055-nucleotide ORF (Figure 1D) is actually expressed in rat testis, we performed RT-PCR using cDNA synthesized from rat testis RNA. The 2055-bp cDNA was indeed amplified by PCR (Figure 3), and we confirmed by DNA sequence that the sequence of the amplified cDNA was completely identical to that of Spergen-3.
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The full-length cDNA sequence and its deduced amino acid sequence are shown in Figures 4 and 5. The identified cDNA contains a single ORF of 2055 nucleotides with 264 nucleotides of 5' untranslated region (UTR) and 119 nucleotides of 3' UTR. In the 5' UTR, nonsense codon TGA was located 171 nucleotides upstream of Met codon ATG at 265–267 nucleotides. There was no Met codon between the nonsense codon and the initial Met codon. A poly(A) signal, 5'-AAUAAA-3', was located 19 nucleotides upstream of the poly(A) site. The ORF of Spergen-3 encodes a protein of 685 amino acids with the predicted molecular mass of 75 825 Da and pI of 8.83. Both hydrophobicity plot and SOSUI (http://sosui.proteosome.bio.tua.ac.jp/sosuiframe0.html) analysis suggested that the gene encodes a soluble protein with no transmembrane region and no signal peptide sequence at the N-terminus. PSORT (a computer program used to predict the sorting and localization of proteins; http://psort.ims.u-tokyo.ac.jp) suggested the presence of a monopartite nuclear localization signal (NLS), RKKP, at 54–57 amino acid residues. Proline-repeat was found in the middle portion of Spergen-3 (amino acids 241–256). A search in NCBI and DDBJ databases employing FASTA and BLAST programs revealed that a RIKEN mouse cDNA clone encoding 681 amino acids (Accession No. AK077155) exhibited high homology to rat Spergen-3. It shared 85.5% identity and 89% similarity to Spergen-3 at the amino acid level (Figure 5), and 91% identity at the nuclear acid level. Expression, localization, and characterization of the mouse Spergen-3 gene have not yet been reported. Of human genes, a hypothetical protein (Accession No. AY327405) revealed 63.2% identity to rat Spergen-3 at the amino acid level.
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NCBI Conserved Domain Search (http://ncbi.nem.nihgov/Structre/ccd/wrpsb.cgi) suggested the presence of 2 EF-hand motifs, putative calcium-binding domains, at 438–461 and 470–498 amino acid residues in Spergen-3 (Figure 5). Amino acid sequences of the motifs in rat Spergen-3 were identical to those of mouse counterpart (Figure 5). On comparison with the consensus sequence of the EF-hand motif (Branden and Tooze, 1991; Heizman and Hunziker, 1991) and the EF-hand sequences of several Ca2+-binding proteins, we found that the second EF-hand motif in Spergen-3 was considered to match, while the first one was not because of deletion of 2 amino acid residues in the first loop (Figure 6).
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Genomic Organization of Spergen-3
To determine the genomic organization of Spergen-3, rat and mouse
Spergen-3 cDNAs were BLAST-searched against their respective data
bases at NCBI. The results obtained indicated that the rat Spergen-3
gene is present in the supercontig NW_047726 and that the mouse
Spergen-3 gene is found in the supercontig NT_039267. In both cases,
the gene is composed of 15 exons spanning 100 kb. Rat Spergen-3 is
mapped at chromosome 5q36, and mouse Spergen-3 is mapped at
chromosome 4E1.
Expression Analysis of Spergen-3 in Rat Testes
We performed RT-PCR to study the developmental expression of
Spergen-3 mRNA in testes of 1- to 8-week-old rats. The PCR product of
598 bp was first detected at 4 weeks in postnatal development and continued to
be detected up to 8 weeks (Figure
7A). We next examined by RT-PCR the expression of
Spergen-3 in various organs of adult rats. It was expressed in
testis, but was undetectable in other organs examined
(Figure 7B). These data
indicate that Spergen-3 is developmentally up-regulated during rat
testis development and that it is exclusively expressed in testis.
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In Situ Localization and Expression of Spergen-3 mRNA
We performed in situ hybridization to determine the cell types expressing
Spergen-3 mRNA in rat testis. Frozen sections of adult rat testis
were hybridized either with a RNA probe having the antisense sequence of
Spergen-3 mRNA or with a sense probe as control. The RNA probe was
synthesized using 598 nucleotide-length cDNA as a template
(Figure 1E). Hybridization with
the antisense probe created strong signals in the inner half layer of the
seminiferous epithelium of adult rat testis. Hybridization signals in the
seminiferous tubules at stage I–III, VII–VIII, and IX–XI are
shown in Figure 8A, B, and C,
respectively. Spergen-3 mRNA was found to be present in round
spermatids (step 1–l7) (Figure 8A and
B) as well as in early elongating spermatids (step 8–12),
the cytoplasm of which protruded into the tubular lumen
(Figure 8C). More advanced
elongated spermatids (step 13–19) showed faint or no staining. Signals
for Spergen-3 mRNA were weak or hardly detectable in somatic Sertoli
cells, spermatogonia, and spermatocytes located in the outer half layer of the
seminiferous epithelium as well as in interstitial cells. Hybridization with
the sense probe for Spergen-3 gave no signal
(Figure 8D). These data
indicate that Spergen-3 mRNA is exclusively expressed in spermatids
(step 1–12) in the seminiferous epithelium of the rat testis.
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Developmental expression of Spergen-3 mRNA in the seminiferous epithelium of rat testes was also examined by in situ hybridization. Spergen-3 mRNA was not detected in any cells in the seminiferous tubes of 2- and 3-week-old rats (Figure 8E and F). It was first detected in round spermatids in parts of the seminiferous tubules at 4 weeks (Figure 8G), the period at which haploid germ cells first appeared in the tubules of the rat testis. The signal was routinely detected in spermatids of most of the tubules at 5 week and thereafter (Figure 8H). These results are consistent with the data of RT-PCR (Figure 7A).
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Discussion |
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Calcium ions exert their effects in part via interaction with a wide
variety of intercellular calcium-binding proteins that are subdivided into 2
groups, the EF-hand protein family and the annexin protein family
(Heizmann and Hunziker, 1991).
The EF-hand proteins share a common calcium-binding motif, the EF-hand, which
is present in multiple copies (2–8) in the proteins and bind calcium
selectively. Each of these motifs consists of a loop of 12 amino acids that is
flanked by 2 hydrophobic 
-helices. Of 12 amino acid residues in the
loop, 5 should be Ca2+-coordinating residues containing oxygen in
their side chains, and the sixth amino acid in the loop must be glycine, which
is highly conserved in most EF-hand proteins
(Branden and Tooze, 1991). In
view of these criteria and alignment of EF-hand motifs of Spergen-3 and other
EF-hand proteins (Figure 6), it
seems to be probable that the second motif of Spergen-3, which seems to match
the consensus sequence, might be functional, while the first one in which
deletion of 2 amino acids is found in the loop may not be. Such a
nonfunctional loop in the EF-hand motif is found in Iba1, an ionized
calcium-binding adaptor protein (Imai et
al, 1996). Although Iba1 might function as a cytoskeleton
modulator by regulating Rac and calcium-signaling pathways in
macrophage/microgria (Ohsawa et al,
2000), we have found that Iba1 is also highly expressed in the
cytoplasm of elongating spermatids (Iida
et al, 2001). Therefore, in addition to Iba1, Spergen-3
might be a new member of the EF-hand family expressed in haploid spermatids in
testis.
It has been widely recognized that translocation of proteins across the
nuclear envelope depends on the classical NLS
(Dingwall and Laskey, 1991). NLS consists of a cluster of basic residues (monopartite) or 2 clusters of
basic residues separated by 10–12 residues (bipartite)
(Kalderon et al, 1984;
Robbins et al, 1991). For
transport of proteins across the nuclear envelope, NLS is recognized by the
heterodimer import receptor complex comprising importin and importin
ß (Gorlich and Kutay,
1999). The definition of a NLS sequence is, however, somewhat
vague owing to the diversity of sequence that can apparently act as a
functional NLS (Dingwall and Laskey,
1991; Tanaka et al,
1999). As shown in Figure
5, Spergen-3 has a monopartite NLS, while it is not found in the
mouse counterpart. It is now under investigation whether Spergen-3 localizes
in the nuclei of haploid spermatids as well as whether the NLS in Spergen-3
works practically as a signal for transport to nucleus.
In conclusion, we discovered a novel rat gene Spergen-3 exclusively expressed in haploid spermatids in rat testis. Because Spergen-3 encodes a soluble protein bearing EF-hand motifs, Spergen-3 might play a role in calcium signaling, which mediates some signals of ionized calcium to other undefined molecules. The physiological functions, the signals mediated by Spergen-3, as well as the subcellular localization of Spergen-3 in spermatogenic cells remain to be clarified.
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Footnotes |
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References |
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Hind III digest are shown in the left column. Molecular masses are shown in
kilobases in the left.
