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From the * Center for Biomedical Research,
Population Council, and Department of Anatomy,
Histology and Embryology, Peking Union Medical College, Beijing, P.R. China;
Department of Chemistry and Center for
Biomolecular Simulation, Columbia University, New York, New York; and
Rockefeller University, 1230 York Avenue, New
York, New York.
| Correspondence to: Dr Ching-Ling C. Chen, Rockefeller University, 1230 York Avenue, New York, NY 10021 (e-mail: chen{at}popcbr.rockefeller.edu). |
| Received for publication December 2, 2003; accepted for publication February 17, 2004. |
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Abstract |
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Key words: Thioredoxin, spermatogenesis, testis, gene cloning
60 spe genes are associated with
spermatogenesis-defective phenotypes in Caenorhabditis elegans (for a
review, see L'Hernault,
1997). Many of the spe mutant genes identified in C. elegans affect developmental stages before the formation of spermatozoa (Varkey et al, 1995) or sperm-oocyte interactions (Singson et al, 1998). The spe-26 gene encodes a cytoskeletal actin-associated protein and is necessary for spermatid formation (Varkey et al, 1995). The spe-9 gene encodes a sperm transmembrane protein with epidermal growth factor–like repeats and has been suggested to play a role in fertilization by sperm-egg interactions (Singson et al, 1998). The spe-11 gene was the first sperm protein gene demonstrated to directly contribute to the development of embryos from fertilized eggs (Hill et al, 1989; Browning and Strome, 1996). The SPE-11 protein was identified in mature sperm and is not detectable in the oocyte. Only short-lived single-celled embryos develop if eggs are fertilized by mutant sperm that lack SPE-11. The spe-11 gene is considered to be a paternal-effect embryonic-lethal gene (Hill et al, 1989), and its protein product, SPE-11, has been referred to as "sperm-supplied protein" (Browning and Strome, 1996).
Our general hypothesis is that the mammalian ortholog of spe-11 is a sperm-specific gene that is involved in early embryo development. To test this hypothesis, we used degenerate oligonucleotides prepared from conserved amino acid sequences of SPE-11 from 3 nematodes to isolate new cDNAs from adult rat testes that exhibited sequence similarity with C. elegans spe-11. We describe the cloning and characterization of the newly identified ssp411 cDNA, an analysis of a thioredoxin-like domain in SSP411, and an examination of the age- and stage-dependent expression of both ssp411 mRNA and protein in the rat testis.
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Materials and Methods |
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Isolation of SPE-11–Related cDNAs from Rat Testis
To identify the mammalian ortholog of spe-11, degenerate
oligonucleotides were prepared from conserved amino acid sequences of SPE-11
from 3 nematodes; these were used to isolate new cDNAs from adult rat testes
that exhibit sequence similarity with C. elegans spe-11
(Browning and Strome, 1996).
Clone 24 was isolated, by reverse-transcription polymerase chain reaction
(RT-PCR), from adult rat testicular RNA using a series of degenerated
oligonucleotides prepared from the amino acid sequences WKRYLRSKWD and
HYKKWLEKK in SPE-11, which are conserved in 3 nematodes
(Browning and Strome, 1996).
The RT-PCR–generated cDNA products that contained 500 bp were
analyzed by hybridization with the C. elegans spe-11 cDNA (provided
by Dr Susan Strome, Indiana University, Bloomington, Ind). Clone 24, which
showed strong positive hybridization to spe-11 cDNA, was subcloned
into pGEM-3Z vector and further analyzed.
Clone 411 was isolated by screening a 5' stretch plus cDNA library of rat testis (BD Biosciences Clontech, Palo Alto, Calif), using a radiolabeled clone 24 cDNA fragment as the hybridization probe. Approximately 0.8–1.0 x 106 phage plaques were screened. Filters were hybridized overnight at 42°C in a solution that contained 5x SSPE (750 mM NaCl, 50 mM NaH2PO4·H20, and 5 mM EDTA [pH 7.4]), 5x Denhardt's solution, 1% sodium dodecyl sulfate (SDS), and 100 µg/mL single-stranded salmon sperm DNA. The filters were washed twice in 2x standard saline citrate (SSC) and 1% SDS at room temperature for 15 minutes, once at 42°C for 30 minutes, and then in 1 x SSC and 1% SDS at 42°C for 15–30 minutes. Phage DNA was prepared from purified positive phage clones (Feng et al, 1995) and subjected to Southern blot analysis. Phage DNA isolated from clone 411 was hybridized to both clone 24 and spe-11 cDNA and was subcloned into pGEM-3Z vector for further analysis. Nucleotide sequences of the isolated cDNA clones were analyzed by the DNA Sequencing Laboratory at the Protein/DNA Technology Center of Rockefeller University.
5'- and 3'-RACE-PCR for the Generation of Full-Length ssp411 cDNA
The 5' end of the ssp411 cDNA was generated by
5'-rapid amplification of cDNA ends (RACE)-PCR
(Kressler et al, 2002) using
the GeneRacer Kit (Invitrogen Corp, Carlsbad, Calif). The first-strand cDNA
was synthesized using 80-day-old rat testicular total RNA as a template. The
ssp411 cDNA was then amplified by PCR using a 44-mer oligonucleotide
provided in the kit and a 26-mer oligonucleotide, GSP1
(5'-CTGTCGGTCCGTGGAGTAGCGGTGAA-3'), derived from clone 411, which
was complimentary to the sequence at 1021–1046 of the ssp411
cDNA. The PCR products were subcloned into pCR4-TOPO using the TOPO TA Cloning
Kit (Invitrogen) and analyzed by nucleotide sequencing.
Similarly, the 3' end of the ssp411 cDNA was also prepared from adult rat testicular RNA using 3'-RACE-PCR with a 25-mer GeneRacer 3' primer provided in the kit and a 26-mer oligonucleotide, GSP2 (5'-GCCCTGCTGGCCCGATCTGAGATCAG-3') derived from clone 411 that was complimentary to the sequence at 735–760 of the ssp411 cDNA. The PCR products were subcloned into pCR4-TOPO vector and characterized by sequencing analysis. A pair of primers containing DNA sequences at positions 1–22 and 2559–2580 nt, which were obtained from the 5'- and 3'-end cDNA fragments, was used to isolate a full-length ssp411 cDNA using RT-PCR.
Computer Analysis of the Amino Acid Sequence Deduced From ssp411 cDNA
Protean software (DNASTAR, Inc, Madison, Wis) and SIM software (Expert
Protein Analysis System [ExPASy]) proteomics tools (available at:
http://us.expasy.org)
were used to analyze the alignment of the deduced amino acid sequence of
SSP411 with human FLJ21347, mouse TISP78, and C. elegans SPE-11. The
N-glycosylation and phosphorylation sites were predicted using ScanProsite
software on the ExPASy proteomics server. In addition, the NetNGlyc 1.0 and
NetOGlyc 2.0 servers of Center for Biological Sequence Analysis (available at:
http://www.cbs.dtu.dk)
were also used for predicting the sites of N- and O-linked glycosylation,
respectively. The theoretical molecular weight was calculated using ExPASy
proteomics tools.
3-Dimensional Structural Analysis of the Thioredoxin-Like Domain in SSP411
The 3-dimensional structure of the SSP411 protein was initially analyzed
using the 3D-PSSM server (available at:
http://www.sbg.bio.ic.ac.uk/~3dpssm/)
by submitting the protein sequence directly
(Kelley et al, 2000). A
thioredoxin fold was found at the N-terminal region of the SSP411 protein. The
3-dimensional structure of the N-terminal domain modeling was next carried out
using the PLOP program (kindly provided by Dr Richard A. Friesner, Department
of Chemistry and Center for Biomolecular Simulation, Columbia University, New
York; Eyrich et al, 1999). The
3-dimensional model of the thioredoxin domain in the SSP411 was further
analyzed by comparing it with the available crystallographic structures of 2
thioredoxin proteins, thioredoxin (Eklund
et al, 1991; Nicastro et al,
2000) and human thioredoxin-like protein
(Jin et al, 2002), using the
ProSup server (available at:
http://lore.came.sbg.ac.at:8080/CAME/CAME_EXTERN/PROSUP/index_html;
Lackner et al, 2000). The root
mean square deviation (RMSD) of structurally equivalent residues was also
calculated, which is a common numerical measure of the difference between 2
protein structures.
Northern Blot Analysis
Total RNA was isolated from various tissues by extraction with TRIzol
Reagent (Invitrogen), as described elsewhere
(Zhang et al, 2002). Twenty
micrograms each of total RNA isolated from rat testes and other tissues were
subjected to Northern blot analysis. The RNA was denatured with 6%
formaldehyde and 50% formamide, fractionated in 1.1% agarose gel, and
transferred onto Nytran-Plus membranes (Schleicher & Schuell, Inc, Keene,
NH; Feng et al, 1998,
2000). Newly isolated
ssp411 cDNA fragments were used to prepare radiolabeled probes for
detecting ssp411 mRNA on the RNA blots. Autoradiograms were obtained
by exposing the RNA blots to x-ray films.
Semiquantitative RT-PCR
The ssp411 mRNA in the rat testes ages 7–90 days and in
various other tissues from adult rats was analyzed by RT-PCR using the
ThermoScript RT/PCR System (Invitrogen;
Feng et al, 1995; Zhang et al, 2002). The RT
reaction was performed at 50°C for 60 minutes in a volume of 20 µl that
contained 2 µg each of total RNA isolated from varied rat tissues, 1 mM
deoxynucleotide triphosphate (dNTP), 50 µM oligo (dT)12 as a
primer, 40 U of the RNase inhibitor RNaseOUT, and 15 U of ThermoScript reverse
transcriptase. An aliquot of 1–5 µl of cDNAs (0.1–0.5 µg RNA
equivalent), synthesized as described above, was used as templates for PCR
analysis that contained 0.2 µM each primer, 0.2 mM dNTPs, and 1 U of
Platinum Taq DNA polymerase High Fidelity (Invitrogen). The cDNAs
were amplified by PCR for 25–35 cycles at 94°C for 30 seconds, at
60°C for 30 seconds, and at 68°C for 2.5 minutes in each amplification
cycle. Different amounts of RNA equivalents (0.1–0.5 µg) and various
numbers of PCR cycles (25–35) were tested for PCR analysis, to ensure
that the results obtained were within the linear portion of the reaction.
A pair of ssp411-specific primers was used for amplification: upstream primer 5'-ATGAGCCACCATTCCCCACCAC-3' and downstream primer 5'-TCATTGGTGTAGCAGCTTTCGTA-3'. These were derived from nucleotide sequence at 63–84 and 2410–2432 of the ssp411 cDNA, respectively. A single species of 2.4-kb RT-PCR product that contained the coding region of the ssp411 cDNA was generated after 28–30 cycles of amplification. The levels of total RNA in each sample used for RT-PCR analysis were quantified by measurement of ß-actin mRNA levels using upstream primer 5'-TTGTAACCAACTGGGACGATATGG-3' and downstream primer 5'-GATCTTGATCTTCATGGTGCTAGG-3', which were purchased from BD Biosciences Clontech. An expected 764-bp ß-actin DNA fragment was obtained by RT-PCR with 20 cycles of amplification (Zhang et al, 2002).
Expression and Purification of Recombinant SSP411 Protein
The full-length ssp411 cDNA was subcloned into pET-28a(+)
(Novagen, Madison, Wis) bacterial expression plasmid, pET-SSP411, which
contained a tag protein of 6 histidine residues at the N-terminus
(Studier et al, 1990;
Apezteguia et al, 1994). The
recombinant protein was produced by transforming the pET-SSP411 expression
plasmid to Escherichia coli BL21 (DE3) (Novagen). An overnight
bacterial culture that contained the pET-SSP411 expression plasmid was diluted
to 1:100 in Luria broth medium and grown at 37°C until A600
reached 0.4–0.6. Induction of the expression of the histidine-tagged
SSP411 protein from T7 promoter was achieved by adding 1 mmol/L isopropyl
ß-D-thiogalactoside (IPTG) and a further 3–5 hours of induction.
The induced bacterial cells were harvested by centrifugation, and the
recombinant SSP411 protein was isolated with BugBuster protein extraction
reagent (Novagen) and purified using a protein refolding kit (Novagen). The
obtained soluble recombinant SSP411 protein, which contains a 1-kD histidine
tag at the N terminus, was further purified by affinity chromatography using a
His•Bind column (Novagen). The purified recombinant protein was dialyzed
against 20 mM Tris-HCl (pH 8.5), concentrated by freeze-drying, and stored at
-20°C until further use.
Production of Polyclonal Antibodies
A synthetic oligopeptide containing 17 amino acids (ERMRRVPVALPEMVRAL)
derived from the C-terminal region at amino acids 681–697 of the SSP411
protein was prepared and conjugated to Keyhole Limpet Hemocyanin (KLH) (SynPep
Corporation, Dublin, Calif) as an immunogen. The high-performance liquid
chromatography–purified KLH-conjugated SSP411 oligopeptide was used to
immunize New Zealand White rabbits. Immunization was carried out by Covance
Research Products, Inc (Denver, Penn): 5 injections of 250 µg each, 3 test
bleedings, and 2 ELISA tests to determine antibody concentrations. Antibody
Ab1065 with high titers was further purified using Econo-Pac Serum IgG
Purification Columns (Bio-Rad Laboratories, Hercules, Calif) and characterized
by Western blot analysis. Recombinant SSP411 protein produced from full-length
ssp411 cDNA as described above was used as a positive control for
analyzing the immunoreactivity of the newly generated antibody and for
characterizing the SSP411 protein in the testis.
Western Blot Analysis
Proteins were extracted from various rat tissues, including testes, using
CytoBuster protein extraction reagent (Novagen). An aliquot of 20 µg of
protein isolated from rat tissues was fractionated on a 10%–12%
SDS-polyacrylamide gel and transferred onto a PVDF membrane (Millipore,
Billerica, Mass). Antibody Ab1065 was used for detecting SSP411 protein in the
testis at a dilution of 1:500 to 1:1000. After incubation with a secondary
antibody, horseradish peroxidase–linked anti-rabbit IgG (Amersham
Pharmacia Biotech, Piscataway, NJ), the SSP411 protein on the Western blots
was visualized using the enhanced chemiluminescence (ECL+plus) detection
system (Amersham Pharmacia Biotech).
Tissue Fixation and In Situ Hybridization Analysis
Testicular tissues were collected from adult rats at 60–65 days of
age, and paraffin sections were prepared using protocols described elsewhere
(van Pelt et al, 1999;
Pusch et al, 2000). Rats were
anesthetized with isoflurane (ABBOTT Laboratories, Abbott Park, Ill), and the
testes were fixed by perfusion through the abdominal aorta with 4%
paraformaldehyde (PFA). The testes were removed, further fixed in 4% PFA at
4°C overnight, dehydrated in graded ethanol, and embedded in paraffin.
Paraffin sections of 7 µm thickness were prepared for in situ hybridization
analysis. After deproteination and acetylation, testicular sections were
hybridized with antisense and sense RNA probes to detect ssp411 mRNA
in the testis. RNA probes were prepared with digoxigenin-UTP (DIG-UTP) by in
vitro transcription with SP6 and T7 RNA polymerase using a DIG RNA labeling
kit (Roche Molecular Biochemicals, Indianapolis, Ind). A cDNA fragment
containing the nucleotide sequence at 331–703 of the ssp411
cDNA was used as a template to prepare antisense RNA for detection of the
ssp411 mRNA on testicular sections. The 331–703 fragment is
contained within the 3 isolated cDNA clones and was not found in other genes.
The primers used to prepare RNA probes from this DNA fragment were selected by
computer analysis with PrimerSelect software (DNAStar). The sense RNA probe
was also prepared as a negative control. Antisense and sense DIG-labeled
ssp411 RNA probes were applied onto testicular sections and incubated
overnight at 55°C. The sections were then washed with high stringency
buffer that contained 50% formamide and 2x SSC at 50°C for 30
minutes. The DIG-labeled RNA probe that bound to testicular sections was
detected by color reaction with incubation of anti-DIG antibody overnight at
4°C, as described in the DIG Nucleic Acid Detection Kit (Roche). At least
10 testicular sections were evaluated for a group of stages.
Immunohistochemical Staining
Paraffin sections (7 µm) were prepared from adult rat testes as
described above and processed for immunostaining. After blocking with 10%
normal goat serum, testicular sections were incubated with IgG-enriched
antibody Ab1065 overnight at 4°C at a dilution of 1:100–1:1000. The
sections were then incubated with a biotinylated goat anti-rabbit secondary
antibody (Vector Laboratories, Inc, Burlingame, Calif) at a dilution of 1:600
for 1 hour at room temperature. The visualization of positive signals was done
using the Vectastain Elite Kit (Vector). As negative controls, antibody Ab1065
was preincubated with an excess of a synthetic oligopeptide of SSP411 at
681–697 for 2 hours at room temperature or overnight at 4°C before
use in immunostaining. In general, 10–20 testicular sections were
evaluated for each stage.
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Results |
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500 bp, as predicted
from the spe-11 cDNA sequence, were analyzed for their ability to
hybridize the C. elegans spe-11 cDNA. As shown in
Figure 1A, clone 24 showed a
strong positive hybridization signal to the spe-11 cDNA; however,
nucleotide sequence analysis revealed that clone 24 (487 bp in length;
Figure 2A) did not contain the
conserved amino acid sequences originally used for the preparation of
degenerate nucleotides (indicated by underlining in
Figure 3).
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Because clone 24 demonstrated the strongest hybridization to the spe-11 cDNA (Figure 1A), it was used as a hybridization probe for the isolation of longer cDNAs from a rat testicular cDNA library. Among the positive clones isolated from the cDNA library (Figure 1B), clone 411 (964 bp) showed the highest intensity of hybridization with clone 24. In addition, clone 411 hybridized to C. elegans spe-11 cDNA (Figure 1B). This clone was thus further characterized by nucleotide sequence analysis. The positions of clones 24 and 411 corresponding to the full-length ssp411 cDNA, which was generated by RACE-PCR, as described below, are indicated in Figure 2A.
The 5'- and 3'-RACE-PCR cloning method was used to generate 2 overlapping 5' and 3' ssp411 cDNA fragments from adult rat testicular RNA (Figure 2). Using primers prepared from cDNA sequences obtained from clone 411 (Figure 2A), a 1.0-kb DNA fragment was generated by 5' RACE-PCR that contained the 5' ssp411 cDNA at positions 1–1046 nt, and a 1.9-kb DNA fragment was obtained by 3' RACE-PCR that contained the 3' ssp411 cDNA from 735–2598 nt (Figure 2B). These RACE-PCR–generated overlapping DNA fragments were isolated and analyzed by nucleotide sequencing and used to prepare primers for the isolation of a 2.6-kb full-length ssp411 cDNA from the rat testis (Figure 2C). An EcoRI restriction-enzyme site was detected at the full-length ssp411 cDNA; both clone 24 and clone 411 are located within the 1.1-kb 5' EcoRI fragment (Figure 2A).
Analysis of the Newly Isolated Rat ssp411 cDNA
The DNA sequence of a total of 2598 nt of the isolated rat ssp411
cDNA was determined and scanned against the database, and all sequences with
significant relatedness to the new sequence were identified. Nucleotide and
protein sequences of the ssp411 cDNA were submitted to GenBank, and
accession numbers AY438568 and AAR12892 were assigned, respectively. The
ssp411 cDNA consists of 62 bp of the 5'-untranslated region
(UTR), 2367 bp of the coding region with an open-reading frame of 789 amino
acids (Figure 3), and 151 bp of
the 3' UTR (Figure 2A).
The amino acid sequence of SSP411 protein can be accessed through the NCBI
Protein Database under accession number AAR12892.
A computer analysis of amino acid sequences deduced from ssp411 cDNA using ExPASy proteomics tools revealed that a putative N-linked glycosylation sequence, Asn-Leu-Ser, at amino acids 374–377, 3 tyrosine kinase phosphorylation sites at 67–75, 147–154, and 397–404, and many potential phosphorylation sites for protein kinase C and casein kinase II had been identified. In addition to the N-linked glycosylation site, 4 potential O-linked glycosylation sites were observed at amino acids 5 and 176 for serine and 54 and 245 for threonine, as analyzed by using the NetNGlyc 1.0 and NetOGlyc 2.0 servers of the Center for Biological Sequence Analysis.
Sequence Similarity With a Human Hypothetical Protein FLJ21347 and Mouse TISP78
Although clone 411 (Figure
1B) hybridized with spe-11 cDNA from C. elegans
(Browning and Strome, 1996),
the SSP411 and SPE-11 proteins shared only 21% identity in their amino acid
sequences. The amino acid sequences that are identical to both SPE-11 and
clone 411 or SSP411 were indicated in
Figure 3; these were mainly
located at the 1.1-kb 5' EcoRI DNA fragment
(Figure 2A). Of interest,
compared with the published draft human genome sequences from the NCBI, the
ssp411 cDNA shares 85% and 87% nucleotide and protein sequence
identity, respectively, with a 2.6-kb cDNA that encoded a new, unnamed human
hypothetical protein, FLJ21347 (GenBank accession number AK025000;
Figure 3). In addition, human
FLJ21347 cDNAs with varied lengths of the 5' end were identified in
several human cancer/tumor cell lines, including pancreas epithelia carcinoma
(AAH25255), hepatoma HepG2 (AK025622), and uterus leiomyosarcoma
(BC017468).
The ssp411 cDNA also shared 92% and 93% identity at the nucleotide and protein levels, respectively, with several uncharacterized cDNAs isolated from mouse testes, the 488-bp transcript increased in spermiogenesis 78 (TISP78) cDNA (AB045721), a 1986-bp cDNA (BC050807), and a 2705-bp cDNA (BC050788) (Figure 3). In addition, the nucleotide sequence of a 2589-bp TISP78 cDNA (XM_354637) was predicted by automated computational analysis of mouse chromosome 11. All of these uncharacterized mouse cDNAs shared 99% sequence identity with each other, which suggests that these cDNAs are derived from the same mouse gene. In addition, they showed 92%–93% identity with rat ssp411.
The comparison of amino acid sequences obtained from rat testicular SSP411, human FLJ21347 (AK025000), and mouse testis protein TISP78 (BC050788) is presented in Figure 3. The ssp411 gene was mapped to chromosome 10 in rats, chromosome 17 in humans, and chromosome 11 in C57Bl/6 mice. All 3 genes contain 16 exons and 15 introns in a span of 8.6, 6.8, and 6.5 kb in human, rat, and mouse, respectively. We therefore suggest that FLJ21347 and TISP78 are the human and mouse orthologs, respectively, of the rat ssp411 gene.
Analysis of a Thioredoxin-Like Domain in SSP411
A computer search for conserved domains revealed that SSP411 contained 2
cysteine residues in C-H-W-C located at amino acids 113–116. The C-X-X-C
motif was shown to be the conserved domain for thioredoxin proteins and a
potential active site for the reduction of proteins from their oxidized form
(Holmgren, 1985,
1989;
Whiteley et al, 1997;
Arner and Holmgren, 2000;
Shigenobu et al, 2000). The
identified thioredoxin-like domain in SSP411 protein was conserved in the
homologs, human protein FLJ21347 and mouse testicular protein TSIP78
(BC050788) (Figure 3). The
C-H-W-C motif or other thioredoxin-like domain were not detected in C.
elegans SPE-11.
To investigate the potential function of the thioredoxin-like domain in
SSP411 protein at the secondary structure, the amino acid sequence of the
SSP411 protein was submitted to the 3D-PSSM server (available at:
http://www.sbg.bio.ic.ac.uk/~3dpssm/)
for structural analysis. A thioredoxin fold was identified at the N-terminal
region of the SSP411 protein. A model for the 3-dimensional structure of
N-terminal SSP411 was next built, using the PLOP program
(Rapps and Friesner, 1999).
Through the Protein Explorer Program, the 3-dimensional structure of the
N-terminal SSP411 was compared with that of a typical thioredoxin protein,
BacTrx (Protein Data Bank code, 1quw, 105 residues), which has a known
tertiary crystallographic structure and function
(Martin, 1995;
Nicastro et al, 2000)
(Figure 4). Like other members
of the thioredoxin family, a typical thioredoxin protein has a common fold,
the thioredoxin fold, which consists of central ß-sheets (blue) flanked
by 
-helices (red) and an active site (green) with a catalytic disulfide
bond (orange) between the sequence C-X-X-C
(Eklund et al, 1991;
Nicastro et al, 2000)
(Figure 4A). In the predicted
3-dimensional structure of the SSP411 protein
(Figure 4B), a similar
thioredoxin fold was clearly identified at amino acids 85–202, and the
potential active site with a disulfide bond was detected at amino acid
113–116, which contains a C-H-W-C thioredoxin-like domain.
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The high conservation of the thioredoxin fold in SSP411 was also confirmed by the results obtained from protein structure alignment between these 2 structures using the ProSup program (Lackner et al, 2000). The ProSup program is a refined and frequently used tool for 3-dimensional protein structure comparison and alignment. It evaluates structure similarity among proteins, primarily by maximizing the number of structurally equivalent residues (Lackner et al, 2000) of 2 proteins while maintaining a certain RMSD cutoff such as 2 Å (Lackner et al, 2000). Between the typical thioredoxin fold and the protein fold identified in the N-terminal SSP411, 101 structurally equivalent residues were found, which accounted for 96% of the residues (101 of 105) in the typical thioredoxin fold (>50% is significant). The RMSD of structurally equivalent residues between these 2 protein structures was 0.44 Å. These results demonstrate extensive structural similarity and fold conservation between SSP411 and a typical thioredoxin protein. Aligned with another thioredoxin-like protein from human, hTRXL (PDB code: 1gh2, 107 residues; Jin et al, 2002), 98 equivalent residues were detected by ProSup program with an RMSD of 1.72 Å. Therefore, the results obtained from structural modeling strongly suggest that SSP411 belongs to the thioredoxin family.
Testis-Specific Expression of the ssp411 Gene
Primers containing nucleotide sequence at the positions of 63–84 and
2410–2432, as described in Figure
2A, were used for the analysis of ssp411 mRNA in
63-day-old rats by semiquantitative RT-PCR. Among 11 tissues examined
(including all of the male reproductive accessory organs), a single band of
2.4 kb ssp411 cDNA was detected at high levels in the testis, and a
faint band was occasionally found in the epididymis
(Figure 5Aa). No other tissues
tested expressed the ssp411 gene. The ß-actin mRNA level in each
RNA sample was also measured by RT-PCR
(Figure 5Ab), to ensure that
the same amount of RNA was loaded for comparisons between sample lanes.
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The tissue specificity of ssp411 gene expression in adult rats was also examined by Northern blot analysis, using clone 411 cDNA as a hybridization probe (Figure 5B). A single species of 2.8-kb ssp411 mRNA was identified exclusively in the rat testis and was absent in other tissues of adult male rats and in the ovaries of immature and adult females (Figure 5B). Identical observations were obtained when another ssp411 cDNA fragment, such as the 1.5-kb 3'-EcoRI fragment shown in Figure 2A, was used for hybridization.
Age-Dependent Expression of the ssp411 Gene
We studied the expression pattern of the ssp411 mRNA in developing
rat testes, using semiquantitative RT-PCR and Northern blot analyses
(Figure 6). Total RNA was
isolated from testes of different ages ranging from 7 to 90 days. The
ssp411 cDNA generated by RT-PCR was first evident at 28 days and
remained at high levels throughout adulthood
(Figure 6Aa). The levels of
ß-actin mRNA in the RNA samples prepared from different-aged animals were
unchanged (Figure 6Ab), which
confirmed that the expression of ssp411 gene in the testis was age
dependent. Similarly, the results of Northern blot analysis
(Figure 6B) showed that
ssp411 mRNA was first detected at age 28 days and was most abundant
at age 
63 days. The age-dependent expression of ssp411 mRNA was
also studied in mouse testes. A 2.8-kb mRNA that hybridized to clone 411 was
detected in 28- and 75-day-old mouse testes but was not observed at 21 days
(data not shown). The developmental profile of ssp411 mRNA expression
in adult rat testes, with no expression at age 
21 days, suggested that
this gene is expressed in spermatids and not in spermatocytes or spermatogonia
of the germ cells.
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Characterization of the SSP411 Protein in Rat Testicular Extracts
To characterize the protein coded by the isolated ssp411 cDNA, a
recombinant SSP411 protein was produced as a histidine-tagged protein by
subcloning the ssp411 cDNA into pET-28a(+) bacterial expression
plasmid (Studier et al, 1990;
Apezteguia et al, 1994). SDS-polyacrylamide gel electrophoresis analysis confirmed that an 89-kD
protein, which contained a 1-kD histidine tag protein at the N terminus, was
detected in the IPTG-treated bacterial culture. The purified 89-kD SSP411
recombinant protein was used as a positive control for the analysis of the
SSP411 protein expressed in rat testis
(Figure 7).
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To identify the protein(s) derived from the newly identified ssp411 mRNA in the testis, we generated a polyclonal antibody against a 17-mer oligopeptide derived from the C-terminal region of the SSP411 protein. The antibody, Ab1065, was shown to immunoreact with the 89-kD SSP411 recombinant protein produced in E. coli (Figure 7, lane 1). To examine further the specificity of the newly raised antibody, protein extracts prepared from various tissues—including testis, ovary, liver, colon, and heart—of 63-day-old rats were subjected to Western blot analysis using antibody Ab1065 for detection. Only testis contained an 88-kD protein that immunoreacted with antibody Ab1065 (Figure 7, lane 2). Because the theoretical molecular weight of SSP411 was calculated to be 88190.97, the 88-kD protein identified in the testis by Western blot analysis is a full-length SSP411 protein.
Localization of the ssp411 mRNA in Rat Spermatids
In situ hybridization was performed to determine the testicular cell types
that express ssp411 mRNA (Figure
8A). DIG-labeled antisense and sense RNA probes were prepared
using a cDNA fragment that contained nucleotides at 331–703 of the
ssp411 cDNA as a template. Paraffin sections of adult rat testes were
hybridized either with an RNA probe that contained the antisense sequence of
the ssp411 mRNA (Figure
8Aa–c) or with a sense probe as a negative control
(Figure 8Ad). Hybridization
with the antisense probe demonstrated strong signals in the seminiferous
epithelium. Intense staining for ssp411 mRNA was observed
predominantly in round spermatids located in the inner half-layer of the
seminiferous epithelium (tubule a, Figure
8Ab–c) as well as in early elongated spermatids whose
cytoplasm protruded into tubular lumen (tubule b,
Figure 8Ab–c).
Hybridization with a sense probe for ssp411 mRNA was negative
(Figure 8Ad), which confirmed
the specificity for the ssp411 cDNA sequence.
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The intensity of the hybridization signals varied markedly between the seminiferous tubules (Figure 8Aa), which suggests the stage-specific expression of ssp411mRNA during the cycle of the seminiferous epithelium. The strongest hybridization was observed in round spermatids at stages VII–VIII and in elongated spermatids at stages IX–XI (Figure 8Ab–c). Weak hybridization signals were detected in round spermatids at stages II–VI and in elongated spermatids at stage XII. These data indicate that ssp411 mRNA is expressed in the spermatids at specific stages of the cycle in the seminiferous epithelium.
Localization of SSP411 Protein in Adult Rat Testes
We next localized the SSP411 protein in the rat testis by
immunohistochemistry using antibody Ab1065 for detection
(Figure 8B). Strong
immunostaining was mainly localized to elongated spermatids
(Figure 8Ba, c, and d). No
staining was detected when preimmune serum was used (data not shown). These
observations suggest that the ssp411 mRNA that is transcribed in
round spermatids might not be translated into protein until later stages of
spermatogenesis, in elongated spermatids. The immunostaining for SSP411
protein on testicular sections was further confirmed by peptide neutralization
(Figure 8Bb). No staining was
detected if antiserum was preincubated with 5- to 8-fold excess of synthetic
oligopeptide at 681–697.
Similar to those observed in ssp411 mRNA by in situ hybridization (Figure 8A), varied intensities of immunostaining were observed in different regions of the seminiferous tubules (tubule a, Figure 8B). Maximal immunostaining of SSP411 was observed at stages V–VI (tubule a, Figure 8Bc). Strong staining was also observed in elongated spermatids at stages VII–VIII (tubule b, Figure 8Bd), with weak signals detected in some residual bodies at stage VIII. Immunostaining for the SSP411 protein was decreased to undetectable levels at stages IX–XI (tubule c, Figure 8Be). Weak immunostaining of the SSP411 protein started to appear again at stages XII–I (tubule d, Figure 8Ba).
The observations shown in Figure 8A B indicate that the transcription and translation of the ssp411 mRNA and SSP411 protein probably occur at different stages in the cycle of the seminiferous epithelium. The transcription of ssp411 gene was most active in both round and elongated spermatids at stages VII–VIII and IX–XII, respectively, and declined after stage XII. On the other hand, the translation of ssp411 mRNA into SSP411 protein in the elongated spermatids was not initiated until stages XII–I, and it continued to increase, reaching maximal levels at stages V and VI. Our observations thus suggested that the ssp411 mRNA transcribed at stages VII–XII might be stored until translational activation occurred at stages XII–I, similar to those reported for protamines (Kleene et al, 1984; Zhong et al, 2001; Giorgini et al, 2002).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The ssp411 cDNA was isolated on the basis of its hybridization to the C. elegans spe-11 cDNA. The expression of ssp411 mRNA and protein in the rat testis are similar in pattern to C. elegans spe-11 (Browning and Strome, 1996): both are expressed in a testis- and stage-specific manner and are predominantly localized to spermatids. The SSP411 protein, however, shares only 21% amino acid sequence identity with that of SPE-11 in C. elegans. Of interest, the SPE-11 observed in 3 Caenorhabditis species, elegans, briggsae, and vulgaris, also had a low protein sequence identity of 55%. Although 60% of the amino acids in SSP411 that shared identity with SPE-11 in C. elegans also showed identity with those in 2 other nematodes, we infer that the newly isolated rat testicular SSP411 is not likely to be a mammalian SPE-11 ortholog. In view of the fact that HCH-1, which is a BMP-1–related protein in C. elegans (NP_510440), shares 20.7% identity in amino acid sequence with rat BMP-3 (NP_058801), we thus suggest that SSP411 is a SPE-11–related protein rather than an ortholog.
The transcriptional and translational profiles, as analyzed by in situ hybridization and immunohistochemistry, respectively, were obtained from observations of at least 10 testicular sections for each stage. We showed that both ssp411 mRNA and the SSP411 protein were present in testicular germ cells in a cell- and stage-specific manner. The ssp411 mRNA is expressed predominantly in round and elongated spermatids at specific stages of the cycle in the seminiferous epithelium of the rat testis. Weak expression of the ssp411 mRNA was first detected in round spermatids at stages II–VI. The expression of the ssp411 mRNA gradually increased and reached highest levels at stages VII and VIII in round spermatids and stages IX–XI in early elongated spermatids. The ssp411 mRNA levels in elongated spermatids declined after stage XII. Immunoreactive SSP411 protein was, however, localized mainly to the elongated spermatids and not in round spermatids, which suggests that the production of SSP411 protein occurred during a later stage of spermatogenesis. Weak immunostaining of SSP411 protein started to appear at stages XII–I. The staining intensity gradually increased through stages II–IV, reached maximal levels at stages V–VI, and continued to stay at high levels at stages VII and VIII. SSP411 was not detectable at stages IX–XI, after spermiation. Our observations suggest that the transcription and translation of the ssp411 mRNA and SSP411 protein occur at different stages in the cycle of the seminiferous epithelium.
The posttranscriptional regulation of sperm protein production has been shown to play a role in gametogenesis. In haploid spermatids, the proteins required for morphogenesis are synthesized from the stored mRNAs that are packaged as ribonucleoprotein particles (mRNPs; Giorgini et al, 2002). For instance, the protamine genes were first transcribed in haploid round spermatids, and the protamine mRNAs were then stored in translationally inert mRNPs for up to 10 days, until translational activation in elongated spermatids (Kleene et al, 1984; Zhong et al, 2001; Giorgini et al, 2002). Similar observations have also been reported for sperm fibrous sheath (FS) protein genes, including spermatid-specific thioredoxin-2, Sptrx-2 (Miranda-Vizuete et al, 2003), FS39 (El-Alfy et al, 1999), and FS75 (Catalano et al, 2001). The FS mRNAs are expressed in round spermatids, stored for several days, and translated in elongating spermatids after the end of transcription, which occurs during nuclear condensation (Padma et al, 2001). As with protamines (Kleene et al, 1984; Zhong et al, 2001; Giorgini et al, 2002) and FS proteins (El-Alfy et al, 1999; Catalano et al, 2001; Miranda-Vizuete et al, 2003), ssp411 mRNA transcribed at stages VII–XII might be stored in translationally inert mRNP particles until translational activation at stages XII–I.
A low level of ssp411 mRNA was detected in the epididymis by RT-PCR but not by Northern blot analysis. Whether ssp411 mRNA and SSP411 protein are present in the epididymis is currently under investigation by using in situ hybridization and immunocytochemistry, respectively. In addition, the nature of the SSP411 protein in the epididymis and mature sperm is also being characterized.
SSP411 contains a thioredoxin-like domain that consists of 2 cysteine residues in C-H-W-C at amino acids 113–116. This sequence was also observed in human protein FLJ21347 and mouse testicular protein BC050788. In addition to the highly conserved sequence W-C-G-P-C (Holmgren, 1985, 1989) in thioredoxin, various thioredoxin-like domains containing a C-X-X-C motif were identified. For instance, C-G-H-C in the protein disulfide isomerase (PDI; Whiteley et al, 1997), C-P-P-C in thiol: disulfide isomerase, and C-V-Y-C and C-P-Y-C in thiol: disulfide interchange protein (Shigenobu et al, 2000) have been demonstrated to exhibit thioredoxin activity. PDI, which contains 2 regions that exhibit sequence homology to thioredoxin (Edman et al, 1985), has been shown to be a substrate for thioredoxin reductase, which indicates that the C-G-H-C domains in PDI are folded and recognized as thioredoxins (Lundstrom and Holmgren, 1990). The SSP411 protein contains a thioredoxin-like domain C-H-W-C at amino acids 113–116. A comparison of the predicted 3-dimensional structure of the SSP411 protein and a known thioredoxin protein suggested that SSP411 is also folded and recognized as a member of thioredoxin family.
Two spermatid-specific thioredoxin proteins, Sptrx-1 and Sptrx-2, identified in human and murine sperm have been shown to be predominantly expressed in round and elongated spermatids, and their proteins were localized to the tail of spermatozoa (Miranda-Vizuete et al, 2001, 2003; Sadek et al, 2001; Jimenez et al, 2002b). Sptrx-1 and Sptrx-2 incorporate to the sperm tail during different steps of sperm maturation. Sptrx-1 transiently associates to the FS during sperm tail assembly but does not remain as a permanent component of the FS in the mature sperm (Yu et al, 2002). In contrast, Sptrx-2 incorporates into the FS during the last steps of spermatid development and remains as an integral FS component in mature epididymal spermatozoa (Miranda-Vizuete et al, 2003). Another thioredoxin protein found in spermatids, thioredoxin-like 2 (Txl-2) is apparent at the spermatid nucleus and tail during the spermatid elongation phase (Sadek et al, 2003). Further studies using immunofluorescence microscopy and confocal microscopy will be used to determine whether SSP411 protein is localized to the sperm nucleus, as are Trl-2 (Sadek et al, 2003) and C. elegans SPE-11 (Browning and Strome, 1996), and/or in the tail of mature sperm, as are Sptrx-1 and Sptrx-2 (Miranda-Vizuete et al, 2001, 2003; Sadek et al, 2001; Jimenez et al, 2002a,b) and Trl-2 (Sadek et al, 2003).
The number of functions assigned to the different thioredoxins is increasing as new members of the family are discovered. Thioredoxins catalyze the reduction of protein disulfide bonds and are involved in various biological functions, including posttranslational modification, protein turnover, chaperones, cell growth and differentiation, and the immune response (Holmgren and Bjornstedt, 1995; Arner and Holmgren, 2000; Tanaka et al, 2000; Sadek et al, 2003). In addition, thioredoxins serve as electron donors for essential enzymes such as ribonucleotide reductase, regulators of transcription factor DNA binding activity, modulators of apoptosis, antioxidant defense, and participants in the regulation of the protein folding process (for reviews, see Arner and Holmgren, 2000; Nordberg and Arner, 2001; Powis and Montfort, 2001). The 3 thioredoxin proteins—Sptrx-1, Sptrx-2, and Txl-2—found in the sperm tail have been suggested to play a role in the regulation of flagellar movement (Yu et al, 2002; Miranda-Vizuete et al, 2003; Sadek et al, 2003), and Txl-2 has been shown to have microtubule-binding activity (Sadek et al, 2003). A thioredoxin homolog in Drosophila has been demonstrated to be involved in female meiosis and early embryo development (Salz et al, 1994). Moreover, a targeted disruption of the mouse thioredoxin (Trx) gene was lethal for the embryo; homozygous mouse embryos die immediately after implantation. The early lethality was caused by impaired DNA replication resulting from thioredoxin depletion (Matsui et al, 1996). The addition of thioredoxin to human sperm incubation media before in vitro fertilization increased the rate of blastocyst formation during early embryo development (Kuribayashi and Gagnon, 1996). A time-dependent increase in sperm membrane sulfhydryl groups exposed to the extracellular space was observed during the first hour of capacitation, which indicates that an important rearrangement of sulfhydryl-containing proteins occurs during the initiation of capacitation (de Lamirande and Gagnon, 1998, 2003). The protein sulfhydryl-disulfide status might be important for the regulation of human sperm capacitation and early embryo development (Kuribayashi and Gagnon, 1996; de Lamirande and Gagnon, 1998, 2003).
In summary, we have isolated a novel spermatid-expressed gene, ssp411, in rats, and we have identified 2 ortholog proteins in human and mouse. We also demonstrated that the expression of ssp411 mRNA and protein in the testis is tissue specific and age dependent. Moreover, SSP411 was present in a stage-dependent manner during the cycle of the seminiferous tubules. Because the proteins involved in fertilization are expressed during late stages of spermatogenesis, the expression of the SSP411 protein in late elongated spermatids is indicative of a function in fertility regulation. From our analysis of the SSP411 protein, we conclude that this protein is a member of a new spermatid-specific thioredoxin family.
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Acknowledgments |
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Footnotes |
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