| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ri3
Laboratory of Zoology,3 Graduate School of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
Molecular Bioscience of Roche Diagnostics,4 Tokyo 105-0014, Japan
Department of Developmental Anatomy,5 Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan
Department of Anatomy and Developmental Biology,6 Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
gametogenesis, sperm maturation, spermatid, spermatogenesis, testis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To investigate the molecular mechanisms regulating the drastic morphological changes from round spermatids to spermatozoa, we used differential display in combination with cDNA cloning technique to isolate genes that are predominantly expressed in haploid spermatids. By this approach, we recently isolated several novel genes that may be involved in the differentiation of spermatogenic cells. Spergen1 is a small protein of 154 amino acids that is associated with mitochondria of both elongating spermatids and mature spermatozoa [6]. It might be involved in mitochondrial sheath formation during spermiogenesis by working as an adhesive molecule between mitochondria [7]. In addition to Spergen1, we recently isolated Spergen2, encoding a 56-kDa nuclear protein [8], and Spetex1, encoding a 63-kDa cytoplasmic protein of elongating spermatids [9]. In the present study, we report a novel gene family, SPETEX2 (spermatid-expressing gene-2), which contains multicopied genes mapped in a cluster on rat chromosome 15p16. These genes are rat-specific and encode a 23-kDa protein localized in the cortical cytoplasm of haploid spermatids. We hypothesize that SPETEX2 might be correlated with cell differentiation of spermatids in rat testis.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Investigations were conducted in accordance with the National Research Council publication Guide for Care and Use of Laboratory Animals. Total RNAs were isolated from testis, epididymis, lung, kidney, intestine, liver, stomach, brain, heart, and spleen of 8-wk-old Wistar rats as previously described [6, 10]. Total testis RNAs also were isolated from Wistar rats at 1, 2, 3, 4, 5, 6, 7, and 8 wk of age.
The mRNA differential display method [11, 12] was carried out using Delta Differential Display Kit (Clontech Laboratories, Palo Alto, CA). The RNAs isolated from Wistar rats of 2, 3, 4, 5, and 6 wk of age were reverse transcribed with oligo-(dT) primers anchored to the beginning of the poly(A) tail. The resulting cDNAs were amplified with T primers and P primers (arbitrary primers) in the kit. The cycling parameters were as follows: 94°C for 30 sec, 40°C for 2 min, and 72°C for 30 sec for 40 cycles. The amplified cDNAs were separated on 6% urea-polyacrylamide gels, fixed, and stained by the silver-sequence system (Promega, Madison, WI). Complementary DNA fragments, the expression levels of which were developmentally increased, were recovered directly by cutting out the gel slices. After elution by boiling the gel slices in distilled water for 15 min, cDNA fragments were reamplified using the same primers as those in the initial polymerase chain reaction (PCR) for differential display. The cDNA fragments were then purified by electrophoresis, cloned into the pGEM-T easy vector (Promega), and sequenced using a DNA sequencer (Applied Biosystems, Foster City, CA).
Northern Blot Analysis
A Northern blot membrane loaded with 15 µg of total RNA was hybridized with the 200-base pair (bp) DNA that was the reamplified PCR fragment described above. The probe was gel-purified and labeled with digoxigenin (DIG)-dUTP according to the manufacturer's instructions (Roche Molecular Biochemicals, Mannheim, Germany). Hybridization was performed as previously reported [13, 14]. Messenger RNA hybridized with the probe was immunologically detected by anti-DIG antibody conjugated with alkaline phosphatase (1:5000 dilution) and then recorded on x-ray films with the chemiluminescence substrate CSPD (Roche Molecular Biochemicals). Ribosomal RNAs were visualized by staining the membranes with methylene blue.
In Situ Hybridization
In situ hybridization was carried out as previously reported [6, 10]. In brief, frozen sections of adult rat testis were preincubated for 30 min 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 of BSA, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 1 mg/ml of herring sperm DNA) and then hybridized for 5 h at 42°C in the hybridization buffer containing a DIG-labeled sense or antisense RNA probe of 200 nucleotides in length. After hybridization, the sections were washed for 1 h in 2x SSC (1x SSC: 0.15 M sodium chloride and 0.015 sodium citrate) with 50% formamide at 42°C and incubated for 30 min at 37°C with RNase A (20 µg/ml), after which bound cRNA was detected using the anti-DIG alkaline phosphatase-conjugated antibody (1:500 dilution; Roche Molecular Biochemicals) and visualized with nitroblue tetrazolium-5-bromocresyl-3-indolylphosphate (Roche Molecular Biochemicals).
cDNA Cloning
To obtain the full-length cDNAs encoding the rat genes, plaque hybridization was performed by the standard method [15]. Rat testis 5'-stretch plus cDNA library was obtained from Clontech Laboratories. The probe for plaque hybridization was a PCR-reamplified, 200-bp DNA fragment that was labeled with DIG-dUTP by DIG High Prime DNA Labeling Kit (Roche Molecular Biochemicals). The hybridized probe was immunodetected by the anti-DIG antibody conjugated with alkaline phosphatase and then recorded on x-ray films with CSPD. Complementary DNAs of isolated clones were sequenced using a DNA sequencer (Applied Biosystems).
Reverse Transcription-PCR
Complementary DNA strands were synthesized from 2 µg of total RNA by using a first-strand synthesis kit (Amersham Pharmacia Biotech, Little Chalfont, U.K.) with random primers. Using the synthesized cDNAs as templates, we performed PCR to amplify the target gene. The primers used to amplify the gene were 5'-ATG TTT CAC CAG CTG CCTC AAG CTA G-3' (forward) and 5'-TTA CTC AAA AGT AAC AGG ACA CAA G-3' (reverse). The cycling parameters were as follows: 94°C for 30 sec, 64°C for 30 sec, and 72°C for 2 min for 30 cycles. The PCR-amplified DNA of 585 bp in length was cloned into pGEM-T easy vector and sequenced using a DNA sequencer (Applied Biosystems). The PCR products were examined by agarose-gel electrophoresis. Primers for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) 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).
Antibody Production
The peptide used for raising antibody is derived from the hydrophilic region of SPETEX2A (EEGKRFCEEASK) (see Fig. 4). The peptide was coupled to keyhole limpet hemocyanin (KLH; Pierce, Rockford, IL). The peptide coupled to KLH (total dose, 1 mg) was dissolved in 1 ml of saline, emulsified with 1 ml of Freund complete adjuvant, and injected at multiple sites on the back of a rabbit as described previously [14, 16]. The antiserum was collected within 2 wk after the third injection.
|
Preparation of Glutathione S-Transferase-Fusion Proteins
A 585-nucleotide, full-length Spetex2 was amplified by PCR and cloned in frame to the COOH terminus of glutathione S-transferase (GST) using pGEX-4T-1 system (Amersham Pharmacia Biotech). Recombinant protein was expressed in Escherichia coli (JM109 or BL21 strain), released from E. coli by sonication, and purified onto glutathione-Sepharose (Amersham Pharmacia Biotech) as previously described [6, 8]. The GST-fused recombinant proteins (RAB3A, RAB6, IBA1, MRF1, SPETEX1, and SPERGEN3) were similarly expressed in E. coli and purified. These recombinant proteins were used for immunoblot analysis.
Immunoblot Analyses
Seminiferous tubules were taken from the testes of ether-anesthetized, adult Wistar rats and washed in PBS at 4°C for 20 min with gentle agitation. Spleen was taken from rats as well and dissected into small pieces. These tissues were homogenized in RIPA buffer (50 mM Tris [pH 7.2], 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Nonidet P-40, protease inhibitors [1 mU/ml of aprotinin, 0.1 mmol/L of leupeptin, and 0.5 mmol/L of phenylmethylsulfonyl fluoride]) and centrifuged at 15 000 rpm for 20 min. Clarified supernatants were used as samples for immunoblot analysis. Proteins prepared for SDS-PAGE were separated on 12% acrylamide gel, and separated proteins were either stained with Coomassie brilliant blue or transferred to nitrocellulose sheets. The sheets were incubated for 2 h with the anti-SPETEX2 antibody diluted 1:1000 with a blocking buffer (PBS containing 5% nonfat milk and 0.1% Tween-20), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) G (Bio-Rad, Richmond, CA) diluted 1:2000 in the same buffer. Antigen-antibody complexes were visualized using an ECL Plus Detection Kit (Amersham Pharmacia Biotech). For controls, the primary antibody was omitted.
Immunohistochemistry
Adult rat testes were fixed in 4% paraformaldehyde in PBS at 4°C for 4 h, washed three times in PBS, incubated in PBS containing NH4Cl for 30 min, and then rinsed in PBS. After infiltration of 20% (w/v) sucrose in PBS, the testes were immersed in OCT compound (Tissue-Tek; Miles, Inc., Elkhart, IN) and immediately frozen by liquid nitrogen. Frozen sections (thickness, 8 µm) were cut by a cryostat (CM1850; Leica, Nussloch, Germany). The sections were washed in PBS, exposed to a blocking buffer (PBS containing 5% nonfat milk) for 15 min, and then incubated for 2 h with the anti-SPETEX2 antibody and the MN-7 monoclonal antibody that recognizes the acrosomal protein Acrin 1 [17]. After washing with PBS, the samples were incubated for 1 h with goat anti-rabbit IgG conjugated with Cy3 (Amersham Pharmacia Biotech) and anti-mouse IgG labeled with fluorescein isothiocyanate (FITC; Amersham Pharmacia Biotech). The samples were then washed with PBS and examined by a confocal laser-scanning microscope (LSM-GB 200; Olympus, Tokyo, Japan). For controls, the primary antibody was replaced by preimmune serum.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To identify gene transcripts that are increased during rat spermatogenesis, cDNAs derived from transcripts of the testes of 2- to 6-wk-old rats were examined by differential display screening using 45 different combinations of primer pairs, by which we isolated 42 DNA fragments with expressions that were up-regulated during testicular development. One of the fragments, which was 200 nucleotides in length, was expressed after 4 wk of postnatal development (Fig. 1). The DNA fragment was eluted from the gel and reamplified by PCR using the same primers used for the initial PCR in the differential display. The amplified DNA fragment was used as a probe for Northern blot analysis. The probe hybridized to a transcript of approximately 0.7 kilobase on the blot to which 15 µg of RNAs from 4-, 5-, and 6-wk-old rat testes were transferred, whereas the probe created faint or no signal on the blot of 15 µg of RNA derived from 2- and 3-wk-old rat testes (Fig. 2). We operationally named the gene 36a.
|
In Situ Localization of 36a mRNA
We performed in situ hybridization to determine the cell types expressing 36a mRNA. Frozen sections of adult rat testis were hybridized either with an RNA probe having the antisense sequence of 36a mRNA or with a sense probe as a control. Hybridization with the antisense probe created strong signals in the inner half-layer of the seminiferous tubules in adult rat testis (Fig. 3A), whereas the sections hybridized with the sense probe were virtually devoid of positive signal (Fig. 3B). At higher magnification, signals for 36a mRNA were found in round spermatids at steps 1– 7 (Fig. 3C). In addition, we detected recognizable, but less intense, signals in elongating spermatids at steps 8–13 (Fig. 3D). The 36a mRNA was hardly detectable in more advanced elongated spermatids (steps 15–19) present in the seminiferous tubules at stages I–VIII (Fig. 3, A and C). Both spermatogonia and spermatocytes located in the outer half-layer of the seminiferous epithelium as well as Sertoli cells were devoid of reaction product (Fig. 3, A and C). Interstitial cells were also negative (Fig. 3A). These data indicate that the 36a gene we isolated is expressed in haploid spermatids at steps 1–13.
Complementary DNA Cloning
Using the 200-nucleotide cDNA fragment of 36a gene as a probe, we next performed plaque hybridization to obtain the full-length cDNA from the rat testis cDNA library. After four rounds of screening, we isolated eight positive clones. One of the clones, which contained 672 bp, seemed to be a full-length of the gene with 5'- and 3'-untranslated regions (UTRs). This size was in agreement with the length of mRNA as detected by Northern blot analysis. The full-length cDNA sequence and its deduced amino acid sequence are shown in Figure 4. The identified cDNA contained a single open reading frame (ORF) of 582 nucleotides, with 45 nucleotides of 5'-UTR and 45 nucleotides of 3'-UTR. A poly(A) was located 23 nucleotides downstream from the termination codon, TAA. A typical consensus poly(A) signal (AAUAAA) was not found in the 3'-UTR sequence. The length of 5'-UTR was not extended by 5'-rapid amplification of cDNA ends. Because the gene is predominantly expressed in spermatids in rat testis (Fig. 3), it was designated as Spetex2A (spermatid-expressed gene-2A; accession no. AB180076, this and all accession numbers below have been deposited with GenBank).
The ORF of Spetex2 encodes a protein of 194 amino acids with the predicted molecular mass of 23 083 Da and a pI of 8.21. Both hydrophobicity plot analysis and the hydrophobic analysis program, SOSUI (http://sosui.proteosome.bio.tua.ac.jp/sosuiframe0.html), suggested that the gene encodes a soluble protein. Neither a signal peptide at the N-terminus nor the transmembrane region was found in SPETEX2. Using the National Center for Biotechnology Information (NCBI) Conserved Domain Search Program (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), a putative nuclear localization signal (NLS) of the bipartite type was observed in SPETEX2 (Fig. 4). In addition, the DUF622 domain, the function of which remains unclear, was found in amino acid residues 34–117 in SPETEX2. The domain also is observed in the SPEER (spermatogenesis-associated glutamate [E]-rich proteins) family [18].
To determine the genomic organization, the full-length sequence of rat Spetex2 cDNA was BLAST searched against rat genome databases at NCBI. The search unexpectedly resulted in 132 hits with high identities on chromosomes 4, 7, 8, 9, 14, 15, and X, of which 117 hits (i.e., 117 exons) were found on chromosome 15p16. Rat Spetex2 is composed of four exons, and a complete set of Spetex2' exons, which consisted of successively aligned four exons of Spetex2, were found in a cluster of at least 18 loci on rat chromosome 15p16 (Fig. 5), spanning approximately 1.7 megabases. These genes are present in the supercontig NW_047446. These results suggested that Spetex2 genes are multicopied and present on rat chromosome 15.
|
To examine whether Spetex2 family genes are, indeed, transcribed in rat testis, we performed reverse transcription (RT)-PCR by using rat cDNAs as templates. The PCR-amplified, full-length Spetex2 genes were subcloned into pGEM-T easy vector, and 45 independent cDNA clones were sequenced. The genes that exhibited identical sequence in more than two cDNAs were picked up and assigned to eight groups: Spetex2B (accession no. AB180077), Spetex2C (accession no. AB180078), Spetex2D (accession no. AB180079), Spetex2E (accession no. AB180080), Spetex2F (accession no. AB180081), Spetex2G (accession no. AB180082), and Spetex2H (accession no. AB180083). The cDNA derived from library screening was designated as Spetex2A. Alignment of the amino acid sequences of these Spetex2 gene products is shown in Figure 6. At the amino acid level, SPETEX2A revealed identities of 99% to SPETEX2B, 96% to SPETEX2C, 95% to SPETEX2D, 94% to SPETEX2E, 93% to SPETEX2F, 94% to SPETEX2G, and 92% to SPETEX2H. A search in the databases at NCBI and the DNA Data Bank of Japan employing the FASTA and BLAST programs revealed that SPETEX2 family proteins exhibited a marked homology to many molecules deposited in the rat databases. The sequences of all these molecules in the databases were predicted by automated computational analysis using gene prediction method (GenomeScan). Identities at the amino acid level between these molecules and SPETEX2A are as follows (identities are in parentheses): XP_344299 (95%), XP_344291 (94%), XP_344307 (94%), XP_299514 (94%), XP_344304 (94%), and XP_229505 (93%). Orthologue of the rat Spetex2 gene was not found in the human cDNA and genome databases. Also, no mouse counterpart highly homologous to rat Spetex2 was detected in the mouse databases.
|
Developmental and Tissue-Dependent Expression of Spetex2 genes
We performed RT-PCR to examine the developmental expression of Spetex2 mRNA in testes of 1- to 8-wk-old rats. We found that Spetex2 mRNA was detected first at 4 wk of postnatal development and continued to be expressed up to 8 wk (Fig. 7A). We next examined by RT-PCR the expression of Spetex2 mRNA in various organs of adult rats. Spetex2 was strongly expressed in testis as well as in spleen, but it was undetectable in other tissues examined (Fig. 7B).
|
Immunoblot Analyses
To examine the expression and the localization of SPETEX2 protein in rat testis, a polyclonal antibody was raised against the synthetic peptide (EEGKRFCEEASK) of SPETEX2 (see Fig. 4). Specificity of the anti-SPETEX2 antibody was examined on the blot to which GST-fused full-length SPETEX2 protein (49 kDa) as well as several GST-fused recombinant proteins were transferred (Fig. 8A). As shown in Figure 8B, the anti-SPETEX2 antibody specifically recognized GST-fused SPETEX2 protein but did not react with other GST-fusion proteins, indicating that the antibody is specific for SPETEX2 protein. On the blots to which whole extracts of rat seminiferous tubule and spleen were transferred, the anti-SPETEX2 antibody recognized a single protein migrating at approximately 21–22 kDa (Fig. 8C), which is close to the 23 kDa calculated from the SPETEX2 amino acid sequence deduced from the cDNA sequence. Omission of the anti-SPETEX2 antibody resulted in no detection of specific band on the blot. These data indicated that the antibody, which was raised against the synthetic peptide of SPETEX2, specifically recognizes SPETEX2 protein expressed in rat testis and spleen.
|
Immunocytochemical Detection of SPETEX2
Using the anti-SPETEX2 antibody, we intended to determine by confocal laser-scanning microscopy the cell types expressing SPETEX2 protein in frozen sections of adult rat testis. Tissue sections were double-immunostained by the anti-SPETEX2 antibody and MN-7 monoclonal antibody that recognizes the acrosomal protein Acrin 1 (a marker for haploid spermatids) [17]. SPETEX2 was visualized by Cy-3-conjugated anti-rabbit IgG (red color), and Acrin 1 was probed by FITC-labeled anti-mouse IgG (green).
Figure 9, A–D, demonstrates SPETEX2 immunostaining patterns in the seminiferous tubules at stages I–III, IV–V, VI–VII, and X–XIII, respectively. SPETEX2 immunostaining was detected at the cell periphery of round spermatids at steps 1–3 (Golgi phase) (Fig. 9A), round spermatids at steps 4–7 (Cap phase) (Fig. 9, B and C), as well as elongating spermatids at steps 10–13 (acrosomal phase) (Fig. 9D). As shown in Figure 9E at high magnification, SPETEX2 immunolabeling appeared as dot-like staining at the cell periphery of round spermatids containing MN-7-positive acrosomal cap. Replacement of the anti-SPETEX2 antibody with preimmune serum gave no specific staining (Fig. 9F). SPETEX2 immunostaining was difficult to detect in spermatids at steps 15—19 present in seminiferous tubules at stages I–VIII (Fig. 9, A–C). It also was undetectable in spermatogonia and spermatocytes as well as in interstitial cells (Fig. 9, B and C). Although spermatid proteins, such as IBA1 and SPERGEN1, are discarded and engulfed as residual bodies by Sertoli cells at the spermiation stage [6, 10], residual bodies exhibiting SPETEX2 immunolabeling were not found in Sertoli cells. Taken together, the immunocytochemical data indicate that immunostaining for SPETEX2 is restricted to the cell periphery of round spermatids (steps 1–8) and elongating spermatids (steps 9–13) in the seminiferous epithelium.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The Rat Genome Sequencing Project (RGSP) revealed that the size of the rat genome is 2.75 gigabases and contains 28 516 transcripts, including 1176 pseudogenes [19]. According to estimation of RGSP, the proportion of 1:1 orthologues in the rat and mouse genomes seems to lie between 86% and 94%, and a similar proportion (89–90%) of rat genes possess a single orthologue in the human genome. The remaining genes are associated with expansions or contractions of lineage-specific gene family.
A BLAST search against rat genome databases at NCBI revealed that Spetex2 was mapped in a cluster of at least 18 loci on rat chromosome 15p16, indicating that the genes occur as a repeated tandem array over a long stretch of genomic DNA. Because only a short DNA fragment homologous to rat Spetex2 was found in the mouse genome and no Spetex2 homologous DNA was detected in the human genome, it naturally is thought that Spetex2 genes are rat-specific. It is plausible that rat Spetex2 gene clusters have arisen recently, via a rapid burst of gene duplication since the rat-mouse split, which seems to be interesting from the viewpoint of evolution. Such species-specific genes that are exclusively expressed in testis also have been reported in the mouse and human. The SPEER family, comprising nine genes and five pseudogenes, is expressed in haploid germ cells of the testis in the mouse but not in other species [18]. The FAM family, which comprises three genes and three pseudogenes that are localized as repeats at Xp22.3 [20], is expressed in human testis, but not in rodents [18]. Although the physiological functions of these genes in testis remain to be elucidated, some of these genes expressed in the species-specific manner may be connected with diversified biochemical and morphological differences of spermatozoa between species [21].
Confocal laser-scanning microscopy revealed that immunoreactivity for SPETEX2 protein was restricted to the cell periphery of round spermatids (steps 1–8) and elongating spermatids (steps 9–13). Because neither a signal peptide at the N-terminus nor the transmembrane region was found in SPETEX2, it is unlikely that SPETEX2 is an integrated membrane protein of spermatids. It might be a soluble protein localized in the cortical cytoplasm of spermatids. Restricted expression of SPETEX2 at the cell periphery of haploid spermatids suggests that the properties of spermatid cortical cytoplasm are distinct from those of spermatogonia and spermatocytes.
Expression of SPETEX2 protein was not detected in spermatids at steps 15–19. If SPETEX2 is retained in spermatids until spermiation (step 19), it should be excluded as residual cytoplasm that ultimately is engulfed as residual bodies into Sertoli cells [22, 23], as demonstrated previously for IBA1 [10] and SPERGEN1 [6]. SPETEX2 immunoreactivity, however, was not detected within the residual bodies in Sertoli cells. This suggests that SPETEX2 expressed in spermatids at steps 1–13 might gradually degrade and disappear as they come up to the spermiation stage.
It has been widely recognized that translocation of proteins across the nuclear envelope depends on the "classical" NLS [24]. The NLS consists of either a cluster of basic residues (i.e., monopartite) or two clusters of basic residues separated by 10 residues (i.e., bipartite) [25–27]. The definition of an NLS sequence, however, is somewhat vague because of the diversity of sequences that can apparently act as a functional NLS [24, 28]. SPETEX2 possesses a putative bipartite NLS, RK(X)10EKRIK, where X = any amino acid residues. As Figure 6 shows, Arg (underlined) is changed to His in SPETEX2F. In addition, immunocytochemical studies demonstrated that SPETEX2 is a cytoplasmic molecule, not a nuclear protein. It therefore is plausible that the NLS in SPETEX2 might be nonfunctional.
In conclusion, in the present study we isolated rat-specific multicopied genes, the SPETEX2 family mapped on rat chromosome 15p16, that appear to have arisen via a rapid burst of gene duplication since the rat-mouse split. The genes encoded proteins of 194–196 amino acid residues, which were exclusively expressed as a cytoplasmic molecule localized at the cell periphery of haploid spermatids. Although the physiological functions of SPETEX2 remain to be determined, further studies of SPETEX2 may shed more light on the molecular mechanisms regulating spermatid maturation as well as gene evolution in the rat. In addition, SPETEX2 could be used as a marker to monitor rat spermatogenic cells when transplanted into mouse testis by recently developed transplantation techniques [29].
|
FOOTNOTES |
|---|
2 Correspondence: Hiroshi Iida, Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, Higashiku Hakozaki 6-10-1, Fukuoka 812-8581, Japan. FAX: 92 642 2804; iidahiro{at}agr.kyushu-u.ac.jp
Received: 16 June 2004.
First decision: 29 June 2004.
Accepted: 3 September 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  H. Kurio, E. Murayama, T. Kaneko, Y. Shibata, T. Inai, and H. Iida Intron Retention Generates a Novel Isoform of CEACAM6 That May Act as an Adhesion Molecule in the Ectoplasmic Specialization Structures Between Spermatids and Sertoli Cells in Rat Testis Biol Reprod, December 1, 2008; 79(6): 1062 - 1073. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Murayama, M. Katoh, A. Kanebayashi, T. Kaneko, Y. Shibata, T. Inai, and H. Iida Germ cell-less like-2 protein is a new component of outer dense fibers in rat sperm flagella Reproduction, December 1, 2007; 134(6): 749 - 756. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
