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RESEARCH |
Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, Higashiku Hakozaki 6-10-1, Fukuoka 812-8581, Japan and 1 Department of Developmental Anatomy, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Fukuoka 812-8582, Japan
Correspondence should be addressed to H Iida; Email: iidahiro{at}agr.kyushu-u.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Recent high-throughput genomics projects have focused on the identification of organism- and tissue-specific transcriptomes by which fundamental insights into biological processes, such as spermatogenesis, are expected to be clarified. Databases for expressed sequence tags (ESTs) provide important information for the discovery of novel genes with tissue-specific expression profiles (Hong et al. 2005). Recently, microarray has been utilized to understand the changes of gene expression during spermatogenesis. A genome-wide transcriptional analysis of developing mouse testis was performed by Shima et al. (2004) using Affymetrix GeneChip system, and stage-specific transcripts were listed as the supplemental data at the Griswold Lab homepage (http://www.wsu.edu/~griswold/microarray). Out of the 215 round spermatid-specific transcripts listed in the database, we selected 10 genes and analyzed their expression profiles in terms of testis development and organ specificity. Among the ten genes, we focused our attention on one gene, a rat homolog of Drosophila germ-cell less (gcl) gene, which encodes a protein of 57.6 kDa that is predominantly expressed in elongating spermatids and integrated into the middle piece of sperm flagella as a component of outer dense fibers (ODF).
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Results |
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Sequence and genomic organization of gcl-2 gene
A search in NCBI and DNA Data Bank of Japan (DDBJ) databases employing the FASTA and BLAST programs revealed that the sequence of the gene described above was identical to that of mouse germ-cell less like-2 (mgcl-2, accession no. NM_027955). Rat ortholog gene (rgcl-2 gene, accession no. XM_228797) was found in the NCBI database. Based on the sequence data of the rat rgcl-2 gene, we made primers for PCR, and performed RT-PCR to obtain full-length rgcl-2, which resulted in amplification of 1497 base-length DNA. The open reading frame (ORF) of rgcl-2 encodes a protein of 498 amino acid residues (Fig. 1
) with the predicted molecular mass 57 578 Da and pI 8.45. SMART (http://smart.embl-heidelberg.de/) and NCBI Conserved Domain Search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) suggested the presence of a BTB (broad-complex, tramtrack and bric-a-brac) domain at 80–191 amino acid residues in rgcl-2. The BTB domain, which is also known as the POZ (pox virus and zinc finger) domain, mediates homomeric dimerization and in some instances heteromeric dimerization. A search in the NCBI and DDBJ databases employing the FASTA and BLAST programs revealed that the rat rgcl-2 (XM_228797) shared 79.72% identity with mouse mgcl-2 (accession no. NM_027955) at the amino acid level and 84.84% at the nucleic acid level.
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). These two rgcl-2 were designated as rgcl-2A (accession no. XM_228797) and rgcl-2B (accession no. BC083583) in this study, both of which encode 498 amino acid residue-length proteins. Rgcl-2A showed 94.39% identity with rgcl-2B at the nucleic acid level and 90.56% identity at the amino acid level (Fig. 1
). The molecular masses of rgcl-2A and rgcl-2B are 57.6 and 57.7 kDa respectively and the pI values of these proteins are 8.45 and 8.15 respectively. These paralog genes seem to be produced by gene duplication during the course of evolution.
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, they exhibited similar expression pattern in the testis of 1- to 8-week-old rats. They were detected at 4 weeks postnatal development and continued to be detected up to 8 weeks, and both the genes were predominantly expressed in the testis. In addition to the testis, the rgcl-2B gene was also expressed in the brain.
Immunoblot analyses
To examine rgcl-2 protein, an antibody against rgcl-2 was raised in rabbits. Since there is a high similarity of the amino acid sequence between rgcl-2A and rgcl-2B, we produced the antibody recognizing the amino acid sequence shared by the two proteins (Fig. 1). Specificity of the affinity-purified anti-rgcl-2 antibody was first examined on the blot to which several glutathione S-transferase (GST)-fused recombinant proteins were transferred. As shown in Fig. 3A, the anti-rgcl-2 antibody specifically recognized GST-rgcl-2 protein without cross-reactivity to other GST-fusion proteins, indicating that the antibody is specific for rgcl-2. This anti-rgcl-2 antibody was used throughout this study.
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). The reason for the difference in the apparent molecular size between them is unclear at present. In spermatozoa, a minor band migrating at 43 kDa, which might represent degraded rgcl-2, was also detected by the antibody.
Localization of rgcl-2 in rat testis
Using the anti-rgcl-2 antibody, we examined the localization of rgcl-2 in frozen sections of adult rat testis by confocal laser scanning microscopy. As shown in Fig. 4A and C, rgcl-2 was detected in elongating spermatids (steps 15–18) located in the luminal compartment of ST. Rgcl-2 immunostaining was virtually undetectable in round spermatids (steps 1–7) as well as in elongating spermatids of steps 8–14. Spermatogonia, spermatocytes, Sertoli cells and interstitial cells were also devoid of immunofluorescence for rgcl-2 (Fig. 4C). Replacement of the anti-rgcl-2 antibody with preimmune serum gave no specific staining (Fig. 4B). At higher magnification, rgcl-2 immunofluorescence was found to be present in the cytoplasm of elongating spermatids as well as in the flagella-like structures protruding from elongating spermatids (Fig. 4D).
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). Although a weak rgcl-2 immunosignal was also observed in the sperm heads, it might be non-specific because of detection of similar immunosignals in the control samples labeled with the preimmune serum (Fig. 4F).
Immunoelectron microscopy
To determine the precise localization of rgcl-2 in sperm flagella, sonicated rat spermatozoa, which were permeable to antibodies, were processed for pre-embedding immunoelectron microscopy using the anti-rgcl-2 antibody. The majority of gold particles complexed with the antibody were present on the surface of ODF without immunolabeling of residual mitochondrial sheath and perforatorium (Fig. 5A). In cross-sections of partially disrupted or fractured flagella (Fig. 5B and C), immunogold labeled the abaxial (convex) surface of ODF that faces mitochondrial sheath. Inner (axonemal) side of ODF was devoid of immuno-labeling (Fig. 5C). A similar staining pattern of immunogolds was identified in scattered ODF in which immunogolds predominantly labeled the abaxial (convex) surface of ODF with rare labeling on the adaxial (concave) surface (Fig. 5A, inset). Fibrous sheath was never labeled by immunogolds (not shown). Little or no labeling was noticed in the control samples in which the primary antibody was replaced by preimmune serum (Fig. 5D).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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The Drosophila germ cell-less (gcl) gene product, which is a component of the germ plasma, is required for primordial germ cell formation (Jongens et al. 1992, 1994, Robertson et al. 1999). A mouse homolog of gcl, mgcl-1, is expressed ubiquitously, with the highest levels of expression occurring in the course of spermatogenesis and localized to the nuclear envelope (Kimura et al. 1999, Leatherman et al. 2000). Defects of mgcl-1 in mice impaired fertility and produced abnormal sperm morphology, such as insufficient chromatin condensation, abnormal acrosome structures, and defects of the sperm tail (Kimura et al. 2003). In this study, we investigated the expression profiles of ten genes chosen from a database. Among them, we directed our attention to one gene, rat gcl-2 (rgcl-2), which is predominantly expressed in the testis and its expression level was up-regulated during testis development. Rgcl-2 protein contains BTB/POZ domain, which is also retained in both Drosophila gcl and mgcl-1. Mouse mgcl-1 protein (AF163665) has 47.3% identity with mouse mgcl-2 (NM_027955), and rat rgcl-1 (AY862307) has 47.1% identity with rat rgcl-2A (XM_228797). Thus, BTB/POZ domain is found in both gcl-1 and gcl-2, but identity of the amino acid sequence between them is relatively low.
Rat rgcl-1 gene is present at 4q34 of chromosome 4 as a single copy gene, whereas rat genome contains duplicate rgcl-2 genes, rgcl-2A and rgcl-2B, both of which are located at q13 of the X chromosome. RT-PCR analysis showed that the two genes exhibited similar expression patterns. These intron-less paralog genes are probably produced by gene duplication during evolution. In addition, rgcl-2A and rgcl-2B showed 94.39% identity at the nucleic acid level and 90.56% identity at the amino acid level. The similarity of these two molecules makes it difficult to distinguish them using the antibody produced in this study.
Rgcl-2 protein was not only detected in the cytoplasm of elongating spermatids of steps 15–18 in rat testis but also retained in spermatozoa as a flagellar component after spermiation. Confocal laser scanning microscopy clearly revealed that rgcl-2 is predominantly localized in the middle piece of flagella of rat spermatozoa without distinct expression in the principal and end pieces. These data suggest that rgcl-2 is synthesized in the cytoplasm of elongating spermatids, and at least a part of it is integrated into the middle piece of spermatozoa during spermiogenesis. To further elucidate the molecular localization of rgcl-2 in mature spermatozoa, we performed pre-embedding immunogold electron microscopy by which we discovered that rgcl-2 is present at the cortex of ODF and not on the adaxial (concave) surface of ODF. Therefore, we concluded that rgcl-2 is a new component of ODF in sperm flagella. However, we could not exclude the possibility that rgcl-2 is also present at the medulla of ODF since the anti-rgcl-2 antibody is not able to penetrate into ODF in the samples prepared for pre-embedding immunoelectron microscopy.
Although the physiological function of ODF remains to be determined, several hypotheses have been proposed. It has been noted that ODF are either lacking or incompletely developed in non-mammalian species and that the appearance of ODF during evolution seems to coincide with the development of internal fertilization. Therefore, it can be speculated that ODF have evolved to overcome a greater resistance to sperm locomotion in the female reproductive tract and act as structural reinforcements that enhance the tensile strength of sperm flagella (Fawcett & Ito 1965, Baltz et al. 1990). Another possibility is that ODF work as force multipliers to increase the tensile strength of the sperm tail as well as improve the bending torque of the tail (Lindemann 1996).
There is a narrow space between ODF and mitochondrial sheath in which an electron-dense matrix, called the submitochondrial reticulum (SMR), exists (Olson & Winfrey 1990). The SMR is attached to the overlaying outer mitochondrial membrane and extends from the connecting piece to the annulus in spermatozoa. In addition, we have recently reported that Tektin4 and Tektin5, new members of the TEKTIN family, are associated with the cortex of ODF and the inner surface of mitochondria sheath respectively (Iida et al. 2006b, Murayama et al. 2007). It is therefore probable that Tektin4, Tektin5, and rgcl-2 interact either with each other or with some molecules in SMR to tether the axoneme–ODF complex to the mitochondrial sheath by which physiological connection between flagellar components is maintained. Further studies of these molecules should provide additional information and understanding for the molecular architecture and complexity of sperm flagella.
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Materials and Methods |
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Antibody production
The peptide used for raising the antibody is derived from N-terminal hydrophilic region of rgcl-2 (SGQSGSRKRKLSN; Fig. 1). The peptide was coupled to keyhole limpet hemocyanin (KLH, Pierce, Rockford, IL, USA). The peptide coupled to KLH (1 mg total dose) was dissolved in 1 ml saline, emulsified with 1 ml Freund’s complete adjuvant, and injected at multiple sites on the back of a rabbit as described previously (Katafuchi et al. 2000, Doiguchi et al. 2002a). The antiserum was collected within 2 weeks after the third injection. Affinity purification of the antibody was carried out over a matrix of the peptide coupled to Formyl-Cellulofine (Seikagaku Kougyo, Nagoya, Japan), as described previously (Matsuyama et al. 2005, Iida et al. 2006a).
Preparation of GST-fusion proteins
Rat rgcl-2 gene has an ORF of 1497 nucleotides encoding 498 amino acid residues. A 183 amino acid residue length (17–199 amino acids) of rgcl-2A was PCR-amplified and cloned in frame to the COOH terminus of GST using pGEX-4T-1 system (GE Healthcare BioScience Corp). Recombinant protein was expressed in Escherichia coli and purified onto glutathione-Sepharose (GE Healthcare BioScience Corp.), as described previously (Iwamoto et al. 2005). The molecular size of GST-rgcl-2A is ~47 kDa. The GST-fused recombinant proteins, rab3A, rab3D, rab6 (Iida et al. 1999), syntaxin 2 (Katafuchi et al. 2000), spetex-2 (Iwamoto et al. 2005), iba1, mrf (Iida et al. 2001), and hsp40 (Doiguchi et al. 2007), were similarly produced and purified. These recombinant proteins were used for immunoblot analysis.
Immunoblot analysis
Testes of ether-anesthetized adult Wistar rats were immersed in PBS on ice, and the capsule (tunica albuginea) was torn away by tweezers. ST released from testes were untangled and washed several times in PBS at 4 °C with gentle agitation to remove interstitial cells. Spermatozoa, which were collected from the epididymis of adult rats, were purified by a Percoll density gradient centrifugation as reported previously (Katafuchi et al. 2000, Doiguchi et al. 2002b). Both ST and purified spermatozoa were solubilized directly in a SDS-PAGE sample buffer followed by centrifugation at 16 000 g (15 000 r.p.m.) for 20 min. Clarified supernatants were used as samples for SDS-PAGE and immunoblot analysis. The sample proteins of spermatozoa and ST 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 3 h with the affinity-purified anti-rgcl-2 antibody diluted 1:10 000 with a blocking buffer (PBS containing 5% nonfat milk and 0.05% Tween-20), followed by incubation with HRP-conjugated goat anti-rabbit IgG (Bio-Rad) diluted 1:3000 in the same buffer. Antigen–antibody complexes were visualized using an ECL-Plus detection kit (GE Healthcare Bioscience Corp). GST-fused proteins were similarly processed for immunoblot analysis.
Immunocytochemistry
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 50 mmol/l 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, USA) and immediately frozen by liquid nitrogen. The frozen sections of 8 µm thickness were cut by a cryostat (CM1850; Leica, Nussloch, Germany). The sections were washed in PBS, exposed to 0.1% Triton X-100 in PBS, and then incubated with the anti-rgcl-2 antibody diluted 1:400 with the blocking buffer followed by incubation with goat anti-rabbit IgG conjugated with Cy3 (GE Healthcare BioScience Corp). For DNA staining, immunostained samples were incubated for 30 min with PBS containing SYTOX Green (1:10 000 dilution, Molecular Probes, Eugene, OR, USA). The samples were then washed with PBS and examined by a confocal laser scanning microscope (Olympus LSM-GB 200, Tokyo, Japan). Purified spermatozoa were fixed, washed in PBS, attached to poly-L-lysine coated glass slides, and then processed for immunohistochemistry, as described above. For controls, the primary antibody was replaced by preimmune serum.
Pre-embedding immunoelectron microscopy
Samples for immunoelectron microscopy were prepared by the method described previously (Iida et al. 2006b). In brief, purified rat spermatozoa were exposed at 4 °C for 60 min to 50 mmol/l sodium borate buffer (pH 8.0) containing 1% Triton X-100 to remove the plasma membrane. The mitochondrial sheath was partially disrupted by this treatment. After extraction, the samples were centrifuged at 1000 g and the pellet was washed two times with 50 mmol/l Tris–HCl (pH 8.0). The samples in 50 mmol/l Tris–HCl (pH 8.0) were probesonicated at maximum power for four 10-s bursts, and centrifuged at 5000 g for 10 min to make pellets. The pellets were fixed in PBS containing 4% paraformaldehyde and 0.2% glutaraldehyde at 4 °C for 2 h, washed in PBS, and incubated for 30 min in PBS containing 0.2% gelatin and 0.2% BSA. The samples were immunostained overnight with the anti-rgcl-2 antibody, followed by incubation for 3 h with 10 nm gold-conjugated goat anti-rabbit IgG (GE Healthcare BioScience Corp). For controls, the primary antibody was replaced by preimmune serum. After immunostaining, the samples were washed in PBS and fixed in 3% glutaraldehyde in 0.1 mol/l cacodylate buffer (pH 7.4), postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanols, and embedded in epoxy resin as reported previously (Katafuchi et al. 2000). Ultrathin sections were examined in a Hitachi H-600 electron microscope after staining with uranyl acetate and lead citrate.
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Acknowledgements |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Footnotes |
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Received 22 June 2007
First decision 27 July 2007
Accepted 5 September 2007
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References |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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