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Hormone Research Center,3 Department of Molecular Endocrinology, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea
Department of Anatomy,4 College of Medicine, Seonam University, Namwon 590-711, Republic of Korea
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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testis, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The polymerase chain reaction (PCR)-coupled subtractive hybridization technique of representational difference analysis (RDA) has been used to identify differences in mRNA expression between two populations of cells [3, 4]. The procedure can be divided into three main phases: 1) PCR generation of amplicons representative of the starting populations of RNA molecules being compared; 2) the two-step subtractive hybridization of these representations, leading to the enrichment of amplified fragments of differentially expressed genes and the sequential depletion of cDNA sequences common to both populations; and 3) the purification, cloning, and sequencing of the resulting cDNA products [5]. Therefore, RDA compares two populations of mRNA and obtains the cDNA clones that are expressed in one population but not in the other [3].
Steroidogenic acute regulatory (StAR) protein-related lipid transfer (START) domain is believed to bind to hydrophobic lipid molecules [6] to promote intracellular lipid shuttling between subcellular compartments, lipid metabolism, and cell-signaling events [7]. Many multidomain signaling protein molecules contain this START domain [6]. The most striking feature of the START domain structure is a predominant hydrophobic tunnel, which is used to bind a single molecule of large lipophilic compounds such as cholesterol [8]. The prototype protein containing such a domain is the StAR protein, which transfers cholesterol to mitochondria in steroid hormone producing cells [9]. X-ray crystallographic structures have been resolved for MLN64 START domain [10], Stard4 [11], and phosphatidylcholine transfer protein [12]; all have been found to share the same helix-grip fold START domain structure, which forms an internal hydrophobic cavity [13].
Stard6 is a new member of the Stard4 subfamily and grouped under the START domain-containing protein family. The Stard4 subfamily also includes Stard4 and Stard5 protein members, which share 26%–32% amino acid identity with Stard6 and with one another [14]. This protein subfamily has been characterized by the presence of 205– 233 amino acid residues consisting almost entirely of the START domain [14], with no additional N-terminal domain, as found in other multidomain START family proteins [10]. In this study, we report the cloning of rat Stard6 cDNA and the spatial and temporal localization of this gene at the mRNA transcript level as well as at the protein level in developing testes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal experimentation was conducted in accordance with the guidelines of Chonnam National University's Animal Care and Use Committee, and the National Research Council Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats and New Zealand rabbits were purchased from Daehan Biolink (Korea). RNAzol B reagent was purchased from Biotecx Laboratories. Nylon and nitrocellulose membranes were purchased from Sigma Chemical Corporation. Restriction enzymes were purchased from DCC-BIONET (Korea). The [
-32P] dCTP (3000 µCi/mmol) was purchased from Amersham Biosciences.
Total RNA Isolation
Total RNA was isolated from 100 to 600 mg of tissue using the acid phenol guanidinium method [15]. Tissues were submerged in RNAzol B and homogenized with a Biomixer (Nissie, Japan) for 1–2 min at 2000 x g. One-tenth volume of chloroform was added to the homogenate, and the mixture was vortexed vigorously for 20 sec. The suspension was centrifuged for 15 min at 14 000 x g, and the upper aqueous phase was removed. An equal volume of isopropanol was then added to the aqueous solution; the sample was kept on ice for 15 min then centrifuged at 14 000 x g for 15 min. The RNA pellet was washed with 75% ethanol by centrifuging for 10 min at 14 000 x g. All centrifugations were done at 4°C. Finally, the pellet was dried for another 10 min, resuspended in diethylpyrocarbonate-treated water by vortexing, and quantified by optical density measurement.
Construction of Subtracted cDNA Library
To identify the specific genes expressed only in adult rat testis, a subtracted cDNA library was constructed using RDA [4]. RDA was carried out with a commercial kit (PCR Select; BD Biosciences Clontech). A driver cDNA population (D-cDNA) and a tester cDNA population (T-cDNA) were prepared from prepubertal (2-wk-old) and adult (8-wk-old) male rat testis, respectively. Briefly, 2 µg of cDNA was digested with RsaI and the digested T-cDNAs were then divided into two fractions and ligated to two different primers with T4 DNA ligase. Hybridization was then carried out by adding excess D-cDNA to each T-cDNA fraction in the thermal cycler at 98°C for 1.5 min, then at 68°C for 8 h. This was followed immediately by a second hybridization with fresh denatured D-cDNA at 68°C overnight. Multiple PCR reactions were set up to generate the initial amplicons (representations). Each 25-µl reaction contained 1 µl of diluted subtracted sample, 1x PCR buffer, 10 mM dNTP mix, 10 µM PCR primer or primers, and Advantage cDNA polymerase mix (#8430-1; BD Biosciences Clontech). After the 3' ends of the adapters were filled, 27 cycles of amplification were then performed at 94°C for 10 sec, 66°C for 30 sec, and 72°C for 1.5 min. During amplification, cDNAs, presumably from the adult testis, were exponentially amplified. The final PCR products were resuspended in 1x TE (10 mM Tris·Cl pH 8.0, 1 mM EDTA) at 0.5 µg/ µl. The adaptors were removed from the representations and further characterizations of the PCR products were conducted. About 100 independent clones from the subtracted library were sequenced, and GenBank was searched for their nucleotide and amino acid sequences. One clone (tsg-105), whose size was about 200 base pairs (bp), revealed significant homology with StAR and MLN64 proteins at the amino acid level.
Molecular Cloning of Rat Stard6 cDNA
The above-mentioned 200-bp fragment (tsg-105) was used to screen the rat testis cDNA library (RL300a; BD Clontech) to obtain full-length cDNA clones [16]. Briefly, about one-half million plaques were plated on top of 10 LB agarose plates at a density of 5 x 105 plaques per plate. When the diameter of the plaques reached 0.5–0.7 mm, the phage particles were transferred onto a nylon membrane, after which the DNA was denatured and neutralized. The DNA was then cross-linked to the membranes by UV exposure. The membranes were incubated in prehybridization solution (6x saline-sodium-citrate [SSC], 0.1% SDS, 5x Denhardt solution, and 100 µg/ml denatured salmon sperm DNA) at 65°C for 2 h. Then, [-32P]-labeled 200-bp cDNA was added to the fresh prehybridization solution (1 x 106 cpm/ml) and incubated at 65°C for 20 h. The random priming procedure was used to radiolabel the DNA [17]. Membranes were subjected to two sequential 30-min washes with 2x SSC/0.1% SDS at room temperature followed by two sequential 30-min washes with 0.3x SSC/0.1% SDS at 50°C, and then exposed to x-ray film at –80°C for autoradiography.
Subcloning, Sequencing, and Database Searching
One positive clone (
CG4.2) was obtained and subcloned into pBluescript vector. The clone was named pCG4.2. The nucleotide sequences of pCG4.2 were determined with a Sanger dideoxy chain termination procedure, and their nucleotide and deduced amino acid sequences were compared with the GenBank database by using the PSI BLAST programs (http://www.ncbi.nlm.nih.gov).
Northern Blot Analysis
Total RNA (10 µg) from each sample was fractionated in a 1% formaldehyde/agarose gel and transferred on to a nylon membrane and UV cross-linked [18]. Northern blots were preincubated in a solution containing 50% formamide, 50 mM NaH2PO4, 5x Denhardt solution, 5x SSC, 0.1% SDS, 2% dextran, 1 mM EDTA, and 100 µg/ml denatured salmon sperm DNA at 42°C for 8 h. Hybridization was performed with the [-32P]-labeled cDNA probe (pCG4.2) prepared by random priming procedure as previously described [17] under the same conditions of preincubation for 24 h. After hybridization, blots were washed in 2x SSC/0.1% SDS for 10 min at room temperature; 1x SSC/0.1% SDS for 30 min at 65°C, and in 0.1x SSC/0.1% SDS for 30 min at 65°C. After washing, the membrane was exposed to x-ray film for 24 h for autoradiographic signals.
In Situ Hybridization
In situ hybridization was carried out as described [19]. Adult rat testis was fixed at 4°C for 6 h in 4% paraformaldehyde in PBS followed by overnight immersion in PBS containing 0.5 M sucrose. Cryostat (Leica) sections of the testis (14 µm thickness) were mounted onto microscope slides coated with poly-L-lysine, fixed in 4% paraformaldehyde solution, and stored at –80°C until analyzed. Sections were pretreated serially with 0.2 M HCl, 2x SSC, pronase (0.13 mg/ml), paraformaldehyde, and acetic anhydride in triethanolamine. Hybridization was carried out at 52–55°C overnight in the mixture containing [35S]-labeled pCG4.2 insert as a probe (108 cpm/ml), 50% formamide, 0.3 M NaCl, 10 mM Tris-Cl, 5 mM EDTA, 1x Denhardt solution, 10% dextran sulfate, 1 µg/ml carrier transfer RNA, and 10 mM dithiothreitol. Posthybridization washing was performed under stringent conditions, which included ribonuclease A (25 µg/ml) treatment at 37°C for 30 min and 0.1x SSC. Slides were dipped into NTB-2 emulsion (Eastman Kodak Co.) and exposed at 4°C until developed after 2 wk. The slides were stained with hematoxylin, counterstained with eosin, and examined under a light microscope with bright field and dark field illumination.
Preparation of 47.5-kDa Recombinant Stard6 Protein and Raising of Polyclonal Anti-Stard6 Antibodies
A Stard6 cDNA fragment (nucleotides 402 to 1086) containing the entire open reading frame (ORF) region was cloned in-frame with glutathione S-transferase (GST) into the pGEX-4T-1 vector (Amersham-Pharmacia). Recombinant fusion protein GST-Stard6 ORF was expressed in Escherichia coli (DH5) and eluted from a 10% SDS-PAGE gel with brief modifications as described previously [20]. The purity of the 47.5 Mr x 10–3 GST-Stard6 fusion protein was checked by SDS-PAGE, followed by Coomassie blue staining. The protein concentration was assessed with the BCA (Pierce) protein assay method. Five milligrams of purified GST-Stard6 protein was dissolved at a concentration of 0.2 mg/ml in physiological saline. Two New Zealand rabbits were immunized with 0.2 mg of fusion protein per single immunization, four times at intervals of 2 wk. After three booster injections of antigen, blood was collected.
Immunoaffinity Purification of Anti-Stard6 Antibodies from Rabbit Sera
Antibodies raised against GST-Stard6 protein were immunopurified from rabbit serum as described previously with little modification [21, 22]. Briefly, the GST alone or GST-Stard6 proteins were prepared in E. coli. The proteins were cross-linked to a cyanogen bromide activated sepharose 4B (Sigma) chromatography matrix. The affinity matrix containing only GST protein thus obtained was incubated first with the rabbit antisera overnight at 4°C. The flow-through from the column was collected, added to the second affinity matrix containing GST-Stard6 fusion protein, and incubated overnight at 4°C. The column was then washed extensively with PBS and the bound immunoglobulin G (IgG) molecules were eluted. The eluted IgG molecules were used for Western blot analysis and immunohistochemical studies.
Western Blot Analysis
Rat tissue homogenates from 8-wk-old rats were fractionated as described [23] and subjected to 12% SDS-PAGE [24]. Proteins were electroblotted to nitrocellulose membranes (Sigma) using a Trans Blot Cell (Bio-Rad Laboratories). Membranes were hybridized with Stard6 (1:1000 dilution), -tubulin (1:2000 dilution) antisera (Sigma), or preimmune serum. Primary antibodies were detected using anti-rabbit IgG (1:3000 dilution) conjugated with horseradish peroxidase (Sigma). Reactive protein bands were visualized by enhanced chemiluminescence using the manufacturer's protocol (Amersham Pharmacia).
Immunohistochemistry
Testicular tissues from 3-, 5-, and 9-wk-old rats were postfixed in 4% neutral buffered paraformaldehyde (Sigma) solution and embedded in paraffin according to standard procedure [25]. Testis cross sections (6 µm thickness) were dewaxed in two changes of xylene for 10 min and rehydrated through a series of alcohol to single-strength PBS. Sections were then incubated in 3% H2O2 (in methanol) for 30 min at room temperature and rinsed in PBS three times for 5 min each. A blocking step was performed by placing slides in 2% normal rabbit serum in PBS for 1 h, and then rinsing in PBS three times (for 5 min each). Sections were incubated overnight at 4°C with rabbit anti-Stard6 antibodies and then were diluted (1:1000) in blocking solution. Slides were rinsed in PBS and incubated with rabbit anti-goat IgG (Vector Laboratories) for 1 h at room temperature. The slides were rinsed in PBS and incubated with ABC reagent (Vecta-stain Kit; Vector Laboratories) for 1 h. Finally, the slides were incubated with diaminobenzidine (Sigma) until a brown color developed. The color reaction for the control (with preimmune rabbit serum) slide was terminated at the same time as for the slides treated with primary antibody. Slides were cleared with xylene and mounted under Permount (Fisher Scientific). Images were made using Image Pro Express software (MagnaFire SP Imaging, Sciscope Instrument Company).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To obtain the genes that are specifically expressed in adult rat testis and not in prepubertal testis, a cDNA library was constructed using PCR-based RDA. About 100 clones from the subtracted cDNA library were selected randomly and their nucleotide sequences were determined. To screen out the novel genes from the library, a homology search on the GenBank database (http://www.ncbi.nlm.nih.gov) was carried out with the sequence information. Of several clones, one clone (tsg-105) with a nucleotide length of 200 bp showed significant homology with StAR and MLN64 proteins at the amino acid level. Therefore, this clone was studied further.
To obtain the full-length cDNA clone corresponding to tsg-105, the rat testis cDNA library was screened with tsg-105 partial cDNA fragment (200 bp) as a probe; one independent lambda clone (CG4.2) was isolated. The cDNA clone (GenBank accession number AY555189) obtained from the rat testis library screening was 1146 bp long and generated the longest ORF of 227 amino acids from nucleotide 402 to nucleotide 1086 (data not shown). The nucleotide sequence of the clone and the deduced amino acid sequence are shown in Figure 1. The cDNA was found to contain a long 5' untranslated region (UTR) with multiple initiation codons and a short 3' UTR with polyadenylation signal at nucleotide 1098 followed by the poly(A) tail (Fig. 1).
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The genomic sequence of rat Stard6 was analyzed by using publicly available resources through the National Center for Biotechnology Information and was found to reside in chromosome 18. However, a part of its cDNA also hit a match in chromosome 12 of the rat genome. The genomic exon-intron structure of rat Stard6 was found to be conserved in the mouse counterpart. The amino acid sequence analysis of the rat Stard6 protein revealed the START domain from amino acid 43 to amino acid 208 (data not shown) without any additional signaling domain, and therefore is regarded as a typical member of the Stard4 subfamily.
A database search revealed 88% homology at the nucleotide level and 81% homology at the amino acid level with the previously cloned mouse Stard6. However, the search revealed 64% homology with the human counterpart of Stard6 in the amino acid sequence. The sequence alignment of rat mouse and human Stard6 amino acids is shown in Figure 2. The C-terminal region of Stard6 homologues was found to be very much divergent among these three species. Homologies with START family proteins rat StAR, MLN64, Stard4, and Stard5 were obtained as 21%, 26%, 28%, and 38%, respectively, at the amino acid level (data not shown).
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Northern Blot Analysis
To investigate the tissue expression pattern of rat Stard6 mRNA, rat multiple tissue blot was hybridized with rat Stard6 cDNA as a probe. A transcript of 1.6 kilobase (kb) was exclusively found in mature testis (Fig. 3A). A further study was conducted to check the developmental expression pattern of Stard6 mRNA transcript in testis. Total RNA extracted from testes of rats 7, 21, 30, 40, and 68 days of age were subjected to Northern blot analysis. There was a faint signal in the testis after 21 days, and stronger signals were detected at 30 days; the signal continued to increase up to adulthood (Fig. 3B). Taken together, these data indicated that the expression of the Stard6 gene may be regulated at both spatial and temporal levels.
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In Situ Hybridization of Stard6 Gene in Rat Testis
In situ hybridization was used to check the cellular distribution of Stard6 mRNA in the adult rat testis. Positive signals were detected exclusively in the developing germ cells. Moreover, the expression level of Stard6 mRNA was detectable from mid pachytene to early elongated spermatids, with maximum expression found in round spermatids (Fig. 4C). The signals were detected as dark and white spots in the bright fields (Fig. 4, A and C) and dark fields (Fig. 4B), respectively, of photoradiographs. A negative control section hybridized with a sense Stard6 probe showed no signal (Fig. 4D).
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Western Blot Analysis of Stard6 Protein
Rabbit polyclonal Stard6 antibodies were raised against GST-Stard6 fusion protein. The antibodies were affinity-purified and the specificity of the antibodies was confirmed by Western blot analysis using in vitro translated Stard6 protein (data not shown). The 684-bp-long ORF of Stard6 having 227 amino acid residues was predicted to produce a protein of approximately 25.5 kDa. However, a protein of approximately 28 kDa, a little bigger than the expected protein size, was detected in the testis (Fig. 5A). Western blot analysis was also carried out to check the expression pattern of Strad6 at various developmental stages of rat testis. There was significant expression of Stard6 protein from 3-wk rat testis, and the expression level was similarly increased up to adulthood (Fig. 5B), as found in Northern blot analysis (Fig. 3B).
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Localization of Stard6 Protein During Spermatogenesis
Sections of rat testis at various developmental stages were immunostained with rabbit polyclonal anti-Stard6 antibodies. Because Stard6 mRNA (Fig. 3B) and Stard6 protein (Fig. 5B) were detected in 3-wk rat testis, immunostaining was carried out in 3-wk-old (Fig. 6A), 5-wk-old (Fig. 6B), and 9-wk-old (Fig. 6C) rat testes. Significant immunostaining was detected in germ cells of prepubertal (3-wk and 5-wk) and adult (9-wk) testes. Adult testis sections showed strong signals in well-differentiated seminiferous tubules mainly in round spermatids and to some extent in elongated spermatids, as seen in the in situ hybridization result (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Northern blot analysis showed rat Stard6 mRNA to be expressed only in adult testis (Fig. 3A), and was thereby found to be consistent with the expression pattern of mouse Stard6 mRNA [14]. The mRNA transcript in the Northern blot was around 1.5 kb (Fig. 3); however, we could only clone about 1.1 kb cDNA. To obtain the full-length cDNA clone, further screening of the cDNA library was carried out, but the effort was not successful. Because Stard6 mRNA transcript was found to be expressed only in testis and not in any other steroidogenic tissues, such as ovary and adrenal gland, in situ hybridization study was therefore necessary to reveal its spatial distribution in adult rat testis. The Stard6 mRNA expression appeared to be germ cell-specific because it was found predominantly in the postmeiotic germ cells in adult rat testis. Moreover, the expression level gradually increased from the mid pachytene stage to early elongated spermatid stage, with the maximal expression found in round spermatids (Fig. 4). Such stage-specific expression of Stard6 mRNA may reflect its strict transcriptional regulation in germ cells during spermatogenesis.
Testis-specific Stard6 protein with a molecular weight of approximately 28 kDa was detected in tissue in Western blot against the anti-Stard6 antibodies (Fig. 5A). However, the protein size appeared to be about 3 kDa larger than the expected size. An additional Western blot analysis with the in vitro translated Stard6 protein was carried out and the data revealed the predicted size (data not shown) for Stard6 protein, as deduced from its amino acid sequence. Such difference in molecular mass between the testicular Stard6 and the in vitro translated Stard6 protein suggested some posttranslational modifications of this protein in its native form. Indeed, computational database analysis revealed few potential sites for O-ß-GlcNAc attachment (http://www.cbs.dtu.dk/services/YinOYang) in Stard6 protein. However, such posttranslational modifications of Stard6 protein have yet to be confirmed. Tissues such as kidney and spleen revealed immunopositive signals in Western blot (Fig 5A), but the protein size was found to be much greater than the predicted size of Stard6 protein and hence was considered to be nonspecific. The appearance of such nonspecific bands might be due to the cross-reactivity of our polyclonal anti-Stard6 antibodies with other unknown proteins in these tissues. Finally, to compare the cellular localization of Stard6 protein with its mRNA localization, immunohistochemistry was carried out in rat testis of different ages. Immunopositive signals were detected only in the developing germ cells at different stages of the developmental testis (Fig. 6), and this result was in concert with the in situ hybridization (Fig. 4).
The present study strongly suggests male germ cell-specific expression of Stard6 at the mRNA transcript level as well as at the protein level. This indicates Stard6 to be a functional gene, expressing Stard6 protein, which may play a pivotal role in the process of spermatogenesis in adult testis. The staining pattern of Stard6 protein suggested that its expression appeared to be localized in the nucleus rather than in the cytoplasm. However, further studies must be carried out to obtain information about the subcellular localization of this protein.
The putative lipid-binding START domain of Stard6 protein spans the entire length of the protein. However, there have been no reports of its natural ligand until now. The protein may act as a cytosolic lipid carrier such as PCTP [29] because it contains only the START domain and lacks any other additional N-terminal signaling domain or other localization signals [30]. Absence of Stard6 protein expression in any of the steroidogenic cells, such as testicular Leydig cells, ovarian granulosa cells, or adrenal cells, suggests that this protein may play a role as a signaling molecule or help in lipid metabolism rather than being involved in steroidogenic activity as is StAR or MLN64 protein [9].
The lipid profile of testis suggests that postlanosterols are surprisingly abundant in testis, including epididymis, where they may play a role in sperm maturation [31]. The first postlanosterol enzyme CYP51 (Lanosterol 14-demethylase) of the cholesterol biosynthetic pathway and NADPH P450 reductase were also detected in round and elongated spermatids [32], which suggested their possible roles in synthesizing testis-meiosis activating sterols (T-MAS) [33], even in postmeiotic spermatids. Thus, similar kinds of stage-specific expression of Stard6 protein suggest that it could possibly bind to T-MAS or other sterols, and therefore might be responsible for sperm differentiation and maturation. To our knowledge, this is the first study indicating that Stard6, a member of the Stard4 subfamily, is expressed only in developing germ cells in rat testis. The present state of knowledge does not provide the exact description of the in vivo role of Stard6. Therefore, further characterization of the protein is needed to predict its involvement in mammalian spermatogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Jaemog Soh, Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea. Fax: 82 62 530 2199; jmsoh{at}chonnam.ac.kr
Received: 8 June 2004.
First decision: 29 June 2004.
Accepted: 14 October 2004.
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