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Testis |
Cell Biology Section,2 Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709
School of Molecular Biosciences,3 Washington State University, Pullman, Washington 99164
Department of Physiology,4 University of Massachusetts Medical School, Worcester, Massachusetts 01655
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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null mice, which have lowered numbers of round spermatids in their testes. These results indicate the possibility that Tsp57 protein plays a role in the postmeiotic phase of germ cell differentiation. Tsp57 contains two putative nuclear localization signals: NLS1 and NLS2. Examination of the cellular localization showed that the green fluorescent protein-Tsp57 fusion protein localized to both cytoplasm and nucleus. After deletion of NLS1 but not NLS2, Tsp57 localized solely to the cytoplasm, indicating a role for NLS1 in the nuclear localization of Tsp57. The localization suggests a nuclear function for Tsp57. Pull-down analysis demonstrated that Tsp57 and RAP80 form a complex in intact cells.
gamete biology, male reproductive tract, spermatid, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The identification of cell type- and stage-specific genes provide excellent tools to dissect the differentiation program and to study the mechanisms by which spermatogenesis is controlled. Although the molecular mechanism of cell type- and stage-specific gene expression is still poorly understood, recent studies have begun to provide insight into some of the regulatory mechanisms. For example, the germ cell-specific transcription factor cAMP-responsive element modulator (CREM) appears to be a key factor in the regulation of the expression of a number of postmeiotic genes [4]. Several members of the nuclear receptor family, including estrogen, retinoid, and peroxisome proliferator-activated receptors [5–7], have been implicated in the regulation of specific stages of spermatogenesis. However, the functions of the nuclear orphan receptors TAK1 and retinoid-related testis-associated receptor (RTR) [8–11], which are highly expressed in pachytenes and round spermatids, respectively, have yet to be determined.
Recently, we identified a novel protein referred to as receptor-associated protein 80 (RAP80) [12]. RAP80 is a nuclear protein containing two putative zinc finger motifs in its carboxyl terminal region. Although RAP80 mRNA can be detected in many tissues, it is most abundantly expressed in testis, where its expression is associated with germ cells. RAP80 mRNA is, however, not differentially regulated during spermatogenesis. Although its function is not yet precisely established, RAP80 appears to play a role in the regulation of gene expression based on evidence that it is able to interact with RTR in mammalian two-hybrid analysis [10, 12].
In an attempt to obtain greater insight into the function of RAP80, we set out to identify proteins interacting with RAP80 using yeast two-hybrid screening with full-length RAP80 as bait. This analysis yielded one in-frame cDNA clone encoding a novel protein, referred to as testis-specific protein (Tsp) 57, not reported previously. The Tsp57 gene maps to human chromosome 11q21 and mouse chromosome 9A1 and contains 11 exons and 10 introns. This gene encodes a basic protein with a molecular mass of 56.8 kDa. Northern blot analysis demonstrated that Tsp57 mRNA is almost exclusively expressed in testis. Analysis of Tsp57 expression during testicular development and in different purified subpopulations of germ cells indicated that Tsp57 mRNA was most highly expressed in haploid round spermatids. This expression profile was confirmed by in situ hybridization analysis. Future studies have to determine the precise function of this protein during this specific phase of spermatogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The yeast-two hybrid system was purchased from Clontech (Palo Alto, CA), and library screening was conducted according to the manufacturer's instructions. The bait construct pGBKT7-RAP80 was generated by cloning full-length RAP80 into the EcoRI and BamHI sites of the vector pGBKT7. Saccharomyces cerevisiae strain AH109(MAT) was then transformed with pGBKT7-RAP80. Yeast two-hybrid library screening was carried out by the yeast-mating method using S. cerevisiae Y187(MAT) pretransformed with a pACT2 mouse embryo (E17) MATCHMAKER cDNA library (Clontech). After mating, positive clones were selected on minimal Synthetic Dropout medium (-Trp/-Leu/-His) containing 25 mM 3-amino-1,2,4-triazole. About 2 x 106 independent pACT2 clones were screened. One positive in-frame cDNA clone, referred to as Tsp57, was identified and characterized in this study. Sequence was submitted to GenBank under accession no. AY251192. Comparison of Tsp57 sequence with those in GenBank revealed a human cDNA sequence (GenBank NM_014679) highly similar to Tsp57. The sequences of the mouse and human Tsp57 genes have not been reported previously.
DNA Sequencing
Plasmids were purified using Wizard miniprep or midiprep kits (Promega, Madison, WI). Automatic sequencing was carried out using a Dynamic ET Terminator Cycle Sequencing Ready reaction kit (Perkin-Elmer, Foster City, CA) and an ABI Prism 377 automatic sequencer (Perkin-Elmer). DNA and deduced protein sequences were analyzed by the SeqWEB sequence analysis software package (GCG Wisconsin Package; Accelrys, San Diego, CA).
Plasmids
The mammalian expression construct pcDNA4/HisMax-Tsp57 was derived by polymerase chain reaction (PCR) amplification using Tsp57-specific 5' and 3' primers containing a BamHI and a XhoI restriction site, respectively. This PCR product was then cloned into the BamHI and XhoI sites of pcDNA4/HisMax (Invitrogen, Carlsbad, CA). To generate pEGFP-Tsp57, full-length Tsp57 was amplified using specific 5' and 3' primers containing a XhoI and a SalI site, respectively. After digestion with SalI, the PCR product was filled in using Klenow DNA polymerase (New England Biolabs, Beverly, MA) and cloned into the XhoI and SmaI sites of pEGFP-C1. The pEGFP-Tsp57 deletion constructs were generated by cloning various restriction fragments of Tsp57 into pEGFP-C1. pEGFP-Tsp57 was cut with BamHI and SmaI, and after the fill-in reaction with Klenow polymerase the products were self-ligated to yield pEGFP-Tsp57
C229. To obtain pEGFP-Tsp57C337, pEGFP-Tsp57F was cut with BamHI/HindIII and self-ligated after the fill-in reaction with Klenow polymerase. pEGFP-Tsp57N336 was generated by cloning the HindIII/BamHI fragment released from pEGFP-Tsp57 into pEGFP-C1. The 3XFlag-RAP80 expression vector was created by insertion of the EcoRI/BamHI fragment of pEGFP-RAP80 [12] into the p3XFlag-CMV10 vector (Sigma, St. Louis, MO) using the same restriction sites.
Cell Culture
Embryonic stem cells and embryonal carcinoma cell lines were grown as described previously [13]. The human leukemia cell line K562, the human choriocarcinoma cell line JEG3, the mouse Leydig cell-derived cell line TM3, and the Sertoli-like cell line TM4 were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and grown using the protocols provided by ATCC.
Northern Blot Analysis
A multitissue blot containing 25 µg of total RNA from 14 different mouse tissues was purchased from Seegene (Seoul, Korea). For the developmental blot, RNA was prepared from testes of mice at different stages of development. Different subpopulations of spermatogenic cells were prepared from either immature or mature mouse testes as described previously [14]. The purity of the different isolated cell populations was 
90%. RNA was extracted using Tri-Reagent (Sigma) according to the manufacturer's protocol. The same procedure was used for the isolation of RNA from the different cell lines. Total RNA (20 µg) was separated by formaldehyde/1.2% agarose gel electrophoresis, blotted to Hybond N+ membrane (Amersham, Piscataway, NJ), and crosslinked with ultraviolet light. The membranes were then hybridized to a 32P-radiolabeled probe for Tsp57. Hybridization was carried out at 68°C for 3 h, and the membranes were then washed twice in 2x saline sodium citrate (SSC) and 0.1% SDS at room temperature for 30 min and then in 2x SSC and 0.1% SDS at 58°C for 15 min. Autoradiography was carried out with Hyperfilm-MP (Amersham) at -70°C.
Animals
Adult female B6-129 mice were placed on a vitamin A-deficient (VAD) diet (Harland Teklad, Madison, WI) for 3 wk before they were mated with males that had been maintained on a normal diet. The females were continued on a VAD diet through gestation and until pups were weaned. After weaning, male offspring were fed the VAD diet for an additional 12 wk to obtain VAD male mice. Retinol-replenished VAD male mice were generated by treating the VAD male mice with 17 mg/kg retinol (Sigma) in 50% ethanol by i.p. injection. Mice heterozygous for RAR were acquired from Dr. Pierre Chambon [15] and mated to produce the RAR wild type, heterozygous, and knockout mice. Animal experimentation was approved by the Institutional Animal Care and Use Committee and conducted in accordance with the highest standards of humane animal care as outlined in the National Institutes of Health guide for the Care and Use of Laboratory Animals.
Real-time PCR
RNA from testes of VAD and RAR-deficient mice was collected using an RNaqueous kit (Ambion, Austin, TX). Real-time PCR primers were designed for mouse Tsp57 and for a housekeeping gene, ribosomal S2, utilizing Primer Express software version 2.0 (Applied Biosystems, Foster City, CA). The forward primer for Tsp57 was 5'-AGCCCTGTGCAATGATCGA-3', and the reverse primer was 5'-GGAAGGAGGCACCACTGACTT-3'. The forward primer for ribosomal S2 was 5'-CTGACTCCCGACCTCTGGAA-3', and reverse primer was 5'-GAGCCTGGGTCCTCTGAACA-3'. Complementary DNA was synthesized from RNA samples using Superscript II RNase H-minus reverse transcriptase (Invitrogen) according to the manufacturer's protocol. Subsequently, cDNA was used as template for real-time PCR assays of Tsp57 mRNA levels with a Gene Amp 7000 thermocycler (Applied Biosystems). Threshold (Ct) values for Tsp57 and S2 were determined using Prism SDS software version 1.0 (Applied Biosystems), and the level of Tsp57 was evaluated using the 2-(Ct) method [16]. Specifically, the Ct value for Tsp57 was normalized to that for S2 in each sample, and then the fold change for Tsp57 was calculated relative to the level in RAR wild-type mice. The real-time PCR was conducted on cDNA in triplicate. Statistical analysis of Tsp57 levels consisted of one-way ANOVA followed by pairwise comparison of the means by the Tukey method (Prism; Graphpad, San Diego, CA).
Subcellular Localization of Tsp57 Protein
To study the subcellular localization of Tsp57, we transfected CV-1 cells with pEGFP-Tsp57, pEGFP-Tsp57C229, pEGFP-Tsp57C337, and pEGFP-Tsp57N336 plasmid DNA using Fugene 6 transfection reagent (Roche, Indianapolis, IN). After 36 h of culture, cells were examined in a confocal microscope (LSM 510 NLO; Zeiss, Thornwood, NY). The excitation and emission frequencies of Enhanced Green Fluorescent Protein (EGFP) were 488 nm and 507 nm, respectively.
In Situ Hybridization Analysis
Adult mouse testes were fixed in 4% paraformaldehyde and embedded in paraffin, and sections (5 µm thick) were prepared for in situ hybridization analysis. A 463-base pair fragment (nucleotides 1007–1469) of Tsp57 was amplified by PCR and cloned into the vector pGEM3Zf(+) (Promega). Plasmid DNA was linearized with EcoRI to generate the sense probe and with HindIII for the antisense probe. Digoxigenin (DIG)-labeled antisense and sense RNA probes were prepared using the DIG RNA-Labeling Kit (Roche). In situ hybridization was carried out as described previously [17]. Instead of active diethyl pyrocarbonate treatment, sections were treated with 0.25% acetic anhydride in 10 mM triethanolamine for 15 min at room temperature. Hybridizations were carried out at 50°C for 24 h, and bound probes were detected using alkaline phosphatase-conjugated anti-DIG antibody with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates. Counterstaining was performed with 0.05% safranin O.
Pull-Down Analysis
To check for the interaction of Tsp57 with RAP80, p3XFlagCMV-RAP80 and pcDNA4/HisMax-Tsp57 plasmids were transfected into CV-1 cells using Fugene 6. After 48 h, cells were harvested and lysed in RIPA buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS). Subsequently, half of the cell lysate was incubated with anti-Flag M2 agarose resin (Sigma) for 3 h at 4°C with agitation. The resin was then washed five times with PBS. The pulled-down protein complexes were then examined by Western blot analysis using anti-Xpress antibody (Invitrogen). Proteins in the other half of the cell lysate were mixed with sample buffer and examined by Western blot analysis using either an anti-Xpress antibody or an anti-Flag M2 antibody (Sigma).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In an attempt to identify proteins interacting with the nuclear protein RAP80, yeast two-hybrid library screening was carried out using RAP80 as bait. This screening yielded one clone encoding a protein not reported previously. This protein was named Tsp57. Analysis of the Tsp57 sequence revealed a long open reading frame that starts with a putative initiation codon at nucleotide 155 and ends with a putative stop codon at nucleotide 1657 (Fig. 1). A polyadenylation signal was found at nucleotide 2340. Tsp57 encodes a novel basic protein of 500 amino acid residues with an isoelectric point of 9.23 and a molecular mass of 56.8 kDa. Sequence of the apparent human homologue of mTsp57 (NM_014679) was retrieved from GenBank through sequence comparison. The sequence of human TSP57 has not been reported previously. Human TSP57 exhibits an 87% identity with mouse Tsp57 (Fig. 1A). Tsp57 did not have any similarity with any other known protein. Analysis of its amino acid sequence by Motifscan, PSORTII and identified several putative protein kinase C and protein kinase A phosphorylation sites and two putative bipartite nuclear localization signals, NLS1 and NLS2. NLS1 is located in the middle of Tsp57 (258RKIKKKKSKPPEKKGSR274), and NLS2 is located in the C-terminal region of Tsp57 (418RKYQAQLEKQNIDKQKK435).
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Gene Structure and Chromosomal Localization of Tsp57
A search in GenBank for genomic sequences for the Tsp57 gene identified one contig (GenBank NW_000351.1) encoding the complete mouse Tsp57 gene and one contig (GenBank AP000771.2) encoding part of the human TSP57 gene. From this comparison, we were able to deduce the genomic structure of TSP57. The mouse Tsp57 gene spans >16.5 kilobases (kb) and consists of 11 exons and 10 introns (Fig. 1B). The locations of the exon/intron junctions are indicated in Figure 1A by arrowheads. The chromosomal localization of these contigs predicted that the TSP57 gene maps to human chromosome 11q21 and mouse chromosome 9A1.
Tissue Distribution of Tsp57 mRNA
To examine in which tissues Tsp57 is expressed, we performed Northern blot analysis using RNA from mouse placenta and 13 different adult tissues, including brain, heart, lung, kidney, testis, and skin. The radiolabeled Tsp57 probe hybridized to a single 2.4-kb transcript that was highly expressed in testis (Fig. 2A). Little expression was observed in the other tissues examined.
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A number of genes, selectively expressed in testis, are also expressed in embryonic stem cells, embryonal carcinoma cells, and trophoblasts. We therefore examined the expression of Tsp57 in embryonic stem cells and several related cell lines. Although embryonic stem cells and most embryonal carcinoma cells expressed Tsp57 mRNA, the levels of expression were much lower than those observed for testis (Fig. 2B). Similarly, weak expression of Tsp57 was observed in the mouse Leydig and Sertoli cell lines TM3 and TM4, respectively. Little or no expression was found in human choriocarcinoma JEG-3 and leukemia K562 cells.
Developmental Onset of Tsp57 mRNA Expression in Testis
To determine whether the expression of Tsp57 mRNA is developmentally regulated, we analyzed Tsp57 mRNA expression during postnatal testicular development. Total RNA samples were prepared from testes isolated from juvenile mice (7–30 days old) and examined by Northern blot analysis. Tsp57 mRNA was expressed at low levels in testis from 7-day-old mice, and levels were steady through Day 17 (Fig. 3). Tsp57 mRNA expression was enhanced at Day 21 and was dramatically increased by Day 25 of postnatal testicular development. These results demonstrate that Tsp57 is developmentally regulated and that its expression is induced when haploid spermatids begin to accumulate (Days 20–30).
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In Situ Hybridization of Tsp57 in Testis
Because the presence of Tsp57 mRNA was restricted to testis, we examined Tsp57 mRNA expression using in situ hybridization analysis with cross sections from adult mouse testis and a DIG-labeled antisense Tsp57 riboprobe (Fig. 4). This analysis revealed that the hybridization signal was associated with the seminiferous tubules and was limited to certain populations of germ cells. Weak or no signal was detectable in Sertoli cells, Leydig cells, and spermatogonia. This finding is in agreement with the low level of Tsp57 mRNA expression observed in the Leydig and Sertoli cell lines TM3 and TM4, respectively, compared with whole testis (Fig. 2B). No hybridization signal was observed when a sense Tsp57 probe was used.
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During spermatogenesis in the adult mouse, germ cell differentiation advances in highly ordered waves along the axis of the seminiferous tubule, and each cross section of a tubule can represent 1 of the 12 stages of spermatogenesis [18, 19]. The observed variation in the intensity of the hybridization signal among tubules appears to be related to the different stages of the spermatogenic cycle (Fig. 4). Seminiferous tubules corresponding to stages VI–VIII of the cycle had a strong hybridization signal, whereas tubules representing other stages had a weak signal. After observing many tubules at various stages, we concluded that Tsp57 mRNA was most abundant in round spermatids.
Tsp57 Expression in Different Subpopulations of Spermatogenic Cells
To confirm the expression of Tsp57 in spermatids, we examined the pattern of Tsp57 expression in several purified spermatogenic subpopulations (Fig. 5). The expression of Tsp57 mRNA was very low in spermatogonia and preleptotene spermatocytes and was somewhat increased in prepubertal (early) pachytenes. Expression of Tsp57 mRNA was somewhat enhanced in late pachytene spermatocytes and reached its highest level in haploid round spermatids. Because the enriched cell populations have a purity of 90%, the small increase in Tsp57 mRNA seen in late pachytene spermatocytes may be due to contamination with round spermatids. The observed pattern of expression is in agreement with the time of induction of Tsp57 during testicular development and the pattern of Tsp57 expression observed with in situ hybridization.
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Expression of Tsp57 mRNA in VAD and RAR Null Mice
Vitamin A plays a vital role in spermatogenesis [6, 18]. Vitamin A deficiency blocks spermatogenesis at an early stage, and only spermatogonia and preleptotene spermatocytes can be observed in the testis. Similarly, RAR null mice with a nonfunctional RAR protein, one of the receptors that mediate the action of vitamin A, exhibit degeneration of the germinal epithelium similar to that in VAD rats [19]. More precisely, the testes of RAR null mice display varying degrees of germinal epithelium degeneration, from severe to moderate, in different sections of the seminiferous tubules. Real-time PCR was performed to determine the level of Tsp57 mRNA in VAD and RAR null mice (Fig. 6). When the Tsp57 mRNA level was normalized to an internal control (the level of ribosomal S2 mRNA in each sample), we found that practically no mRNA was detectable in the testes from VAD mice compared with those from wild-type mice. The level of Tsp57 mRNA also did not increase with retinol treatment of VAD mice. A control experiment was conducted to determine whether retinol-replenished testes are responsive to retinol. The mRNA levels of retinol-regulated genes increased in the testes from retinol-replenished mice (data not shown). In addition, the level of Tsp57 mRNA in testes from normal mice (RAR wild type) was similar to that in RAR heterozygous mice. In contrast, there was a significant decrease (30%) in the level of Tsp57 mRNA in the testes from RAR null mice compared with normal mice.
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Subcellular Localization of EGFP-Tsp57 Protein
The identification of two putative nuclear localization signals, NLS1 and NLS2 suggested that Tsp57 might function as a nuclear protein. In addition, the Reinhardt's method for cytoplasmic/nuclear prediction indicated a 94% probability for Tsp57 to be a nuclear protein. To investigate this question further, we generated several expression vectors encoding different EGFP-Tsp57 fusion proteins. EGFP-Tsp57C229 contains only NLS1, EGFP-Tsp57C337 lacks both putative NLSs, and EGFP-Tsp57N336 contains only NLS2. Plasmid DNA was transfected into CV-1 cells and 36 h later the localization of the fusion proteins was analyzed by confocal microscopy (Fig. 7). The results demonstrated that full-length Tsp57 localized to both the cytoplasm and nucleus. In contrast, EGFP-Tsp57C229 and pEGFP-Tsp57C337 were exclusively localized to the cytoplasm while EGFP-Tsp57N336 was found in both the cytoplasm and nucleus. These results suggest that the carboxyl terminus containing the NLS2 is necessary for the nuclear localization of Tsp57. These observations are in agreement with the conclusion that Tsp57 may have a function in the nucleus.
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Tsp57 and RAP80 Are Part of a Complex
Yeast two-hybrid analysis demonstrated that Tsp57 and RAP80 interact. To determine whether Tsp57 and RAP80 are able to form a complex in intact mammalian cells, we expressed Flag-RAP80 and HisMax-Tsp57 in CV-1 cells and performed immuno-pull-down with the isolated protein lysates. Flag-RAP80 was able to pull down HisMax-Tsp57, suggesting that in intact cells the two proteins are in a complex with each other (Fig. 8). The pDNA4/HisMax-TSP57 expression vector allowed only weak expression of TSP57 in CV-1 cells, which explains the low level of TSP57 protein observed after immuno-pull-down.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Analysis of the tissue-specific expression of Tsp57 revealed that Tsp57 mRNA is expressed exclusively in testis. Little or no expression was detectable in all other tissues examined. Embryonic stem cells and embryonal carcinoma cells, which have been reported to express a number of testis-specific genes, contain only very low levels of Tsp57 mRNA. Analysis of Tsp57 expression during postnatal testicular development demonstrated that Tsp57 was developmentally regulated. At Day 7 of postnatal development, seminiferous tubules contain only Sertoli cells and early spermatogenic cells in which spermatogenesis has not yet progressed and express low levels of Tsp57 mRNA. The level of Tsp57 mRNA only increased dramatically between Day 20 and Day 25 of testicular development, a period in which round spermatids are appearing. These observations suggest that expression of Tsp57 mRNA is associated with the postmeiotic phase of spermatogenesis and with the generation of haploid round spermatids in particular. Two other lines of evidence supported this conclusion: in situ hybridization analysis and examination of Tsp57 expression in purified subpopulations of spermatogenic cells. In the adult testis, each cross section of a tubule can represent 1 of the 12 stages of the spermatogenic cycle [18, 19]. Our in situ hybridization analysis demonstrated that the hybridization signal was maximum in seminiferous tubules at stages VI–VIII of the cycle. These seminiferous tubules contain a variety of spermatogenic cells: spermatogonia, pachytenes, round spermatids, and elongated spermatids. The highest hybridization signal was observed in regions of round spermatids. This finding was corroborated by experiments examining the expression of Tsp57 in purified subpopulations of spermatogenic cells. Tsp57 mRNA was expressed at low levels in spermatogonia, preleptotenes, and early pachytene spermatocytes, increased in late pachytenes, and reached highest levels of expression in round spermatids. These observations suggest that Tsp57 has a functional role during the postmeiotic phase of spermatogenesis.
Through binding to nuclear receptors, vitamin A/retinoic acid plays a critical role in the regulation of spermatogenesis [6, 15, 20]. The virtual absence of expression of Tsp57 mRNA observed in testes from VAD mice is in agreement with the absence of spermatocytes beyond the preleptotene stage of spermatogenesis. Retinol treatment of VAD mice did not increase Tsp57 mRNA levels consistent with the absence of spermatocytes or round spermatids in the retinol-replenished testis. Postpreleptotene spermatocytes and round spermatids are not found in VAD testis 24 h after retinol replenishment (data not shown). These observations, however, do not eliminate the possibility that expression of Tsp57 in pachytene spermatocytes or round spermatids is regulated by retinol. The 30% reduction in Tsp57 expression observed in RAR null mice is consistent with the varying degrees of degeneration of the germinal epithelium and reduction in spermatids reported for testes of these mice [15]. These results support the hypothesis that Tsp57 is primarily expressed in pachytene spermatocytes and round spermatids.
The function of Tsp57 in spermatids has yet to be determined. Examination of the subcellular localization of EGFP-Tsp57 showed that this fusion protein localizes to both the cytoplasm and nucleus. The size of the fusion protein is such that it probably does not enter the nucleus by inactive transport. Analysis of its localization by the Reinhardt method predicted a 94% probability that Tsp57 functions as a nuclear protein. This hypothesis was supported by the presence of two putative nuclear localization signals, NLS1 and NLS2. Deletion mutant analysis demonstrated that the nuclear localization of TSP57 required NLS1 but not NLS2. The requirement of NLS1 for the nuclear localization of Tsp57 supports the hypothesis that Tsp57 has a nuclear function. The cDNA encoding Tsp57 was identified by yeast two-hybrid screening using the nuclear protein RAP80 as bait. In vivo pull-down analysis confirmed that Tsp57 and RAP80 form a complex in intact cells. Although RAP80 mRNA is expressed in several tissues, it is most abundantly expressed in testis, where it can be found in all spermatogenic cells. Studies are in progress to further characterize this interaction.
The expression pattern of Tsp57 in testis is very similar to that of the nuclear orphan receptor RTR. The coexpression of RTR and Tsp57 is intriguing in two different ways. First, Tsp57 was isolated by yeast two-hybrid screening using RAP80 as bait. Because RAP80 also interacts with RTR [12], the three proteins may form a multimeric complex. Alternatively, RTR could be involved in the regulation of Tsp57 expression in round spermatids. Although analysis of the 3-kb Tsp57 promoter flanking region for the presence RTR response elements (RTREs) did not reveal any DNA sequence resembling the RTRE consensus (not shown) [21], this finding does not necessarily rule out regulation by RTR. CREM, which mediates its transcriptional activation through interaction with cAMP-response elements (CREs) in target genes, is another trancription factor highly expressed in round spermatids. It has been reported to participate in the testis-specific promoter activation of numerous haploid-expressed genes [22]. Because CREM and Tsp57 are both highly expressed in round spermatids and the 3.0-kb Tsp57 promoter flanking region contains several CREs (not shown), CREM might be involved in the regulation of Tsp57.
Here, we describe the identification and partial characterization of the novel testis-specific gene Tsp57. The stage-specific expression of Tsp57 mRNA indicates that it has a very specific role during the haploid phase of spermatogenesis. Its subcellular localization suggests a function for Tsp57 in the nucleus; however, a dual role in cytoplasm and nucleus cannot be ruled out. Its specific pattern of expression suggests that its promoter region might be a useful tool in gene targeting experiments including Cre/Lox-mediated targeted knockout. Further study is required to determine the physiological function of Tsp57 protein.
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FOOTNOTES |
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Received: 5 May 2003.
First decision: 15 June 2003.
Accepted: 28 August 2003.
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Ct) method. Methods 2001 25:402-408[CrossRef][Medline]
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0.01) from the mean value of a. c denotes mean values significantly different (P 
