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a Department of Biochemistry, Molecular Biology & Cell Biology, Northwestern University, Evanston, Illinois 60208
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We identified a novel testis-specific protein from human and mouse testes, which contains a leucine-rich repeat domain in the N-terminus. The abundant expression in late pachytene and diplotene cells and significant decline after the first meiotic division implies a functional association of this protein with spermatocytogenesis or prophase of meiosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Serum #629 from an infertile woman was provided by Dr. Gary Clarke (Royal Women's Hospital, Carlton, Victoria, Australia). The serum was absorbed with Escherichia coli lysate, diluted 1:1000 and used to screen a human testis 
gt11 cDNA library according to the method of Young and Davis [8]. From about 1.2 x 106 phage plaques, 7 positive clones were isolated. One of them (operationally termed LRTP for leucine-rich testis protein), which was 1.5 kilobases (kb) in size was subcloned into pBluescript for sequencing. To isolate the full length LRTP, a 600-base pair (bp) fragment was released from the 1.5-kilobase clone by EcoRI digestion and used as probe to screen a human testis 5' stretch plus cDNA library. A 2.3 kb cDNA encoding the full-length open reading frame of human LRTP was isolated and sequenced.
To identify the mouse homolog of LRTP, a gt11 mouse testis pachytene cell cDNA library was obtained courtesy of Dr. E.E. Eddy and screened with a human LRTP cDNA fragment labeled with [32P]dCTP and corresponding to the C terminal 220 amino acids. In about 2 x 105 plaques, an 800-bp cDNA clone was isolated, and the insert was amplified by PCR with gt11 forward and reverse primers. The 800-bp fragment was used to screen a pachytene cell Unizap cDNA library (a gift from Dr. John McCarrey). Among 1 x 105 plaques, 2 positive clones, with overlapping sequences, were isolated. The larger clone was inserted into a pBluescript plasmid, and the 2.1-kb insert was sequenced.
Northern Analysis
Total RNA was extracted from CD-1 mouse testis with guanidinium thiocyanate. Equal amounts of RNA were loaded in each lane of a 1% agarose gel containing 0.5% formaldehyde, single-strength 3-(N-morpholino)butanesulfonic acid (MOPS) running buffer (20 mM MOPS, 8 mM sodium acetate, 1 mM EDTA). The fractionated RNA was transferred to a nylon membrane, UV cross-linked, and hybridized with [
-32P]dCTP-labeled cDNA. The nylon membrane was immersed in hybridization buffer for 6 h and then the radiolabeled probe was added and hybridized over night. After hybridization, the membrane was washed in single-strength, 0.5-strength, and 0.1-strength SSC at room temperature for 20 min for each wash, 0.1-strength SSC for 30 min at 55°C, followed by x-ray film exposure. Labeled human ß actin was used as a control probe for Northern analysis.
Mouse RNA Blot
An RNA dot blot containing normalized loading of poly(A)+ RNA from different mouse tissues and containing control RNAs and DNAs was purchased from Clontech (Palo Alto, CA). The blot was hybridized as described above for Northern analysis and according to instructions from the supplier.
In Situ Hybridization
An 800-bp and a 600-bp cDNA fragment was PCR amplified and subcloned into pBluescript. The plasmids were linearized, and sense and antisense riboprobes were transcribed and labeled with digoxigen UTP with a riboprobe kit from Promega (Madison, WI). The probes were ethanol precipitated and resuspended in DEPC water for hybridization.
Adult CD-1 mouse testis was frozen on dry ice, and 15-µm sections were fixed in 4% paraformaldehyde for 5 min, double-strength SSC for 5 min, 0.25% anhydride in 0.1 M TEA for 10 min, dehydrated in ethanol, and air dried. Hybridization was performed in buffer (50% formamide, 0.3 M NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, single-strength Denhardt's, 10% dextran sulfate, 10 mM DTT, 0.5 mg/ml tRNA, 0.5 mg/ml poly A RNA) with the sense or antisense probe at 47°C over night. After hybridization, sections were washed in single-strength, 0.5-strength, 0.1-strength SSC at 50°C for 30 min each. The sections were then incubated with blocking buffer (double-strength SSC, 0.05% Triton X-100, 0.1% BSA) for 1 h at room temperature, and anti-digoxigenin monoclonal antibody Fab for another hour. After a wash with buffer 1 (100 mM Tris pH 7.5, 150 mM NaCl) and 2 (100 mM Tris pH 9.5, 100 mM NaCl, 50 mM MgCl2), the sections were incubated with nitroblue tetrazolium (250 µg/ml) and 5-bromo-4-chloro-3-indolyl indolyl phosphate (225 µg/ml).
Preparation of Anti-mLRTP-C Antiserum
A 600-bp fragment, encoding the C terminal 100 amino acids of mouse LRTP plus 3' untranslated region, was PCR amplified with pfu polymerase (Stratagene, La Jolla, CA), and ligated into the EcoRI site of the plasmid pGEX 4T1 (Amersham Pharmacia, Piscataway, NJ) through adaptive EcoRI linkers in the PCR primers. The E. coli were transformed, and positive clones sequenced. The recombinant-transformed cells were induced for 3 h with 0.5 mM IPTG. Cells were collected and resuspended in PBS, with lysozyme and NP-40 added to a final concentration of 100 µg/ml and 0.1%, respectively. The cells were sonicated, cell debris removed by centrifugation, and the supernatant run through a glutathione sepharose 4B (Amersham Pharmacia) column. After extensive washing with PBS, the fusion protein was eluted with 10 mM glutathione. To separate the fusion partners, the column was incubated with 500 units of thrombin (Sigma, St. Louis, MO), instead of glutathione, overnight at room temperature, and the mLRTP-C was eluted with PBS. The purified mLRTP-C fragment was used to immunize rabbits.
Rabbit antiserum was affinity purified as follows: purified mLRTP-C antigen was isolated by SDS-PAGE and transferred to a nitrocellulose membrane, which then was incubated with protein A purified IgG from anti-mLRTP serum. After incubation overnight, the nitrocellulose membrane was washed extensively with PBS, the antibody was released from the membrane by 50 mM glycine pH 2.5, and the eluate was neutralized, dialyzed against PBS, and concentrated for use in Western blot analysis and immunohistochemistry.
For Western blots, various fresh CD-1 mouse tissues were homogenized in PBS and centrifuged, and the supernatant was collected. SDS sample loading buffer, 1:1, was added. Equal amounts of sample were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Anti-mLRTP-C affinity purified antiserum (diluted 1:50 000), preabsorbed with mouse liver extract, was used as primary antibody, 1:30 000 HRP-labeled goat anti-rabbit IgG was used as secondary antibody, and immunoreactivity was visualized with the ECL detection system (Amersham Pharmacia).
Immunohistochemistry
Adult CD-1 mice were euthanized by CO2 inhalation, and dissected testes were fixed in Bouin's fluid overnight. Prior to paraffin embedding, picric acid was removed by extensive washing in 70% ethanol. Tissue was dehydrated in ethanol, embedded in paraffin, sectioned at 5 µm, and rehydrated. Immunohistochemistry was performed according to the procedure described by the manufacturer (Zymed, South San Francisco, CA). Briefly, sections were incubated with 3% H2O2 in methanol to block endogenous peroxidase, blocked with goat serum, incubated with 1:500 mLRTP-C antiserum overnight at 4°C, and then incubated with biotinylated second antibody. After incubation with streptavidin-HRP conjugate, sections were color-reacted with AEC as chromogen.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our attempts to identify sperm antigens, a gt11 human testis cDNA expression library was screened [9] with sera that contained spermagglutinating activities. A testis-specific cDNA, designated LRTP (leucine-rich testis-specific protein), was identified, and a cDNA probe detected an 800-bp fragment in a mouse testis cDNA library. This was then used to isolate a full-length clone from a pachytene cell cDNA library. A 2.1-kb cDNA, containing the complete open reading frame of LRTP, was obtained.
Figure 1A shows the amino acid sequence deduced from the LRTP clone. LRTP is a polypeptide with 473 amino acids that contains a leucine-rich repeat (LRR) domain in the N-terminal region. The LRR domain is represented as tandem repeats of leucines in the sequence shown in Figure 1B. Mouse LRTP has about 5 LRRs, which span more than 100 amino acids in the amino terminus.
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The protein also contains several charged, acidic regions (underlined) rich in glutamic acid, aspartic acid, and glutamine. These sequences are not homologous to any known functional domains represented in GenBank. However, the structural features of the sequence do show certain similarities to sds22+ and LANP. Figure 1C shows the partial sequence comparison between LRTP and sds22+, and LRTP and LANP.
Computer-assisted sequence analysis showed that LRTP contains three potential glycosylation sites and several PKC and cAMP-dependent phosphorylation sites. The DNA sequence of LRTP also matches an EST mouse genome sequence (AA138956) deposited in GenBank.
Testis-Specific Expression of LRTP
To demonstrate the testis specificity of LRTP, total RNA was extracted from mouse tissues, and an equal amount of RNA from each tissue was analyzed by a Northern blot with the full-length mouse LRTP cDNA as probe. As shown in Figure 2 (top), mRNA signals, 2–2.4 kb in size, were detected exclusively in mouse testis and not in any somatic tissues.
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The multiple tissue RNA blot that was hybridized with the same probe as the Northern blot confirmed testis specificity and in particular demonstrated that LRTP mRNA is not in ovarian tissue (Fig. 2, bottom). There are some tissue RNAs that do show a very weak signal with this probe after prolonged exposure of the x-ray film. This cross reactivity is not unexpected because of the highly conserved LRR and acidic amino acid sequences of LANP encoded by the RNAs.
Expression Pattern of LRTP During Testis Development
Northern analysis of RNA from 10-day-old to adult mouse testis reveals LRTP mRNA is expressed initially at Day 14 after birth (Fig. 3), corresponding to the appearance of cells at mid-pachytene. The cDNA probe hybridized to two bands, each of which followed distinctive time courses of mRNA expression. Prior to Day 20 postpartum, the smaller mRNA is much less abundant, but a similar expression level is reached in the adult testis.
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Western Analysis of Testis Specificity
A GST fusion protein containing the C terminal 100 amino acids of mouse LRTP was used to generate antibodies. Both GST and GST-mLRTP-C fusion protein were recognized by GST antibody, while the mLRTP-C antiserum only bound the fusion protein, but not the GST partner (Fig. 4A). The anti-mLRTP-C serum cross-reacted with a somatic component as well and therefore was absorbed with mouse liver extract before use in the Western analysis illustrated in Figure 4B. Consistent with Northern analysis, the LRTP protein, about 60 kDa in size, was detected only in testis.
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Stage-Specific Distribution of Mouse LRTP
Figure 5 shows the results of in situ hybridization localizing LRTP mRNA to the seminiferous tubule. The mRNA distributed within the cytoplasm of pachytene cells has a ring-like appearance. Different tubules show different levels of expression, and no signal is observed with the control sense probe.
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The cell type and stage-specific distribution of LRTP protein in mouse testis is shown in Figure 6. Figure 6, A and B, show four seminiferous tubules at different stages of the cycle. For comparison, the junction between two adjacent tubules is magnified in Figure 6, C and D. In Figure 6C, the most abundant expression was found in pachytene cells in the stage VIII tubule, which is characterized by late pachytene cells. In contrast, early pachytene cells in the stage III tubule were barely labeled. In Figure 6D, the LRTP protein localizes to the diplotene cells of a stage XI tubule, but there is a dramatic decrease in content in the adjacent stage XII tubule, which is characterized by the completion of meiosis I and the presence of meiotic figures.
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Figure 6F shows an unique stage XII seminiferous tubule. The cell type transition of a meiotic division can be visualized with diplotene cells and secondary spermatocytes appearing in a single tubule. LRTP protein expression peaks in the diplotene cells, is lower in meiotic figures, and has mostly disappeared in secondary spermatocytes.
There was a low level expression of LRTP in round spermatids before stage VIII (Fig. 6, A and B) and no signal detected after stage IX of spermatogenesis (Fig. 6E).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Towards determining a function for LRTP, we noted that the time course of expression is similar to that of several cell cycle regulators, such as CDC25, CDC2, and cyclin B [12–14]. For example, CDC25c is expressed in pachytene cells and round spermatids, peaking in diplotene cells and decreasing during meiotic division [12]. The time course of CDC25c expression overlaps that of LRTP. The CDC25a mRNA also reached a maximum at diplotene, and the protein was found in the cytoplasm in all spermatocyte stages and in all spermatids except elongated spermatids [12]. The substrate of CDC25, CDC2 transcription product was found to be most abundant in late pachytene and diplotene cells as well [15]. These characteristic distributions indicate that these regulators are involved in the meiotic division processes of spermatocytes. Despite the observation that the LRTP expression pattern overlaps these cell cycle regulators, any direct or indirect functional relationships with these control elements has not been established.
A GenBank search revealed that LRTP is a member of the family of leucine-rich repeat proteins. There are a great number of proteins that contain a leucine-rich repeat domain [16]. The numbers of repeats differ among them, with some having as many as 30 repeats, such as Chaoptin [17], and some having fewer, such as human Trk, a receptor protein kinase-NGF, which contains only two tandem repeats [18]. These proteins have very divergent tissue distributions and species origins, from yeast, to drosophila, to man. They are involved in various biological functions, such as morphogenesis, signal transduction, etc.; however, these functions are usually attributed to functional domains other than the leucine-rich repeat domain. Nevertheless, all proteins containing these repeats are thought to be involved in protein-protein interactions. The crystal structure of ribonuclease inhibitor, one of the LRR proteins, revealed that the LRR domain is involved in ß sheet formation [19]. There are two LRR proteins, SDS22+ and LANP, that have some identity to the LRTP protein, both of these are related to protein phosphatase regulators. Meiosis involves cycles of phosphorylation and dephosphorylation orchestrated by protein kinases and phosphatases. The association of LRTP with germ cell stages of meiosis suggests that the protein may play a regulatory role as a protein phosphatase inhibitor. For example, phosphorylation of CDC25 is required for its activation. Dephosphorylation by PP1 could be blocked by LRTP, thus permitting the cascade of reactions in the G2 to M transition during meiosis. Our attempts to demonstrate LRTP inhibition of PP1 have been hampered by the poor solubility and, therefore, low yield of recombinant protein expressed in E. coli.
SDS22+, identified from yeast pombe, contains 12 LRRs that span almost the whole sequence of the protein. It was found to be involved in positive regulation of protein phosphatase 1, and the deletion of any single repeat made the protein nonfunctional. SDS22+ is required for cell division. The disruption of this gene results in a lethal mitotic defect [20, 21]. To investigate a potential functional similarity of LRTP to SDS22+, we expressed mouse LRTP protein in cells of a Saccharomyces cerevisiae strain with an SDS22+ deletion. The expressed LRTP failed to rescue mitosis in the mutant cell line (data not shown).
LANP is a leucine-rich nuclear protein expressed abundantly in cerebellum during its active proliferative period and possibly involved in morphogenesis [22]. The same protein, but termed Ipp2a [23], was identified later from bovine kidney by another group and shown to have an inhibitory effect on protein phosphatase 2A. Furthermore, a protein named PP32, which is expressed abundantly in active self-renewing tissues (such as intestinal crypt cells) and in neoplastic cells, shares the same sequence with LANP [24]. LANP's distribution indicates its association with cell proliferation. The LRTP protein shares similar structural features in two respects. First, both proteins contain five or six LRR domains in the amino terminus; second, they are both rich in glutamic acid in the C terminus, resulting in an acidic protein. However, LANP contains more glutamic acid residues than does LRTP. Functional similarities between LRTP and LANP remain to be established. It is likely that in order to arrive at a function for LRTP, it will be necessary to isolate the protein from germ cells or to express it in a eukaryotic vector or as a transgene.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NICHD Sub-5-U54-HD29099 and by P30 HD28048. 
2 Correspondence: Erwin Goldberg, Department of Biochemistry, Molecular Biology & Cell Biology, Northwestern University, 2153 North Campus Drive, Evanston, IL 60208. FAX: 847 467 1380; erv{at}nwu.edu
Accepted: December 31, 1999.
Received: September 8, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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