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Keywords:

  • Prss21;
  • serine protease;
  • spermatogenesis;
  • testis

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The recently characterized human serine protease, Testisin, is expressed on premeiotic testicular germ cells and is a candidate type II tumor suppressor for testicular cancer. Here we report the cloning, characterization and expression of the gene encoding mouse Testisin, Prss21. The murine Testisin gene comprises six exons and five introns and spans ≈ 5 kb of genomic DNA with an almost identical structure to the human Testisin gene, PRSS21. The gene was localized to murine chromosome 17 A3.3-B; a region syntenic with the location of PRSS21 on human chromosome 16p13.3. Northern blot analyses of RNA from a range of adult murine tissues demonstrated a 1.3 kb mRNA transcript present only in testis. The murine Testisin cDNA shares 65% identity with human Testisin cDNA and encodes a putative pre-pro-protein of 324 amino acids with 80% similarity to human Testisin. The predicted amino-acid sequence includes an N-terminal signal sequence of 27 amino acids, a 27 amino-acid pro-region, a 251 amino-acid catalytic domain typical of a serine protease with trypsin-like specificity, and a C-terminal hydrophobic extension which is predicted to function as a membrane anchor. Immunostaining for murine Testisin in mouse testis demonstrated specific staining in the cytoplasm and on the plasma membrane of round and elongating spermatids. Examination of murine Testisin mRNA expression in developing sperm confirmed that the onset of murine Testisin mRNA expression occurred at ≈ day 18 after birth, corresponding to the appearance of spermatids in the testis, in contrast to the expression of human Testisin in spermatocytes. These data identify the murine ortholog to human Testisin and demonstrate that the murine Testisin gene is temporally regulated during murine spermatogenesis.

Abbreviations
EST

expressed sequence tag

mTestisin

murine Testisin

RT-PCR

reverse transcription-polymerase chain reaction

Human Testisin is a recently identified protein that is expressed by spermatocytes and may function as a nonclassical type II tumor suppressor gene [1]. Testisin is a member of the large multigene family of serine proteases, enzymes characterized by a triad of histidine, aspartate and serine residues necessary for catalytic activity [2]. Testisin mRNA was also identified from human eosinophils using reverse transcription polymerase chain reaction (RT-PCR) and called esp-1 [3]. The human Testisin gene (PRSS21) has been localized to human chromosome 16p13.3 [1,4], a region associated with high genomic instability. Testisin belongs to an emerging group of serine proteases that possess a hydrophobic C-terminal domain which acts as a direct anchor for membrane attachment, likely via a glycosyl-phosphatidylinositol anchor. This structural feature is also present in the serine proteases CAP1, a putative mediator of sodium channel activity [5], prostasin, which is thought to function in the activation of cell surface proteins [6], and the murine serine proteases TESP1 and TESP2 [7].

Testicular germ cell maturation, or spermatogenesis, is a complex process by which diploid spermatogonial stem cells differentiate into haploid spermatozoa [8], and requires tight regulation of developmental genes and extensive morphological changes. The process involves initial proliferation of spermatogonia, followed by a phase of differentiation generating tetraploid spermatocytes. These cells proceed through two successive meiotic divisions to form haploid round spermatids which undergo extensive morphological restructuring resulting in elongate sperm [8]. Spermatogenesis is generally highly conserved among mammals. Differences largely involve the rate at which maturing germ cells progress through each spermatogenic phase and the outcomes of morphological differentiation which occurs after meiosis. The most obvious example of the latter is the shape of the head and the length of the tail.

The morphological restructuring of testicular germ cells is a dynamic process, requiring cell−cell communication and localized cell–extracellular matrix interactions [9]. Proteases are likely to play a key role in these processes. Cell surface proteolytic activities control the activation of functionally diverse effector molecules, such as cytokines, growth factors and cell surface receptors. While there is indirect evidence for proteolytic activities during testicular germ cell maturation [10,11], only a few germ cell serine proteases have been shown to play functional roles, and these generally act during the later stages of spermatogenesis. This is the case for acrosin and the murine serine proteases TESP1 and TESP2, which are present in the sperm acrosome and are activated during the acrosome reaction [7,12]. A primary function of acrosin is to accelerate the dispersal of acrosomal components during the acrosome reaction [12].

In this study we isolated and characterized a cDNA for mouse Testisin (mTestisin), established the structure of the mouse gene, localized it to mouse chromosome 17 and examined its expression during spermatogenesis. We found that it is expressed abundantly only in testis and its expression is temporally regulated. It is almost identical in gene organization to the human Testisin gene.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

mTestisin cDNA and 5′-RACE

An expressed sequence tag (EST) clone containing a partial mTestisin cDNA (959 bp) in pBluescript SK (accession no. AA144961), identified using the human Testisin cDNA sequence [1] as a query sequence against the GenBank EST database, was purchased from Genome Systems Inc. The cDNA was extended by 5′-RACE (Gibco-BRL). Briefly, 500 ng of adult mouse testis total RNA was reverse transcribed using a mTestisin-specific primer (R5) (5′-AGTCAGTTCGGTTCTCAA-3′). The PCR product was poly(C) tailed using terminal deoxynucleotidyl transferase TdT (Gibco-BRL) prior to PCR amplification using a bridging anchor primer (5′-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3′) and a nested mouse Testisin-specific primer (R3) (5′-TGGGGCTCAGGAAAATATCT-3′). The resultant amplicon (433 bp) was purified and sequenced.

Genomic cloning

A murine 129SV strain genomic library constructed in LambdaFIX II (Stratagene) was screened with a 427-bp probe spanning nucleotides 214–640 of the mTestisin cDNA. Approximately 1.5 × 106 recombinant phages were screened [13], and three plaques that hybidized with the probe were isolated for further characterization. One genomic clone (λSC1) containing both the 5′-end and 3′-end of mTestisin cDNA was identified by PCR analysis.

Genomic PCR amplification

To obtain fragments of the mTestisin gene, 10 sets of primers, yielding overlapping fragments, were designed according to the cDNA sequence of EST clone AA144961, the known structure of the human Testisin gene or partial mTestisin genomic sequences. mTestisin gene fragments were amplified directly from murine 129SV genomic DNA using the Expand High Fidelity PCR system (Boehringer-Mannheim). Cycling conditions were 94 °C for 10 s, 68 °C for 1–3 min (depending on the fragment size) repeated for 35 cycles. For direct sequencing, PCR products were purified and sequenced using appropriate primers.

DNA sequence analysis and database searches

DNA sequencing was performed with Dye Deoxy Terminator Cycle Sequencing (Applied Biosystem) and sequences were analyzed on the Applied Biosystems DNA Sequencing System (Model 373A). GenBank databases were searched using the algorithms available at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/). Similarities between polypeptide sequences were determined using the BestFit algorithm and the multiple sequence alignment was performed using pileup.

Northern blot and RT-PCR analyses

Murine tissues were obtained from ARC Swiss mice and frozen immediately in liquid N2. Total cellular RNA (10 µg) was prepared after homogenization using the Trizol reagent (Gibco-BRL) and separated on 1% agarose gels. RNA was transferred to Hybond N+ nylon membranes (Amersham) and hybridized overnight at 65 °C with EST clone AA144961 randomly labeled with [α-32P]dCTP. Washes were carried out at 65 °C in 2 × NaCl/Cit, 1 × NaCl/Cit and 0.5 × NaCl/Cit, respectively, with 0.2% SDS for 30 min each prior to exposure on film.

RNA (2 µg) was annealed to oligo(dT) (0.5 µg) and reverse transcribed using Superscript™ II reverse transcriptase (Gibro-BRL). PCR was performed on 3 µL of cDNA in a 25-µL solution containing 2.5 µL of 10 × reaction buffer (without MgCl2), 1 µL of 10 mm dNTP mix (Amresco) and 0.5 units of Red Hot DNA polymerase (Advanced Biotechnologies). PCR for β-actin was performed using the forward primer, 5′-GACATGGAGAAGATCTGGCA-3′ and the reverse primer, 5′-GGTCTTTAC-GGATGT-CAACG-3′ yielding a product of 637 bp. PCR for mTestisin was performed using the forward primer, 5′-AACCTTGCTCAACCGCCGC-3′ and the reverse primer, 5′-CGCAAACCATGTCTCCCCAG-3′ yielding a product of 455 bp. Both primer sets spanned introns, so that PCR products derived from cDNA could be distinguished from those derived from any contaminating genomic DNA. The PCR program was as follows: 94 °C for 5 min followed by 25 cycles (β-actin) or 35 cycles (murine testisin) of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 90 s with a final extension of 72 °C for 10 min. The PCR products were analyzed by agarose gel electrophoresis and stained with ethidium bromide. DNA markers were φX174 RF DNA HaeIII digest (New England Biolabs).

Anti-peptide Ig production

Polyclonal rabbit anti-(mTestisin peptide) Ig were produced as described previously [1]. Antibodies were directed against a peptide synthesized commercially (Auspep, Australia). The peptide encompassed a region of moderate hydrophilicity and designated A4: Ser-Met-Cys-Asn-His-Met-Tyr-Lys-Lys-Pro-Asp-Phe-Arg-Thr-Asn-Ile-Cys (residues 212–227; numbering as in Fig. 1). A cysteine residue was incorporated at the C-terminus of the peptide to enable conjugation to keyhole limpet hemocyanin using µ-maleimidobenzoic acid N-hydroxysuccinimide ester prior to immunizations. Antibody titers were monitored by ELISA and the anti-A4 serum was affinity-purified using an A4 peptide-linked Sulfolink coupling gel (Pierce, Rockville, IL, USA).

image

Figure 1. Nucleotide sequence of the mTestisin cDNA and amino-acid sequence of the encoded protein. (A) Nucleotides are numbered on the left and amino acids on the right. Catalytic residues are circled. Predicted disulfide bond forming cysteines are boxed. Potential N-glycosylation sites are indicated by a pentagon. The putative activation site is indicated by a filled triangle. The putative N-terminal signal sequence and C-terminal hydrophobic region are underlined. The polyadenylation signal is double underlined. (B) Hydropathy plot of mTestisin amino-acid sequence showing hydrophobic N-terminus and C-terminus.

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Production and purification of glutathione S-transferase−mTestisin fusion protein

A 566-bp Sau3AI fragment (corresponding to amino-acid residues 102–290) of the mTestisin cDNA (from EST clone AA144961) was ligated into the BamHI site of pGEX-1 (Pharmacia Biotech, Sweden). The predicted fusion protein would encode a polypeptide of 48 kDa. Ligated products were transformed into the Escherichia coli host DH5α by electroporation and expression of the fusion protein induced with 0.5 mm isopropyl thio-β-d-galactoside.

Western blotting

Proteins were separated under reducing conditions by SDS/PAGE on a 10% gel then transferred electrophoretically to a Hybond-P membrane (Amersham, Aylesbury, UK). The membrane was blocked using 5% skim milk in NaCl/Tris (10 mm Tris/HCl, 0.9 m NaCl; pH 7.4) and incubated with affinity purified anti-(mTestisin peptide-A4) Ig (1 : 1500), developed with donkey anti-rabbit Ig horseradish peroxidase conjugate (Amersham) (1 : 2000) and visualized using enhanced chemiluminescence (Amersham).

Immunohistochemistry

Testes were removed from ARC Swiss mice at 5, 6, 8, 10, 12, 14, 16, 18, 20, 25 and 44 (adult) days after birth, fixed in Bouins fixative and embedded in paraffin. Sections were dewaxed and rehydrated prior to incubation with 1% H2O2 (10 min) to remove endogenous peroxidase activity. Nonspecific binding was blocked with 4% nonfat skim milk in NaCl/Tris (10 mm Tris/HCl, 0.9 m NaCl; pH 7.4) for 15 min followed by 10% normal goat serum (Bioscientific, NSW, Australia) (20 min). Sections were incubated with affinity purified anti-(mTestisin peptide-A4) Ig (3.5 µg·mL−1) overnight in a humidified chamber at room temperature. Controls included sections incubated with no primary antibody or antibody which had been preabsorbed for 2 h at room temperature with 1 µg of the A4 peptide. Following incubation with prediluted biotinylated goat anti-rabbit Ig (Zymed, San Francisco, CA, USA) for 30 min, streptavidin−horseradish peroxidase conjugate (Zymed) was applied for 15 min and color developed using the chromogen 3,3′-diaminobenzidine with hydrogen peroxide as substrate. The sections were counterstained in Mayers’ hematoxylin.

Chromosomal localization of the mTestisin gene (Prss21)

EST clone AA144961 was used as a probe to localize the mTestisin gene to mouse splenic lymphocytic chromosomes, using standard methods of radioactive in situ hybridization [14]. The probe was labeled to a specific activity of 3.4 × 107 c.p.m.·µg−1 using a nick translation kit and tritiated dATP, dCTP and dTTP (Amersham) then hybridized to the chromosomes of BALB/c and C57BL male mice. The slides were coated with Ilford L4 emulsion, exposed for 41–61 days and stained through the processed emulsion to produce GBG-banded (G-banding by early S-phase incorporation of 5-BrdU and Giemsa staining) chromosomes. Silver grain signals were recorded on a G-banded idiogram of murine chromosomes.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Isolation and characterization of mTestisin cDNA

A murine EST clone containing nucleotides 117–1073 of the mTestisin cDNA was identified by screening the EST database with human Testisin cDNA. Additional 5′-sequence was obtained by 5′-RACE and from a genomic clone. The final mTestisin cDNA of 1073 nucleotides contained an open reading frame of 972 nucleotides and a 3′-UTR of 83 nucleotides with a polyadenylation signal 13 nucleotides upstream from the poly(A) sequence (Fig. 1A). The sequence surrounding the proposed start codon matched the Kozak consensus for eukaryotic translation initiation [15]. The amino-acid sequence deduced from the largest open reading frame predicts a polypeptide of 324 residues, including pre-, pro- and catalytic regions, with a molecular mass of 36.2 kDa and possessing four potential N-glycosylation sites at Asn170, Asn177, Asn210 and Asn283 (Fig. 1A). A hydropathy plot [16] of the deduced protein sequence revealed a hydrophobic region spanning amino acids 6–24 that conformed with the consensus for a typical N-terminal secretory signal sequence [17] with cleavage likely following Leu27 (Fig. 1B). In addition, a second hydrophobic region was present at the C-terminus at amino acids 303–324. The presence of these hydrophobic regions is consistent with mTestisin being an extracellular protein anchored to the plasma membrane, possibly via a glycosyl-phosphatidylinositol anchor.

The deduced mTestisin protein possesses the hallmark features of the serine protease family. It is a putative zymogen containing pro- and proteolytic domains of 27 and 270 residues, respectively (Fig. 1), delineated by a classic serine protease activation motif Arg54-Ile-Val-Gly-Gly, with cleavage predicted to occur between Arg54 and Ile55. The predicted mTestisin amino-acid sequence shares 80% similarity to human Testisin [1], 44% to human prostasin [6], and 42, 35 and 30% to the murine serine proteases mouse mast cell protease-6 [18], TESP2 and TESP1 [7], respectively. Alignment of these serine proteases revealed a number of common structural features (Fig. 2). In particular, the catalytic domain of mTestisin contains the triad of His95, Asp147 and Ser248 residues (mTestisin numbering) in positions and surrounding motifs that are required for the proteolytic activity of serine proteases. mTestisin has an aspartate (Asp242) at the bottom of the substrate-binding pocket and is therefore predicted to cleave target substrates following an arginine or lysine [19]. A conserved Ser267-Trp-Gly motif is predicted to be located at the top of the mTestisin binding pocket and is likely to participate in the correct orientation of the scissile bond of the substrate. Based on the alignment and the structure of other serine proteases, the mTestisin cysteines predicted to form disulfide bonds are: Cys46-Cys167, Cys80-Cys96, Cys181-Cys254, Cys214-Cys233 and Cys244-Cys272. The first of these cysteine pairs is predicted to link pro- and catalytic domains, whereas the remainder are likely to form disulfide bonds within the catalytic domain.

image

Figure 2. Alignment of mTestisin with closely related serine proteases. Residues that are identical between at least four of the enzymes are boxed. Catalytic residues are indicated by filled boxes. Disulfide bond-forming cysteines are indicated by filled circles. The residue located at the bottom of the substrate binding pocket is indicated by a circle. The activation site is indicated by a filled triangle. The alignment was performed using the pileup program. GenBank accession nos: mTestisin, AY005145; human Testisin (short isoform), AF058300; TESP1, AB008910; TESP2, AB008911; prostasin, NM_002773; murine mast cell protease-6 (MCP-6), M57625.

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Localization, isolation and structural characterization of the mTestisin (Prss21) gene

The chromosomal localization of the mTestisin gene (Prss21) was mapped by in situ hybridization using tritium-labeled mTestisin cDNA to mouse mitotic chromosomes. The gene was localized to murine chromosome 17 with strongest signals over bands 17 A3.3-B (average grain density of 15.3 on an overall background grain density of 2.0, data not shown). The results from C57BL mice were similar to those scored for BALB/c. Bands A3.3-B of murine chromosome 17 are syntenic with a homeologous segment of human chromosome 16p13.3 [20,21], the region to which the human Testisin gene (PRSS21) has been mapped previously [1].

Sequence of Prss21 was determined by genomic PCR amplification and from clones identified from a murine genomic library. Prss21 is ≈ 5 kb in length and contains six exons (I−VI), ranging from 27 to 320 bp, and five introns (A−E) ranging from 101 to 2716 bp (Table 1). The exon sizes show near identity with exons in the human Testisin gene and the intron sizes are very similar. The putative signal peptide was encoded by exon I and the pro-peptide spanned exons II and III. Consistent with human Testisin and other serine protease genes, the catalytic triad of His, Asp and Ser residues were encoded by different exons (exons III, IV and VI, respectively). Exon VI contained the stop codon and the 3′-UTR. All exon−intron boundaries conformed with the GT-AG rule [22]. The codon phases were identical to those identified in the human gene [23]: introns A and B, phase 1; intron C, phase 2; intron D, phase 1; and intron E, phase 0.

Table 1.  Characteristics of the mTestisin gene Prss21a and comparison with the human Testisin gene, PRSS21.
Exon5′-splice donor exon−intron junctionMouse exon (bp)Human exon (bp)Intron3′-splice donor sequence at exon−intron junctionMouse intron (bp)Human intron (bp)Amino acids interrupted (codon phase)
  • a

    Exon and intron sequences are in capitals and lower case, respectively.

  • b 

    Sizes in parenthesis are coding sequence and 3′UTR, respectively.

I GAGAAACCCGgtgagctgct > 103161A ttccacccagAACTGCAGGA  101  97Pro-G/lu-Leu (1)
II CTATTGTCAGgtaaggtgct 27 27B ctcctcacagGGCCCTGCGG  291 344Ser-G/ly-Pro (1)
III GCTTCCAAAAgtgagtctgg 166166C tctctgtcagGGATAACGAT  559 710Gln-Ly/s-Asp (2)
IV GAAGATGAGAgtgagactgg 284293D ccccacacagGTCTGCCATC 27161985Glu-S/er-Leu (1)
V TGCCTGCTTTgtaagtgtca 155155E tgcttctcagGGTGACTCGG  269 256Phe-/-Gly (0)
VI 320 (240, 80)b354 (240 114)b     

Tissue distribution of mTestisin mRNA

Northern blot hybridization analyses of total RNA from a range of adult murine tissues was carried out with a radiolabeled mTestisin probe. A mTestisin mRNA transcript of 1.3 bp was detected exclusively in the testis (Fig. 3A). The broad signal suggests the presence of multiple RNA species. RT-PCR analysis of mTestisin gene expression confirmed strong expression only in testis (Fig. 3B). Interestingly, mTestisin was not detected in bone marrow, which could suggest that mTestisin is not expressed in murine eosinophils, unlike human Testisin which has been identified from human eosinophils [3]. However, a weak signal for mTestisin was detected in spleen, a significant hematopoietic organ in mouse. Given the lack of expression in bone marrow, the weak signal for mTestisin in spleen may be associated with mTestisin expression in more mature hematopoietic cells.

image

Figure 3. Expression of mTestisin. (A) Northern blot analysis of total RNA (10 µg) from a range of murine tissues obtained from adult mice probed with the 32P-labeled mTestisin cDNA. Tissues are as indicated. Levels of 18S rRNA are shown as a measure of loading. (B) RT-PCR analysis of testisin expression in mRNA from a range of adult murine tissues. Tissues are as indicated and include resident peritoneal (RP) macrophages and thioglycolate-elicited (TE) peritoneal macrophages. β-Actin mRNA was amplified as a measure of RNA loading. Conditions and primers are as described in Materials and methods. (C) Western blot analysis of lysates from E. coli DH5α cells transformed with GST−Testisin expression construct: N, uninduced; I, induced with 0.5 mm isopropyl thio-β-d-galactoside for 3 h. The blot was probed with affinity purified polyclonal anti-(mTestisin peptide-A4) Ig.

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mTestisin expression is associated with post-meiotic testicular germ cells

Polyclonal antibodies were generated against a unique peptide derived from a nonhomologous, hydrophilic region of the mTestisin polypeptide sequence in order to investigate its expression and localization in mouse testis. This region was contained within the serine protease catalytic domain between the conserved Asp212 and Ser227 amino-acid residues. Immunoblot analysis of a recombinant GST−mTestisin fusion protein expressed in E. coli showed a strong immunoreactive band following induction (Fig. 3C). The antibody did not cross-react with an expressed GST−human Testisin fusion protein [1] (data not shown), demonstrating its specificity for mTestisin.

mTestisin expression was examined using immunohistochemistry in adult murine testis using the mTestisin specific anti-A4 serum. Figure 4A shows that mTestisin is expressed in specific cells within the germ cell lineage. As illustrated in Fig. 4BD, mTestisin expression was detected in round (RS) and elongating (ES) spermatids. Staining was diffuse within the cytoplasm of these cells with some accentuated staining at the plasma membrane, consistent with the identified C-terminal extension being involved in anchoring of Testisin on the cell surface. No detectable staining was apparent in spermatogonia (Sp), primary spermatocytes (PS), Sertoli cells or other cells of the testicular interstitium. Control samples using no antibody (Fig. 4E) or the A4 polyclonal antibody in the presence of competing A4 peptide (Fig. 4F) showed absence of this specific staining pattern. This staining pattern was different to that seen previously for human Testisin in human testis, where specific staining was associated with primary spermatocytes, with the most intense immune-specific staining seen in late pachytene and diplotene spermatocytes [1].

image

Figure 4. Analysis of mTestisin expression during male germ cell maturation. Immunohistochemical staining of mouse testis tissues was performed using the affinity purified polyclonal anti-(mTestisin peptide-A4) Ig as primary antibody. (A) Adult (44 days old) mouse testis at 100× magnification, (B) adult mouse testis at 200× magnification, (C) adult mouse testis at 600× magnification, (D) adult mouse testis at 500× magnification, (E) as (D) without the addition of antibody, (F) as (D) in the presence of competing A4 peptide, (G) day 18 after birth at 150× magnification, (H) day 18 after birth at 600× magnification, (I) day 25 after birth at 600× magnification. Arrows indicate spermatogonia (Sp), primary spermatocytes (PS), round spermatids (RS) and elongating spermatids (ES).

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mTestisin is temporally regulated during spermatogenesis

The temporal onset of mTestisin expression during testicular development was investigated by immunohistochemical analysis of testes from prepubertal mice (5–25 days after birth). No staining for mTestisin was detected at days 5–16 (data not shown). However, at day 18, weak cell-specific staining was detected in round spermatids, indicating the onset of mTestisin protein expression (Fig. 4G,H). Because spermatocytes begin to differentiate into haploid, round spermatids at ≈ 18 days after birth [24,25], these data suggest that mTestisin is expressed in a stage-specific manner in germ cells correlating with the early stages of spermiogenesis. By day 25, strong staining was detected in all the postmeiotic germ cells, showing a pattern similar to the adult testis for these cells (Fig. 4I). This pattern of expression differs from that of human Testisin, which is expressed in primary spermatocytes during the first meiotic prophase [1].

To confirm that mTestisin expression initiates after the first meiotic division, mTestisin mRNA expression was examined in total RNA isolated from testes of 5- to 25-day-old mice. As shown in Fig. 5, mTestisin transcripts were first detectable at 18 days after birth. A strong, diffuse band was detected from day 18 to day 25 (Fig. 5). If mTestisin was expressed during the first meiotic prophase, it should be detectable at ≈ day 10 after birth. mTestisin RNA was not detected in murine ovarian tissue from 6, 14 or 20 days after birth. These data demonstrate that mTestisin is temporally regulated during murine spermatogenesis, and highlight a difference between human and mouse Testisin expression.

image

Figure 5. Expression of mTestisin mRNA during male germ cell maturation. Total RNA (10–20 µg), isolated from the testes of male mice at the ages indicated, were analyzed by Northern blot analysis using a mTestisin cDNA probe. Ovary specimens were obtained from female mice at the indicated ages (given as days after birth). Total RNA isolated from the human cervical adenocarcinoma cell line HeLa S3, is the positive control (lane 13).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We isolated and characterized the cDNA and gene, and determined the gene localization and expression of the mouse ortholog of human Testisin [1]. The mouse and human Testisin genes have maintained a high degree of conservation at the genomic level. Both PRSS21 and Prss21 are ≈ 5 kb in length, span six exons with five intervening introns and have syntenic gene locations. The exon sizes and intron–exon boundary positions of PRSS21 and Prss21 are highly conserved and are distinct from most other known members of the serine protease family. Southern blot analysis of genomic mouse DNA probed with both human and mTestisin cDNAs showed identical banding patterns (data not shown), providing additional confirmation that the gene described here encodes the murine ortholog of human Testisin.

Human Testisin and mTestisin predicted polypeptides share features that strongly suggest orthologous roles. These features include a common abundant expression in testis, expression by maturing male germ cells, high sequence similarity (80%) and apparent cell-surface locations with membrane attachment likely via a glycosyl-phosphatidylinositol anchor. mTestisin, in a manner similar to human Testisin, possesses all the features of a functional serine protease. These include catalytic histidine, aspartate and serine residues in highly conserved motifs, conserved disulfide bond forming cysteines and a Ser-Trp-Gly motif necessary for correct orientation of the scissile bond of the substrate. As observed for human Testisin, mTestisin is predicted to cleave target substrates following a basic residue such as Arg or Lys. Because mTestisin is predicted to be a zymogen requiring activation following an arginine, it is possible that this protease will have autocatalytic activity. While mTestisin and human Testisin share three putative N-glycosylation sites, mTestisin possesses one additional site not found in the human ortholog.

mTestisin shows a very restricted tissue distribution and is expressed in abundance in murine testis. The diffuse band observed for mTestisin mRNA in Northern blot experiments suggests the existence of multiple mRNA species. Variations in mRNA transcript sizes are frequently observed in transcripts expressed by haploid spermatozoa. This is because transcripts which contain large poly(A) tracts are accumulated during meiosis until they are selectively activated by deadenylation during later stages, e.g. spermiogenesis [26,27]. It is also possible that the mTestisin mRNA transcript may be initiated at multiple sites, as reported for the human Testisin gene [23].

Spermatogenesis in the mouse develops synchronously with the production of specific cell types as the animal reaches puberty; no meiotic cell types are present in mice before ≈ day 10 after birth, whereas postmeiotic cell types are not present before day 18 after birth. Temporal studies of mTestisin gene and protein expression demonstrate that mTestisin is expressed postmeiotically as mTestisin mRNA from day 18 after birth and protein expression is present in the cytoplasm and on the plasma membrane of postmeiotic haploid spermatids. This expression pattern is in contrast to human Testisin which is associated with premeiotic germ cells [1].

Male germ cell maturation among mammals has a number of similarities, but there are some documented differences. The transition from an immature germ cell to a mature sperm, in addition to meiotic events, entails concomitant movement and morphological changes as germ cells pass from the seminiferous tubule basal membrane to the adlumenal compartment. With the exception of several key androgens, regulation of this process is not well understood [28]. Humans undergo the most inefficient spermatogenesis, a process that encompasses 63 days for one complete cycle with the meiotic component spanning 24 days. In mice, however, spermatogenesis encompasses only 35 days for one complete cycle, with the meiotic and spermiogenesis stages spanning 12 and 14 days, respectively [29]. In addition to this difference in cycle duration, structural relationships are different between human and mouse. Mouse spermatogenesis is found to be synchronous within a cross-section of the seminiferous tubule, whereas human spermatogenesis shows a sectoring pattern. Thus the cross-section of a human seminiferous tubule contains a near total repertoire of maturing germ cells. The reasons for these differences are not known, and whether Testisin plays a functional role related to these phenomena remains to be determined.

Expression of mTestisin in spermatids is likely to be determined, at least in part, at the transcriptional level. Cell-specific and stage-specific elements in the promoters of testis-specific genes have been described for pachytene-specific expression [30,31] and spermatid-specific expression [32,33]. These elements may vary between mouse and human orthologs [34]. There is increasing evidence that the chromatin structure [35] and methylation of CpG islands [36] may also be integral to the regulation of testis-specific genes. The human Testisin gene, PRSS21, contains a CpG island that spans the 5′-region of the gene [23], however, a similar CpG rich region does not appear to be present in the mTestisin gene (data not shown).

The expression pattern of mTestisin is consistent with a specialized role during spermatogenesis. If Testisin performs orthologous roles in mice and humans, it is unlikely to be involved in events directly related to meiosis, but rather in other processes associated with male germ cell development. Testisin could play a role in proteolytic cleavage and release of specific biologically active molecules required for spermatogenesis or may participate in proteolytic events required for migration of maturing germ cells within the adlumenal space of the seminiferous tubule. Such functions would be novel and may be elucidated through targeted disruption of the mTestisin gene.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by AMRAD Operations Pty. Ltd, Melbourne, Australia and the National Health and Medical Research Council of Australia. J. D. H. was supported by a John Earnshaw Scholarship from the Queensland Cancer Fund. We are grateful to Dr Kate Loveland for histological assessments and advice, and to Dr David Nicol for helpful discussions. We wish to thank Dr Shirley Smith for providing samples of murine tissue RNA. The LambdaFIXII library was provided by Dr Graham Kay and Dr Ian Tonks of the Transgenic and Gene Targeting Laboratory at the Queensland Institute of Medical Research. The Award of a part-time Senior Research Fellowship from the Research Foundation of The Queen Elizabeth Hospital, and the use of the laboratory facilities of Dr Cynthia Bottema, is gratefully acknowledged by G. C. W.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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Footnotes
  1. Present address: Center for Molecular Biotechnology, Queensland University of Technology, Gardens Point, Brisbane 4000, Australia.

  2. Note: the novel nucleotide sequence data published here have been submitted to the EMBL sequence data bank and are available under accession numbers AY005145(mTestisin cDNA) and AF304012(mTestisin gene).