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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.
Human Testisin is a recently identified protein that is expressed by spermatocytes and may function as a nonclassical type II tumor suppressor gene . 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 . Testisin mRNA was also identified from human eosinophils using reverse transcription polymerase chain reaction (RT-PCR) and called esp-1 . 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 , prostasin, which is thought to function in the activation of cell surface proteins , and the murine serine proteases TESP1 and TESP2 .
Testicular germ cell maturation, or spermatogenesis, is a complex process by which diploid spermatogonial stem cells differentiate into haploid spermatozoa , 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 . 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 . 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 .
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.
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- Materials and methods
We isolated and characterized the cDNA and gene, and determined the gene localization and expression of the mouse ortholog of human Testisin . 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 .
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 .
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 . 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 . 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 . There is increasing evidence that the chromatin structure  and methylation of CpG islands  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 , 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.