Molecular Human Reproduction, Vol. 7, No. 12, 1133-1142,
December 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Perforin-independent expression of granzyme B and proteinase inhibitor 9 in human testis and placenta suggests a role for granzyme B-mediated proteolysis in reproduction
1 Department of Biochemistry and Molecular Biology, Monash University, Clayton, 3800, 2 The Peter MacCallum Institute, East Melbourne, 3002 and 3 Monash Institute of Reproduction and Development, Monash University, Clayton, 3168, Australia
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Granzyme B (graB) plays a pivotal role in cytotoxic lymphocyte granule-mediated apoptosis through cleavage of intracellular proteins in target cells. Proteinase inhibitor-9 (PI-9) is a potent inhibitor of graB and is highly expressed in cytotoxic lymphocytes. Here, we show by immunohistochemistry that PI-9 is also abundantly expressed in human testicular Sertoli cells and placental syncytial trophoblasts. Postulating that PI-9 protects these tissues from graB-producing auto- or allo-reactive cytotoxic lymphocytes, we also stained sections for graB. Unexpectedly, graB was observed in non-cytotoxic cells in both tissues. In the adult human testis, graB was present in spermatogenic cells within the seminiferous tubule, and this was verified by in-situ hybridization and reverse transcription–polymerase chain reaction (RT–PCR). Immunohistochemical analysis of term placentae demonstrated graB in syncytial trophoblasts, and this was confirmed by RT–PCR on primary trophoblasts from term placenta. Perforin, which is co-produced with graB by activated cytotoxic lymphocytes and is required for graB release into the target cell, was not detected in either testis or placenta. We postulate that, in these organs, graB has a perforin-independent role, involving hydrolysis of extracellular matrix components. In the testis, graB may facilitate migration of developing germ cells, while in the placenta, it may contribute to extracellular matrix remodelling during parturition.
extracellular matrix remodelling/granzyme B/perforin/proteinase inhibitor 9 (PI-9)/serpin
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Introduction |
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The serine proteinase granzyme B (graB) is highly expressed in cytotoxic T cells (CTLs) and natural killer (NK) cells. It is stored in granules of activated CTLs and NK cells with a number of other cytotoxins, including the pore forming protein perforin. When CTLs or NK cells recognise and adhere to virus-infected or malignant cells, these granules migrate to the site of cell contact and their contents are released into the intercellular space. Cytotoxins are endocytosed by the target cell and perforin mediates their release into the cytoplasm by disrupting the endocytic vesicles. Once released from endocytic vesicles into the cytoplasm, graB initiates apoptosis by cleaving the Bcl-2 family member, BID (Alimonti et al., 2000
; Barry et al., 2000; Heibein et al., 2000; Sutton et al., 2000), as well as caspases (Andrade et al., 1998; Yang et al., 1998). GraB also has substrates in the nucleus (Pinkoski et al., 1996; Trapani et al., 1998) and is necessary for rapid fragmentation of target cell DNA (Heusel et al., 1994).
At the end of an immune response, excess CTLs and NK cells are removed from the circulation by activation-induced cell death. However, during target cell killing it is important that CTLs and NK cells are resistant to their own cytotoxins so that they do not undergo premature apoptosis. To protect against misdirected graB they produce an intracellular serpin, proteinase inhibitor 9 (PI-9), which is a member of a large superfamily of metazoan and viral proteinase inhibitors. We have shown that PI-9 is a potent graB inhibitor and that expression of PI-9 in the cytoplasm and nuclei of cells affords protection from graB-mediated apoptosis (Bird et al., 1998). PI-9 inhibitory function is mediated through a C-terminal reactive centre loop (RCL) which resembles a graB substrate (Sun et al., 2001). Cleavage of the PI-9 RCL by graB causes a rapid conformational change in the serpin, resulting in the formation of a stable serpin–proteinase complex (Sun et al., 1996). The recent crystallization of a serpin–proteinase complex has shown that the proteinase is substantially distorted during complex formation and that catalysis of the RCL is not completed, leaving the two molecules covalently linked and inactive (Huntington et al., 2000).
As well as protecting cytotoxic lymphocytes, PI-9 may also protect bystander cells or antigen-presenting cells likely to be exposed to graB during an immune response. The presence of PI-9 in non-cytotoxic cells such as B cells (Sun et al., 1996), monocytes (Young et al., 2000) and endothelial and mesothelial cells (Buzza et al., 2001) is consistent with such a role. Furthermore, very high levels of PI-9 transcripts are present in placenta and testis, suggesting a role in maintaining the privileged immune status of these tissues (Sun et al., 1996).
To further understand the physiological role that PI-9 plays in bystander protection and immune privilege, it is important to identify specific cells that express PI-9. In this study we have surveyed various human tissues for PI-9, focusing on testis and placenta. We show that PI-9 is present in Sertoli cells and syncytial trophoblasts, consistent with a role in maintaining immune privilege. Surprisingly we also detect perforin-independent expression of graB in and around these cells. This is the first description of graB expression outside lymphocytes, and suggests a novel role for this proteinase in reproductive function.
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Materials
All reagents were of analytical grade and were purchased from either Sigma Chemicals, St Louis, MI, USA or Merck BDH Chemicals, Darmstadt, Germany unless otherwise specified. All reagents were obtained from local subsidiaries or agents of the manufacturers. The recombinant human serpins PI-9, PI-8 and PI-6, monocyte-neutrophil elastase inhibitor (MNEI), murine serine proteinase inhibitor 6 and 3 (SPI-6 and SPI-3) and the viral serpin cytokine response modifier A (CrmA) were expressed in Pichia pastoris and purified following methods described previously (Sun et al., 1995
). Recombinant human plasminogen activator 2 (PAI-2) was obtained from Dr R.Medcalf (Monash University Department of Medicine, Box Hill Hospital). Recombinant human squamous carcinoma cell antigens 1 and 2 (SCCA-1 and -2) were a kind gift of Dr D.M.Worrall (Department of Biochemistry, University College, Dublin). Dr S.Bottomley and Dr R.Pike provided purified human 
1-antitrypsin (1-AT), 1-antichymotrypsin (1-AC) and antithrombin (AT), J.Irving provided recombinant myeloid and erythroid nuclear termination stage specific protein (MENT) (Department of Biochemistry and Molecular Biology, Monash University). Dr H.Abts provided recombinant Hurpin (Department of Dermatology, Heinrich-Heine-University). YT, a human NK-like cell line (Wano et al., 1984), was cultured in RPMI 1640 with 10% heat inactivated fetal calf serum, 2 mmol/l L-glutamine, 50 IU/ml penicillin and 50 µg/ml streptomycin. Antibodies used to detect graB were 2C5 (Apostolidis et al., 1995) and rabbit polyclonal anti-graB (Edwards et al., 1999). GrB-7 was obtained from Chemicon, Temecula, CA, USA (Kummer et al., 1993), and goat polyclonal anti-graB (C-19) was from Santa Cruz Biotechnologies, Santa Cruz, CA, USA.
Monoclonal antibody production
BALB/c mice were immunized by intraperitoneal injections of recombinant PI-9 (rPI-9) at 2 week intervals. The initial injection consisted of rPI-9 emulsified in Freund's complete adjuvant, the second of rPI-9 emulsified in incomplete Freund's, and the third of sodium dodecyl sulphate (SDS)-denatured rPI-9 mixed with phosphate-buffered saline (PBS). Three days after the final inoculation, splenocytes were recovered and fused to NS-1 myeloma cells as described previously (Apostolidis et al., 1995). HAT resistant hybridomas were screened by ELISA and immunoblotting against purified rPI-9. Positive hybridomas were expanded and cloned by limiting dilution.
Specificity of PI-9 monoclonal antibodies
The specificity of the hybridoma 7D8 to PI-9 was determined by immunoblotting against a panel of purified serpins. The indicated serpins (100 ng of each) were separated on duplicate 12.5% SDS-polyacrylamide gels under reducing conditions. One gel was silver-stained (Rapid-Ag-Stain, ICN, Costa Mesa, CA, USA) to verify equal loading of proteins. Proteins on the second gel were transferred to nitrocellulose and immunoblotted with hybridoma supernatant. Bound antibody was detected by horse-radish peroxidase (HRP) conjugated sheep anti-mouse IgG (Chemicon) and enhanced chemiluminescence (NEN DuPont, Boston, MA, USA).
Preparation of tissue from normal human adult testis and term placenta
Samples of human adult testes were obtained with informed consent following the guidelines of the Monash Human Ethics Committee. Immediately upon collection, part of the testis was fixed for 5 h in Bouin's fixative, then rinsed and stored in 70% ethanol before dehydration and paraffin embedding. The histological specimens were cut into 5 µm sections, floated on diethyl pyrocarbonate (DEPC)-treated MilliQ water and dried onto slides (Superfrost Plus, Biolabs Scientific, Clayton, Vic, Australia). These sections were used for both immunohistochemistry and in-situ hybridization analyses. Another sample of testis material was snap frozen immediately upon collection and stored at –70°C until use for RNA analysis. S.Black (Monash University Department of Medicine, Box Hill Hospital) provided formalin-fixed paraffin-embedded term placental blocks obtained from routine Caesarean sections. Sections of 5 µm were cut onto glass slides (Menzel-Glaser, Braunscheig, Germany) and used for immunohistochemistry.
Immunohistochemistry
Various tissue blocks (other than listed above) were obtained from the Box Hill Hospital, Department of Anatomical Pathology archive. Endometrial tissue sections were provided by Dr P.Rogers (Monash University Department of Obstetrics and Gynaecology, Monash Medical Centre). Sections were dewaxed, rehydrated and treated with 0.3% hydrogen peroxide. Antigen retrieval was then performed by heating sections at 100°C for 10 min in 50 mmol/l glycine, pH 3.5 (testis sections), or 10 mmol/l citric acid, pH 6.0 (placental sections). Slides were washed three times in Tris-buffered saline (TBS: 10 mmol/l Tris-HCl, 150 mmol/l NaCl, pH 7.6), and these washes were repeated between each incubation step. Sections were blocked in 5% normal sheep serum diluted in TBS with 0.1% bovine serum albumin (BSA; testis sections), or with 2% BSA in TBS (placental sections), for 20 min. Primary antibodies were diluted in TBS/0.1% BSA and incubated overnight in a humidified container. Bound antibody was detected by biotinylated sheep anti-mouse or anti-rabbit IgG (Chemicon) for 1h, followed by HRP conjugated streptavidin (Chemicon) for 1h or in some cases Vectastain® Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA), and visualized using the Liquid DAB Chromogen Substrate System (DAKO). Sections were counterstained with Harris' haematoxylin and mounted in DPX mounting fluid (Merck BDH).
In-situ hybridization
Riboprobes for PI-9 were generated from nucleotides 827–1388 of a human PI-9 cDNA subcloned into the SacI/EcoRI sites of pBluescript II KS- (Stratagene) by digestion with SacI and EcoRI. Riboprobes for graB 3' UTR were generated by amplifying nucleotides 747–873 of human graB with primers JT 300 and JT 301 (Table I). This fragment was subcloned into pCR®-Blunt (Invitrogen), excised with EcoRI and cloned into the EcoRI site of pBluescript II KS-. Digoxigenin-labelled riboprobes for both graB and PI-9 were generated following the methods outlined in the dUTP-DIG Labelling Kit (Roche Molecular Biochemicals, Mannheim, Germany). The in-situ hybridization was performed essentially as previously described (Meinhardt et al., 1998).
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Reverse transcription–polymerase chain reaction (RT–PCR) for PI-9, graB and perforin
Primary placental cytotrophoblasts were isolated from fresh term placentae by S.Black by enzymatic digestion and Percoll gradient centrifugation, essentially as previously described (Kliman et al., 1986
), except that the cells were cultured in RPMI 1640. Most cells had formed syncytial structures 48 h after plating. Prior to RNA extraction the cells were washed thoroughly with PBS to remove any contaminating non-adherent leukocytes or erythrocytes and observed microscopically to verify purity. RNA isolated from testicular germ cell tumour lines was provided by Dr M.Pera (Monash Institute of Reproduction and Development). Total RNA was extracted from YT cells, normal testis tissue and placental syncytial trophoblasts using RNAzolTM B (Tel-Test) according to manufacturer's instructions. M-MuLV reverse transcriptase (New England Biolabs) and oligo dT (Amersham) were used to synthesize cDNA from 1 µg of total RNA. PCR was performed using 20 pmol of the indicated primer in 50 mmol/l KCl, 10 mmol/l Tris-HCl, pH 9.0, 0.1% Triton X-100 containing 2.5 mmol/l MgCl2, 200 µmol/l dNTPs and 2 IU of Taq polymerase (GeneWorks, Thebarton, SA, Australia) for 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 45 s. The PCR primers were designed to amplify across intron/exon boundaries, thereby allowing discrimination of genomic DNA contamination in the cDNA. PCRs were performed with the primers indicated in Table I
and amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control for reverse transcription reactions. Amplified products were resolved on a 2% Agarose gel by electrophoresis and transferred to nitrocellulose under alkaline conditions. Membranes were hybridized with [
-32P] ATP-labelled oligonucleotides specific for PI-9, graB or perforin that were internal to the amplifying primers (PB 286, PB 279 and JT 219 respectively). |
Results |
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Production and characterization of monoclonal antibodies to PI-9
Due to the high sequence and structural similarities between serpins, a polyclonal antiserum raised to one serpin will often cross-react with other family members. To reliably identify cells expressing PI-9 by immunohistochemistry, monoclonal antibodies were generated by immunizing mice with recombinant PI-9. Four hybridomas producing antibodies to PI-9 were identified by ELISA and screened for specificity to PI-9 by indirect immunofluorescence, immunoblotting and immunoprecipitation against a panel of serpins. One antibody, designated 1F3, recognised the majority of the serpins tested. Further analyses indicated that 1F3 recognises the highly conserved serpin proximal hinge region (data not shown).
Of the three other hybridomas, 2E7 and 8D3 reacted poorly against denatured PI-9 as assessed by immunoblotting and indirect immunofluorescence on acetone/methanol fixed cells. By contrast, 7D8 detected PI-9 under denaturing conditions, suggesting that it would be suitable for immunohistochemistry. 7D8 (IgG1 
) was assessed for cross-reactivity to a range of serpins including members of the ov-serpin family to which PI-9 belongs (Sun et al., 1996). Following immunoblotting, a 42 kDa protein was detected only in the lanes containing rPI-9, demonstrating that 7D8 is specific for PI-9 (Figure 1).
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Expression of PI-9 and graB in the testis
To identify cells that produce PI-9, an immunohistochemical survey of normal human tissues was performed using the specific monoclonal antibody 7D8. As indicated in Table II
, PI-9-expressing cells were observed in a variety of tissues. In lymphoid tissue, PI-9 was restricted to lymphocytes and dendritic cells, while in reproductive and other tissues PI-9 was found in a number of epithelial and stromal cells. This distribution is consistent with its proposed role in cytotoxic cells and bystander protection during an immune response (Bird et al., 1998; Buzza et al., 2001). As previously indicated by RNA analysis (Sun et al., 1996), PI-9 was also observed in testis and placenta. As these are known to be sites of immune privilege, they were the focus of our subsequent investigations.
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Analysis of normal human testis by immunohistochemistry indicated that PI-9 is present within seminiferous tubules (Figure 2A
ii). Under higher magnification, the most prominent staining for PI-9 was apparent in Sertoli cells (Figure 2Aiii) extending from the basement membrane to the lumen. There was also pronounced nuclear staining for PI-9, which we have previously observed in many cell types (Bird et al., 2001). As the cytoplasmic extensions of the Sertoli cell wrap around the developing germ cells, it was difficult to identify cell boundaries and definitively rule out PI-9 expression in germ cells in a single section. However, careful analysis of a number of serial sections indicated that within the seminiferous tubule, PI-9 is restricted to Sertoli cells. In some sections however, staining was also observed in Leydig cells and lymphocytes within the interstitium.
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As Sertoli cells form the blood–testis barrier, the presence of PI-9 in these cells suggested that PI-9 contributes to the maintenance of the immune privileged status of the testis. By expressing PI-9, Sertoli cells would be resistant to graB produced by activated cytotoxic lymphocytes responding to the developing and potentially immunogenic germ cells. To further explore this role for PI-9 in immune privilege, we performed immunohistochemistry to determine if graB-expressing lymphocytes are normally present in the testis. Surprisingly, immunohistochemistry on normal human testis using a monoclonal antibody to graB indicated expression of the proteinase within the seminiferous tubule (Figure 2A
v). As with PI-9, graB was observed in Sertoli cells, but in contrast, it also appeared in a number of different cells of the spermatogenic lineage (Figure 2Avi). The intensity of staining for graB varied between cell types; Sertoli cells had a diffuse cytoplasmic distribution while primary spermatocytes and round spermatids had a more intense cytoplasmic staining. No graB was visible in the nuclei of these cells. To determine if graB was present in mature spermatozoa, immunoblotting was performed on spermatozoa isolated from seminal fluid; however, no graB was detected in these samples (data not shown). As graB is thought to be restricted to CTLs and NK cells (Caputo et al., 1988; Trapani et al., 1988), we confirmed the immunohistochemistry using three other antibodies to human graB (both monoclonal and polyclonal) and all three demonstrated graB within the seminiferous tubule (data not shown).
Analysis of PI-9 and graB transcripts in the testis
To eliminate the possibility that the graB antibodies were in fact recognising some highly homologous but as yet uncharacterized serine proteinase in the testis, we examined whether graB and PI-9 transcripts could be detected by in-situ hybridization using riboprobes, or by RT–PCR performed on RNA extracted from normal adult testis.
In-situ hybridization was performed using probes specific for either PI-9 or graB on normal human adult testis. The PI-9 probe encompassed the region including the reactive centre loop, which is the most variable region of a serpin gene. The graB probe was generated from the 3' untranslated region, which is most divergent from other serine proteinases. In-situ hybridization confirmed the results of the immunohistochemistry, localising both PI-9 and graB to cells within the testis. Careful examination of multiple sections from both the immunohistochemistry and in-situ hybridization studies indicated that PI-9 is present in Sertoli cells within the tubules and Leydig cells and capillary endothelial cells within the interstitium. Endothelial cell expression of PI-9 has been previously observed (Buzza et al., 2001).
Analysis by in-situ hybridization indicated that graB is expressed in Sertoli cells as well as in germ cells at various stages of development (Figure 2Bii). There was also some staining of interstitial cells within the testis consisting of scattered lymphocytes and endothelial cells. Precise identification of the spermatogenic cells expressing graB was difficult due to variation between samples, and the fact that the cytoplasmic extensions of Sertoli cells are intimately involved with the developing germ cells, making it difficult to delineate intercellular boundaries. Nevertheless, cell types positive for graB included some spermatogonia and pre-leptotene spermatocytes with the most obvious staining found in pachytene spermatocytes (Figure 2Bii). No signal was detected in the various stages of spermatid development, indicating that graB is restricted to germ cells in the early stages of meiotic division.
The presence of both graB and PI-9 transcripts in normal adult testis was confirmed by RT–PCR. Amplification using PI-9-specific primers resulted in a product of the expected size (188 bp), which was confirmed as PI-9 by Southern blotting with an internal oligonucleotide (Figure 3A). RT–PCR using graB primers resulted in several products, one of which was of the correct size (561 bp) and was confirmed to be graB by Southern blotting with an internal oligonucleotide (Figure 3A). To exclude the possibility that the graB transcripts were derived from contaminating lymphocytes within the tissue sample, we tested for co-expression of perforin, which is produced with graB by activated lymphocytes (Liu et al., 1989). No products were detected by PCR either visually (Figure 3B upper panel) or by Southern blotting with an internal oligonucleotide to perforin (Figure 3B lower panel). By contrast, perforin was amplified from cDNAs generated from the NK-like cell line YT at the same time as the testis cDNA (Figure 3B). The lack of perforin expression confirmed that graB is produced by non-cytotoxic cells within the testis, and indicates that in this context graB may not act as a cytotoxic proteinase, as its entry into cells is perforin-dependent (Jans et al., 1996; Shi et al., 1997).
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Dysregulation of graB and PI-9 in disease
To further elucidate the role of graB and its inhibitor, PI-9, in testicular function, we examined their expression in abnormally developed testes. In the maldescent testis, either one or both of the testes fails to descend from the abdominal space into the scrotal sac, and developing germ cells are exposed to higher temperatures causing loss of germ cell progenitors. Due to the interplay between the developing germ cells and Sertoli cells, this also results in abnormal Sertoli cell development. The expression of both graB and PI-9 (as determined by immunohistochemistry) was decreased in the aberrant Sertoli cells of maldescent testis (data not shown).
Analysis of graB and PI-9 gene expression was also performed on RNA extracted from four human testicular germ cell tumour lines (GCT 27C4, 27X1, 48 and 72) (Pera et al., 198719881989). Although GAPDH cDNA was detected by RT–PCR, neither graB nor PI-9 were detected by RT–PCR or Southern blotting with an internal oligonucleotide, indicating that these proteins are absent from testicular germ cell tumours (data not shown). The lack of PI-9 is consistent with its absence from germ cells in normal testis, but the absence of graB suggests that it may be lost during tumorigenesis.
Expression of PI-9 and graB in placental trophoblasts
We have previously shown that high levels of PI-9 mRNA are produced in human placenta (Sun et al., 1996). To identify the cells expressing PI-9, we performed immunohistochemical analysis of normal term placentae using PI-9 specific antibodies. As shown in Figure 2C, PI-9 was highly expressed in the multi-nucleated syncytial trophoblast layer (syncytium) of the chorionic villi and in endothelial cells (Figure 2Cii,iii).
Given the unexpected finding of graB in the human testis, we also examined term placentae for graB expression, and it was detected in syncytial trophoblasts (Figure 2Cv,vi), with weak staining of endothelial cells occasionally observed. It is unlikely that the graB staining was due to antibody cross-reaction with a similar protein, as graB was detected in these cells with three other antibodies. To confirm this finding, we performed RT–PCR for graB on primary cultured syncytial trophoblasts. Primary cytotrophoblasts were isolated from human term placenta and after 48 h of culture, most had fused to form multi-nucleated syncytial trophoblasts. RT–PCR using RNA extracted from these cells yielded appropriately sized products for both PI-9 and graB, which were verified by Southern blotting with internal oligonucleotides (Figure 4). To eliminate the possibility that the graB products arose from RNA of contaminating lymphocytes in the trophoblast preparation, RT–PCR for perforin was performed and no product was detected (Figure 4).
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Discussion |
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This study has demonstrated the presence of both graB and its cognate inhibitor, PI-9, in adult human testis and term placenta, in contradiction to the current dogma that graB is confined to NK cells and activated CTLs. The view that graB is present only in cytotoxic lymphocytes has arisen because the original analysis of graB mRNA expression was carried out on a panel of haemopoietic cell lines (Trapani et al., 1988
). Subsequent immunohistochemical analyses have also focused exclusively on tissues of the immune system (Held et al., 1990; Hameed et al., 1991; Ebnet et al., 1995; Kummer et al., 1995). A comprehensive survey of human graB expression has apparently never been carried out, so its presence in non-immune tissues and any additional physiological functions has not been considered.
GraB has a preference for cleavage after acidic amino acids, particularly aspartic acid (Poe et al., 1991). It is this `Asp-ase' activity that allows cleavage of BID, caspases and other intracellular components during apoptosis. However, a number of studies have indicated that graB is also released into the circulation during inflammation (e.g. rheumatoid arthritis) (Tak et al., 1999; Ronday et al., 2001) and have identified extracellular substrates of graB. For example, graB has the ability to degrade components of the extracellular matrix such as the proteoglycan, aggrecan (Froelich et al., 1993), and it is able to cause detachment of adherent tumour cell lines (Sayers et al., 1992). In CD34+ peripheral blood progenitor cells, graB may play a role in detachment from bone marrow stromal cells (Berthou et al., 1995). Thus, it is evident that graB has the ability to exert extracellular effects and may have a perforin-independent role in extracellular matrix remodelling. Our finding that graB is expressed in the testis and placenta in the absence of perforin suggests a non-cytotoxic role for graB in reproduction. As discussed below, in the testis graB may contribute to migration of developing germ cells through Sertoli cell tight junctions, while in the term placenta syncytial trophoblast expression of graB may contribute to extracellular matrix remodelling during parturition.
GraB is produced as a zymogen which is activated by the cysteine proteinase dipeptidyl-peptidase-I (DPP-I, cathepsin C). DPP-1 is present in cytotoxic lymphocyte granules and removes an N-terminal Gly-Glu dipeptide from the graB zymogen (Brown et al., 1993; Smyth et al., 1995). Although studies on DPP-1 have focused on its role in activation of serine proteinases in immune and inflammatory cells, it has nonetheless been demonstrated in primary spermatocytes (Chung et al., 1998) and in human placenta (Lampelo et al., 1987; Rao et al., 1997). Thus, the mechanism for graB activation exists in both germ cells and placenta, further supporting the idea that graB-mediated proteolysis occurs in these tissues.
Proteases and their inhibitors play an important role in testicular development and germ cell maturation (Fritz et al., 1993). Controlled proteolytic activity is essential to the remodelling and restructuring of the seminiferous tubule during the migration of germ cells from the basement membrane to the lumen of the tubule. The plasminogen activators t-PA (tissue type) and u-PA (urokinase) and their cognate inhibitors (PAI-1 and PAI-2) play a role in the degradation of tight junctions between Sertoli cells during testis development (Lacroix et al., 1977). The plasminogen activators are also present in seminal plasma (Astedt et al., 1979) and spermatozoa (Smokovitis et al., 1992), and PAI-1 and PAI-2 are expressed within the seminiferous tubule (Gunnarsson et al., 1999). Acrosomal serine proteinases such as acrosin, TESP-1 and TESP-2 (Kohno et al., 1998) have also been implicated in the degradation of the zona pellucida and fertilization of the ova.
Serine proteinases of unknown function are also present in human testis. The proteolytic activity of leydin and testisin are unknown. However, their restricted expression to Leydig cells and pachytene spermatocytes respectively, implicates them in testicular function (Hooper et al., 1999; Poorafshar and Hellman, 1999). Loss of testisin expression has also been associated with the development of testicular germ cell tumours. Interestingly, graB shares a similar expression pattern to testisin, as it is also expressed in pachytene spermatocytes and is absent from germ cell tumour lines. Perhaps loss of graB is also associated with germ cell tumour progression.
The expression of graB in cells that represent the transition from mitotic to meiotic division suggests that graB is involved in germ cell maturation. Developing germ cells must also migrate through the blood–testis barrier in a process that involves proteolytic degradation of the tight junctions between Sertoli cells and it is possible that graB is involved in this process. However, the lack of graB in mature spermatozoa suggests that it is not involved in fertilization.
Serine proteinases are intimately involved in placental development, particularly during implantation. During this process the trophectoderm (which comprises the outer layer of the blastocyst) and invasive cytotrophoblasts secrete a myriad of extracellular matrix degrading proteases. Of these, the serine proteinases uPA, tPA, kallikrein, tryptase and elastase contribute to the extensive matrix remodelling required for invasion of the blastocyst through the endometrial wall and stroma, and eventually into maternal blood vessels. Expression of the serpins PAI-1 and PAI-2, and tissue inhibitors of metalloproteinases (TIMPs) by trophoblasts and endometrial/decidual cells is thought to regulate the extent of invasion (Salamonsen, 1999; for review). It will be of interest to determine whether graB is expressed by invasive cytotrophoblasts and contributes to implantation. In the term placenta, matrix remodelling proteases and their inhibitors are also important during parturition. Here, matrix metalloproteinases and TIMPs, as well as plasminogen activators and their inhibitors, are thought to regulate this process (Tsatas et al., 1998; Athayde et al., 1999; Hu et al., 1999; Riley et al., 1999), and graB may also contribute to this process.
The presence of PI-9 in Sertoli cells and the syncytial trophoblast layer, which form the blood–tissue barriers, is consonant with its role in cytoprotection against graB. PI-9 may provide protection against graB produced by maternal or self-reactive cytotoxic lymphocytes and thus contribute to immune privilege. Although the entry of graB into the cytoplasm of cells normally requires perforin, other endosomolytic agents or events can mediate its release (Froelich et al., 1996; Browne et al., 1999). Thus the presence of PI-9 in the cytoplasm and nuclei of cells producing graB (or in closely associated cells) may guard against inappropriate apoptosis in testis and placenta induced by misdirected graB.
Finally, it is possible that the role of graB and PI-9 in reproduction or immune privilege may be further elucidated by careful study using rodent models. At present it is not known whether graB or PI-9 orthologues are expressed in mouse testis or placenta. However, using polyclonal antisera raised against PI-9 we have demonstrated staining in rat and mouse seminiferous tubules (data not shown). These findings should be tempered by the observation that, compared with humans, rodents have a significantly expanded repertoire of both granzymes (Smyth et al., 1996) and serpins (Sun et al., 1997), and that the functions of these genes may be duplicated, shared or redundant. This difficulty is illustrated by the graB-null mice which have an immune deficiency but are fertile (Heusel et al., 1994), implying that in rodents either graB is unimportant for reproduction or it is functionally redundant. Nonetheless it should be noted that these mice have not been thoroughly analysed for reproductive function in terms of organ or embryonal development, fecundity or litter size. As was the case with FSH-null mice, FSH was predicted to be essential for spermatogenesis. However, more detailed investigations revealed that FSH-deficient males were fertile despite having markedly reduced testis weight (Kumar et al., 1997). Similar investigations may uncover a reproductive defect in the graB-null mice.
In conclusion, our work clearly suggests roles for graB and PI-9 beyond the immune system in human reproductive function. The targets and precise function of graB in this context remain to be determined, but it is likely to involve extracellular matrix remodelling in germ cell maturation and trophoblast invasion.
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Acknowledgements |
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We are grateful to Dr P.Hosking (Department of Anatomical Pathology, Box Hill Hospital) for providing archival tissues, S.Black (Monash University, Box Hill Hospital) for the placental trophoblasts and tissue blocks, Dr P.Rogers (Monash University, Monash Medical Centre) for the endometrial sections and Prof. D.M.de Kretser (Monash Institute of Reproduction and Development) for access to the testis material. We would also like to thank T.Meehan for help with the preparation of figures. This work was funded by the National Health and Medical Research Council of Australia. C.Hirst and M.Buzza are recipients of the Australian Postgraduate Award.
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4 To whom correspondence should be addressed at: Department of Biochemistry and Molecular Biology, Building 13B, Room G09, Monash University, Clayton, 3800, Australia. E-mail: phil.bird{at}med.monash.edu.au
* Both authors contributed equally to this work
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References |
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Submitted on May 11, 2001; accepted on September 12, 2001.
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