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RESEARCH |
1 , Departamento de Biología Celular and 2 Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional No. 2508 (CINVESTAV-IPN), PC 07360, México, DF, México3 Area Académica de Medicina, Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, PC 42090, Pachuca, Hgo. México and4 Posgrado en Ciencias Biológicas, Facultad de Ciencias, Universidad Nacional Autónoma de México (UNAM), PC 04510, México, DF, México
Correspondence should be addressed to A Mújica; Email: adelam{at}cell.cinvestav.mx
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Regarding the mechanism and molecular components involved in the process of fertilization, there is a considerable amount of information. ADAMs (disintegrin and metalloprotease domain proteins) are among the candidates that may be the binding entities at the egg membrane surface (Almeida et al. 1995, Evans et al. 1997, Bigler et al. 2000, Evans 2001, Takahashi et al. 2001). ADAMs are transmembrane glycoproteins with a characteristic domain organization composed of a signal sequence, pro-, metalloprotease, and disintegrin domains, a cysteine-rich region, a transmembrane domain, and a cytoplasmic tail. This protein family is implicated in a number of cellular processes, which include fertilization, neurogenesis, and myogenesis, in the shedding of membrane-bound receptors and also in the activation of cytokines. Expression of ADAMs can vary considerably in mammals; many of them such as ADAMs 2, 7, 18, 20, 21, 29, and 30 are expressed exclusively in the testis. Other members (ADAMs 8, 9, 10, 11, 12, 15, 17, 19, 22, 23, 28, and 33) manifest a broader distribution (reviewed by Seals & Courtneidge 2003; for expression, refer to the table of ADAMs at http://www.people.virginia.edu/~jw7g/).
Earlier evidence suggested that ADAMs 1, 2, and 3, also referred to as fertilin 
, fertilin β and cyritestin respectively, participate in fertilization (Cho et al. 1998, 2000, Shamsadin et al. 1999, Zhu et al. 2000, Nishimura et al. 2001). Fertilin, which is a heterodimeric protein complex composed of and β subunits present on the sperm surface, has been thought to mediate adhesion and fusion between the sperm and the egg plasma membranes (Blobel et al. 1992, Cho et al. 1998, Evans et al. 1998). In mice, two different isoforms of ADAM1, ADAM1a and ADAM1b, are synthesized in the testes, while only ADAM1b has been found on the plasma membrane of epididymal sperm (Kim et al. 2003). Recently, it has been shown that mutant male mice lacking ADAM1b are fertile and that sperm lacking ADAM1b can still bind to and fuse with eggs, demonstrating that ADAM1b and ADAM2 are not necessary for sperm–egg fusion, as was formerly proposed (Kim et al. 2006).
Fertilin is composed of two subunits, fertilin and β (Primakoff et al. 1987, Blobel et al. 1990, 1992, Wolfsberg et al. 1993). Both subunits consist of large precursors that are proteolytically processed during various physiological stages of the sperm, eliminating the fertilin pro- and metalloprotease domains. Fertilin is processed within the testis (Lum & Blobel 1997), whereas fertilin β processing occurs during the transit of spermatozoa along the epididymis (Blobel et al. 1990, Hunnicutt et al. 1997). In mature spermatozoa, the fertilin heterodimer expresses the disintegrin domain at its N-terminus in the case of both fertilin subunits.
There is evidence that ADAM disintegrin domains, behaving as integrin ligands, may be important for sperm–egg interaction (Bigler et al. 2000, Zhu et al. 2000, Takahashi et al. 2001). ADAM15 may be one of these ADAMs, although their identities have not been fully recognized. ADAM15 has been implicated in cell–cell and cell–matrix interactions and in the proteolysis of molecules on the cell surface or the extracellular matrix (Nath et al. 1999, Eto et al. 2000, Fourie et al. 2003, Seals & Courtneidge 2003). The function of ADAM15 in cell–cell adhesion has been attributed to the disintegrin domain as integrin ligand (Seals & Courtneidge 2003), while functions involving ectodomain processing have been attributed to its metalloprotease domain (Fourie et al. 2003). Besides this, Eto et al. (2002) demonstrated that the disintegrin domains from ADAM2 and ADAM15 inhibit the binding of sperm to mouse egg in a dose-dependent manner, whereas that from ADAM17 does not affect the sperm–egg binding. Although this result may indicate a possible role played by the ADAM15 disintegrin domain in sperm–egg binding, the presence of ADAM15 in sperm has not yet been described.
The current work reports the presence of ADAM15 in mouse spermatozoa and describes how ADAM15 is processed during epididymal maturation and AR as well as the possible participation of ADAM15 in sperm–egg interaction.
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Localization of ADAM15 in mouse spermatozoa
In order to localize ADAM15 within mouse sperm, indirect immunofluorescence (IIF) assays with anti-ADAM15-CT antibody were performed in fixed and permeabilized spermatozoa during three physiological steps: the first sample was formed by non-capacitated sperm that are morphologically differentiated, but unable to fertilize; the second consisted of capacitated sperm that are capable of binding to the ZP; and the third was formed by acrosome-reacted (AR) sperm that are able to pass through ZP, and bind and fuse with the egg plasma membrane (Yanagimachi 1994). ADAM15 was located in the acrosome cap and flagellum of non-capacitated and capacitated sperm, still with acrosome (Fig. 3A and B respectively). When acrosomal exocytosis was induced with the calcium ionophore A23187
[GenBank]
, the pattern of fluorescence of the acrosome was lost, but the entire head and flagellum remained labeled diffusely (Fig. 3C). As expected, no signal was detected when pre-immune serum was used (Fig. 3D). The location of ADAM15 in the head of acrosome-reacted sperm suggests that ADAM15 may be involved in the binding of sperm to the egg plasma membrane. IIF assays were performed using antibody against the N-terminal of the ADAM15, which recognized the pro-domain: a similar pattern was detected in the samples from non-capacitated and capacitated sperm with acrosome reaction (Fig. 3E and F), but only faint fluorescence. However, in acrosome-reacted sperm, the ADAM15 precursor was not detected (Fig. 3G). These results indicate that ADAM15 was present in mouse sperm and may undergo changes during the acrosome reaction.
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80% when 500 µM dodecapeptide was used and 55% of sperm–egg fusion inhibition was observed at 300 µM peptide (Fig. 5C). In order to evaluate the inhibition specificity obtained with this peptide, a control peptide was used: of the same composition but different sequence (DREPDLPCFDPT; McLaughlin et al. 2001) and no inhibition was observed (Fig. 5D). These results suggest that the disintegrin domain of ADAM15 may play a role in sperm–egg membrane adhesion.
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Mouse testis ADAM15 contains the different domains present in ADAMs proteins
The protein cloned in this work demonstrates that ADAM15 from mouse testis has multi-functional domains consisting of a metalloprotease, a disintegrin, and a cysteine-rich region.
Processing of ADAM15 during sperm epididymal maturation
During the ADAMs protein maturation, proteolytic cleavage exposed different ADAMs domains, such as the metalloprotease or the disintegrin domain that potentially play a role in cell-surface proteolysis or cell adhesion (Seals & Courtneidge 2003). ADAM15, a protein involved in cell–cell interaction (Nath et al. 1999, Eto et al. 2000), is known to lose the pro-domain while still inside the cell, probably when in the Golgi complex; ADAM15 is located on the plasma membrane, with the metalloprotease domain exposed (Lum et al. 1998). The present results analyze ADAM15 processing during mouse sperm maturation. Analysis of the proteins recognized by the anti-ADAM15 antibodies suggests that the pro-domain of ADAM15 is removed during processing in a manner similar to that of Testase 1 (Zhu et al. 2001). ADAM15 is partially processed in the mouse testis and cleaved after it has reached the sperm surface; this takes place during the sperm's transit through the epididymis. Consequently, metalloprotease and disintegrin domains of ADAM15 are present in cauda mouse sperm and non-capacitated sperm. ADAM15 is further processed during the acrosome reaction, possibly by acrosomal proteolytic enzymes, exposing the disintegrin domain.
When the pro- and the metalloprotease domains are absent in ADAM15, this molecule may play a role during sperm–egg binding through its disintegrin domain. However, what the function of ADAM15 is, prior to the acrosome reaction in mouse sperm, still remains unsolved. ADAM15 includes the metalloprotease domain that is activated by cleavage of the pro-domain (Lum et al. 1998). Primakoff has proposed that testase 1 has proteolytic activity on the sperm surface, which modifies other proteins during the later stages of epididymal maturation and/or fertilization (Zhu et al. 2001). Similar functions might be proposed for ADAM15.
Although the processing mechanism of ADAM15 is unknown, our results suggest that in mice, this processing requires alternative proteases. The ADAM15 pro-domain may be processed by a pro-protein convertase, present in testis because ADAM15 contains a pro-protein convertase site, located between the pro- and metalloprotease domains. We suggest that the ADAM15 metalloprotease domain in mouse sperm is removed in a way, similar to that of the metalloprotease domain of fertilin-. Blobel has proposed that the pro-protein convertase PC4 may cleave the metalloprotease domain of fertilin- (Lum & Blobel 1997, Blobel 2000). The processing of ADAM15 during sperm maturation in the epididymis may be performed by proteases, secreted from the epididymis, although this still needs to be clarified because the proteases present in the epididymal fluid have not been extensively studied.
ADAM15 may play a role in fertilization
It has been postulated that at least three ADAMs participate in fertilization, ADAM1 (fertilin ), ADAM2 (fertilin β), and ADAM3 (cyritestin). Studies employing synthetic peptides and anti-disintegrin loop antibodies appear to indicate that fertilin β and cyritestin use their disintegrin domain to interact with the egg membrane (Evans 2001). This work upholds the premise that ADAM15 may also participate in the interaction between the sperm and the egg membrane because: (1) ADAM15 is present in mouse acrosome-reacted sperm at the point of fertilization; (2) following the acrosome reaction, ADAM15 with its disintegrin domain exposed was located throughout the head, where the post-acrosomal region and equatorial segment are involved in the interaction of the sperm with the egg plasma membrane (Yanagimachi 1994); (3) the sequence determined for mouse ADAM15 indicates that the DCD motif is contained in its disintegrin domain; and (4) our assays for fertilization manifested the following results: the disintegrin peptide from mouse testis ADAM15 inhibited (80%) sperm binding to zona-free mouse egg and sperm–egg fusion (55%). This evidence points towards the possible participation of ADAM15 in the fertilization process. Similar results were presented for fertilin β and cyritestin, using short fertilin β or cyritestin-based peptide mimics, which have been found to cause very substantial inhibition of sperm–oocyte interactions. However, the gene knockout experiments in mice do not support this conclusion. For example, cyritestin-deficient sperm were still able to bind the oolemma (Shamsadin et al. 1999). McLaughlin et al. (2001) have proposed that these inconsistencies are due to the use of peptides that affected unintended targets. Furthermore, they present data indicating that a single XCD motif-containing peptide (where X represents one of a limited, but as yet undefined, subset of amino acid residues) is able to inhibit mouse sperm–oolemma binding and fusion. These results indicate that the flanking sequences do not confer any sequence specificity in the in vitro assays. In our studies, which utilized a disintegrin ADAM15 peptide that contains the DCD motif, sperm–oolemma binding was affected, whereas a scrambled ADAM15 peptide that lacked the DCD inhibition failed to exhibit any effect.
These results implicate ADAM15 in binding to the oolemma, although still the in vivo role of ADAM15 remains to be defined because it is not known whether the peptide affects other targets. Regarding the possible role of ADAM15 in sperm–oolemma binding, we agree with the hypothesis forwarded by McLaughlin et al. (2001), which proposes that the concerted effects of several ADAMs result in maximum ‘fertilizing ability’ even if individual proteins are dispensable, implying some degree of functional redundancy.
The ADAMs disintegrin domains are homologous to snake venom disintegrins and represent potential integrin ligands. The disintegrin domain from human ADAM15 binds to the integrins 9β1, vβ3, and vβ1 (Zhang et al. 1998, Nath et al. 1999, Eto et al. 2000, 2002), but He et al. (2003) demonstrated that none of the integrins reported in mouse eggs or known as ADAM receptors are essential for sperm–egg binding and fusion. This finding raises the question whether other possible players are involved in the fertilization process.
ADAM15 is also present along the sperm flagellum, but its function is still unknown. As a rule, the entire sperm tail is incorporated into the egg (Yanagimachi 1994); therefore, the interaction that takes place between the plasma membranes from sperm tail and egg during fertilization may require the adhesion of molecules such as ADAM15.
In conclusion, this is the first time that ADAM15 has been observed in mammalian sperm, and our results, together with previously reported data (Eto et al. 2002), suggest the participation of ADAM15 in the fertilization process. Moreover, the role of ADAM15 in the human reproductive process should be considered of major relevance because the human spermatozoid does not express fertilin or cyritestin (Jury et al. 1997, Frayne & Hall 1998). The detection of ADAM15 opens new possibilities to improve our understanding of the mechanisms that regulate fertilization.
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Materials and Methods |
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Adam15 cDNA from testis was cloned by RT-PCR
Total RNA from NMRI mouse testis was isolated using the method described by Martinez-Perez et al. (2005). RT specific for Adam15 mRNA was performed with 500 ng primer LA15, 5 µg total RNA from testis and ThermoScript Reverse Transcriptase (Invitrogen), following the manufacturer's instructions. Reaction conditions were 62 °C for 55 min. The cDNA from heart was obtained, as previously described for external control RT from Adam15, and purified as reported by Martinez-Perez et al. (2005).
A 1800 bp DNA fragment of the Adam15 gene was obtained by means of RT-PCR, using the primers UMet-LA15 and LA15. These primers were designed in the function of the long Adam15 cDNA sequence from M. musculus (GenBank Accession number NM_009614, Fig. 1A). The PCR conditions consisted of 15 cycles of denaturing at 96 °C for 20 s, annealing at 55 °C for 20 s, and extending to 72 °C for 2.15 min. Second touchdown was at 96 °C for 15 s, annealing at 60 °C for 15 s, and extending to 72 °C for 2.15 min, for 25 cycles. The PCR products were separated by electrophoresis in 1% agarose gels and visualized by staining the gel with ethidium bromide. The Adam15 RT-PCR products were excised and eluted from agarose, using silica gel particles, following the manufacturer's instructions (Quiagen).
To confirm the identity of the RT-PCR product as Adam15, a nested PCR was performed and each product was sequenced. The products were separated and visualized by electrophoresis in 2% agarose gel, as previously described. The RT-PCR products were purified using silica gel columns according to the manufacturer's instructions (Quiagen). Subsequently, they were subcloned in the vector PCR 4.0 with TOPO isomerase, using a TOPO TA cloning, following the manufacturer's instructions (Invitrogen). Ligation reaction was used to transform Escherichia coli DH 5 cells. The positive clones were purified and sequenced using a deoxy-sequencing kit (Biosystem Prysm Dye) and an ABI PRISM 310 Genetic Analyzer automatic sequencer (Applied Biosystems, Foster City, CA, USA). The nucleotide sequences were compared with other GenBank reported sequences, using the BLAST program. The conceptual translation was carried out with the program ‘Traduccion multiple’ from the infobiogen database (www.infobiogen.fr/).
Peptides
Peptides consisting of 12 amino acid residues that correspond to the predicted disintegrin loop of mouse ADAM15, as well as a scrambled sequence thereof, were prepared by (Invitrogen). The sequences were: ADAM15, RPPTDDCDLPEF; scrambled ADAM15, DREPDLPCFDPT.
Antibody preparation
A rabbit polyclonal antibody against the active site of the disintegrin domain of mouse ADAM15 (483RPPTDDCDLPEF494) was prepared in rabbits, using the peptide conjugated to BSA. A well-known immunization program was employed and the antibody was purified by means of affinity chromatography on a peptide–sepharose column (Drenckhahn et al. 1993). The antibody was called anti-ADAM15-DD.
Preparations of testicular cells and epididymal sperm from mice
Testicular cells were isolated from adult mice (12-week-old male NMR-1), as described by Phelps et al. (1990). Animals were anesthetized with chloroform and killed by cervical dislocation; the testis was quickly removed and stripped of its fat pad and epididymis. The tissue was chopped into small pieces using a razor blade and the fragments were vigorously pipetted using a wide bore pipette. The extract was filtered through a nylon mesh to remove connecting tissue and debris. After dissection, epididymal spermatozoa were removed from three regions of the epididymis: the caput, corpus, and cauda, and from vas deferens. Sperm were washed twice in PBS, by a process of resuspension–centrifugation.
In vitro capacitation and acrosome reaction in mice sperm
Mouse sperm from the cauda epididymis and vas deferens were incubated at 37 °C, under 5% CO2 in air for 3 h in M16 medium, containing 3% BSA for in vitro capacitation and acrosome reaction. In order to induce the acrosome reaction, spermatozoa were incubated for 15 min in M16 and then the medium was supplemented with 10 µM of A23187
[GenBank]
(Sigma Chemical Co.) for 15 min. Successful induction of the acrosome reaction was verified by staining smears of formaldehyde-fixed spermatozoa, for 15–20 min with 0.2% Coomassie brilliant blue R250 (Larson & Miller 1999). After washing with bi-distilled water, the stained spermatozoa were air dried and the preparation was mounted.
Sperm immunofluorescence
Non-capacitated sperm, capacitated gametes (with and without spontaneous acrosome reaction), and acrosome-reacted sperm, induced by a treatment with the calcium ionophore A23187
[GenBank]
, were fixed (v/v) with 3.0% formaldehyde in PBS. Twenty minutes later, the spermatozoa were collected. Pelleted spermatozoa (at 600 g for 3 min) were incubated in 50 mM NH4Cl for 10 min, then rinsed twice in PBS, and once in bi-distilled water and finally resuspended in bi-distilled water. This suspension was spread on microscope slides and air dried at room temperature. Subsequently, the cells were permeabilized with absolute acetone for 7 min at –20 °C and washed in PBS. The presence of ADAM15 was revealed using anti-ADAM15-CT or with anti-ADAM-NT, both of which were diluted (1:100) in blocking solution, 1% BSA in PBS, and incubated for 1 h at 37 °C. Samples were washed in PBS, then incubated for 2 h at 37 °C with the TRITC-conjugated anti-rabbit or TRITC-conjugated anti-goat antibodies, and diluted (1:100) in blocking solution. After being washed thrice in PBS, a drop of glycerol: PBS (1:1) was added to the samples that were placed on glass slides. These were covered with a coverslip and sealed with nail polish. Cells were viewed using a Zeiss photomicroscope equipped with phase-contrast and epifluorescent optics.
Live sperm were stained with anti-ADAM15-DD at a final dilution of 1:50, but they were neither permeabilized nor fixed. After a 2-h incubation at 37 °C with the antibody, sperm samples were washed thrice with PBS. Goat anti-rabbit conjugated with rhodamine was added to the sperm in PBS, at a dilution of 1:100 and incubated for 1 h at 37 °C, together with antibody. Sperm were washed thoroughly with PBS beforehand and were later fixed with 1.5% formaldehyde for 10 min. Microscopy slide smears were prepared and observed, as described previously.
Western blot analysis
Jurkat cells, testicular cells, epididymal sperm, and non-capacitated and acrosome-reacted sperm were collected by means of spin cell suspensions at 600 g for 3 min. They were then washed twice with PBS, dissolved in lysis buffer (1% NP-40 in PBS; cell-extracts), and supplemented with the following set of protease inhibitors: 1 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml pepstatin, 50 µg/ml benzamidine, 1 µg/ml soybean trypsin inhibitor, 10 µg/ml iodoacetamide, and 300 µl of a commercial mixture of protease inhibitors (a complete tablet dissolved in H20) for a 10 ml sperm suspension. Samples were incubated on ice for 20 min and then centrifuged at 10 000 g and 4 °C for 20 min.
The supernatants were collected and their protein concentration was measured (Bradford 1976). Supernatant aliquots were boiled for 5 min in sample buffer (Laemmli 1970), containing 720 mM of 2-mercaptoethanol for protein disulfide reduction and applied to SDS-PAGE gels. Following electrophoresis, the proteins were transferred to nitrocellulose membranes, which were then blocked using 5% fat-free dried milk in TBS–Tween (10 mM Tris–HCl, 150 mM NaCl, 1% Tween (pH 7.5)). Membranes were incubated overnight at 4 °C, along with anti-ADAM15-CT (1:500 dilutions), anti-ADAM15-NT (1:400 dilutions), anti-ADAM15-DD (1:100 dilutions), or anti-actin (1:100 dilutions). After five washes (7 min each) with TBS–Tween, the membranes were incubated with appropriate secondary antibodies, coupled to HRP (1:7500 dilutions). Immune-reactive proteins were detected by chemiluminescence using an ECL kit.
Superovulation and egg recovery
Eggs were obtained from the oviducts of 6- to 8-week-old NMRI female mice. Superovulation was induced by s.c. injection of 15 IU PMSG, followed by 15 IU of hCG after a 48-h period. The animals were killed 15–17 h post-hCG. The uterine ovary–salpinge–horn complexes were dissected and eggs were removed from the ampulla of the oviduct and collected in M16 medium, supplemented with 3% BSA. The ampulla of each oviduct was punctured, the cumulus mass extruded, and placed in 0.1% hyaluronidase in M16 medium for 7 min at 37 °C in order to remove cumulus cells. Cumulus-free eggs were then washed four times in M16 medium, for 3 min each. The ZP was removed by incubating briefly in low pH buffer (Chen & Sampson 1999). The ZP-free eggs were then immediately washed thrice with M16 medium and then the eggs were allowed to recover for 60 min in M16 medium at 37 °C under 5% CO2/air. Only mature eggs with a detectable first polar body were used for fertilization.
In vitro fertilization and assessment of sperm binding and fusion
The ZP-free eggs were initially incubated with the peptide corresponding to the active site of the disintegrin domain of ADAM15 or with control peptide. Different peptide concentrations were assayed (100–500 µM) in M16 medium with 3% BSA, 30 min prior to co-incubation with capacitated sperm. None of the peptide concentrations tested altered sperm motility. Eggs and capacitated sperm (see above) were co-incubated in 200 µl drops (20 eggs per drop, with the addition of 10 µl capacitated sperm suspension (2x106 cells/ml). A 30-min co-incubation period was employed for the determination of binding, but only after 3–4 h was it possible to clearly observe swollen sperm nuclei in the egg cytoplasm. Following the co-incubation of gametes, the eggs were washed thrice in M16 medium containing 3% BSA in order to remove any loosely attached sperm. Eggs were then fixed (v/v) with freshly prepared 3.0% formaldehyde in PBS for 1 h. Eggs were washed twice in PBS and stained with Hoechst 33342 (20 µg/ml PBS) for 30 min, washed 3–4 times (15 min each time) in PBS, and mounted on a glass slide with 50% glycerol–PBS. Sperm–egg binding was observed in phase-contrast images. Fluorescent images of decondensed spermatozoa nuclei stained with Hoechst 33342 were recorded, in order to define whether the sperm had been incorporated into the egg cytoplasm.
Statistical analyses
Data were analyzed using a one-way ANOVA, followed by Tukey's honest significant differences and probability was set at P<0.05.
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Footnotes |
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Received 26 June 2007
First decision 11 March 2008
Revised manuscript received 7 September 2007
Accepted 26 March 2008
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References |
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