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Characterization of a membrane-boundangiotensin-converting enzyme isoform in crayfishtestis and evidence for its release into the seminal fluidJuraj Simunic, Daniel Soyez and Ne´dia KamechEquipe Biogene`se des Signaux Peptidiques, ER3, Universite´Pierre et Marie Curie, Paris, FranceIntroductionAngiotensin-converting enzyme (ACE; dipeptidyl car-boxypeptidase; EC 3.4.15.1) is an enzyme that belongsto the family of M2 peptidases with a zinc chelatormotive HEXXH-23(24)-E. Substrate hydrolysis inACE is activated by chloride ions, which is a uniquefeature among metalloproteases. However, the mole-cular mechanism behind this is unclear. In vertebrates,it is present as two isoforms, somatic and testicular,which are both transcribed from the same gene underthe control of tissue-specific promotors [1,2]. TheKeywordsangiotensin-converting enzyme; crayfish;Crustacea; spermatogenesis; testisCorrespondenceN. Kamech, Equipe Biogene`se des SignauxPeptidiques, ER3, Universite´Pierre et MarieCurie, 7 Quai Saint Bernard, 75251 Paris,Cedex 05, FranceFax: +33 1 44 27 23 61Tel: +33 1 44 27 22 58E-mail: nedia.kamech@upmc.fr(Received 4 May 2009, revised 18 June2009, accepted 25 June 2009)doi:10.1111/j.1742-4658.2009.07169.xIn the present study, an isoform of angiotensin-converting enzyme wascharacterized from the testis of a decapod crustacean, the crayfish Asta-cus leptodactylus. Angiotensin-converting enzyme cDNA, obtained by3¢-to5¢ RACE of testis RNAs, codes for a predicted one-domain proteinsimilar to the mammalian germinal isoform of angiotensin-convertingenzyme. All amino acid residues involved in enzyme activity are highlyconserved, and a potential C-terminus transmembrane anchor may bepredicted from the sequence. Comparison of this testicular isoform withangiotensin-converting enzyme from other crustaceans, namely Carci-nus maenas, Homarus americanus (both reconstituted for this study fromexpressed-sequence tag data) and Daphnia pulex, suggests that membrane-bound angiotensin-converting enzyme occurs widely in crustaceans, con-versely to other invertebrate groups where angiotensin-converting enzymeis predominantly a soluble protein. In situ hybridization and immunohisto-chemistry performed on testis sections show that angiotensin-convertingenzyme mRNA is mainly localized in spermatogonias, whereas protein ispresent in spermatozoids. By contrast, in vas deferens, immunoreactivity isdetected in the seminal fluid rather than in germ cells. Accordingly, angio-tensin-converting enzyme activity assays of testis and vas deferens extractsdemonstrate that the enzyme is present in the membrane fraction in testis,but in the soluble fraction in vas deferens. Taken together, the resultsobtained in the present study suggest that, during the migration ofspermatozoids from testis to vas deferens, the enzyme is cleaved from themembrane of the germ cells and released into the seminal fluid. To ourknowledge, this present study is the first to report such a maturationprocess for angiotensin-converting enzyme outside of mammals.AbbreviationsACE, angiotensin-converting enzyme; Asl, Astacus leptodactylus; DIG, digoxigenin; EST, expressed-sequence tag; gACE, germinal isoform ofangiotensin-converting enzyme; tACE, testicular isoform of angiotensin-converting enzyme.FEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government works 4727somatic isoform exhibits two catalytic domains and ispresent in numerous tissues, such as on the surface ofendothelial cells in the lung, myocardium, liver, intestineand testis, as well as in the epithelial cells of the kidneyand intestine [3]. Its role in the regulation of the renin–angiotensin–aldosterone system has been well character-ized [4]. The enzyme cleaves angiotensin I to produceangiotensin II, a powerful vasosuppressor. It alsocleaves the vasodilatator peptide bradikinin, and thuscontributes to the augmentation of blood pressure.On the other hand, the role of the testicular isoform(tACE), also called germinal ACE (gACE), is not soclear. In mice, the testis ACE protein is first detectedin step 10 spermatids, whereas ACE mRNA is firstdetected in developmentally younger cells, the pachy-tene spermatocytes, implying a delay in ACE transla-tion [5]. A similar phenomenon was described inhuman testes, with mid-pachytene spermatocytesexpressing the mRNA and stage III spermatids con-taining the protein, which corresponds to a delay byone germ cycle [6].The physiological role of tACE is still a matter ofdebate, but numerous studies point to the importanceof this enzyme in male and female reproduction; forexample, mice males with a ‘knockout’ for the Acegene show extremely reduced fertility [7].Analogues of ACE have also been identified in manyinvertebrate species, most notably insects, and havebeen shown to play a major role in reproduction.Indeed, as in mice, males of Drosophila with a knock-out for Ace show a dramatic decrease in fertilitybecause the developing spermatozoids cannot completethe phase of individualization and demonstrate anabnormal morphology [8]. In Lepidoptera, the treat-ment of adults with the ACE inhibitor, captopril,causes a decrease in egg-laying [9]. In Haematobia irri-tans exigua, a blood meal initiates the strong synthesisof ACE in the testes, but not in the ovaries [10]. Bycontrast, in female Anopheles stephensi, a dramaticincrease in ACE activity is observed in the ovary aftera blood meal, with a maximum just prior to egg-lay-ing, and ACE is completely transferred to newly-laideggs [11]. Similar results were obtained from thetomato moth Lacanobia oleracea [12]. Recently, such atransfer of ACE from males to females was reportedto take place during copulation in Drosophila melanog-aster [13].Even if the implication of ACE in reproductionappears to be well established, the possible substratesinvolved remain to be determined. To date, the onlysubstrate identified in vivo in invertebrates is an 11-merpeptide (Neb-ODAIF) isolated from the ovaries of thefly Neobellieria bullata [14].In previous studies, RT-PCR and northern blottingon RNAs from several tissues of the crayfish Asta-cus leptodactylus (hepatopancreas, haemolymph andtestis) revealed the presence of four different ACE iso-forms, including two from the hepatopancreas [15].Correlatively, an ACE-like activity was demonstratedin membrane fractions from hepatopancreas and testis,as well as in haemocytes.In the present study, we present the molecular char-acterization of ACE from the crayfish testes. The cellu-lar expression of the enzyme was explored using in situhybridization and immunohistochemistry on testis andvas deferens sections. We have established that, in thetestis, the ACE RNAs are detected in germ cells at anearly stage of development (spermatogonia), whereasthe protein is mainly present in later stages (spermato-zoids). Conversely, in vas deferens, ACE immunoreac-tivity was found in the seminal fluid rather than incells. Accordingly, activity assays have demonstratedthat ACE activity shifts from the insoluble (i.e. mem-brane) fraction in testis to the soluble fraction in vasdeferens. To our knowledge, this is the first demonstra-tion of a dynamic maturation process of ACE in inver-tebrates, similar to that already described in mammals.ResultsMolecular characterization of A. leptodactylustesticular ACEIn our previous studies, the partial cDNA sequence ofthe region surrounding the testicular ACE active sitewas obtained [15]. To complement this cDNAsequence, 5¢-to3¢ RACE was performed. The exten-sion to the 3¢ end of the cDNA was realized success-fully, which was not the case for the 5¢ end.Consequently, new specific primers were designed,based on the ACE cDNA sequence reconstructed fromlobster (Homarus americanus) and crab (Carcinus mae-nas) expressed-sequence tags (ESTs) (see below). Fromthe alignment of these two cDNAs, we synthesized twodegenerate primers based on the 5¢ region of these twosequences. One of those primers (sequence provided inthe Experimental procedures) provided a satisfyingresult and the A. leptodactylus (Asl)-tACE cDNAsequence obtained has a length of 2.3 kb, with the firststop codon at 1.9 kb (accession number: FN178630).The deduced amino acid sequence comprised 635amino acids (Fig. 1). This protein had a predictedhydrophobic region of 26 amino acids near the C-ter-minus, suggesting that the enzyme is anchored to thecellular membrane. The predicted molecular weightwas 73.7 kDa, with an isoelectric point (pI) value ofAngiotensin-converting enzyme in crayfish testis J. Simunic et al.4728 FEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government worksFig. 1. Alignment of the predicted amino acid sequence of the A. leptodactylus testicular ACE with the D. melanogaster AnCE and humantesticular tACE. Important residues are indicated as: active site (bold underlined), zinc-binding residues (green), chloride-binding residues(orange for the first chloride ion and blue for the second one), sites of glycosylation (red), and cysteine residues forming disulfide bridges(boxed). The predicted transmembrane anchor is shown in underlined italics.J. Simunic et al. Angiotensin-converting enzyme in crayfish testisFEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government works 47296.24. The native protein could have a higher molecularweight because one N-glycosylation site was predictedat Asn288. This site is conserved when compared withthe D. melanogaster AnCE sequence. Eight cysteinylresidues, probably involved in the formation of fourdisulfide bonds, are conserved in both AnCE and Asl-tACE (Cys110-Cys118, Cys312-Cys330, Cys444-Cys590and Cys499-Cys517).The ACE active site motif HEXXH with two zinc-binding histidines (His343 and His347) was conservedin Asl-tACE and additional coordination was providedby the third zinc-binding ligand (Glu371), 24 aminoacid residues downstream, which is also conservedwhen compared with the Drosophila AnCE sequence.In silico reconstitution of ACE cDNA sequencesfrom three crustacean speciesThree new ACE sequences have been deduced byin silico methods. The Daphnia pulex ACE sequencewas obtained by the blast of the genome using the As-tacus sequence (GeneID: NCBI_GNO_452254),whereas Homarus and Carcinus sequences were recon-stituted from ESTs (accession numbers: BN001300 andBN001299, respectively). Comparison of Asl-tACEwith three other crustacean sequences shows that Asl-tACE has 79% sequence identity with Homarus, 75%with Carcinus and 53% with Daphnia (Fig. 2). Boththe Carcinus and Daphnia sequences contain a pre-dicted transmembrane region, whereas the EST assem-bly of Homarus is incomplete in its 3¢-terminus.Furthermore, all cysteines implicated in disulfidebridge formation are conserved, as well as the residuesinvolved in the coordination of chloride ions.A. leptodactylus testicular ACE expression andtissue localizationAs shown in Fig. 3, the A. leptodactylus testis is com-posed of three lobes. One vas deferens exits from eachof the two lateral lobes. During spermatogenesis,mature spermatozoids accumulate and are maintainedin vas deferens until fertilization, which results in adramatic size increase. In the resting period, the vasdeferens are atrophied and are barely visible. At thecellular level (Fig. 4A), the testis is composed of acinithat open into collector canals and finally into a vasdeferens. The acini contain mesodermal cells and sper-matogonia in different stages of development, as wellas mature spermatozoids.To provide more detailed information about whichcells in the testes are involved in both Asl-tACEmRNA synthesis and protein expression, we performedin situ hybridization and antibody staining. For in situhybridization, an antisense 158 bp long digoxigenin(DIG)-labelled cRNA probe was used. Tissue sections(5 lm) were prepared from testes taken from animalsin active spermatogenesis, as indicated by the presenceof a highly developed vas deferens filled with seminalfluid. The results show a specific hybridization ofmRNAs in cells that morphologically correspond tospermatogonia (Fig. 4B). A weak signal was alsodetected in some spermatozoids (Fig. 4C). No signalwas observed in negative controls performed using asense probe (not shown).To localize the expressed protein, labelling of 5 lmsections was performed using an antibody developedagainst a synthetic peptide designed from the Asl-tACE sequence obtained by cloning.The protein distribution is the inverse of the expres-sion pattern of mRNAs, namely the strongest signal isdisplayed on the cytoplasm membranes of spermato-zoids, whereas the spermatogonia exhibit very faintstaining only (Fig. 4D). The thickness of the stainingis the result of the morphology of spermatozoid inAstacus. Indeed, the spermatozoid is almost devoid ofcytoplasm, and the cell membrane is highly invagi-nated, forming crests that enter deeply into nucleo-plasm. The spermatozoid is surrounded by a periodicacid-Schiff positive casing of finely granular material,suggesting the presence of complex carbohydrates suchas in mucus [16,17]. This most likely has resulted inthe thick staining of spermatozoid membrane that weobserved on our preparations. Similarly, on vasdeferens sections, some staining was also present onthe outer membrane of spermatozoids, although thesignal was much weaker than in testis. By contrast, astrong signal was found in the seminal fluid itself(Fig. 4E, F). Control testis sections incubated withpreabsorbed antibodies failed to exhibit any signal(not shown).A. leptodactylus testicular ACE activity assaysTo determine the enzymatic activity of the testicularA. leptodactylus ACE, an activity assay with a radioac-tive substrate was performed (see Experimental proce-dures). We tested the activity in testes and in vasdeferens separately (Fig. 5), which were sampled fromanimals in the reproductive period and in genital rest.In testes sampled during spermatogenesis, a strongenzymatic activity was found in the insoluble fractionthat contains membranes, whereas the soluble fractionshowed very little enzymatic activity. In the vas defer-ens, the activity was as strong, but, interestingly, itwas present mostly in the soluble fraction. In animalsAngiotensin-converting enzyme in crayfish testis J. Simunic et al.4730 FEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government worksFig. 2. Alignment of A. leptodactylus testicular ACE with the H. americanus, C. maenas and D. pulex ACE sequences. Predicted signal pep-tides are shown in bold. Active sites are underlined, with zinc coordinating residues shown in green. The predicted transmembrane anchoris shown in underlined italics.J. Simunic et al. Angiotensin-converting enzyme in crayfish testisFEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government works 4731sampled during the resting period, no significant activ-ity was found in testes and, because the vas deferensalmost completely disappears during this period, thistissue was not tested.DiscussionThe present study aimed to provide a detailed charac-terization of the ACE isoform found in the testes ofthe crayfish A. leptodactylus (Asl-tACE). By perform-ing RT-PCR and 5¢-to3¢ RACE on testis RNAs,using degenerated primers deduced from crustaceanACEs, we were able to clone a 2.3 kb cDNA. This sizecorresponds to the values obtained previously bynorthern blotting (i.e. in the range 2–2.5 kb for theACE mRNA from the crayfish testis) [15].In silico translation of the cloned cDNA has shownthat the encoded protein of 635 amino acid residuesshares most of the common characteristics of ACEsfrom other invertebrate species: all the residues puta-tively important for the coordination of two chlorideions (Arg147, Tyr185, Trp445, Arg449 and Arg482)were conserved, as were the positions of the cysteinylresidues that are probably involved in the formation offour disulfide bonds.Based on the crystal structure of AnCE bound tocaptopril and lisinopril [18], we found that the residuesimplicated in inhibitor binding are highly conserved(Glu123, Thr127, Gln242, His313, Ala314, Asn336,Thr340, Glu344, Lys471, His473, Tyr480 and Tyr483),apart from Asp146 and Asp360 in Drosophila, whichare replaced by Glu123 (as in human ACEs) andAsn336, respectively.Comparison with the human tACE sequence showsthat Asl-tACE has conserved residues that are impli-cated in the coordination of two chloride ions. Thefirst chloride ion is coordinated by Arg147, Arg450and Trp446 and the second one is bound to Tyr186and Arg483.In addition to these features common to mostACEs, Asl-tACE displays some more specific ones.Especially, it appears to be a membrane-bound proteinbecause a potential hydrophobic transmembraneanchor comprising 26 amino acid residues was foundin the C-terminal region of the molecule. Conversely,most of the invertebrate ACEs described to date aresoluble proteins, with the exception of two Anophe-les gambiae ACEs (AnoACE7 and AnoACE9), whichappear to be membrane-bound enzymes [19]. However,these forms do exhibit two catalytic domains, such assomatic mammalian ACE, whereas Asl-tACE, with lessthan 700 residues, is likely to display only one catalyticdomain, such as mammalian gACE.Interestingly, data mining and reconstruction ofputative ACEs from other crustacean species, namelyESTs from C. maenas and H. americanus, and thewhole D. pulex genome, indicate the presence of a sim-ilar transmembrane C-terminal region, in addition toother conserved features (Fig. 2). Accordingly, anACE-like activity was reported in the membranes ofC. maenas gills, which may be easily solubilized bydetergent application [20]. At present, it is not possibleto speculate whether, in crustaceans, in contrast toother groups, ACE isoforms are always membrane-bound proteins because no genome has been sequencedfrom a crustacean species other than Daphnia.InA. le-ptodactylus, several different ACE isoforms have beenidentified [15] and it will prove very informative todetermine whether or not every isoform displays atransmembrane region. Knowing whether all isoformsin Astacus are membrane bound proteins is interestingbecause it raises questions about both the evolution ofACE in different groups of animals and the physiologi-cal significance of membrane bound isoform comparedto the soluble form, which was almost exclusivelyfound in other invertebrate groups.Subsequent to early studies conducted in the rat andpig [21,22], it is well established that ACE is present ingerm cells. This has been described for several insectspecies, including Drosophila [8]. Accordingly, ourin situ hybridization experiments peformed on testissections show that ACE mRNA is mainly present inspermatogonia (i.e. in the early stages of spermato-genesis), whereas only a small amount or even anabsence of RNA was detected in mature spermato-zoids, and no signal was ever observed in mesodermalcells or in vas deferens. Immunolocalization of theACE protein using a hapten-specific antibody revealeda very different distribution pattern; in the testes, theprotein appeared to be present on the external side ofthe cytoplasmic membrane of spermatozoids, butnot on spermatogonia. By contrast, in vas deferens,VasdeferensTestislobes1 cmFig. 3. Morphology of A. leptodactylus testis during spermatogene-sis. The testis is composed of three lobes. Two vasa deferentia arewell developed and contain mature spermatozoids.Angiotensin-converting enzyme in crayfish testis J. Simunic et al.4732 FEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government worksimmunoreactivity on germ cell membranes was muchweaker, with a strong signal being present in theseminal fluid.The fact that no ACE protein was detectable inearlier stages of spermatogenesis when correspondingRNAs are present suggests the existence of a transla-tional arrest, a phenomenon already known to occurin the expression of testicular ACE in mice [5]. On theother hand, in Astacus during spermatid differentia-tion, the nucleus undergoes several stages of reorgani-zation in which chromatin density and pattern ofdistribution change on several occasions. In the laststage, there is actually a decrease in density of nuclearmaterial [16].When the ACE activity was assayed in both solubleand insoluble (membrane) fractions from testis and vasdeferens homogenates, it was observed that maximalactivity is associated with membranes in testis, butwith soluble material in vas deferens. During genitalrest, where no spermatozoids are present in the germi-nal tract, no significant activity was detected in thetestis extract.Taken together, the results of in situ hybridization,immunohistochemistry and activity assays stronglysuggest a shift of the enzyme from the germ cell mem-brane to the seminal fluid when the spermatozoidsmigrate from the testes to the vas deferens during sper-matogenesis.AB C D E F Fig. 4. In situ hybridization and immunohistochemical localization of A. leptodactylus testicular ACE expression in testis and vas deferens.Morphology of testis during spermatogenesis: (A) mesodermal cells (mc), spermatogonia (g) and spermatozoids (spz). In situ hybridization:(B) strong mRNA signal in the spermatogonia (g); (C) weak ⁄ no signal in the spermatozoids (spz). Immunohistochemistry: (D) In the testis,staining is present on the membranes of mature spermatozoids (spz), whereas spermatogonia are unstained. (E, F) In the cross section ofvas deferens, staining is strong in the seminal fluid (sf), with weak staining also being present in the spermatozoid membranes (spz). Thewalls (w) of the vas deferens are not stained.J. Simunic et al. Angiotensin-converting enzyme in crayfish testisFEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government works 4733In mammals, gACE was shown to be anchored inthe cytoplasmic membrane of spermatozoids and to bereleased in the epididymal fluid during the transit ofthe sperm in the epididymis [23]. This cleavage involvesa serine protease (sheddase) [24]. Such a maturationprocess has never been described for ACE in animalgroups other than mammals until our present study,which clearly indicates the presence of a similar mecha-nism of protein ectodomain shedding in crayfish testis.The similarity between the maturation process ofcrustacean and mammalian ACE could imply the pres-ence of an unknown protease in crayfish testis, whichmay cleave the Asl-tACE from spermatozoid mem-brane; however, the cleavage site (-Arg-Leu-) identifiedin mammalian tACE [24] was not found in the Asl-tACE sequence.To date, the physiological role of testis ACE rem-ains a matter of debate. In mammals, experiments withAce ‘knockout’ mice have shown that ACE plays arole in fertilization because the absence of testicularACE leads to defects in sperm transport in oviducts aswell as in binding to zonae pellucidae, without modifi-cation of sperm morphology and counts [7]. Becausethere is no apparent ACE substrate involved in fertil-ization, the molecular mechanism for this effectremains unknown, with one possible explanation beingthat tACE could be involved in the distribution ofADAM3, a protein essential for sperm–zonae pelluci-dae interactions [25]. The results obtained in otherstudies [26] indicate that ACE could have a glycosyl-phosphatidylinositolase activity that is unrelated to itspeptidase active site, although this hypothesis isstrongly debated [27,28].In invertebrates, ACE has an important role in bothreproduction and development. In dipteran insects, ithas been reported that inhibition of ACE activityaffects different aspects of reproduction [29] and thatACE inhibition by dietary administration of inhibitorsreduces oviposition in A. stephensi female mosquitoes.In males of the same species, inhibitor feeding resultsin an 80% loss of fecundity, which is expressed as thereduction in the number of eggs laid by blood-fedfemales mated with ACE-inhibited males. It has beensuggested that Drosophila Ance, which is present insecretion vesicles in spermatocytes, may have a func-tion in the maturation of bioactive peptides duringspermatogenesis [8]. In the lepidopteran species L. oler-acea, it was shown that ACE is transferred from themale to the female during mating [12]. In addition,through activity assays including ACE inhibitors andHPLC analysis, ACE was demonstrated to be animportant protease among the peptide-degradingenzymes present in the female spermatophore ⁄ bursacopulatrix. Regarding its physiological function, it ishypothesized that ACE, along with other peptidasespresent in the spermatophore ⁄ bursa copulatrix, couldprovide dipeptides or amino acids that are necessaryfor different metabolic pathways. However, to date, noexperimental evidence is available to support thishypothesis. Such a hypothesis is unlikely in A. lepto-dactylus because the spermatozoids lack a flagellumand therefore are not mobile. Nevertheless, it cannotbe excluded that Asl-tACE may play a role in sperma-tozoid metabolism. Indeed, during mating, the malecrayfish deposits sperm near the openings of the femalegonoducts (i.e. at the base of the third periopods),using the two first pair of pleopods that are modifiedto copulatory appendices for guidance of the sperminto the female spermatheca, where it may be kept fora period of up to several months before fertilization.During this period, it is possible that Asl-tACE couldplay a role in metabolic pathways that are importantfor spermatozoid maintenance and survival.In conclusion, the results obtained in the presentstudy demonstrate that the ACE maturation process byprotein shedding is similar in crayfish and mammaliantestis. It remains to be elucidated whether, similarly,the function of the testicular ACE, which still remainsobscure, is conserved throughout animal evolution.Fig. 5. ACE activity in testes and vas deferens. Activity (c.p.m. permg) was determined by an in vitro assay using tritiated hippuryl-gly-cyl-glycine as substrate (see Experimental procedures). The barsrepresent the difference between c.p.m. values per mg of proteinof the [3H]hippurate obtained after incubation with and without10 lM captopril. Soluble (SOL) and insoluble (INSOL) fractions weretested in animals in active spermatogenesis and in resting period.Three experiments were conducted. P-values between the solubleand insoluble fractions, as calculated using Student’s t-test, were0.01 and 0.002 for the testis and vas deferens, respectively.Angiotensin-converting enzyme in crayfish testis J. Simunic et al.4734 FEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government worksExperimental proceduresAnimalsCrayfish A. leptodactylus were obtained from a commercialsupplier. They were kept in the laboratory in recirculatedfiltered water and fed twice a week with cat food pellets(Friskies pellets, Nestle Purina PetCare SAS, Rueil-Malmai-son, France). Before dissection, animals were anesthetizedin crushed ice and ice-cold water.Male crayfish exhibit only one spermatogenesis cycle peryear, from spring until late summer. There are no externalsigns to indicate whether the animal is in active spermato-genesis or in a resting period and, within a tank, the repro-ductive cycles are not fully synchronized. Therefore, thereproductive status of the animal could only be estimatedaccurately after dissection.Males with well-developed vas deferens filled with semi-nal fluid (Fig. 3) were considered to be in active spermato-genesis, whereas those with vas deferens reduced to a thinwhitish cord were considered as males in the resting period.Molecular characterizationReconstitution of three crustacean ACE cDNA fromEST and genomic dataA cDNA ACE of the green shore crab C. maenas wasreconstituted from four ESTs of multiple tissues [30].The Genbank accession numbers of those ESTs are:DW249183, DY657025, DN203050 and DV944439.An ACE cDNA of the lobster H. americanus was alsoreconstituted from five ESTs of multiple tissue (corre-sponding Genbank accession numbers: FF277412,CN854003, EH401278, CN854103 and EG949475).D. pulex sequence was found by blast of D. pulexgenome assembly (JGI-2006-09): scaffold_25 positionfrom 1 040 210 to 1 042 148. Multiple sequence align-ments were performed with clustalw2 [31] at theEuropean Bioinformatics Institute (http://www.ebi.ac.uk/Tools/clustalw2/index.html).Cloning the testicular ACE isoform of crayfishSpecific upper and lower primers were selected from thepartial Asl-tACE sequence previously obtained to deter-mine the 3 ¢- and 5¢ regions of the protein. Total RNAused for the amplification was isolated from spermato-genic testes by the SV Total RNA Isolation Systemaccording to the manufacturer’s instructions (Promega,Madison, WI, USA). The first strand cDNA for3¢-RACE was synthesized from 1 lg of total RNA usinga SMARTÔRACE cDNA Amplification Kit (Clontech,Mountain View, CA, USA) according to the manufac-turer’s instructions. The 3¢-RACE was performedbetween the upper primer T3U1 5¢-GGGACTTCTGTAATGGCAAAG-3¢ and the Universal Primer A Mix(UPM), provided with the kit. The PCR product wasdirectly sequenced using the T3U1 primer by CogenicsGenome Express (Cogenics, Meylan, France).To determine the 5¢ region of the mRNA, a degener-ate upper primer was synthesized in the 5¢ region ofACE sequences from H. americanus and C. maenas,deduced by EST assembly as described above. Thesequence of this primer was: 5¢-AGGARCTTCCTGMASGAGWTGGAC-3¢. The lower primer had thesequence: 5¢-GGTCTTGTTTGGGAAGGGCAGCTGTGC-3¢.PCR products were purified from 1.5% agarose gelsand subcloned into the transcription vector pGEM-TEasy vector (pGEMÒ-T Easy Vector System II; Pro-mega) and propagated in JM-109 Escherichia colibacteria. Recombinant plasmids were purified withWizardÒPlus SV Minipreps kit (Promega). Sequencingwas performed by Cogenics Genome Express using thedideoxy chain termination method. Another degenerateprimer was synthesized in the signal peptide region,although it failed to produce satisfactory results.In silico analysis of sequencesPossible glycosylation sites were identified with thenetnglyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/). The pI and molecular weight were cal-culated with the compute pI ⁄ Mw Tool (http://www.expasy.ch/tools/pi_tool.html).Prediction of signal peptides was performed usingthe signalp 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/) and prediction of transmembrane regionswas performed using the sosui engine, version 1.11(http://bp.nuap.nagoya-u.ac.jp/sosui/).Tissue preparation for in situ hybridization andimmunohistochemistryTestis and vas deferens tissue were dissected and immedi-ately immersed in Bouin’s fixative solution (75% picricacid, 20% formaldehyde, 5% acetic acid) for 24 h at roomtemperature. After dehydration in graded ethanol solutions(2 · 70%, 3 · 95%, 1 · 100%), the tissue was embedded inparaffin wax according to conventional histological proce-dures. Five micrometer sections were cut and alternatelymounted on poly(l-lysine)-coated slides (Polysine, Menzel-Glaser, Germany). The slides were deparaffinized in EZ-DeWax deparaffinization solution (InnoGenex, San Ramon,CA, USA), hydrated and used for in situ hybridization orimmunohistochemistry.J. Simunic et al. Angiotensin-converting enzyme in crayfish testisFEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government works 4735In situ hybridizationA 158 bp probe was obtained by amplification of the frag-ment spanning nucleotides 950–1107 of the Asl-tACEcDNA with the upper primer 5¢-GGGACTTCTGTAATGGCAAAG-3¢ and the lower primer 5¢-GAACCCTGGGTTGGCTCCGCTTCG-3¢. The cDNA was cloned in the tran-scription vector pGEM-T Easy vector (pGEMÒ-T EasyVector System II; Promega). After linearization of the tem-plate DNA, in vitro transcription reactions were carried outin the presence of DIG-UTP (DIG RNA labelling Kit;Roche Diagnostics, Meylan, France), with T7 and SP6polymerases for antisense and sense probes, respectively.The template was degraded with RNase-free DNase (RocheDiagnostics). The DIG-labelled RNA probes were purifiedby ethanol and sodium acetate precipitation and stored at)20 °C in 0.1% diethyl pyrocarbonate (Sigma-Aldrich, StLouis, MO, USA)-treated water until used for in situhybridization. All solutions and glassware were RNase-free.The sections were treated with 0.1% pepsin (Roche Diag-nostics) in 0.2 m HCl (37 °C for 10 min). Postfixation wasperformed by treating the sections with a fresh solution of2% paraformaldehyde in NaCl ⁄ Pi (10 mm sodium phos-phate, pH 7.4, 0.1 mm KCl, 0.8% NaCl) for 4 min, andimmersed in 1% hydroxylammonium hydrochloride inNaCl ⁄ Pi for 15 min. The sections were then dehydratedwith successive ethanol washings. For hybridization, ahumid chamber with 4 · SSC (1 · SSC: 150 mm NaCl,15 mm sodium citrate, pH 7.4) was prepared. DIG RNAprobes (antisense or sense) were diluted to a final concentra-tion of 50 ngÆl L)1in the hybridization mixture [50% form-amide, 10% dextran sulfate, 4 · SSC, 1 · Denhardt’ssolution (0.1% Ficoll 400, 0.1% polyvinylpyrrolidone, 0.1%BSA in water), 0.1% yeast tRNA and 0.1% salmon spermDNA], denatured at 70 °C for 5 min and cooled on ice.One hundred and twenty microlitres of this mix was placedon each tissue section. Sections were covered with coverslips and placed in the humid chamber at 45 °C overnight(16 h). Post-hybridization washes in consecutive stringencybaths of SSC (reducing concentrations; · 2, · 1, · 0.5,· 0.2, · 0.1; 20 min each bath) were used to remove non-specifically bound probes. The sections were treated withanti-DIG alkaline phosphatase-conjugated IgGs (RocheDiagnostics) for 30 min, washed and, finally, the phospha-tase substrate (nitro blue tetrazolium ⁄ 5-bromo-4-chloro-3-indolyl phosphate; Sigma-Aldrich) was added with 1 mmlevamisole to block possible endogenous alkaline phospha-tase. After satisfactory colour development ( 1 h), theslides were washed carefully in tap water and mounted withglycerol ⁄ gelatin (Sigma-Aldrich) preheated at 42 °C.ImmunohistochemistryThe sections were washed with NaCl ⁄ Pi ⁄ Triton 0.5% ⁄ goatserum (Sigma-Aldrich) 3% buffer before incubatingovernight with rabbit polyclonal antibodies raised againstthe synthetic peptide RENYGEEHVSRRGP, locatedbetween R217and P230of the Asl-tACE sequence. Duringsynthesis, the R217-P230peptide was extended at the C-ter-minus by a cysteine residue to facilitate coupling to keyholelimpet haemocyanin. The antibodies were produced in tworabbits by GenScript Corporation (Piscataway, NJ, USA).Negative controls were performed by incubating the sec-tions with antiserum adsorbed with R217-P230peptide.As secondary antibody, Alexa Fluor 568 goat anti-rabbitIgG (H+L) (Molecular Probes, Carlsbad, CA, USA) wasused. Analyses were performed on a confocal laser-scanningmicroscope (TCS4D confocal imaging system; Leica,Heidelberg, Germany) with an argon-krypton ion laser.They were scanned sequentially at an excitation wavelengthof 568 nm. A series of confocal sections (thickness in therange 0.1–2 lm) was collected for each specimen. Focal ser-ies were then processed to produce single composite imagesor montages, combining high spatial resolution and highfield depth (nih image, version 1.63; NIH Image, BethesdaMD, USA). Micrographs were processed and assembledwith Adobe photoshop 8.0 (Adobe Systems Inc., San Jose,CA).ACE activity assaysTestis and vas deferens tissue from males in active sper-matogenesis were dissected out and weighed. One hundredmilligrams of each tissue were prepared separately. Afterhomogenization in 700 lL of assay buffer (50 mmHEPES-HCl buffer with 300 mm NaCl, pH 8.3), thehomogenate was centrifuged (9200 g for 20 min at 4 °C).The Asl-tACE activity was tested in the supernatant (solu-ble fraction) and in the pellet which was resuspended in600 lL of assay buffer (insoluble fraction). The proteincontent of each fraction was estimated by the Bradfordmethod using the Bio-Rad Protein Assay reagent(Bio-Rad Laboratories GmbH, Muenchen, Germany) withBSA as standard. Activity assays were performed inaccordance with a previously described protocol [15].Briefly, the enzyme activity was determined by incubatingtissue samples with the ACE radiolabelled substrate[phenyl-4(n)-3H-hippuryl-glycyl-glycine (282 mCiÆmmol)1;Amersham, Little Chalfont, UK)].3H-labelled hippurate,the product of ACE hydrolysis, was separated by ethylacetate extraction and the radioactivity was assayed for2 min with a b-IV scintillation counter (Kontron Instru-ments, Watford, UK) to obtain the ‘total c.p.m.’ value.To discriminate ACE activity from other peptidase activi-ties, a reaction assay was performed in the same condi-tions, except that 10 lm captopril, a specific ACEinhibitor, was added to the tube, giving the ‘captoprilc.p.m.’ value. ACE activity was calculated as: (total c.p.m.– captopril c.p.m.) ⁄ mg protein. Each data point wasassayed in quadruplicate.Angiotensin-converting enzyme in crayfish testis J. Simunic et al.4736 FEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government works[...]... enzyme (ANCE), suggesting a role for the peptide-processing enzyme in seminal fluid J Exp Biol 210, 3601–3606 Vandingenen A, Hens K, Baggerman G, Macours N, Schoofs L, De Loof A & Huybrechts R (2002) Isolation and characterization of an angiotensin converting enzyme substrate from vitellogenic ovaries of Neobellieria bullata Peptides 23, 1853–1863 Kamech N, Simunic J, Franklin SJ, Francis S, Tabitsika M... Franke FE (2003) Isoforms of angiotensin I-converting enzyme in the development and differentiation of human testis and epididymis Andrologia 35, 32–43 7 Hagaman JR, Moyer JS, Bachman ES, Sibony M, Magyar PL, Welch JE, Smithies O, Krege JH & O’Brien DA (1998) Angiotensin-converting enzyme and male fertility Proc Natl Acad Sci USA 95, 2552– 2557 8 Hurst D, Rylett CM, Isaac RE & Shirras AD (2003) The. .. (2003) Angiotensin I-converting enzyme (ACE) activity of the tomato moth, Lacanobia oleracea: changes in levels of activity during development and after copulation suggest roles during metamorphosis and reproduction Insect Biochem Mol Biol 33, 989–998 Rylett CM, Walker MJ, Howell GJ, Shirras AD & Isaac RE (2007) Male accessory glands of Drosophila melanogaster make a secreted angiotensin I-converting enzyme. ..J Simunic et al Angiotensin-converting enzyme in crayfish testis Acknowledgements We thank Dr Lawrence Dinan for his critical reading and correction of the manuscript 12 References 1 Kumar RS, Thekkumkara TJ & Sen GC (1991) The mRNAs encoding the two angiotensin-converting isozymes are transcribed from the same gene by a tissuespecific choice of alternative transcription initiation sites J Biol... Evidence for an angiotensin-converting enzyme (ACE) polymorphism in the crayfish Astacus leptodactylus Peptides 28, 1368–1374 Moses MJ (1961) Spermiogenesis in the crayfish (Procambarus clarkii) II Description of stages J Biophys Biochem Cytol 10, 301–333 Anderson WA & Ellis RA (1967) Cytodifferentiation of the crayfish spermatozoon: acrosome formation, transformation of mitochondria and development of. .. Gatti JL (2005) Shedding of the germi- FEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS No claim to original French government works 4737 Angiotensin-converting enzyme in crayfish testis nal angiotensin I-converting enzyme (gACE) involves a serine protease and is activated by epididymal fluid Biol Reprod 73, 881–890 25 Yamaguchi R, Yamagata K, Ikawa M, Moss SB & Okabe M (2006) Aberrant... distribution of ADAM3 in sperm from both angiotensin-converting enzyme (Ace)- and calmegin (Clgn)-deficient mice Biol Reprod 75, 760–766 26 Kondoh G et al (2005) Angiotensin-converting enzyme is a GPI-anchored protein releasing factor crucial for fertilization Nat Med 11, 160–166 27 Leisle L, Parkin ET, Turner AJ & Hooper NM (2005) Angiotensin-converting enzyme as a GPIase: a critical reevaluation Nat Med... Expression of angiotensin-converting enzyme- related carboxydipeptidases in the larvae of four species of fly Insect Biochem Mol Biol 27, 451–460 11 Ekbote U, Coates D & Isaac RE (1999) A mosquito (Anopheles stephensi) angiotensin I-converting enzyme 13 14 15 16 17 18 19 20 21 22 23 24 (ACE) is induced by a blood meal and accumulates in the developing ovary FEBS Lett 455, 219–222 Ekbote UV, Weaver RJ & Isaac... Angiotensin-converting enzyme- like activity in crab gills and its putative role in degradation of crustacean hyperglycemic hormone Arch Insect Biochem Physiol 68, 171–180 Yotsumoto H, Sato S & Shibuya M (1984) Localization of angiotensin converting enzyme (dipeptidyl carboxypeptidase) in swine sperm by immunofluorescence Life Sci 35, 1257–1261 Strittmatter SM & Snyder SH (1984) Angiotensin-converting enzyme in the male... et al 28 Fuchs S et al (2005) Male fertility is dependent on dipeptidase activity of testis ACE Nat Med 11, 1140– 1142; author reply 1142-3 29 Isaac RE et al (2007) Angiotensin-converting enzyme as a target for the development of novel insect growth regulators Peptides 28, 153–162 30 Towle DW & Smith CM (2006) Gene discovery in Carcinus maenas and Homarus americanus via expressed sequence tags Integr . Characterization of a membrane-bound angiotensin-converting enzyme isoform in crayfish testis and evidence for its release into the seminal fluid Juraj Simunic, Daniel Soyez and Ne ´ dia Kamech Equipe. 2009) doi:10.1111/j.1742-4658.2009.07169.x In the present study, an isoform of angiotensin-converting enzyme was characterized from the testis of a decapod crustacean, the crayfish Asta- cus leptodactylus. Angiotensin-converting enzyme. cDNA, obtained by 3¢-to5¢ RACE of testis RNAs, codes for a predicted one-domain protein similar to the mammalian germinal isoform of angiotensin-converting enzyme. All amino acid residues involved
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