Báo cáo khoa học: Characterization of a membrane-bound angiotensin-converting enzyme isoform in crayfish testis and evidence for its release into the seminal fluid

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 Biogene` se des Signaux Peptidiques, ER3, Universite´ Pierre et Marie Curie, Paris, France

Keywords angiotensin-converting enzyme; crayfish; Crustacea; spermatogenesis; testis

Correspondence N. Kamech, Equipe Biogene` se des Signaux Peptidiques, ER3, Universite´ Pierre et Marie Curie, 7 Quai Saint Bernard, 75251 Paris, Cedex 05, France Fax: +33 1 44 27 23 61 Tel: +33 1 44 27 22 58 E-mail: nedia.kamech@upmc.fr

(Received 4 May 2009, revised 18 June 2009, accepted 25 June 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¢- to 5¢ 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 in enzyme activity are highly conserved, and a potential C-terminus transmembrane anchor may be predicted from the sequence. Comparison of this testicular isoform with angiotensin-converting enzyme from other crustaceans, namely Carci- nus maenas, Homarus americanus (both reconstituted for this study from expressed-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 enzyme is predominantly a soluble protein. In situ hybridization and immunohisto- chemistry performed on testis sections show that angiotensin-converting enzyme mRNA is mainly localized in spermatogonias, whereas protein is present in spermatozoids. By contrast, in vas deferens, immunoreactivity is detected in the seminal fluid rather than in germ cells. Accordingly, angio- tensin-converting enzyme activity assays of testis and vas deferens extracts demonstrate that the enzyme is present in the membrane fraction in testis, but in the soluble fraction in vas deferens. Taken together, the results obtained in the present study suggest that, during the migration of spermatozoids from testis to vas deferens, the enzyme is cleaved from the membrane of the germ cells and released into the seminal fluid. To our knowledge, this present study is the first to report such a maturation process for angiotensin-converting enzyme outside of mammals.

Introduction

Abbreviations ACE, angiotensin-converting enzyme; Asl, Astacus leptodactylus; DIG, digoxigenin; EST, expressed-sequence tag; gACE, germinal isoform of angiotensin-converting enzyme; tACE, testicular isoform of angiotensin-converting enzyme.

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Angiotensin-converting enzyme (ACE; dipeptidyl car- boxypeptidase; EC 3.4.15.1) is an enzyme that belongs to the family of M2 peptidases with a zinc chelator motive HEXXH-23(24)-E. Substrate hydrolysis in ACE is activated by chloride ions, which is a unique feature 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 under [1,2]. The the control of tissue-specific promotors

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Angiotensin-converting enzyme in crayfish testis

In previous studies, RT-PCR and northern blotting on RNAs from several tissues of the crayfish Asta- cus leptodactylus (hepatopancreas, haemolymph and testis) revealed the presence of four different ACE iso- including two from the hepatopancreas [15]. forms, Correlatively, an ACE-like activity was demonstrated in membrane fractions from hepatopancreas and testis, as well as in haemocytes.

somatic isoform exhibits two catalytic domains and is present in numerous tissues, such as on the surface of endothelial cells in the lung, myocardium, liver, intestine and testis, as well as in the epithelial cells of the kidney and 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 produce angiotensin II, a powerful vasosuppressor. It also cleaves the vasodilatator peptide bradikinin, and thus contributes to the augmentation of blood pressure.

On the other hand, the role of the testicular isoform (tACE), also called germinal ACE (gACE), is not so clear. In mice, the testis ACE protein is first detected in step 10 spermatids, whereas ACE mRNA is first detected in developmentally younger cells, the pachy- tene spermatocytes, implying a delay in ACE transla- tion [5]. A similar phenomenon was described in human testes, with mid-pachytene spermatocytes expressing the mRNA and stage III spermatids con- taining the protein, which corresponds to a delay by one germ cycle [6].

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 situ hybridization and immunohistochemistry on testis and vas deferens sections. We have established that, in the testis, the ACE RNAs are detected in germ cells at an early stage of development (spermatogonia), whereas the protein is mainly present in later stages (spermato- zoids). Conversely, in vas deferens, ACE immunoreac- tivity was found in the seminal fluid rather than in cells. Accordingly, activity assays have demonstrated that ACE activity shifts from the insoluble (i.e. mem- brane) fraction in testis to the soluble fraction in vas deferens. 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.

The physiological role of tACE is still a matter of debate, but numerous studies point to the importance of this enzyme in male and female reproduction; for example, mice males with a ‘knockout’ for the Ace gene show extremely reduced fertility [7].

Results

Molecular characterization of A. leptodactylus testicular ACE

this

for case the 5¢

[11]. Similar

Analogues of ACE have also been identified in many invertebrate species, most notably insects, and have been shown to play a major role in reproduction. Indeed, as in mice, males of Drosophila with a knock- for Ace show a dramatic decrease in fertility out because the developing spermatozoids cannot complete the phase of individualization and demonstrate an abnormal 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 synthesis of ACE in the testes, but not in the ovaries [10]. By in female Anopheles stephensi, a dramatic contrast, increase in ACE activity is observed in the ovary after a blood meal, with a maximum just prior to egg-lay- ing, and ACE is completely transferred to newly-laid results were obtained from the eggs tomato moth Lacanobia oleracea [12]. Recently, such a transfer of ACE from males to females was reported to take place during copulation in Drosophila melanog- aster [13]. Even if

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In our previous studies, the partial cDNA sequence of the region surrounding the testicular ACE active site was obtained [15]. To complement cDNA sequence, 5¢- to 3¢ RACE was performed. The exten- sion to the 3¢ end of the cDNA was realized success- fully, which was not end. the Consequently, new specific primers were designed, based on the ACE cDNA sequence reconstructed from lobster (Homarus americanus) and crab (Carcinus mae- nas) expressed-sequence tags (ESTs) (see below). From the alignment of these two cDNAs, we synthesized two degenerate primers based on the 5¢ region of these two sequences. One of those primers (sequence provided in the Experimental procedures) provided a satisfying result and the A. leptodactylus (Asl)-tACE cDNA sequence obtained has a length of 2.3 kb, with the first stop codon at 1.9 kb (accession number: FN178630). The deduced amino acid sequence comprised 635 amino acids (Fig. 1). This protein had a predicted hydrophobic region of 26 amino acids near the C-ter- minus, suggesting that the enzyme is anchored to the cellular membrane. The predicted molecular weight was 73.7 kDa, with an isoelectric point (pI) value of the implication of ACE in reproduction appears to be well established, the possible substrates involved remain to be determined. To date, the only substrate identified in vivo in invertebrates is an 11-mer peptide (Neb-ODAIF) isolated from the ovaries of the fly Neobellieria bullata [14].

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Fig. 1. Alignment of the predicted amino acid sequence of the A. leptodactylus testicular ACE with the D. melanogaster AnCE and human testicular 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.

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6.24. The native protein could have a higher molecular weight because one N-glycosylation site was predicted at Asn288. This site is conserved when compared with the D. melanogaster AnCE sequence. Eight cysteinyl residues, probably involved in the formation of four disulfide bonds, are conserved in both AnCE and Asl- tACE (Cys110-Cys118, Cys312-Cys330, Cys444-Cys590 and Cys499-Cys517).

in situ hybridization and antibody staining. For in situ hybridization, an antisense 158 bp long digoxigenin (DIG)-labelled cRNA probe was used. Tissue sections (5 lm) were prepared from testes taken from animals in active spermatogenesis, as indicated by the presence of a highly developed vas deferens filled with seminal fluid. The results show a specific hybridization of mRNAs in cells that morphologically correspond to spermatogonia (Fig. 4B). A weak signal was also detected in some spermatozoids (Fig. 4C). No signal was observed in negative controls performed using a sense probe (not shown).

The ACE active site motif HEXXH with two zinc- binding histidines (His343 and His347) was conserved in Asl-tACE and additional coordination was provided by the third zinc-binding ligand (Glu371), 24 amino acid residues downstream, which is also conserved when compared with the Drosophila AnCE sequence.

To localize the expressed protein, labelling of 5 lm sections was performed using an antibody developed against a synthetic peptide designed from the Asl- tACE sequence obtained by cloning.

In silico reconstitution of ACE cDNA sequences from three crustacean species

sequence

is cysteines

Three new ACE sequences have been deduced by in silico methods. The Daphnia pulex ACE sequence was obtained by the blast of the genome using the As- tacus (GeneID: NCBI_GNO_452254), whereas Homarus and Carcinus sequences were recon- stituted from ESTs (accession numbers: BN001300 and respectively). Comparison of Asl-tACE BN001299, with three other crustacean sequences shows that Asl- tACE has 79% sequence identity with Homarus, 75% with Carcinus and 53% with Daphnia (Fig. 2). Both the Carcinus and Daphnia sequences contain a pre- dicted transmembrane region, whereas the EST assem- bly of Homarus incomplete in its 3¢-terminus. Furthermore, all implicated in disulfide bridge formation are conserved, as well as the residues involved in the coordination of chloride ions.

A. leptodactylus testicular ACE expression and tissue localization The protein distribution is the inverse of the expres- sion pattern of mRNAs, namely the strongest signal is displayed on the cytoplasm membranes of spermato- zoids, whereas the spermatogonia exhibit very faint staining only (Fig. 4D). The thickness of the staining is the result of the morphology of spermatozoid in Astacus. Indeed, the spermatozoid is almost devoid of cytoplasm, and the cell membrane is highly invagi- nated, forming crests that enter deeply into nucleo- plasm. The spermatozoid is surrounded by a periodic acid-Schiff positive casing of finely granular material, suggesting the presence of complex carbohydrates such as in mucus [16,17]. This most likely has resulted in the thick staining of spermatozoid membrane that we observed on our preparations. Similarly, on vas deferens sections, some staining was also present on the outer membrane of spermatozoids, although the signal was much weaker than in testis. By contrast, a strong signal was found in the seminal fluid itself (Fig. 4E, F). Control testis sections incubated with preabsorbed antibodies failed to exhibit any signal (not shown).

A. leptodactylus testicular ACE activity assays the two lateral

As shown in Fig. 3, the A. leptodactylus testis is com- posed of three lobes. One vas deferens exits from each lobes. During spermatogenesis, of mature spermatozoids accumulate and are maintained in vas deferens until fertilization, which results in a dramatic size increase. In the resting period, the vas deferens are atrophied and are barely visible. At the cellular level (Fig. 4A), the testis is composed of acini that open into collector canals and finally into a vas deferens. The acini contain mesodermal cells and sper- matogonia in different stages of development, as well as mature spermatozoids.

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interestingly, To determine the enzymatic activity of the testicular A. leptodactylus ACE, an activity assay with a radioac- tive substrate was performed (see Experimental proce- dures). We tested the activity in testes and in vas deferens separately (Fig. 5), which were sampled from animals in the reproductive period and in genital rest. In testes sampled during spermatogenesis, a strong enzymatic activity was found in the insoluble fraction that contains membranes, whereas the soluble fraction showed very little enzymatic activity. In the vas defer- ens, the activity was as strong, but, it was present mostly in the soluble fraction. In animals To provide more detailed information about which cells in the testes are involved in both Asl-tACE mRNA synthesis and protein expression, we performed

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Fig. 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 anchor is shown in underlined italics.

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Vas deferens

Testis lobes

1 cm

Fig. 3. Morphology of A. leptodactylus testis during spermatogene- sis. The testis is composed of three lobes. Two vasa deferentia are well developed and contain mature spermatozoids.

a potential hydrophobic

In addition to these features common to most ACEs, Asl-tACE displays some more specific ones. Especially, it appears to be a membrane-bound protein because transmembrane anchor comprising 26 amino acid residues was found in the C-terminal region of the molecule. Conversely, most of the invertebrate ACEs described to date are soluble proteins, with the exception of two Anophe- les gambiae ACEs (AnoACE7 and AnoACE9), which appear to be membrane-bound enzymes [19]. However, these forms do exhibit two catalytic domains, such as somatic mammalian ACE, whereas Asl-tACE, with less than 700 residues, is likely to display only one catalytic domain, such as mammalian gACE.

sampled during the resting period, no significant activ- ity was found in testes and, because the vas deferens almost completely disappears during this period, this tissue was not tested.

Discussion

in crustaceans,

The present study aimed to provide a detailed charac- terization of the ACE isoform found in the testes of the crayfish A. leptodactylus (Asl-tACE). By perform- ing RT-PCR and 5¢- to 3¢ RACE on testis RNAs, using degenerated primers deduced from crustacean ACEs, we were able to clone a 2.3 kb cDNA. This size to the values obtained previously by corresponds northern blotting (i.e. in the range 2–2.5 kb for the ACE mRNA from the crayfish testis) [15].

Interestingly, data mining and reconstruction of putative ACEs from other crustacean species, namely ESTs from C. maenas and H. americanus, and the whole D. pulex genome, indicate the presence of a sim- ilar transmembrane C-terminal region, in addition to other conserved features (Fig. 2). Accordingly, an ACE-like activity was reported in the membranes of C. maenas gills, which may be easily solubilized by detergent application [20]. At present, it is not possible to speculate whether, in contrast to other groups, ACE isoforms are always membrane- bound proteins because no genome has been sequenced from a crustacean species other than Daphnia. In A. le- ptodactylus, several different ACE isoforms have been identified [15] and it will prove very informative to determine whether or not every isoform displays a transmembrane region. Knowing whether all isoforms in Astacus are membrane bound proteins is interesting because it raises questions about both the evolution of ACE in different groups of animals and the physiologi- cal significance of membrane bound isoform compared to the soluble form, which was almost exclusively found in other invertebrate groups.

In silico translation of the cloned cDNA has shown that the encoded protein of 635 amino acid residues shares most of the common characteristics of ACEs from other invertebrate species: all the residues puta- tively important for the coordination of two chloride ions (Arg147, Tyr185, Trp445, Arg449 and Arg482) were conserved, as were the positions of the cysteinyl residues that are probably involved in the formation of four disulfide bonds.

Based on the crystal structure of AnCE bound to captopril and lisinopril [18], we found that the residues implicated 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, which are replaced by Glu123 (as in human ACEs) and Asn336, respectively.

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Comparison with the human tACE sequence shows that Asl-tACE has conserved residues that are impli- cated in the coordination of two chloride ions. The first chloride ion is coordinated by Arg147, Arg450 and Trp446 and the second one is bound to Tyr186 and Arg483. Subsequent to early studies conducted in the rat and pig [21,22], it is well established that ACE is present in germ cells. This has been described for several insect including Drosophila [8]. Accordingly, our species, in situ hybridization experiments peformed on testis sections show that ACE mRNA is mainly present in spermatogonia (i.e. in the early stages of spermato- genesis), whereas only a small amount or even an absence of RNA was detected in mature spermato- zoids, and no signal was ever observed in mesodermal cells or in vas deferens. Immunolocalization of the ACE protein using a hapten-specific antibody revealed a very different distribution pattern; in the testes, the protein appeared to be present on the external side of spermatozoids, but the cytoplasmic membrane of in vas deferens, not on spermatogonia. By contrast,

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C

B

A

D

F

E

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 of vas deferens, staining is strong in the seminal fluid (sf), with weak staining also being present in the spermatozoid membranes (spz). The walls (w) of the vas deferens are not stained.

immunoreactivity on germ cell membranes was much weaker, with a strong signal being present in the seminal fluid.

When the ACE activity was assayed in both soluble and insoluble (membrane) fractions from testis and vas deferens homogenates, it was observed that maximal activity is associated with membranes in testis, but with soluble material in vas deferens. During genital rest, where no spermatozoids are present in the germi- nal tract, no significant activity was detected in the testis extract.

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The fact that no ACE protein was detectable in earlier stages of spermatogenesis when corresponding RNAs are present suggests the existence of a transla- tional arrest, a phenomenon already known to occur in the expression of testicular ACE in mice [5]. On the in Astacus during spermatid differentia- other hand, tion, the nucleus undergoes several stages of reorgani- zation in which chromatin density and pattern of distribution change on several occasions. In the last stage, there is actually a decrease in density of nuclear material [16]. Taken together, the results of in situ hybridization, immunohistochemistry and activity assays strongly suggest a shift of the enzyme from the germ cell mem- brane to the seminal fluid when the spermatozoids migrate from the testes to the vas deferens during sper- matogenesis.

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Fig. 5. ACE activity in testes and vas deferens. Activity (c.p.m. per mg) was determined by an in vitro assay using tritiated hippuryl-gly- cyl-glycine as substrate (see Experimental procedures). The bars represent the difference between c.p.m. values per mg of protein of the [3H]hippurate obtained after incubation with and without 10 lM captopril. Soluble (SOL) and insoluble (INSOL) fractions were tested in animals in active spermatogenesis and in resting period. Three experiments were conducted. P-values between the soluble and insoluble fractions, as calculated using Student’s t-test, were 0.01 and 0.002 for the testis and vas deferens, respectively.

site, although this hypothesis that tACE could be involved in the distribution of ADAM3, a protein essential for sperm–zonae pelluci- dae interactions [25]. The results obtained in other studies [26] indicate that ACE could have a glycosyl- phosphatidylinositolase activity that is unrelated to its peptidase active is strongly debated [27,28].

among

In mammals, gACE was shown to be anchored in the cytoplasmic membrane of spermatozoids and to be released in the epididymal fluid during the transit of the sperm in the epididymis [23]. This cleavage involves a serine protease (sheddase) [24]. Such a maturation process has never been described for ACE in animal groups 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 of crustacean and mammalian ACE could imply the pres- ence of an unknown protease in crayfish testis, which may cleave the Asl-tACE from spermatozoid mem- brane; however, the cleavage site (-Arg-Leu-) identified in mammalian tACE [24] was not found in the Asl- tACE sequence.

In invertebrates, ACE has an important role in both reproduction and development. In dipteran insects, it has been reported that inhibition of ACE activity affects different aspects of reproduction [29] and that ACE inhibition by dietary administration of inhibitors reduces oviposition in A. stephensi female mosquitoes. In males of the same species, inhibitor feeding results in an 80% loss of fecundity, which is expressed as the reduction in the number of eggs laid by blood-fed females mated with ACE-inhibited males. It has been suggested that Drosophila Ance, which is present in secretion vesicles in spermatocytes, may have a func- tion in the maturation of bioactive peptides during spermatogenesis [8]. In the lepidopteran species L. oler- acea, it was shown that ACE is transferred from the male to the female during mating [12]. In addition, through activity assays including ACE inhibitors and HPLC analysis, ACE was demonstrated to be an the peptide-degrading important protease enzymes present in the female spermatophore ⁄ bursa copulatrix. Regarding its physiological function, it is hypothesized that ACE, along with other peptidases present in the spermatophore ⁄ bursa copulatrix, could provide dipeptides or amino acids that are necessary for different metabolic pathways. However, to date, no experimental evidence is available to support this hypothesis. Such a hypothesis is unlikely in A. lepto- dactylus because the spermatozoids lack a flagellum and therefore are not mobile. Nevertheless, it cannot be excluded that Asl-tACE may play a role in sperma- tozoid metabolism. Indeed, during mating, the male crayfish deposits sperm near the openings of the female gonoducts (i.e. at the base of the third periopods), using the two first pair of pleopods that are modified to copulatory appendices for guidance of the sperm into the female spermatheca, where it may be kept for a period of up to several months before fertilization. During this period, it is possible that Asl-tACE could play a role in metabolic pathways that are important for spermatozoid maintenance and survival.

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the molecular mechanism for this To date, the physiological role of testis ACE rem- ains a matter of debate. In mammals, experiments with Ace ‘knockout’ mice have shown that ACE plays a role in fertilization because the absence of testicular ACE leads to defects in sperm transport in oviducts as well as in binding to zonae pellucidae, without modifi- cation of sperm morphology and counts [7]. Because there is no apparent ACE substrate involved in fertil- ization, effect remains unknown, with one possible explanation being In conclusion, the results obtained in the present study demonstrate that the ACE maturation process by protein shedding is similar in crayfish and mammalian testis. It remains to be elucidated whether, similarly, the function of the testicular ACE, which still remains obscure, is conserved throughout animal evolution.

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Experimental procedures

Animals

Crayfish A. leptodactylus were obtained from a commercial supplier. They were kept in the laboratory in recirculated filtered water and fed twice a week with cat food pellets (Friskies pellets, Nestle Purina PetCare SAS, Rueil-Malmai- son, France). Before dissection, animals were anesthetized in crushed ice and ice-cold water.

instructions. The 3¢-RACE was performed turer’s between the upper primer T3U1 5¢-GGGACTTCTG TAATGGCAAAG-3¢ and the Universal Primer A Mix (UPM), provided with the kit. The PCR product was directly sequenced using the T3U1 primer by Cogenics Genome Express (Cogenics, Meylan, France).

Male crayfish exhibit only one spermatogenesis cycle per year, from spring until late summer. There are no external signs 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, the reproductive status of the animal could only be estimated accurately 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 thin whitish cord were considered as males in the resting period.

To determine the 5¢ region of the mRNA, a degener- ate upper primer was synthesized in the 5¢ region of ACE sequences from H. americanus and C. maenas, deduced by EST assembly as described above. The sequence of this primer was: 5¢-AGGARCTTCCTG MASGAGWTGGAC-3¢. The lower primer had the sequence: 5¢-GGTCTTGTTTGGGAAGGGCAGCTG TGC-3¢.

Molecular characterization PCR products were purified from 1.5% agarose gels and subcloned into the transcription vector pGEM-T Easy vector (pGEM(cid:3)-T Easy Vector System II; Pro- mega) and propagated in JM-109 Escherichia coli bacteria. Recombinant plasmids were purified with Wizard(cid:3) Plus SV Minipreps kit (Promega). Sequencing was performed by Cogenics Genome Express using the dideoxy chain termination method. Another degenerate primer was synthesized in the signal peptide region, although it failed to produce satisfactory results.

In silico analysis of sequences

Possible glycosylation sites were identified with the netnglyc 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 using the signalp 3.0 Server (http://www.cbs.dtu.dk/services/ SignalP/) and prediction of transmembrane regions was performed using the sosui engine, version 1.11 (http://bp.nuap.nagoya-u.ac.jp/sosui/).

Institute Reconstitution of three crustacean ACE cDNA from EST and genomic data A cDNA ACE of the green shore crab C. maenas was reconstituted 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 also reconstituted 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. pulex genome assembly (JGI-2006-09): scaffold_25 position from 1 040 210 to 1 042 148. Multiple sequence align- ments were performed with clustalw2 [31] at the (http://www.ebi. European Bioinformatics ac.uk/Tools/clustalw2/index.html). Tissue preparation for in situ hybridization and immunohistochemistry

Cloning the testicular ACE isoform of crayfish

Testis and vas deferens tissue were dissected and immedi- ately immersed in Bouin’s fixative solution (75% picric acid, 20% formaldehyde, 5% acetic acid) for 24 h at room temperature. After dehydration in graded ethanol solutions (2 · 70%, 3 · 95%, 1 · 100%), the tissue was embedded in paraffin wax according to conventional histological proce- dures. Five micrometer sections were cut and alternately mounted 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 or immunohistochemistry.

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Specific upper and lower primers were selected from the partial Asl-tACE sequence previously obtained to deter- mine the 3¢- and 5¢ regions of the protein. Total RNA used for the amplification was isolated from spermato- genic testes by the SV Total RNA Isolation System according to the manufacturer’s instructions (Promega, Madison, WI, USA). The first strand cDNA for 3¢-RACE was synthesized from 1 lg of total RNA using a SMART(cid:2) RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA) according to the manufac-

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synthetic peptide RENYGEEHVSRRGP,

spanning nucleotides 950–1107 of

overnight with rabbit polyclonal antibodies raised against the located between R217 and P230 of the Asl-tACE sequence. During synthesis, the R217-P230 peptide was extended at the C-ter- minus by a cysteine residue to facilitate coupling to keyhole limpet haemocyanin. The antibodies were produced in two rabbits by GenScript Corporation (Piscataway, NJ, USA).

Negative controls were performed by incubating the sec-

tions with antiserum adsorbed with R217-P230 peptide.

(TCS4D confocal

As secondary antibody, Alexa Fluor 568 goat anti-rabbit IgG (H+L) (Molecular Probes, Carlsbad, CA, USA) was used. Analyses were performed on a confocal laser-scanning microscope imaging system; Leica, Heidelberg, Germany) with an argon-krypton ion laser. They were scanned sequentially at an excitation wavelength of 568 nm. A series of confocal sections (thickness in the range 0.1–2 lm) was collected for each specimen. Focal ser- ies were then processed to produce single composite images or montages, combining high spatial resolution and high field depth (nih image, version 1.63; NIH Image, Bethesda MD, USA). Micrographs were processed and assembled with Adobe photoshop 8.0 (Adobe Systems Inc., San Jose, CA).

In situ hybridization

assay buffer

using

the Bio-Rad Protein Assay

((cid:2) 1 h),

A 158 bp probe was obtained by amplification of the frag- the Asl-tACE ment cDNA with the upper primer 5¢-GGGACTTCTGTAATG GCAAAG-3¢ and the lower primer 5¢-GAACCCTGGGTT GGCTCCGCTTCG-3¢. The cDNA was cloned in the tran- scription vector pGEM-T Easy vector (pGEM(cid:3)-T Easy Vector System II; Promega). After linearization of the tem- plate DNA, in vitro transcription reactions were carried out in the presence of DIG-UTP (DIG RNA labelling Kit; Roche Diagnostics, Meylan, France), with T7 and SP6 polymerases for antisense and sense probes, respectively. The template was degraded with RNase-free DNase (Roche Diagnostics). The DIG-labelled RNA probes were purified by ethanol and sodium acetate precipitation and stored at )20 (cid:4)C in 0.1% diethyl pyrocarbonate (Sigma-Aldrich, St in situ Louis, MO, USA)-treated water until used for hybridization. All solutions and glassware were RNase-free. The sections were treated with 0.1% pepsin (Roche Diag- nostics) in 0.2 m HCl (37 (cid:4)C for 10 min). Postfixation was performed by treating the sections with a fresh solution of 2% paraformaldehyde in NaCl ⁄ Pi (10 mm sodium phos- phate, pH 7.4, 0.1 mm KCl, 0.8% NaCl) for 4 min, and immersed in 1% hydroxylammonium hydrochloride in NaCl ⁄ Pi for 15 min. The sections were then dehydrated with successive ethanol washings. For hybridization, a humid chamber with 4 · SSC (1 · SSC: 150 mm NaCl, 15 mm sodium citrate, pH 7.4) was prepared. DIG RNA probes (antisense or sense) were diluted to a final concentra- tion of 50 ngÆlL)1 in the hybridization mixture [50% form- amide, 10% dextran sulfate, 4 · SSC, 1 · Denhardt’s solution (0.1% Ficoll 400, 0.1% polyvinylpyrrolidone, 0.1% BSA in water), 0.1% yeast tRNA and 0.1% salmon sperm DNA], denatured at 70 (cid:4)C for 5 min and cooled on ice. One hundred and twenty microlitres of this mix was placed on each tissue section. Sections were covered with cover slips and placed in the humid chamber at 45 (cid:4)C overnight (16 h). Post-hybridization washes in consecutive stringency baths 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 with anti-DIG alkaline phosphatase-conjugated IgGs (Roche Diagnostics) 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 mm levamisole to block possible endogenous alkaline phospha- tase. After satisfactory colour development the slides were washed carefully in tap water and mounted with glycerol ⁄ gelatin (Sigma-Aldrich) preheated at 42 (cid:4)C.

except

ACE activity assays

Testis and vas deferens tissue from males in active sper- matogenesis were dissected out and weighed. One hundred milligrams of each tissue were prepared separately. After (50 mm homogenization in 700 lL of HEPES-HCl buffer with 300 mm NaCl, pH 8.3), the homogenate was centrifuged (9200 g for 20 min at 4 (cid:4)C). The Asl-tACE activity was tested in the supernatant (solu- ble fraction) and in the pellet which was resuspended in 600 lL of assay buffer (insoluble fraction). The protein content of each fraction was estimated by the Bradford method reagent (Bio-Rad Laboratories GmbH, Muenchen, Germany) with standard. Activity assays were performed in BSA as [15]. accordance with a previously described protocol Briefly, the enzyme activity was determined by incubating samples with the ACE radiolabelled substrate tissue (282 mCiÆmmol)1; [phenyl-4(n)-3H-hippuryl-glycyl-glycine Amersham, Little Chalfont, UK)]. 3H-labelled hippurate, the product of ACE hydrolysis, was separated by ethyl acetate extraction and the radioactivity was assayed for 2 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- that 10 lm captopril, a specific ACE tions, inhibitor, was added to the tube, giving the ‘captopril c.p.m.’ value. ACE activity was calculated as: (total c.p.m. – captopril c.p.m.) ⁄ mg protein. Each data point was assayed in quadruplicate.

The sections were washed with NaCl ⁄ Pi ⁄ Triton 0.5% ⁄ goat incubating serum (Sigma-Aldrich)

3% buffer before

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Immunohistochemistry

J. Simunic et al.

Angiotensin-converting enzyme in crayfish testis

Acknowledgements

(ACE) is induced by a blood meal and accumulates in the developing ovary. FEBS Lett 455, 219–222.

12 Ekbote UV, Weaver RJ & Isaac RE (2003) Angiotensin

We thank Dr Lawrence Dinan for his critical reading and correction of the manuscript.

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