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 ppt

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
Introduction
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
the control of tissue-specific promotors [1,2]. The
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:
(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¢-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 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.
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.
FEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government works 4727
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].

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].
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-
out for Ace show a dramatic decrease in fertility
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
contrast, in female Anopheles stephensi, a dramatic
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
eggs [11]. Similar results were obtained from the
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 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].
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-
forms, including two from the hepatopancreas [15].
Correlatively, an ACE-like activity was demonstrated
in 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 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.
Results
Molecular characterization of A. leptodactylus
testicular ACE
In our previous studies, the partial cDNA sequence of

the region surrounding the testicular ACE active site
was obtained [15]. To complement this cDNA
sequence, 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 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
Angiotensin-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 works
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.
J. Simunic et al. Angiotensin-converting enzyme in crayfish testis
FEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government works 4729

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).
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.
In silico reconstitution of ACE cDNA sequences
from three crustacean species
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 sequence (GeneID: NCBI_GNO_452254),
whereas Homarus and Carcinus sequences were recon-
stituted from ESTs (accession numbers: BN001300 and
BN001299, respectively). Comparison of Asl-tACE
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 is incomplete in its 3¢-terminus.
Furthermore, all cysteines 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
As shown in Fig. 3, the A. leptodactylus testis is com-
posed of three lobes. One vas deferens exits from each
of the two lateral lobes. During spermatogenesis,
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.
To provide more detailed information about which
cells in the testes are involved in both Asl-tACE
mRNA synthesis and protein expression, we performed
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).
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.
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
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, interestingly, it
was present mostly in the soluble fraction. In animals
Angiotensin-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 works
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.
J. Simunic et al. Angiotensin-converting enzyme in crayfish testis
FEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government works 4731
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
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¢-to3¢ RACE on testis RNAs,
using degenerated primers deduced from crustacean
ACEs, we were able to clone a 2.3 kb cDNA. This size
corresponds to the values obtained previously by
northern blotting (i.e. in the range 2–2.5 kb for the

ACE mRNA from the crayfish testis) [15].
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.
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.
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 a potential hydrophobic 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.
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 crustaceans, 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.InA. 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.
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
species, including Drosophila [8]. Accordingly, our
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
the cytoplasmic membrane of spermatozoids, but
not on spermatogonia. By contrast, in vas deferens,
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.
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 works
immunoreactivity on germ cell membranes was much
weaker, with a strong signal being present in the
seminal fluid.

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
other hand, in Astacus during spermatid differentia-
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].
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.
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.
A
B
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 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.
J. Simunic et al. Angiotensin-converting enzyme in crayfish testis
FEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government works 4733
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.
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, the molecular mechanism for this effect
remains unknown, with one possible explanation being
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 site, although this hypothesis is
strongly debated [27,28].
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

important protease among the peptide-degrading
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.
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.
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 [
3
H]hippurate obtained after incubation with and without
10 l
M 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.
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 works
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.
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.
Molecular characterization
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
European Bioinformatics Institute (.
ac.uk/Tools/clustalw2/index.html).
Cloning the testicular ACE isoform of crayfish
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
Ô

RACE cDNA Amplification Kit (Clontech,
Mountain View, CA, USA) according to the manufac-
turer’s instructions. The 3¢-RACE was performed
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).
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¢.
PCR products were purified from 1.5% agarose gels
and subcloned into the transcription vector pGEM-T
Easy vector (pGEM
Ò
-T Easy Vector System II; Pro-
mega) and propagated in JM-109 Escherichia coli
bacteria. Recombinant plasmids were purified with
Wizard
Ò
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 ( />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 ( />SignalP/) and prediction of transmembrane regions
was performed using the sosui engine, version 1.11
( />Tissue preparation for in situ hybridization and
immunohistochemistry
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.
J. Simunic et al. Angiotensin-converting enzyme in crayfish testis
FEBS Journal 276 (2009) 4727–4738 Journal compilation ª 2009 FEBS. No claim to original French government works 4735
In situ hybridization
A 158 bp probe was obtained by amplification of the frag-
ment spanning nucleotides 950–1107 of the Asl-tACE
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
Ò
-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 °C in 0.1% diethyl pyrocarbonate (Sigma-Aldrich, St
Louis, MO, USA)-treated water until used for in situ
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 °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 ngÆl L
)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 °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 °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 ( 1 h), the
slides were washed carefully in tap water and mounted with
glycerol ⁄ gelatin (Sigma-Aldrich) preheated at 42 °C.
Immunohistochemistry
The sections were washed with NaCl ⁄ Pi ⁄ Triton 0.5% ⁄ goat
serum (Sigma-Aldrich) 3% buffer before incubating
overnight with rabbit polyclonal antibodies raised against
the synthetic peptide RENYGEEHVSRRGP, located
between R
217
and P
230
of the Asl-tACE sequence. During
synthesis, the R
217
-P

230
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 R
217
-P
230
peptide.
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 (TCS4D confocal 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).
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
homogenization in 700 lL of assay buffer (50 mm

HEPES-HCl buffer with 300 mm NaCl, pH 8.3), the
homogenate 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 in
600 lL of assay buffer (insoluble fraction). The protein
content of each fraction was estimated by the Bradford
method using the Bio-Rad Protein Assay reagent
(Bio-Rad Laboratories GmbH, Muenchen, Germany) with
BSA as standard. Activity assays were performed in
accordance with a previously described protocol [15].
Briefly, the enzyme activity was determined by incubating
tissue samples with the ACE radiolabelled substrate
[phenyl-4(n)-
3
H-hippuryl-glycyl-glycine (282 mCiÆmmol
)1
;
Amersham, Little Chalfont, UK)].
3
H-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-
tions, except that 10 lm captopril, a specific ACE
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.
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
Acknowledgements
We thank Dr Lawrence Dinan for his critical reading
and correction of the manuscript.
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