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First published online October 5, 2007
Journal of Experimental Biology 210, 3601-3606 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.009035
Male accessory glands of Drosophila melanogaster make a secreted angiotensin I-converting enzyme (ANCE), suggesting a role for the peptide-processing enzyme in seminal fluid
1 Institute of Integrative and Comparative Biology, Faculty of Biological
Sciences, University of Leeds, Leeds LS2 9JT, UK
2 Institute of Molecular and Cellular Biology, Faculty of Biological
Sciences, University of Leeds, Leeds LS2 9JT, UK
3 Department of Biological Sciences, Lancaster University, Lancaster, LA1
4YQ, UK
Author for correspondence (e-mail:
r.e.isaac{at}leeds.ac.uk)
Accepted 26 July 2007
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Summary |
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Key words: Drosophila, male accessory gland, angiotensin I-converting enzyme, dicarboxypeptidase, ANCE
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Introduction |
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). The
enzyme also cleaves a dipeptide from the C terminus of the circulating
vasodilator bradykinin, which inactivates this peptide hormone
(Erdos, 1979). In mammals,
there are two isoforms of ACE; a two-domain somatic protein and a smaller
single domain germinal isoform that is found exclusively in male germ cells
(Corvol et al., 2004). Although
somatic ACE is mainly found on the surface of endothelial cells, soluble
enzyme is found in body fluids, including blood, cerebrospinal fluid and
seminal fluid (Corvol et al.,
2004). The precise function of germinal ACE is not known, but mice
deficient in germinal ACE are infertile
(Hagaman et al., 1998).
Somatic ACE and other components of the RAS are also found in male
reproductive tissues (Leydig cells, seminiferous tubules, epididymis, testis
and prostate gland) with the prostate being a likely source of the angiotensin
II that has been found in human seminal fluid
(Leung and Sernia, 2003;
O'Mahony et al., 2005;
O'Mahony et al., 2000).
Angiotensin II might affect fertility in the human male because of its ability
to stimulate sperm motility, induce the acrosome reaction and increase oocyte
penetration (Kohn et al.,
1997; Kohn et al.,
1998; Vinson et al.,
1996; Vinson et al.,
1995). By contrast, the pronounced male infertility of mice
lacking germinal ACE is independent of the RAS and probably results from
failure to cleave a peptide distinct from angiotensin I
(Fuchs et al., 2005).
ACE is an evolutionarily ancient dicarboxypeptidase found in a range of
invertebrates (e.g. arthropods and annelids) and even in some bacteria
(Rawlings et al., 2006). In
insects, ACE is a soluble secreted enzyme that is strongly expressed in the
male reproductive tissues (Isaac et al.,
2007). In the testes of Drosophila melanogaster, germ
cells are the major site of ACE (known as ANCE) biosynthesis and male flies
homozygous for hypomorphic alleles of Ance are infertile
(Tatei et al., 1995). This
infertility results from a failure in spermiogenesis, suggesting that one of
the functions of germinal ANCE in D. melanogaster is the processing
of a regulatory peptide required for spermatid differentiation
(Hurst et al., 2003). Another
possible role for ANCE is in the processing of peptides in seminal fluid, as
has been proposed for human prostate ACE. In D. melanogaster, the
male accessory glands (AG) are responsible for the production and secretion of
a large number of proteins into the seminal fluid that mix with sperm on
ejaculation (Ram and Wolfner,
2007). These include several peptide and protein hormones as well
as enzymes, stress response proteins and immune defence proteins. The
peptide/protein hormones are responsible for a variety of physiological and
behavioural responses in the post-mated female, including increased rate of
ovulation, loss of receptivity to males, improved sperm storage and increased
appetite (Carvalho et al.,
2006; Chapman and Davies,
2004).
We now show that in addition to expression in the testes, ANCE is also produced in the AG of adult male D. melanogaster and that the peptidase is localised to giant vesicles of the secondary cells. ACE-like peptidase activity of the AGs is reduced in the post-mated male, presumably as a result of transfer to the mated female of the AG products as part of the seminal fluid. We speculate that the AG ANCE functions to cleave the C terminus of peptides with a role in reproduction to alter their biological activity.
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Materials and methods |
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Insects
Wild-type D. melanogaster Meigen (Oregon R) were maintained on
oatmeal–molasses–agar medium at 25°C
(Ashburner and Thompson,
1978).
Immunohistochemistry and in situ hybridisation
Antibodies were raised against recombinant ANCE expressed in Pichia
pastoris as described elsewhere
(Houard et al., 1998). For
immunohistochemistry, AGs were dissected in 10 mmol l–1
phosphate-buffered saline (PBS;
Na2PO4–NaHPO4 buffer, pH 7.4, 150 mmol
l–1 NaCl) and treated with 2% (v/v) hydrogen peroxide in
methanol for 5 min, then washed three times in PBS prior to fixation for 20
min in 4% (w/v) paraformaldehyde in PBS. Tissues were blocked in 1% (w/v)
bovine serum albumin, 10% (v/v) normal goat serum in PBST [PBS, 0.03% (v/v)
Triton X-100] for 1 h at 25°C before incubation in a 1:2000 dilution of
primary antibody (either immune or pre-immune serum) in PBST overnight at
4°C. AGs were washed three times in PBST before treatment with the
Vectastain ABC Kit according to the manufacturer's instructions. For
immunofluorescence assays, AGs were dissected, fixed and incubated with
primary antibody as described above. AGs were then incubated in a 1:2000
dilution of FITC-conjugated goat anti-rat IgG in PBST for 1 h at 25°C.
After three washes in PBST, tissues were mounted in Vectashield and images
were recorded using a Zeiss LSM510 META upright confocal microscope. Samples
were excited with the 488 nm laser line of an argon laser running at 5% power
output; and emission was collected with a long pass LP505 nm filter. 3D
reconstruction and iso-surface rendering were performed in Imaris version
4.0.6 (Bitplane, Zurich, Switzerland) using contours within the Surpass module
of the software.
In situ hybridisation experiments were carried out using
Ance RNA probes according to the method described previously
(Siviter et al., 2000); sense
probes were used as a control.
Measurement of ACE activity with the substrate hippuryl-L-histidyl-L-leucine (Hip-His-Leu)
ACE activity was determined by incubating AG tissue or AG secretions that
had been collected in 5 µl of PBS with the substrate solution (5 mmol
l–1 Hip-His-Leu in 0.1 mol l–1
Tris–HCl, pH 8.3, 0.3 mol l–1 NaCl, 10 µmol
l–1 ZnSO4; total volume 20 µl). After 4 h at
35°C, the enzyme reaction was terminated by reducing the pH to 2.0 with
the addition of 8% (v/v) trifluoracetic acid. The final volume was made up to
260 µl with 0.1% (v/v) trifluoracetic acid and the released hippuric acid
was quantified by HPLC using a 5 µ C18 (150 mmx4.6 mm) column, as
described previously (Lamango et al.,
1996).
Immunoelectrophoresis
AG proteins from both Oregon R and tudor (tud) flies were
extracted and separated on a 10% sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS–PAGE) gel, transferred to a PVDF membrane, and then
incubated with anti-ANCE antibody at a 1:5000 dilution in PBST, 5% (w/v)
non-fat dried milk powder as described previously
(Houard et al., 1998;
Hurst et al., 2003). Bound
anti-ANCE antibody was detected by using a horseradish peroxidase-conjugated
sheep anti-rabbit Fc antibody and the Enhanced Chemiluminescence Detection Kit
(Amersham Pharmacia Biotech Ltd., UK) as described in the manufacturer's
instructions. tud mutant flies were used in addition to wild-type
Oregon R because the tud males possess much lower levels of
testicular ANCE, which might contaminate the protein preparation. Similar
levels of ANCE were detected in AG extracts from the mutant and wild-type
males, but only blots of the tud AGs are presented.
Electron microscopy
Male AGs were dissected from virgin D. melanogaster males of
between 3 and 5 days old and fixed in 2.5% (v/v) gluteraldehyde in 0.1 mol
l–1 Na2PO4–NaHPO4
buffer, pH 6.9, for 3 h. Specimens were then washed twice in the buffer before
post-fixation for 1 h in 1% (w/v) osmium tetroxide in 0.1 mol
l–1 Na2PO4–NaHPO4, pH
6.9. After washing in two changes of buffer, the specimens were dehydrated by
using an ascending ethanol series (five concentrations ranging from
20–100%), each step taking 20 min. After one additional change of 100%
ethanol, the specimens were embedded in Araldite
(Luft, 1961). Sections
(80–90 nm) were cut from the Araldite blocks using an Ultramicrotome
(Reichert-Jung Ultracut-E, Leica, Milton Keynes, UK) and were stained with
uranyl acetate and Reynolds' lead citrate solution
(Reynolds, 1963). Sections
were examined using a Jeol 1200EX transmission electron microscope (Jeol UK
Ltd, Welwyn Garden City, UK) at an 80 kV accelerating voltage.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Lamango et al., 1996). The
distribution of the AG ACE was determined by gently compressing AGs from
unmated males with forceps to discharge the luminal contents into PBS. A
comparison of the ACE activity present in these secretions with the activity
remaining in the compressed AGs showed that around 80% of the active enzyme
was in the AG lumen (Table 1).
When males were allowed to mate with ten females over a 3-day period, the ACE
activity of the AGs was reduced by 70%, consistent with the enzyme being
transferred during copulation from the AG to the female in the seminal fluid
(Table 1).
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Immunological confirmation of the presence of ANCE in male accessory glands
To establish the identity of the ACE-like activity found in the male AGs,
antibodies that specifically recognise ANCE were employed in western blot
analysis of AG proteins (Fig.
1). A single protein band (Mr,
72x103) of the expected size was detected with the ANCE
antiserum, establishing ANCE as an AG product and the enzyme most likely to be
responsible for the ACE-like activity. A second D. melanogaster
ACE-like enzyme called ACER (ACE-related) can also cleave Hip-His-Leu in a
captopril-sensitive manner and therefore ACER might also contribute towards
the dicarboxypeptidase activity of the male AGs. However, ACER was not
detected in western blots using antibodies raised to recombinant D.
melanogaster ACER. We therefore conclude that ANCE, rather than ACER, is
responsible for the ACE-like activity detected in the male AGs.
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). Comparison of
the micrographs with the fluorescent immunocytochemistry images show clearly
that ANCE is localised to the dense large granule of the secondary cell. Three
dimensional reconstruction of the secondary cells using confocal microscopy
found the volume of the vesicles to be on average 103±14
µm3. Around half of this volume (54±15 µm3)
was taken up by the dense core granule
(Fig. 3B).
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In situ hybridisation confirms ANCE expression in the secondary cells
In situ hybridisation using digoxigenin-labelled antisense RNA
probes for Ance revealed strong expression in the secondary cells of
the gland and strong staining in the AG lumen
(Fig. 4). In contrast, the
level of staining in the main cells did not appear to be significantly
stronger than that obtained using control sense probes.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). It was not possible to follow the fate of the D.
melanogaster AG ANCE, since the enzyme was also expressed in the female
reproductive tissues (R. E. Isaac, unpublished). There are six ACE-like genes
in the D. melanogaster genome, but only Ance and
Acer products are considered to be enzymatically active; the other
gene products are predicted to be proteolytically inactive as they are missing
key active site residues (Coates et al.,
2000). Using antibodies that differentiate between ANCE and ACER,
we have shown that the AG dicarboxypeptidase activity can be attributed to
ANCE and not ACER. ANCE is expressed in the secondary cells of the AG and
becomes incorporated into the dense granular mass that half-fills the giant
vesicles of these cells. This is the first identification of a stored
component of this unusual organelle. In situ hybridisation using an
Ance riboprobe was consistent with relatively strong expression in
the secondary cells. It has been suggested previously that the secondary cells
of D. melanogaster AGs discharge their contents by a holocrine
secretory mechanism (Chen,
1984). This mode of secretion might explain our observation of
Ance RNA in the AG lumen.
ANCE is just one of several secreted peptidases produced by the AGs of
D. melanogaster. In total, eleven peptidases including
aminopeptidases, endopeptidases and a 
-glutamyl transpeptidase have
already been identified as AG products in D. melanogaster
(Mueller et al., 2004;
Walker et al., 2006). One of
these peptidases, an astacin-like endopeptidase, is involved in the cleavage
of the male AG ovulin in the female reproductive tract to produce four
products, two of which stimulate ovulation in the first 24 h post-mating
(Ravi Ram et al., 2006). ANCE
might work in concert with other peptidases to process prohormone polypeptides
to biologically active peptides in a similar manner to the local RAS of the
mammalian prostate (Leung and Sernia,
2003; O'Mahony et al.,
2005). The best known biologically active peptide of the male
reproductive tissues is the sex peptide, which stimulates egg-laying and
reduces receptivity to males in the mated female
(Kubli, 2003). However, ANCE
is unlikely to be involved in its biosynthesis because the C terminus of the
proprotein does not undergo proteolytic processing. In contrast, Dup99B, a
peptide made by the male ejaculatory duct, is a potential substrate for ANCE.
The mature Dup99B, the C-terminal amino acid sequence of which is almost
identical to the sex peptide, elicits the same responses in the post-mated
female as the sex peptide when the peptide is injected into virgin female
D. melanogaster (Rexhepaj et al.,
2003; Saudan et al.,
2002). Unlike the sex peptide, the DUP99B proprotein has a basic
dipeptide (Arg-Lys) at the C terminus, which must be proteolytically removed
to expose the C-terminal cysteine. We have shown previously that peptides with
pairs of basic residues at the C terminus make excellent ANCE substrates
(Isaac et al., 1998) and it is
possible, therefore, that ANCE has a role in cleaving the Arg-Lys dipeptide
from the C terminus of a partially processed DUP99B precursor as the AG
products mix with the material secreted by the ejaculatory.
We cannot, at present, reach any conclusions regarding the relative importance of the AG and germinal ANCE for male fertility in D. melanogaster. This level of understanding will require tissue-specific knock-down of Ance expression.
List of abbreviations
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Acknowledgments |
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Footnotes |
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References |
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