Mussel MAP, a major gonad-duct esterase-like protein, is released into sea water as a dual constituent of the seminal fluid and the spermatozoon
1 Developmental Biology Unit, Institute of Health Sciences, University of La
Coruña, Campus de Oza, Building `El Fortín', As Xubias s/n, La
Coruña 15006, Spain
2 Institute of Gene Biology of the Russian Academy of Sciences, Vavilov Str.
34/5, Moscow, Russia
* Author for correspondence (e-mail: margot{at}udc.es)
Accepted 14 October 2002
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Summary |
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Key words: esterase, external fertilizer, seminal fluid protein, spermatozoa, bivalve mollusc, fruitfly, mammal
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Introduction |
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The identification, characterization and potential functions of
seminal-fluid proteins are mainly taken from studies on internal fertilizers,
particularly from mammals (Robert and Gagnon,
1995,
1999
;
Syntin et al., 1996
;
Naaby-Hansen et al., 1997
;
Gerena et al., 1998
;
Mortarino et al., 1998
). It
has been shown that a sperm-coating protein acquired from secretions of the
accessory sex gland can modulate the motility
(Aumüller et al., 1997
)
and plasma membrane organization of mammalian spermatozoa (Soubeyrand and
Manjunatu, 1997; Therien et al.,
1999
). In addition, fertilization-promoting peptides have been
detected in mammalian semen (Kennedy et
al., 1997
). Sophisticated functional implications of seminal-fluid
proteins in reproduction have been demonstrated in fruit flies.
Drosophila seminal fluid contains a bulk of proteins secreted by the
male accessory glands, the ejaculatory ducts and the ejaculatory bulb that
affect the processes of sperm storage and sperm competition, and modulate
female reproductive physiology after mating (reviewed in
Chapman, 2001
;
Wolfner, 2002
).
Some of the seminal-fluid proteins detected in fruit flies and mammals
share the same (or similar) molecular mass, antigenic epitopes and enzymatic
activity (see Torrado et al.,
2000). An essential question in terms of the comparative
physiology of reproduction is how similar seminal-fluid proteins become
important for reproductive fitness in different animal groups (see
Mikhailov and Torrado, 2000
).
We therefore decided that it would be interesting to trace the presence of
such polypeptides identified in internal fertilizers in the seminal fluid of
marine free-spawners. To date studies on seminal fluid components in seawater
external-fertilizers are limited to the identification of the following
compounds: a spawning pheromone in fish
(Carolsfeld et al., 1997
),
arylsulphatase (Moriya and Hoshi,
1979
,
1980
) and proteins with
esterase- and protease-like activities
(Resing et al., 1985
) in sea
urchins, and a sperm-associated fucosidase in bivalve molluscs
(Focarelli et al., 1997
).
We focused our attention on protein composition of the gonad-duct and
seminal fluids in the bivalve mollusc, Mytilus galloprovincialis.
Species of the genus Mytilus are distributed worldwide and can be
found attached by fibrous byssus threads to rock surfaces in open coasts.
Their significant reproductive success is reflected not only in their wide
occurrence along exposed coastlines, but also in their frequent predominance
in intertidal rock habitats (see Gosling,
1992). Although these species are characterized by a strict
gonochoric pattern of sexuality, their gonad system, as in a majority of
bivalves, does not show any structural sign of sexual dimorphism. Neither
accessory sex glands nor copulatory organs were found in these animals. During
the reproductive period, males and females release their gametes in seawater
where fertilization takes place.
In the Mytilus species, the tubular gonad is spread all along the
mantle tissue. Published hand-drawn schemes
(Wilson, 1886;
White, 1937
;
Lubet, 1959
) seem to suggest
that the mussel reproductive-tract system consists of genital ducts, canals
and follicles. However, a major limitation is that these schemes only mimic
natural features of mantle-associated gonad organization. In this report we
present a detailed structural patterning of the M. galloprovincialis
tubular gonad and reproductive tract, required for gonad-duct manipulations
and sampling.
Our previous studies led to the identification of the so-called
`male-associated polypeptide' (MAP), an abundant soluble protein in the male
gonad with low levels in the female, which shows a marked male-predominant
expression in the somatic gonad tissue of M. galloprovincialis
(Mikhailov et al., 1995,
1996
). MAP protein levels are
related to male gonad development and the annual reproductive cycle, reaching
maximum values during the spawning period
(Mikhailov et al., 1998
;
Torrado and Mikhailov, 1998
).
Interestingly, MAP seems to be similar to esterase S, a seminal fluid protein
of D. virilis, as judged by cross-reactivity with antibodies against
esterase S, by its esterase enzymatic activity in vitro, and by its
amino acid composition (Mikhailov et al.,
1997
; Torrado et al.,
1997
). Esterase S belongs to a family of diverse proteins in which
related members are also expressed in the adult male reproductive tract of
different Drosophila species (reviewed in
Korochkin et al., 1990
,
Richmond et al., 1990
;
Oakeshott et al., 1995
,
1999
). Esterase S is expressed
and secreted by the epithelium of the ejaculatory bulb and is transferred
together with sperm to females upon copulation
(Yenikolopov et al., 1983
).
Although a precise function of esterase S remains elusive, there is some
evidence for a possible role in sperm storage and use in the female
reproductive tract (see Korochkin et al.,
1990
).
Given our interest in the comparative analysis of male-reproductive tract esterases in different animal groups and the apparent lack of information on seminal-fluid proteins in marine free-spawners, we undertook a detailed study of MAP presence throughout the mussel gonad-duct tract and at spawning. We found that MAP is a major protein in M. galloprovincialis semen, present in both sperm cells and cell-free seminal fluid. Sperm-associated MAP is localized in the mid-piece region of spawned spermatozoa. This unexpected finding raises the possibility that in bivalves, MAP may play a role in sperm fertility. Of particular interest is the observation that MAP is detected in luminal fluids and sperm throughout the male gonad-duct system, from spermatogenic tubules to the terminal efferent duct. In addition, we show that human semen reveals positive cross-reactivity with antibodies directed against Mytilus MAP and Drosophila esterase S.
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Materials and methods |
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Induction of spawning and semen sampling
No method other than slightly increasing seawater temperature was used to
induce sperm emission in mussels, so that no artificial alteration in the
seminal fluid components, which are physiologically released at natural
spawning, could be expected. Mussels were scrubbed thoroughly to remove
fouling organisms, byssus threads were cut out, and the animals (15-20) were
immersed in a 20 litre tank with filtered (0.45 µm) natural seawater (FSW)
at room temperature (RT). Spawning was induced by increasing seawater
temperature to 25-26°C, which was 2-4°C above RT. Once spawning was
initiated, males were easily identified and transferred to a separate smaller
transparent tank at a lower density in FSW (2-3 animals per 10 liters FSW),
where spawning continued. The transparent tank was located over a piece of
black paper and spawning was monitored during 2-3 h. Photographs were taken
and spawned sperm mass was recollected with minimum dilution in FSW.
Microscopic examination of a small portion of each sample confirmed that they
contained viable and motile spermatozoa. Semen samples were centrifuged (500
g, 10 min, 2°C), and supernatants (i.e. semen-soluble
fractions) were filtered (0.22 µm-membrane, Amicon, Beverly, MA, USA) and
concentrated (3 kDa cut-off units, Amicon, Beverly, MA, USA). Sperm-containing
sediments were resuspended in ice-cold FSW and precipitated again. Final
sperm-cell pellets and cell-free supernatants were kept at -20°C.
Detergent treatment of spawned sperm
Spawned sperm were washed in FSW at 100 g for 1 min to
remove any gross cell debris, assessed for motility and diluted for use. In
all cases, large-orifice (1.5 mm diameter) pipette tips were used to minimize
damage to the sperm membrane. Sperm suspension was divided into portions for
detergent solubilization, and each sample was finally resuspended in the same
volume of 500 µl of FSW, and incubated with a varying concentration
(0.01-2% v/v) of Triton X-100 (Merck, Darmstadt, Germany) for 30 min at
20°C (each experiment was performed in triplicate). Sperm cells were
sedimented (5,000 g, 20 min), and the resulting supernatants
were filtered and concentrated (3 kDa cut-off units, Amicon, Beverly, MA,
USA). Precipitated sperm cells were resuspended and centrifuged again. The
final pellets were immediately extracted by 2x SDS-Laemmli sample
buffer, centrifuged (30,000 g, 30 min, 12°C), and the
supernatants kept at -80°C until use.
Gonad-duct manipulations
The male mantle lobe was removed and placed in a Petri dish. The mantle
epithelium was dissected through a stereomicroscope (Nikon, Tokyo, Japan), and
the content of the spermatogenic tubules and transversal gonad ducts (see
Fig. 3) was aspirated using a
fine capillary pipette or a 5 µl Hamilton syringe as previously described
(Torrado and Mikhailov, 1998).
The samples (10-20 µl) were centrifuged (500 g, 15 min,
2°C), and the top 80% of the supernatants was subjected to a second
centrifugation (60 000 g, 30 min, 2°C). A small volume
(1-2 µl) of the resulting supernatants was examined microscopically to
confirm that all cells had been removed. The sperm-containing pellet (obtained
after the first centrifugation) was resuspended in ice-cold FSW (1:100 v/v)
and precipitated by centrifugation (20 000 g, 15 min,
2°C). The resulting supernatants and sediments were stored at -20°C
until use. For some experiments, fragments of transversal gonad ducts were
microsurgically dissected from the mantle, and isolated samples were shaken in
ice-cold FSW for 30 min in order to remove luminal fluid and sperm cells.
Washed tube fragments were immediately extracted with 2x SDS-Laemmli
sample buffer containing the protease inhibitor cocktail (Roche Diagnostic,
Mannhein, Germany), centrifuged (30 000 g, 30 min, 12°C)
and supernatants stored at -80°C.
|
Protein sample preparation
Sperm pellets, seminal and gonad-duct-derived fluids were mixed with an
equal volume of 2x SDS-Laemmli sample buffer containing a protease
inhibitor cocktail (Roche Diagnostic, Mannhein, Germany), boiled for 5 min,
cooled on ice and centrifuged (30,000 g, 30 min, 12°C).
The supernatants were stored at -80°C. Samples were subjected to SDS-PAGE
followed by Coomassie gel staining, and protein concentration was measured by
gel densitometry using bovine serum albumin (BSA) (Sigma, Madrid, Spain) as
the reference protein. Mantle lobes were homogenized at a 1:1 (w/v) ratio in
ice-cold deionized water (for isoelectrofocusing separations) or a 1:2 (w/v)
ratio in 100 mmol l-1 Tris-EDTA buffer, pH 7.0 (for SDS-PAGE
separations), both containing the protease inhibitor cocktail. After 30 min
extraction on ice, homogenates were centrifuged at 60 000 g
for 60 min at 2°C, and the supernatants used immediately or supplemented
with glycerol (to a final concentration of 50% v/v) and stored at -80°C.
Protein concentration in mantle extracts was measured according to the
Bradford method.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Samples were separated using 5% stacking and 10% resolving Tris-glycine
SDS-polyacrylamide gels (Bio-Rad, München, Germany). Proteins were
visualized by Coomassie Blue staining. The apparent molecular masses (MW) of
the bands were determined by comparing high and low MW calibration kits
(Amersham Biosciences, Uppsala, Sweden) in the same gel.
Isoelectrofocusing (IEF)
Ultra-thin (0.4 mm) 5% polyacrylamide gels, containing a mixture of 5-7,
3-10 Bio-Lytes (Bio-Rad, München, Germany) in a proportion of 4:1 were
electrophoresed in the mini IEF Cell (Bio-Rad, München, Germany)
following the manufacturer's instructions. The visible marker, myoglobin
(Sigma, Madrid, Spain), was loaded at opposite sides (near the cathode and
anode contact zone) of the same gel run; IEF was finished when both myoglobin
bands were colocated. After separation, proteins were detected by Coomassie
Blue staining. The following pI markers (Sigma, Madrid, Spain) were used:
ß-lactoglobulin A (pI 5.1), carbonic anhydrase I (pI 6.6), carbonic
anhydrase II (pI 5.9) and myoglobin (pI 6.8, 7.2).
Western immunoblot
Proteins resolved in SDS-PAGE gels were electrophoretically transferred to
nylon or nitrocellulose membranes (`Nytran' or `Optitran', Schleicher and
Schuell, Dassel, Germany) by routine methods using the mini `Trans-Blot' Cell
(Bio-Rad, Hercules, CA, USA). Proteins resolved in IEF gels were transferred
to nylon membranes as described by Heukeshoven and Dernick
(1995). Protein loading and
localization of MW and pI markers were verified by membrane staining with
Amido Black (Merck, Darmstadt, Germany) or Ponceau S (Sigma, Barcelona,
Spain). Prior to immunodetection, the blots were incubated in a blocking
solution containing 20% v/v normal horse serum (Sigma, Barcelona, Spain) in
100 mmol l-1 Tris-HCl (pH 7.6), 150 mmol l-1 NaCl, 0.1%
Triton X-100, 0.01% NaN3, at room temperature for 1 h. The
following rabbit polyclonal antibodies were used: (1) anti-MAP raised against
the protein isolated from the M. galloprovincialis male gonad
(Mikhailov et al., 1995
), at a
dilution of 1:200; (2) anti-esterase S raised against a fusion-protein
containing over 90% of the coding sequence of the Esterase S gene of
D. virilis (Tamarina et al.,
1991
), at a dilution of 1:200, and (3) anti-pig esterase against
the protein fraction isolated from porcine liver (Polysciences, Eppelheim,
Germany), at a dilution of 1:200. Antibody dilutions were prepared in the
blocking solution. All antibody incubations were followed by six washes (10
min in each) in the same solution without horse serum. Immunoblots were
revealed as described (Mikhailov et al.,
1997
) using peroxidase-labeled anti-rabbit immunoglobulins (Sigma,
Madrid, Spain), at a dilution of 1:3000, and diaminobenzidine tablets (Sigma,
Madrid, Spain).
Densitometric analysis
Gels and blots were scanned using a model GS-700 densitometer (Bio-Rad,
Hercules, CA, USA). Profile analysis of the fractions/bands including peak
identification/quantitation, area integration and MW determination, was
carried out with the software `Molecular Analysis' (Bio-Rad, Hercules, CA,
USA). The images were processed by computer software to remove image noise and
background.
Protein purification and microsequencing
MAP was isolated from the M. galloprovincialis male gonad by IEF
followed by SDS-PAGE. Briefly, a mantle water-soluble extract was separated by
IEF in 5% polyacrylamide slab gels (Amersham Biosciences, Uppsala, Sweden).
The MAP-containing fraction (pI 6.2) was isolated, subjected to SDS-PAGE, and
MAP (39 kDa) was electro-eluted from SDS-gel fragments, using the
electro-eluter model 442 (Bio-Rad, Hercules, CA, USA) following the
manufacturer's instructions. Purified protein preparation was
reelectrophoresed using a 10% resolving SDS-polyacrylamide gel, and MAP was
subjected to in-gel digestion with sequencing grade modified trypsin (Promega,
Madison, WI, USA) following recommendations described in Stensballe and Jensen
(2001) and Soskic and
Godovac-Zimmermann (2001
).
Random trypsinized peptides were microsequenced at the Laboratory of
Mechanisms of Ocular Diseases of the National Eye Institute, NIH, Bethesda,
USA.
Whole-mount histology
Juvenile mussels were fixed in Bouin's solution and extensively rinsed
several times in 70% ethanol. To separate shells, the posterior adductor
muscle was sectioned, internal organs (i.e. foot, hepatopancreas, gills) were
removed and the whole (two-lobe) mantle sheet containing the gonad-tubular
`tree' was carefully micro-dissected and isolated. The isolated mantle
preparation was stained with Hematoxylin-Eosin, dehydrated, mounted on slides,
viewed and photographed with a Nikon stereomicroscope and a Nikon Eclipse 800
microscope.
Immunocytochemistry
Spawned sperm were washed and resuspended in FSW. The sperm suspension was
dropped on slides precoated with 3-aminopropyltriethoxysilane (Sigma),
air-dried and fixed in 4% neutral buffered formalin in FSW for 10 min at RT.
Prior to immunostaining, fixed sperm cells were treated with 0.1% Triton X-100
(v/v) for 30 min, washed and further assayed as described in Torrado and
Mikhailov (1998). Briefly,
sections were pre-incubated with 20% normal horse serum in Tris-buffered
saline, pH 8.0 (TBS: 50 mmol l-1 Tris-HCl, 50 mmol l-1
NaCl) for 1 h and incubated with rabbit anti-esterase S antibodies (at a
dilution of 1:50) for 1 h at RT. After six washing steps in TBS (10 min each),
sections were incubated with fluorescein-labelled anti-rabbit immunoglobulins
(Sigma, Madrid, Spain) at a dilution of 1:1000 for 1 h. After six washing
steps in TBS, sections were mounted in 100 mmol l-1 Tris-HCl (pH
8.6): glycerol (1:9, v/v) containing freshly added 0.1%
p-phenylenediamine (Sigma, Barcelona, Spain) and viewed and
photographed with a Nikon Eclipse 800 microscope (Nikon, Tokyo, Japan)
equipped with epifluorescence. Control experiments included assessing a
fluorescent background of sperm cells treated by anti-esterase S antibodies
pre-absorbed by MAP.
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Results |
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To analyze the protein composition of the mussel semen, cloudy-like as well as compact sperm (before and after precipitation) (see Fig. 1B,C) were collected in a minimum volume of seawater. Cell-containing and cell-free soluble fractions of each semen sample were separated by centrifugation and filtration and analyzed by SDS-PAGE followed by western blot. On Coomassie Blue-stained gels, a band with an apparent molecular mass of 39 kDa (identical to that of the MAP isolated from the male gonad) was found in the sperm-cell fractions of each of the semen samples studied (Fig. 1D, lanes 2,6). A similar polypeptide detected in the cell-free soluble semen fraction was characterized by a slightly faster electrophoretic mobility (Fig. 1D, lanes 3-5). The identity of these bands was confirmed as MAP by western blot using anti-MAP antibodies (Fig. 1E). Of note, the cell-free soluble fraction of the cloudy sperm contains trace amounts of the 39 kDa protein although it was 2-3 times more concentrated than the seminal fluid obtained from both types of semen clots. These results suggest that after spawning, a portion of the seminal-fluid MAP, which is included in sperm aggregates, persists as long as the aggregates keep their compact form.
The densitometric analysis of Coomassie Blue-stained gels shows that MAP represents more than 90% of the protein in the soluble fraction of the spawned semen clots. These findings demonstrate that MAP is the predominant protein constituent of the mussel seminal fluid and that this male gonad-derived polypeptide is excreted together with sperm into seawater during spawning.
The sperm cells sampled at different time periods of spawning displayed
positive immunoblot reactions with anti-MAP antibodies (see
Fig. 1E, lanes 2 and 6). This
raised the possibility that MAP could remain on the sperm surface long after
sperm emission. To check this possibility, sperm suspensions obtained from the
semen streams (see Fig. 1A)
were diluted 50 times in seawater, and spermatozoa were kept in the container
for 12 h at RT. Note that under these conditions any changes in sperm
viability and motility could be detected. At different time-exposure periods,
spermatozoa were sampled (using the so-called `swim up' technique; see
Graczykowski and Siegel,
1991), precipitated by centrifugation and analyzed by SDS-PAGE
followed by western blot. Comparison of MAP immunoblot signals of 0 h- and 12
h-incubated sperm did not reveal any detectable reduction of MAP
immunostaining intensity (data not shown). These results indicate that sperm
cells do not lose MAP after prolonged swimming and survival in seawater, which
suggests that MAP could be tightly associated with spermatozoa.
The association between MAP and sperm cells was evaluated according to its susceptibility to being removed from cells by the non-ionic detergent, Triton X-100. Spawned spermatozoa were treated with serial dilutions of Triton X-100 (from 0% to 2%) in seawater, sedimented by centrifugation and subjected to SDS-PAGE followed by western immunoblot with anti-MAP and anti-esterase S antibodies (see Fig. 2AC, respectively). Although the same results were observed with both types of the antibodies used, the intensity of MAP immunodetection was higher in blots treated with anti-MAP in comparison to that in blots treated with anti-esterase S antibodies. It is likely that anti-esterase S recognizes only a part of MAP-specific epitopes, which are detected by anti-MAP antibodies.
|
A significant portion (up to 30%) of MAP immunoreactivity could be extracted from sperm cells using the lowest detergent concentration (0.01%), and 0.03% Triton X-100 treatment resulted in the removal of approximately 70% of the protein. Further increase in detergent concentration (0.06-0.6%) in the incubation medium did not result in a further significant decrease of the MAP-specific immunoblot signal of the treated sperm. Almost complete extraction (up to 95%) of the protein was achieved at a substantially higher concentration of Triton X-100 (2%) in seawater (Fig. 2E). Note that the observed kinetics of Triton X-100-mediated MAP extraction from sperm cells is relatively selective because a significant part of the other sperm proteins were not solubilised at any of the detergent concentrations used (see Fig. 2A). The reduced MAP signal of the Triton X-100-treated spermatozoa was not due to MAP proteolytic degradation as the protein was demonstrated in western blots of the resulting supernatants of untreated and detergent-treated sperm samples (Fig. 2D). In addition, no signs of protein degradation were observed on the gels or blots of the Triton X-100-treated sperm samples.
The simplest interpretation of these results is that there is more than one type of MAP association with spawned spermatozoa. Over 70% of MAP immuno-reactivity is extracted from the cells at a low Triton X-100 concentration, which could reflect adsorption of the protein on the sperm surface from MAP-containing luminal fluid during sperm transit through gonad collecting and efferent ducts (see below). However, another (`resting') portion of the protein (up to 20%) seems to be more tightly associated with spermatozoa because it could only be solubilized at a very high Triton X-100 concentration.
To study MAP localization in spawned sperm, these were washed, fixed,
permeabilized with 0.1% Triton X-100 (v/v) and assayed by immunofluorescence.
A MAP-specific signal was detected in the mid-piece of spermatozoa
(Fig. 2F,G). Together with our
previous findings that MAP can be detected on the surface of non-permeabilized
spermatozoa, but not in sperm cells
(Torrado and Mikhailov, 1998),
the results suggest that seminal-fluid MAP may be internalized by mature sperm
cells following its compartmentalization in the mid-piece region of spawned
spermatozoa.
MAP is a major protein of the Mytilus male gonad-duct luminal
fluid
The published data contains some contradictory conclusions on the
structural and functional compartmentalization of the male tubular gonad in
Mytilus, so we have studied gonad-duct branching morphogenesis in the
mantle of juvenile mussels. The entire two-lobe mantle sheets were
micro-dissected from post-metamorphic animals, fixed and stained. A
representative whole-mount of the male paired tubular gonads and reproductive
tract settled into a whole mantle sheet is shown in
Fig. 3. Within each mantle
lobe, the corresponding gonad-duct network includes: (1) a bulb-like structure
with gonopore, (2) a major longitudinal gonad duct (LGD), (3) several (5-10)
transversal gonad ducts (TGDs), (4) TGD-ramifications with numerous bud-like
lateral extensions, and (5) terminal end tubules. Microscopic examination
revealed that spermatogenesis takes place mainly in TGD-ramifications and
terminal end tubules and so we propose to call these structures `spermatogenic
tubules' (STs). This is in contrast to the generally accepted interpretation
of such structures as `gonad acinus' or `gonad follicles' (see, for instance,
Gosling, 1992).
In adult mussels, the structural organization of the developed tubular gonad is not as clearly observable as in juveniles. In particular, STs form a complex overlapping tridimensional network, which occupies up to 90% of the mantle volume (consequently, mantle thickness increases significantly from 0.5-1 mm in juveniles to 5-6 mm in adults). Only at the ventral mantle edge is it still possible to see non-overlapping STs (Fig. 3E). Mature sperm was found in the lumen of STs, TGDs and LGD.
Bulb-like structures were observed in the male gonad of both juveniles (Fig. 3D) and adults (Fig. 3F). An adult male was induced to spawn, and immediately following the first release of spermatozoa, its posterior adductor muscle and other tissues were sectioned, which allowed us to observe the sperm mass being ejected from the bulbs as white-colored streams (Fig. 3F).
We then studied the presence of MAP in luminal fluid extracted from different structural compartments of the male gonad-duct network. Using sexually mature adult males, luminal contents were extracted from LGD and TGDs and the corresponding cell-free fluids were subjected to SDS-PAGE. In all samples, a major 39 kDa polypeptide was detected (Fig. 4A). Western blot with anti-MAP antibodies showed that this polypeptide corresponds to MAP (Fig. 4B). Both the immunoblot and the electrophoretic MAP bands of the cell-free luminal fluid appeared as closely migrating doublets, while the male gonad and the sperm displayed clear sharp immuno-stained bands, which may suggest that different post-translationally modified forms of the MAP protein coexist in the luminal fluid.
|
Fig. 5 shows the results of
a comparative analysis of the proportion of MAP to total protein in the
luminal and semen-derived fluids, and in the whole male gonad extract. Fluid
was obtained from 3-5 animals, pooled and separated by SDS-PAGE. Stained gels
were scanned by transmittance. For each gel, a total profile analysis was
carried out, and the proportion of the MAP peak relative to the total protein
scan value was calculated and expressed as a percentage. The MAP fraction
percentage for the ST, LGD and seminal fluid profile was approximately 70%,
80% and 90%, respectively. We consider that these densitometric values reflect
MAP proportions, taking into account that MAP is a major protein component of
the scanned 40 kDa bands, as demonstrated by 2-D electrophoresis followed by
western blot (Mikhailov et al.,
1997). The kinetics of the MAP:total protein ratio can be ascribed
to a progressive `simplification' of the protein composition of the samples
studied, i.e. from a relatively complex protein mixture in the ST-sample to an
almost single-protein pattern in the semen-derived fluid. This, in turn,
suggests that MAP may be the only major protein in the gonad-duct luminal
fluid that is excreted from the gonad at spawning. Note that it was necessary
to concentrate the semen-derived fluid over 20 times to make it suitable for
SDS-PAGE analysis, whereas protein content of the gonad-duct fluids was
sufficient for use without concentration.
|
As we have previously suggested
(Torrado and Mikhailov, 1998),
MAP can be highly expressed in the gonad-duct wall. To investigate this
possibility, fragments of the TGD (see Fig.
6A) were microsurgically dissected, extracted with Laemmli sample
buffer, and analyzed by western blot with anti-MAP antibodies parallel to
luminal sperm and luminal fluid samples obtained from the same TGD
(Fig. 6B). The intensity of the
MAP-specific immunoblot reaction in the TGD-wall sample was threefold higher
than that in the luminal fluid and tenfold higher than in luminal sperm. The
results indicate that the duct network may be a principal source of MAP in the
ripe male gonad and strongly suggest that the protein is secreted by duct
epithelial cells into duct lumen.
|
Mytilus MAP shares sequence similarity with Drosophila
esterase S
The idea that MAP is a bivalve relative of Drosophila
male-specific esterases is based on data from our previous studies on MAP
amino acid composition, enzymatic activity and cross-reactivity with
antibodies against D. virilis esterase S
(Mikhailov et al., 1997;
Torrado et al., 1997
). Here we
present structural evidence to support this suggestion.
A water-soluble extract of the Mytilus male gonad was subjected to IEF followed by western blot. On IEF gels, MAP was identified as a single band at pI 6.2 (Fig. 7A), recognized by three different antibodies against: (1) Mytilus MAP, (2) Drosophila esterase S, and (3) porcine esterase (Fig. 7B). The pI 6.2 band contains a single protein of 39 kDa (Fig. 7C), which was recognized by all three antibodies (Fig. 7D).
|
Trypsinized peptides derived from the purified MAP protein were subjected
to microsequencing. The sequences of seven peptides (5-7 residues each) were
obtained (43 amino acids were identified in total). Using a multiple
alignment, we were able to match all the MAP-derived peptide sequences over
the D. virilis esterase S sequence; the resulting overall identity
and similarity was 58% (25/43) and 67% (29/43), respectively
(Fig. 8A). Note that none of
the MAP-derived peptides are located in the conserved carboxylesterase domains
(Fig. 8B). Taking into account
an extreme structural divergence within the carboxylesterase protein
multi-family (Torrado et al.,
2000), the sequence similarity revealed between MAP and esterase S
is considered highly significant.
|
It has been suggested that during evolution, carboxylesterase-like
polypeptides could be recruited by the male reproductive tract to perform new
sex-specific roles (Mikhailov and Torrado,
1999,
2000
;
Mikhailov et al., 1999
). We
decided to perform immunochemical screening of human semen using antibodies
against MAP and esterase S. Human sperm and seminal fluid were separated by
centrifugation and filtration, and the samples studied by SDS-PAGE followed by
western blot. A single 64 kDa band was detected in the human seminal fluid and
sperm by both types of antibodies used
(Fig. 9).
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Discussion |
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We observed that mussel semen can form clots shortly after spawning,
suggesting that clot-like sperm structures pre-exist in the gonad duct lumen.
This seems to be an efficient way of collecting and transporting sperm mass
from different branches of the reproductive system, and finally projecting the
sperm mass outside. Of note, the sperm manually isolated from the STs exhibit
lower motion activities than those of spawned sperm, as observed in M.
galloprovincialis (Mikhailov and
Torrado, 2000) and the zebra mussel, Dreissena polymorpha
(Mojares et al., 1995
).
Emission of compacted sperm masses may represent an important feature for
reproductive fitness in Mytilus. It has been shown that some marine
free-spawners have evolved mechanisms to prevent or reduce sperm limitation at
sea (Levitan and Petersen,
1995
; Yund, 2000
;
Wedell et al., 2002
). The
release of sperm in viscous fluids was observed in sea urchins, annelids and
algae, apparently to counteract sperm dilution effects
(Thomas, 1994
). We have been
able to show that in M. galloprovincialis MAP is the only major
protein in the fluid-phase fraction of the thread-shaped sperm clots, which
suggests that MAP does indeed represent a protein component that may be
implicated in maintaining such thread-shaped clots.
Indirect results do not support the interpretation that MAP is present in seminal fluid as a result of `shedding' of the protein from the sperm surface, since the former displayed electrophoretic mobility slightly different from that of the sperm polypeptide (see Fig. 1D,E). It is interesting to note that electrophoretic mobility of the seminal-fluid MAP is similar to that of the faster migrating form of the luminal-fluid MAP (see Fig. 4). These electrophoretic patterns indicate that, in the duct lumen, MAP undergoes certain post-translational modifications rather than a proteolytic degradation because we did not observe the low molecular mass products (30 kDa or less) typical for MAP proteolysis on the corresponding blots.
In addition to being a protein component of mussel seminal fluid, MAP is
also associated with spawned sperm. Before spawning, mature sperm cells are
accumulated in the lumen of TGDs and LGD, where the luminal fluid is
characterized by an extremely high level of MAP. We suggest that during
transit through a MAP-enriched luminal environment, sperm cells may
internalize MAP from the luminal fluid. In this way, MAP may become an
integral component of the spermatozoon. The immunofluorescence data on MAP
localization in the mid-piece of the spawned sperm
(Fig. 2) supports this
suggestion. An uptake of MAP by sperm from ductal secretions can only be
deduced if the protein is not in testicular sperm, as demonstrated by
immunocytochemistry and immunocytoblotting
(Torrado and Mikhailov,
1998).
Although MAP isolated from the male gonad is characterized by esterase
activity (Mikhailov et al.,
1997), we could not detect the enzymatic activity in mature sperm
(M. Torrado and A. T. Mikhailov, unpublished observations), which may suggest
that the MAP associated with the mid-piece region of spawned spermatozoa is an
enzymatically inactive protein. Such protein changes have been shown to occur
during mammalian sperm differentiation: mouse selenoprotein called PHGPx is
expressed as a soluble active enzyme in round spermatids in early sperm
development, and later turns into an enzymatically inactive protein,
contributing to the structural integrity of the mid-piece of mature sperm
(Ursuni et al., 1999).
It has been proposed that maturing sperm from luminal fluid internalizes
MAP, so we speculated that relatively high MAP concentrations would be
maintained in the luminal fluid along the male reproductive tract. As part of
our continuing effort to investigate this issue, we studied the presence of
MAP in luminal fluid sampled from different compartments of the mussel tubular
gonad. Unlike most mussel organs, which have completed morphogenesis by the
end of metamorphosis and whose subsequent development is mainly based on the
enlargement of pre-existing structures, the gonad undergoes most of its
morphogenesis in the adult state, depending on the annual reproductive cycle.
Despite considerable efforts (reviewed in
Bayne, 1976;
Seed, 1969
;
Gosling, 1992
), the structural
organization of the M. galloprovincialis gonad has not been studied
in detail. We re-evaluated the gonad pattern formation in mussel mantle
tissue, using juvenile animals as the experimental model. Whole mounts
revealed a clear compartmentalization of the tubular gonad network into STs,
TGDs and the efferent LGD with terminal end bulb-like structures. In keeping
with this pattern, we studied the luminal samples obtained from similar
compartments of the adult male gonad and were able to show that MAP
concentration in the lumen significantly increases en route from STs
to the LGD. In addition, our immunoblot study revealed that the gonad-duct
wall is highly enriched with MAP, which confirms our previous
immunofluorescence results that MAP is localized in the epithelium of the male
gonad ducts (Mikhailov et al.,
1995
; Torrado and Mikhailov,
1998
). It seems likely that, in the mussel gonad, MAP is
synthesized by duct epithelium and may be secreted into the duct lumen. The
precise role of MAP and, in particular, the fluid-phase form of MAP that may
influence sperm functional maturation and motility, merits further
investigation.
Analysis of our previous results and the data of other research on similar
expression patterns of esterases in the male reproductive tract of different
animal groups (i.e. bivalve molluscs, fruitflies and rodents) has prompted us
to formulate the hypothesis (Mikhailov and
Torrado, 1999) that during evolution, esterase-like polypeptides
are recruited by the male reproductive tract to perform new, adapted or
modified, sex-specific roles, taking into account the requirements and
limitations for reproduction in different species. In this paper, we present
additional information on the structural similarity between mussel MAP and
fruit fly esterase S and highlight MAP as a major protein in gonad duct fluid
and spawned sperm. In this context, it should be noted that esterase S is also
secreted into the lumen of the ejaculatory bulb and transmitted with the sperm
to females upon copulation (see Korochkin
et al., 1990
).
Taken together these observations suggest that there is a general trend in
esterase accomplishment of sperm emission in both free-spawners and internal
fertilizers. On the basis of these assumptions, it is reasonable to expect
that esterase-like proteins may be present in the seminal fluid of many
sexually reproducing species. In the present study, the 64 kDa protein
immunochemically similar to MAP and esterase S was identified in human seminal
fluid and spermatozoa. Previous studies on enzyme profiles of human seminal
fluid allowed the detection of uncharacterized protein fractions with broad
esterase-like activities (reviewed in Mikhailov and Torrado,
1999,
2000
). We still do not know
whether the 64 kDa protein, observed by us in human seminal fluid, corresponds
to any of the previously characterized human esterases. Of note, the protein
product of the human liver carboxylesterase gene, HCE-2, has an apparent
molecular mass of 64 kDa (Takai et al.,
1997
). More importantly, the fact that polypeptides similar to
mussel MAP and fruit fly esterase S are also detected in seminal fluid in
humans allows us to hypothesize that such male reproductive-tract-associated
esterases may be essential for sperm fertility. Collectively, the results of
this work as well as the data of other research (reviewed in Mikhailov and
Torrado, 1999
,
2000
), reveal certain
similarities between the mussel and mammalian reproductive system, such as the
production of esterase-like proteins in the somatic gonad and excurrent duct
network, an improvement in motility of sperm within the duct system, and
association of gonad-derived esterases with sperm.
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