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
Mario Torrado1,
María Paz1,
Leonid I. Korochkin2 and
Alexander T. Mikhailov1,*
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
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Fig. 3. Structural organization of the male reproductive system in the mantle of
1-year-old (A—D) and adult (E,F) mussels. (A) Dorsal view of the right
(R) and left (L) shells; a—p, anterior-posterior axis; v—d,
ventral-dorsal axis. (B) Paired tubular gonad and reproductive tract spreading
into a two-lobe mantle sheet (whole-mount histology). (C) View of the tubular
gonad network in one mantle lobe (whole-mount histology). (D) Detailed view of
the ejaculatory bulb-like structure (whole-mount histology). ST, spermatogenic
tubule; TGD, transversal gonad duct; LGD, longitudinal gonad duct; EB,
`ejaculatory' bulb-like structure. (E) The ST-network occupies almost the
entire volume of the mantle lobe in adults. (F) Sperm emission (SP) through
the EB-like structure. G, gills; PAM, posterior adductor muscle. Scale bars, 1
mm (A); 0.5 mm (B,E); 0.2 mm (C); 0.1 mm (D); 2 mm (F).
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Fig. 1. The identification of MAP as a dual component of M.
galloprovincialis semen. (A—C) Sequential phases of spawning (bars,
1.6 cm). (A) Whitish-colored semen streams are emitted by the male. (B) 5-10
min later, one portion of the sperm (II) is dispersed into seawater
(asterisk), whereas the other portion (I) forms compact thread-shaped
structures (black arrow), which are precipitated at the bottom of the tank
(III, white arrow) 30-40 min after sperm emission (C). (D,E) The sperm
suspension (II) and thread-shaped semen (I and III) were sampled, separated
into cell-free seminal fluid (sf) and sperm (sp) fractions and studied by
SDS-PAGE (D) followed by western blot (E) with anti-MAP antibodies. The
protein loading for each lane is indicated. Lane 1, extract from the total
male gonad (15 µg). Lanes 2 and 6, sperm fraction of the semen (15 µg
each). Lanes 3-5, cell-free seminal fluid (2.0 µg, 0.3 µg and 2.0 µg,
respectively). In both the soluble phase (Lanes 3-5) and in the cell fraction
(Lanes 2, 6) of each semen sample, the 39 kDa band was detected on the gel (D)
and it was positively stained by anti-MAP antibodies on the blot (E). The
positions of marker proteins (kDa) are shown.
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Fig. 2. MAP is a sperm-associated protein. Equal samples of spawned sperm (A, Lanes
2-9) were treated with Triton X-100 (TX-100) at final concentrations varying
from 0.01% to 2.00%, respectively. Control (Lane 1) and detergent-treated
spermatozoa (Lanes 2-9) were studied by SDS-PAGE (A) followed by western blot
with anti-MAP (B) or anti-esterase S (C) antibodies. As shown in A, the 20 kDa
band intensity of the treated sperm remains unchanged at all TX-100
concentrations used. (D) Resulting supernatants of control (Lane 1s) and
TX-100-treated spermatozoa (Lanes 2s, 3s and 9s) were concentrated and
subjected to SDS-PAGE followed by western blot with anti-MAP antibodies. (E)
Graphical presentation of the MAP densitometric analyses of both the gel (A)
and blots (B,C), showing the percentage of the protein resistant to extraction
by TX-100. Note that the three profiles (A—C) have the same MAP
retention dynamics. (F) Immunofluorescence localization of MAP in the
mid-piece region of spawned spermatozoa (white arrows) using anti-esterase S
antibodies. (G) Control reaction: no MAP-positive signals were detected in the
sperm cells treated with the antibodies pre-absorbed by MAP. Bars, 3
µm.
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Fig. 4. MAP is a major protein constituent of the luminal gonad-duct fluid.
Representative SDS-gel electrophoresis (A) and western blot (B) revealed with
anti-MAP antibodies. The protein loading for each lane is indicated. Lane 1,
extract from the total gonad/mantle (15 µg). Lanes 2 and 3, cell-free
fraction obtained from the lumen of the longitudinal gonadal duct (LGD) (4
µg per lane). Lanes 4 and 5, cell-free fraction obtained from the lumen of
the transversal gonadal ducts (TGDs) (4 µg per lane). Lane 6, sperm cells
obtained from the lumen of the TGDs (15 µg). Note that the intensity of MAP
immunostaining is much higher in the luminal fluid than in sperm and total
gonad extract. The positions of marker proteins (kDa) are shown.
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Fig. 5. The MAP:total protein ratio (%) is higher in the seminal fluid than in the
spermatogenic tubule (ST) luminal fluid. Representative scan profiles of the
Coomassie-stained proteinograms after SDS-PAGE. The protein loading for each
lane is indicated. (A) Seminal fluid (1.5 µg). (B) Luminal fluid of the
longitudinal gonadal duct (2.5 µg). (C) Luminal fluid of the STs (10
µg). (D) Extract from the whole gonad/mantle (10 µg). Dotted lines
indicate the MAP fractions, 39 kDa.
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Fig. 6. MAP is highly detected in the gonad-duct wall of adult males. (A) One of
the transversal gonadal ducts (TGDs; arrows) located in the posterior mantle
region was microsurgically isolated and assayed as described in Materials and
methods (bar, 0.4 cm). (B) Western blot (after SDS-PAGE) with anti-MAP
antibodies. The protein loading for each lane is indicated. Lane 1, extract
from the total male gonad/mantle (12 µg). Lanes 2 and 3, extract from the
gonad-duct wall (12 and 6 µg, respectively). Lane 4, cell-free luminal
fluid (5 µg). Lane 5, sperm cells from the lumen (10 µg). Note that the
intensity of MAP imunostaining is higher in the gonad-duct wall (Lanes 2, 3)
in comparison with that of the luminal fluid (Lane 4) and sperm (Lane 5).
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Fig. 7. Isolation of MAP by isoelectric focussing (IEF) followed by SDS-PAGE with
immuno-identification of MAP fractions by western blot using three different
antibodies. (A) IEF: Lanes 1-3, water-soluble extract from a whole
gonad/mantle. (B) Western blots after IEF revealed with anti-MAP (Lane 4),
anti-esterase S (Lane 5), and anti-porcine esterase (Lane 6) antibodies. Note
that the same single band of pI 6.2 was detected by all antibodies. (C)
SDS-PAGE of MAP fraction (Lanes 1-3) isolated from IEF-gels. Only 39 kDa bands
were observed in the SDS-gel, which indicates the homogeneity of the MAP
IEF-fraction. (D) Western blots after SDS-PAGE revealed with anti-MAP (Lane
4), anti-esterase S (Lane 5), and anti-porcine esterase (Lane 6) antibodies.
The SDS-fraction contains the only 39 kDa mussel MAP, immunologically similar
to both D. virilis esterase S and pig esterase.
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Fig. 9. A 64 kDa polypeptide is detected in human ejaculate by
anti-Mytilus MAP and anti-Drosophila esterase S antibodies.
Equal amounts (20 µg of total protein) of cell-free (Lane 1) and sperm-cell
(Lane 2) fractions of human semen were separated by SDS-PAGE (A), blotted on
membranes and treated with anti-MAP (B) or anti-esterase S (C) antibodies.
Lane 3, rabbit liver carboxylesterase, 1 µg (Sigma). Note that the
intensity of immunostaining of the 64 kDa band is slightly higher in the sperm
than in the seminal fluid. The positions of marker proteins (kDa) are
shown.
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© The Company of Biologists Ltd 2003
