Accessory Sex Gland Proteins Affect Spermatophore Digestion Rate and
Spermatozoa Acrosin Activity in Eriocheir sinensis
Author(s): Xue-Li Hou, Qian Mao, Lin He, Ya-Nan Gong, Di Qu, and Qun Wang
Source: Journal of Crustacean Biology, 30(3):435-440. 2010.
Published By: The Crustacean Society
DOI: http://dx.doi.org/10.1651/09-3177.1
URL: http://www.bioone.org/doi/full/10.1651/09-3177.1
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and
environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published
by nonprofit societies, associations, museums, institutions, and presses.
Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of
BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use.
Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries
or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research
libraries, and research funders in the common goal of maximizing access to critical research.
JOURNAL OF CRUSTACEAN BIOLOGY, 30(3): 435-440, 2010
ACCESSORY SEX GLAND PROTEINS AFFECT SPERMATOPHORE DIGESTION RATE AND
SPERMATOZOA ACROSIN ACTIVITY IN ERIOCHEIR SINENSIS
Xue-Li Hou, Qian Mao, Lin He, Ya-Nan Gong, Di Qu, and Qun Wang
(XH; QM; LH; YG; DQ; QW, corresponding author, [email protected]) School of Life Science, East China Normal University,
Shanghai 200062, China
ABSTRACT
Accessory sex gland (ASG) in Eriocheir sinensis is the important component of the male reproductive system; its function is still
unknown. Here we report that ASG protein can digest the spermatophore wall effectively. By optimizing the incubation temperatures,
durations, and secreted protein concentrations using the L30 (52) orthogonal method, we find the protein concentration was the important
factor affecting spermatophore wall digestion. In order to find out the digestion effect and spermatozoa quality, we observed the acrosin
activity and survival rate of free spermatozoa obtained by different methods. We found that the survival rate of ASG protein digestion is
the highest (98.06%), and its acrosin activity of free spermatozoa was significantly higher (4.290 6 0.095 mIU/106) than those obtained
by traditional trypsinase digestion (2.446 6 0.251 mIU/106) or mechanical homogenate (1.423 6 0.109 mIU/106) methods, but it was still
significantly lower than those obtained from the spermatheca of mated females (21.729 6 0.138 mIU/106). Thus, there must be some
cooperation between the ASG and other related fertilization organs to improve the acrosin activity of free spermatozoa in vivo. Hence,
we colleted the spermatozoa isolated from spermatophores using the trypsinase method and incubated with homogenates from seminal
vesicle, accessory sex gland, and spermatheca alone or in combination. We observed that acrosin activity was greatest in samples
incubated with an equal mixture of spermatheca and accessory sex gland protein homogenates. We conclude that the accessory sex gland
can effectively digest the spermatophore wall to release free spermatozoa and interact with proteins from spermatheca to increase
acrosomal enzyme activity of free spermatozoa.
KEY WORDS: accessory sex gland protein, acrosomal enzyme, Eriocheir sinensis, sperm
DOI: 10.1651/09-3177.1
2004), but these two methods often induce cellular damage
and affect analytical results. Thus, further studies on
spermatophore breakdown mechanisms in decapod would
not only advance crustacean spermatozoa laboratory
research, but also promote the study of crustacean
reproductive biology.
The ASG is known to have significance in mammals
(Henault et al., 1995), and its secretions contain a variety of
bioactive molecules. These molecules exert wide-ranging
effects on female reproductive activity, and they improve
the male’s chances of siring a significant proportion of the
female’s offspring (Gillott, 2003). A portion of the ASG
proteins work as nutritional contributions for newly
developed spermatozoa, and other yet unidentified factors
are capable of inducing a cascade of spermatozoa
membrane alterations (Polakoski and Kopta, 1982) and
exert influence on spermatozoa vitality (Sostaric et al.,
2008), physiological state (Ying et al., 1998), motility and
capacitation (Setchell et al., 1994; Elzanaty et al., 2002), as
well as fertilization capacity (Henault et al., 1995; Ying et
al., 1998). Eriocheir sinensis (Milne Edwards, 1853) is an
economically important aquaculture species in China.
Research on male reproductive biology of this crab mainly
focuses on the spermatogenesis, spermatophore formation,
spermatozoa ultrastructure, spermatozoa metabolism, and
testicular EST sequence analysis (Du et al., 1987; Du et al.,
1988; Wang et al., 2000). However, research on the ASG
has gotten less attention. The mitten crab has a welldeveloped ASG in post-sexual maturity at the junction of
the ejaculatory duct and seminal vesicles. Why this organ
INTRODUCTION
The spermatozoa of most decapods reside in the male
seminal vesicle in the form of spermatophores. In
Brachyura, spermatophores are delivered into the spermatheca of the female during mating and gradually are broken
down to release free sperm into the spermatheca and,
ultimately, facilitating spermatozoa and egg fusion to
complete fertilization. Several studies have confirmed that
the spermatophore wall is typically comprised of 1-3 layers
of acid mucopolysaccharide protein, chitinase, phenolic
compounds, and other material compositions (Spalding,
1942; Uma and Subramonian, 1979; Brunet, 1980). Early
studies suggest that mechanical stress and water absorption
may be the primary inducers of Brachyura spermatophore
rupture (Hamon, 1937; Spalding, 1942; Hamon and
Mouchet, 1961; Hinsch, 1988). Later studies extended
these findings by identifying low-molecular-weight material in female secretions that may contain yet unknown
factors that function in the breakdown of the spermatophore
wall (Anilkumar and Adiyodi, 1977; Dudenhausen and
Talbot, 1983; Diesel, 1989).
The mechanism of spermatophore breakdown remains
unclear in decapods. Spermatophore rupture and the
subsequent release of free spermatozoa are prerequisites
for both natural and artificial fertilization, so how does one
obtain high quality free spermatozoa is the key to further
research? Until now, two artificial methods were reported,
mechanical homogenization and enzymatic digestion,
(Leung-Trujillo and Lawrence, 1985; Lezcano et al.,
435
436
JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 30, NO. 3, 2010
develops so rapidly in the peak development stage, and
whether it has similar important function in fertilization in
mammals need to be answered. A series of works, such as
establishing an SSH library of the ASG, identifying ASG
protein by two-dimensional electrophoresis and mass
spectrometry, and effectively improving the ASG protein
digestion of the spermatophore wall have been carried out.
In this paper, we test the effect of ASG proteins in
spermatophore rupture and optimize the reaction conditions, including reaction temperature and protein concentration. We compared the different methods, ASG protein
digestion,trypsinase digestion, and mechanical homogenate, by noting survival rate and acrosomal enzyme
activity. Considering that other reproductive organs may
be involved in the process, the efficiency of homogenates
from several reproductive tissues in digestion of the
spermatophore and effect of sperm acrosome enzyme
activity were also analyzed.
MATERIALS AND METHODS
Experimental Animals
Twenty healthy sexual mature female and male crabs of E. sinensis were
purchased from the Changfeng market in Shanghai, China in December 2008.
Crabs were dissected on ice, and the seminal vesicle, accessory sex gland,
spermatheca, and other relevant organs were immediately removed. Tissues
were processed for analysis immediately. All chemicals were purchased from
Sigma (USA) unless otherwise specified, including benzamidine, ficoll400,
hepes, dimethyl sulfoxide (DMSO), trypsinase, Triton X-100, Na-BenzoylDL-Arginine-b-Nitroanilide (BAP-NA), and eosin.
Ultrastructure of Accessory Sex Gland Contents
Transmission Electron Microscopy (TEM).—A single accessory sex gland
was immersed in 2.5% glutaraldehyde and 4% paraformaldehyde, and
fixed at 4uC overnight, post-fixed in 1% osmium tetroxide for 2 h,
dehydrated through a gradient of alcohols followed by acetone, and
embedded in Epon812. Ultrathin slices were obtained with a Reichert-Jung
ultra-thin slicer, double stained with uranyl acetate and lead citrate, and
observed using a Hitachi H800 model transmission electron microscope
for image capture.
Scanning Electron Microscopy (SEM).—A whole accessory sex gland was
pre-fixed for 5 seconds in 2.5% glutaraldehyde, and the contents was
squeezed into the 2.5% glutaraldehyde fixative medium after its wall was
slightly hardened. The contents were then double-fixed in 1% osmium
tetroxide, dehydrated through a series of alcohol and a gradient of acetone,
dried with a HCP-2 critical point dryer, and sprayed gold with EIKO IB-3
ion sputtering sprayer. The contents were then photographed with a JXA840 scanning electron microscope.
Spermatophore Digestion: Orthogonal Tests under Varying Temperatures
and Accessory Sex Gland Protein Concentrations
In order to find the better combination of protein concentration/
temperature to digest the spermatophore, we did the following experiments. Spermatophores were removed from male crab seminal vesicles,
put into 100 mL of Ca2+-FASW (0.4 mol/L NaCl, 15 mmol/L KCl,
8.6 mmol/L boric acid, 4.8 mmol/L NaOH, 41 mmol/L MgSO4, pH7.4),
and mixed by concussion. One milliliter of the middle aqueous layer was
then transferred to a pre-weighed centrifuge tube, and centrifuged at
400 rpm for 10 min. The spermatophores were precipitated and weighted.
ASG protein homogenate with different concentrations (15.40, 7.70, 3.85,
1.93, 0.96, 0.48 mg/mL) were prepared by a gradient dilution method with
Ca2+-FASW and its concentration determined by Folin-phenol method
(Lowry et al., 1951). Five-times that volume of ASG protein was added to
each spermatophore sample to test the digestion efficiency under different
temperatures (18uC, 23uC, 27uC, 32uC, and 37uC). The time required for
complete spermatophore dissolution at each temperature was recorded.
The L30(52) orthogonal method served as the experimental design, with
incubation temperature (A) and accessory sex gland protein concentration
(B) serving as two factors; each experiment was performed in triplicate. A
total of 30 tests were performed [30 is the number of tests, 5 is the level
number of factors, 2 is the number of factors].
Sperm Isolation Methods
Trypsinase Digestion Method.—Spermatophores (100 mg) were digested in
0.2% trypsinase solution (5-6 times spermatophore volume) at 37uC for 5 min.
One milliliter 15% new bovine serum was added to terminate the reaction
(Ca2+-FASW preparation), followed by centrifugation for 10 min at 400 rpm.
The supernatant was isolated and centrifuged for 10 min at 3000 rpm. The
precipitate was then washed three times with Ca2+-FASW, and concussion
mixed after the addition of 2 mL Ca2+-FASW (Ma et al., 2006).
Mechanical Homogenate Method.—Spermatophores (100 mg) were
suspended in 1 mL Ca2+ -FASW, and manually homogenated on ice for
1.5 min until all spermatophores ruptured. We transferred the homogenate
solution into centrifuge tubes. The subsequent treatment process was the
same as the trypsinase digestion method above.
Accessory Sex Gland Protein Digestion Method.—Spermatophores
(100 mg) were incubated with 15.4 mg/mL accessory sex gland protein
solution (5-6 times spermatophore volume) at 32uC for approximately
7 min until the rupture of all spermatophores. Ruptured spermatophores
were then processed as described above.
Spermatozoa Survival Rate and Acrosomal Enzyme Activity
One milliliter of free spermatozoa suspension extracted from spermatophores as described by the methods above was added to an 0.25% eosin
solution and incubated for 10 min. Sperm were identified as live by vital
dye, and the death rate was calculated (Ma et al., 2006). Free spermatozoa
suspensions extracted from spermatophores as described by the methods
above were adjusted to a similar density (4-5 3 106/mL), and the
acrosomal enzyme activity was determined (Kennedy et al., 1989). First,
the 500mL Ficoll (0.11 M Ficoll in 0.12 M NaCl, 0.025 M Hepes, pH 7.6,
Sigma, USA) was added to all density adjusted sperm samples (200 mL),
then centrifuged at 3000 rpm for 20 min at room temperature. The upper
portion of the suspension (approximately 100 mL) was retained. Controls
consisted of 100 mL 500 mM benzamidine (Sigma, USA) in DI water. One
milliliter of the substrate and decontamination buffer mixture solution
(23 mM BAPNA in 1.1g/mL DMSO, 0.01% Triton X-100 in 0.055 M
Hepes, 0.055 M NaCl, pH 8.0, Sigma, USA) was then added to all
samples, mixed, and incubated for 3 h in a 24uC water bath, with
concussion and mixing every hour. Experimental samples then received
100 mL 500 mM benzamidine in DI water. All samples were then
centrifuged at 2000 rpm for 15 min, and 100 mL of the supernatant was
collected on a micro-glass color plate for analysis on a UV-7504 UV-Vis
spectrophotometer (Hitachi) at a 410 nm wavelength. The substrate and
decontamination buffer mixture solution served as a blank. Enzyme
activity was represented in IU, with 1 IU defined as the level of acrosomal
activity capable of hydrolyzing 1.0 mmol BAPNA per minute at 25uC.
Acrosomal activity was calculated in samples by the following:
acrosin activity mIU 106
~
ðsample mean OD{control mean ODÞ|106
ð1485|sperm count ðmillion=mlÞÞ
Spermatozoa Incubation with Different Reproductive Tract Homogenates
Spermatozoa isolated from spermatophores using the trypsinase digestion
method were divided into 5 experimental groups, and each group being
performed in triplicate. The first three groups received a single
homogenate of identical volume and total protein concentration isolated
from one of three tissues, the spermatheca, seminal vesicle wall, or
accessory sex gland. The fourth group received equivalent volumes of
accessory sex gland and spermatheca homogenates, and the fifth group
served as a negative control containing an equivalent volume of Ca2+FASW. The female crabs that had mated 10 h ago were dissected on ice
and the collected spermaitozoa served as a positive control. Each sample
HOU ET AL.: MALE MATTERS IN ERIOCHEIR SINENSIS
437
Fig. 1. Ultrastructure of accessory sex gland contents of the Chinese mitten crab, Eriochier sinensis. A, transmission electron micrograph (3 10,000), B,
scanning electron micrograph (3 2000) (BV 5 big vesicles; SV 5 small vesicles).
was incubated at room temperature for 30 min, and normalized in sperm
density to the positive control before sperm acrosome enzyme activity was
determined. Statistical analysis was determined using SPSS software, and
significance was set at P , 0.05. Data are expressed as mean (x) 6
standard deviation (s). The t test was adopted for inter-group mean
comparison.
RESULTS
Ultrastructure of Accessory Sex Gland Contents
Two sizes of accessory sex gland vesicles, small and large,
were observed in both transmission and scanning electron
micrographs (Fig. 1). Small vesicles were more predominate in all samples. While both sizes of vesicles displayed
similar electron densities, the small vesicles have substantially greater density of the connective matrix than large
vesicles.
Effects of Temperature and Accessory Sex Gland Protein
Concentration on Spermatophore Digestion
In accordance with the orthogonal test method, a total of 30
experimental conditions were compared with regard to the
time required for complete spermatophore digestion (Table 1). Experimental group 19 (A4B1) required the least
amount of time for spermatophore digestion (7 min), while
group 12 required the longest period of time (116.1 min).
Interestingly, when temperature remained constant, the time
required for spermatophore digestion was inversely proportional to accessory sex gland concentration, increasing from
7 min at 15.40 mg/mL to 73.5 min at 0.48 mg/mL at 32uC.
However, temperature-dependent effects were less robust at
a constant accessory sex gland protein concentration.
Results of orthogonal analysis support these observations
(Table 2). As temperature increased, the time required for
complete spermatophore digestion displayed a parabolic
trend, beginning at the maximum duration, declining to the
minimum and subsequently increasing, with the most rapid
spermatophore digestion observed at 32uC (A4). Moreover,
an increase in accessory sex gland protein concentration
resulted in a longer amount of time required for
spermatophore digestion. Thus, independent analysis suggests a combined treatment of A4B1 as the most
advantageous one, which was confirmed experimentally
in treatment group 19 (A4B1). Furthermore, comparing to
temperature (factor A), the larger R-value for the protein
concentration (factor B) confirmed that protein concentration is a more influential parameter.
Effect of Isolation Method on Sperm Mortality and
Acrosomal Enzyme Activity
Spermatozoa mortality and acrosome enzyme activity were
assessed in samples obtained using three different isolation
methods (Table 3) and differed significantly among isolation methods, with greatest mortality observed with the
homogenization method (22.3%), followed by enzyme
digestion (7.36%), and the lowest in spermatozoa isolated
after accessory sex gland protein exposure (1.94%) (P ,
0.05). Results are in agreement with previously reported data
(Ma et al., 2006). Similarly, acrosomal enzyme activity was
significantly lowest with the homogenization method
(1.423 mIU/106) and greatest with the method after accessory
sex gland protein exposure (4.290 mIU/106) (P , 0.05).
Effects of Reproductive Tissue Homogenates on Acrosome
Enzyme Activity
Free spermatozoa isolated by trypsinase digestion were
exposed to different reproductive tissue homogenates, and
the acrosomal enzyme activity was measured (Table 4). As
predicted, the lowest enzyme activity was observed in the
438
JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 30, NO. 3, 2010
enzyme activity as indicators, we compared three methods
to obtain free spermatozoa. We compared impacts of
different tissue homogenates on acrosin activity to confirm
that the accessory sex gland might increase sperm
acrosomal enzyme activity. All these studies proved helpful
to understand the role of accessory sex gland during the E.
sinensis reproduction.
Based on a two factor orthogonal test, we determined
that both temperature and protein concentration had
significant effects on the rate of spermatophore digestion,
although accessory sex gland protein concentration yielded
more robust effects. We identified incubation with 15.4 mg/
mL accessory sex gland protein at 32uC for 7 min as the
prime conditions for rapid and complete spermatophore
digestion. Furthermore, the accessory sex gland protein
method of spermatophore digestion yielded free spermatozoa with low mortality and high acrosin enzyme activity
compared to the traditional methods of trypsinase digestion
and mechanical homogenization. Data demonstrated that
the accessory sex gland homogenate contained endogenous
unidentified substance, we hypothesize some proteins may
be capable of digesting the spermatophore wall. Spermatozoa released via this process retained high viability and
function, thus we hypothesize fertilization would proceed
effectively. Results reported here further elucidate prospective components of the mechanistic pathways to
rupture the walls of the spermatophore in E. sinensis,
although the proteins displaying this activity yet remain to
be isolated, characterized, and identified.
The accessory sex gland, an important component of the
male reproductive system, opens at the junction of the
seminal vesicle and ejaculatory duct. Secretions of the
accessory sex gland, along with spermatophores from the
seminal vesicle and spermatic fluid, enters into the female
spermatheca through the ejaculatory duct during mating.
Within the spermatheca, the spermatophores are gradually
broken down and free spermatozoa are released, facilitating
sperm and oöcyte interaction and leading to fertilization.
There are always a large number of whole spermatophores
(squeezed into the gland lumen from the seminal vesicle)
within the base lumen of the accessory sex gland near the
seminal vesicle, while our prior observations in mated
females indicate spermatophore rupture occurs slowly over
several hours, results reported here illustrate that the
spermatophore wall is completely digested in a matter of
minutes although rapidity is dependent on protein concentration. We speculate that the small and big vesicles
observed in transmission and scanning electron micrographs may contain the enzymatic proteins or other
activation factors required for spermatophore rupture,
which were immediately released during homogenate
isolation and processing. We hypothesize that in a natural
mating context environmental parameters, such as pH or
spermatheca-produced factors, may induce the slow release
Table 1. Experimental effects of temperature and accessory sex gland
protein concentration on spermatophore digestion rate. Note: Parentheses
indicate the number of the gradient level.
Factors of effect
Treatment
no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Factor A:
temperature (uC)
(1)
(1)
(1)
(1)
(1)
(1)
(2)
(2)
(2)
(2)
(2)
(2)
(3)
(3)
(3)
(3)
(3)
(3)
(4)
(4)
(4)
(4)
(4)
(4)
(5)
(5)
(5)
(5)
(5)
(5)
Factor B: protein
concentration (mg/ml)
18
18
18
18
18
18
23
23
23
23
23
23
27
27
27
27
27
27
32
32
32
32
32
32
37
37
37
37
37
37
(1)
(2)
(3)
(4)
(5)
(6)
(1)
(2)
(3)
(4)
(5)
(6)
(1)
(2)
(3)
(4)
(5)
(6)
(1)
(2)
(3)
(4)
(5)
(6)
(1)
(2)
(3)
(4)
(5)
(6)
Time required for
spermatophore digestion (min)
15.40
7.70
3.85
1.93
0.96
0.48
15.40
7.70
3.85
1.93
0.96
0.48
15.40
7.70
3.85
1.93
0.96
0.48
15.40
7.70
3.85
1.93
0.96
0.48
15.40
7.70
3.85
1.93
0.96
0.48
12.1
16.6
18.1
23.8
53.5
112.7
11.6
13.6
16.0
21.4
45.9
116.1
10.0
11.1
15.7
20.2
42.1
81.5
7.0
7.4
10.1
15.1
25.8
73.5
10.4
11.8
12.9
15.4
33.8
110.0
negative control. Enzyme activity after seminal vesicle
homogenate treatment was also low and showed no
significant difference from the negative control. While
treatment with spermatheca homogenate and accessory sex
gland homogenate remarkably and significantly increased
enzyme activity compared to the negative control (P ,
0.05), there is no difference from one another. Further,
combined treatment of spermatheca and accessory sex
gland homogenates resulted in an additive effect on enzyme
activity, although enzyme activity was still lower than
observed in spermatozoa collected from recently mated
females.
DISCUSSION
Our studies have found the accessory sex gland proteins
function in digesting the spermatophore wall. For determining the best spermatophore digestion condition, we
employed the orthogonal test with varying temperatures
and accessory sex gland protein concentrations in this
study. With the spermatozoa mortality and acrosomal
Table 2. Orthogonal analysis of temperature and accessory sex gland protein concentration effects on spermatophore digestion rate.
Factors of effect
K1
K2
K3
K4
K5
K6
A: temperature (uC)
236.8 224.6 180.6 138.9 194.3 —
B: concentration (mg/ml) 51.1 60.5 62.8 95.9 201.1 493.8
K̄1
K̄2
K̄3
K̄4
K̄5
K̄6
R
39.5
10.3
37.4
12.1
30.1
12.56
23.2
19.18
32.4
40.22
—
98.76
16.3
88.46
439
HOU ET AL.: MALE MATTERS IN ERIOCHEIR SINENSIS
Table 3. Comparison of spermatophore digestion methods regarding sperm mortality and acrosin activity (x 6 s).
Methods
Death rate (%)
Quantity of sperm (106/ml)
Acrosin enzyme activity (mIU/106)
Mechanical homogenate method
Trypsinase digestion method
Accessory sex gland protein digestion method
22.28 6 0.48a
7.36 6 0.30b
1.94 6 0.29c
4.23
4.22
4.19
1.423 6 0.109a
2.446 6 0.251b
4.290 6 0.095c
of the vesicle contained proteins or factors. Although
further study is required to explore these hypotheses, it is
important to note that free sperm are crucial for
fertilization, and as a rule, female E. sinensis ovulate in
24 h after mating; thus, premature sperm capacitation
would directly and adversely affect fertilization success
rates.
The acrosin enzyme, which is a trypsin-like protease, is
essential for fertilization, dissolving the zona pellucida and
permitting sperm penetration of the egg (Shi and Roldan,
1995; Gonzalez-Martinez et al., 2002; Meizel, 1997), but it
can be found in the sperm head in an inactive form in vivo.
Thus, the level of acrosin enzyme activity may have a
direct impact on successful fertilization (Howes and Jones,
2002). Sperm acrosome reaction defects are key examples
of sperm dysfunction (Tarlatzis et al., 1995), and both in
vivo and in vitro experiments show that fertilization is
prevented if the acrosome enzyme is inhibited (Bhattacharyya and Zaneveld, 1982). Conversely, the level of the
enzyme activity is positively correlated with sperm quality,
and directly impacts the outcome of fertilization (Gao et al.,
1995; Wang et al., 1999; Gao et al., 2003). Therefore,
sperm acrosin enzyme activity has become an important
indicator and evaluation tool of semen quality (Cui et al.,
2000), and as such is one of the more sensitive markers of
unexplained clinical male infertility (Jiang et al., 2007). In
the present study, trypsinase digested free sperm treated
separately with spermatheca or ASG homogenate significantly improved sperm acrosomal enzyme activity. The
results observed on samples treated with a combination of
both homogenates resulted in an additive increase in sperm
acrosomal enzyme activity imply that secretions from the
accessory sex gland and spermatheca in combination can
effectively activate the acrosin zymogen and greatly
activate the acrosin enzyme, thereby enhancing spermegg fusion and the fertilization rate. More importantly, the
level of sperm acrosomal enzyme activity in samples
collected from the spermatheca of recently mated females
(for 10 h) was significantly greater, further confirming the
above-mentioned observation.
Table 4. Acrosin activity after treatment with different reproductive
tissue homogenates before and after mating (x 6 s).
No. treatment
The negative control group
Seminal vesicle homogenate fluid
Spermatheca homogenate fluid
Accessory sex gland protein
homogenate fluid
Spermatheca and accessory sex
gland homogenate mixture
The positive control group
Quantity of sperm
(106/ml)
Acrosin enzyme activity
(mIU/ 106)
4.20
4.20
4.20
2.306 6 0.089a
2.691 6 0.320a
9.966 6 0.285b
4.20
10.480 6 0.129b
4.20
4.20
14.800 6 0.062c
21.729 6 0.138d
In conclusion, we find that the contents of the accessory
sex gland are the main factors causing spermatophore
natural breakdown, and the mixture of spermatheca and
ASG protein homogenates could increase the sperm
enzyme acrosin vitality. The best condition for spermatophore digestion is incubated with 15.4 mg/mL ASG protein
at 32uC for 7 min. This result provides a convenient,
reliable method for obtaining free sperm, which could
ensure the more effective sperm physiological and
functional studies of this species of crab, but the specific
proteins and its mechanism are unknown.
ACKNOWLEDGEMENTS
This research was supported by grants from the ‘‘Dawn’’ Program of
Shanghai Education Commission (No.08SG24), the National Nature
Science Foundation of China (No. 30972241), and the Scientific Research
Foundation for the Returned Overseas Chinese Scholars, State Education
Ministry.
REFERENCES
Anilkumar, G., and K. G. Adiyodi. 1977. Spermatheca of the fresh water
crab, Paratelphusa hydromous (Herbst), in relation to the ovarian cycle,
pp. 267-274. In, K. G. Adiyodi and R. G. Adiyodi (eds.), Advances in
Invertebrate Reproduction, Vol. l. Peralam-Kenoth, Karivellur, India.
Bhattacharyya, A. K., and L. J. D. Zaneveld. 1982. The sperm head, pp.
119-151. In, L. J. D. Zaneveld and R. J. Chatterton (eds.), Biochemistry
of Human Reproduction. Wiley, New York.
Brunet, R. 1980. La composition des mod’eles dans l’analyse spatiale.
L’espace geographique 4: 253-265.
Cui, Y. H., R. L. Zhao, Q. Wang, and Z. Y. Zhang. 2000. Determination of
sperm acrosin activity for evaluation of male fertility. Asian Journal of
Andrology 2: 229-232.
Diesel, R. 1989. Structure and function of the reproductive system of the
symbiotic spider crab Inachus phalangium: observations on sperm
transfer, sperm storage, and spawning. Journal of Crustacean Biology 9:
266-277.
Du, N. S., W. Lai, and L. Z. Xue. 1987. Acrosome reaction of the sperm in
the Chinese mitten- handed crab, Eriocheir sinensis (Crustacea,
Decapoda). Acta Zoologica Sinica 33: 8-14.
———, ———, and ———. 1988. Studies on the sperm of Chinese
mitten- handed crab, Eriocheir sinensis (Crustacea, Decapoda) II.
Spermatogenesis. Oceanologia et Limnologia Sinica 19: 71-75.
Dudenhausen, E. E., and P. Talbot. 1983. An ultrastructural comparison of
soft and hardened spermatophores from the crayfish Pacifastacus
leniusculus Dana. Canadian Journal of Zoology 61: 182-194.
Elzanaty, S., J. Richthoff, J. Malm, and A. Giwercman. 2002. The impact
of epididymal and accessory sex gland function on sperm motility.
Human Reproduction 17: 2904-2911.
Gao, X. Q., W. Yang, and A. P. Xu. 1995. Relations between intact rate of
acrosome and activity of acrosin in sperm. Journal of Gui Yang Medical
College 20: 18-20.
———, A. P. Xu, X. L. Zhang, F. Mo, and J. Wang. 2003. The detection
and analysis on acrosin in the sperm of 217 cases of male infertility.
Journal of Reproductive Medicine 12: 202-205.
Gillott, C. 2003. Male accessory gland secretions: odulators of Female
Reproductive Physiology and Behavior. Annual Review of Entomology
48: 163-84.
440
JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 30, NO. 3, 2010
Gonzalez-Martinez, M. T., M. A. Bonilla-Hernandez, and A. M. GuzmanGrenfell. 2002. Stimulation of voltage-dependent calcium channels
during capacitation and by progesterone in human sperm. Archives of
Biochemistry and Biophysics 408: 205-210.
Hamon, J., and J. Mouchet 1961. Les vecteurs secondaires du paludisme
humain en Afrique. Médecine Tropicale 21: 643-660.
Hamon, M. 1937. Les mechanismes produisant la déhiscence des
spermatophores d’Eupagurus brideauxi Leach. C. R. Academy of
Sciences. Paris 204: 1504-1506.
Henault, M. A., G. J. Killian, J. F. Kavanaugh, and L. C. Griel. 1995.
Effect of accessory ex gland fluid from bulls of differing fertilities on
the ability of caudal epididymal sperm to penetrate zona-free bovine
oocytes. Biology of Reproduction 52: 390-397.
Hinsch, G. W. 1988. Ultrastructure of the sperm and spermatophores of the
golden crab Geryon fenneri and a closely related species, the red crab G.
quinquedens, from the eastern Gulf of Mexico. Journal of Crustacean
Biology 8: 340-345.
Howes, L., and R. Jones. 2002. Interactions between zona pellucida
glycoproteins and sperm proacrosin/acrosin during fertilization. Journal
of Reproductive Immunology 53: 181-192.
Jiang, X. H., S. Ye, and H. Y. Wang. 2007. Influence of circadian rhythm
gene clock on the fertility of malemice. Space Medicine and Medical
Engineering 20: 354-357.
Kennedy, W. P., J. M. Kaminski, H. H. Van der Ven, R. S. Jeyendran, D.
S. Reid, J. Blackwell, P. Bielfeld, and L. J. Zaneveld. 1989. A simple,
clinical assay to evaluate the acrosin activity of human spermatozoa.
Journal of Andrology 10: 221-231.
Leung-Trujillo, J. R., and A. L. Lawrence. 1985. The effect of eyerallk
ablation on spermatophore and sperm quality in Penaeus vannamei.
Journal of the World Mariculture Society 16: 258-266.
Lezcano, M., C. Granja, and M. Salazar. 2004. The use of flow cytomerty
in the evaluation of cell viability of cryopreserved sperm of the marine
shrimp (Litopenaeus vannamei). Cryobiology 48: 349-356.
Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951.
Protein measurement with the Folin phenol reagent. Journal of
Biological Chemistry 193: 265-275.
Ma, Q., Q. Wang, K. Li, and Y. D. Ding. 2006. Comparative study of
trypsinase digestion and homogenization in acquiring free sperms from
Eriocheir sinensis. Journal of East China Normal University (Natural
Science) 2: 82-87.
Meizel, S., K. O. Turner, and R. Nuccitelli. 1997. Progesterone triggers a
wave of increased free calcium during the human sperm acrosome
reaction. Developmental Biology 182: 67-75.
Milne Edwards, H. 1853. Mémoires sur la famille des Ocypodiens, suite
(1). Annales des Sciences Naturelles série 3, Zoologie 20: 163-228.
Polakoski, K. L., and M. Kopta. 1982. Seminal plasma, pp. 89-116. In, L.
J. D. Zaneveld and R. T. Chatterton (eds.), Biochemistry of Mammalian
Reproduction. John Wiley and Sons, New York.
Setchell, B. P., S. Maddocks, and D. E. Brooks.1994. Anatomy,
vasculature, innervation, and fluids of male reproductive tract, pp.
1063-1175. In, E. Knobil and J. D. Neil (eds.), The Physiology of
Reproduction. Raven Press, New York.
Shi, Q. X., and E. R. Roldan. 1995. Bicarbonate/CO2 is not required for
zona pellucida- or progesterone-induced acrosomal exocytosis of mouse
spermatozoa but is essential for capacitation. Biology of Reproduction
52: 540-546.
Sostaric, E., M. Aalberts, B. M. Gadella, and T. A. E. Stout. 2008. The
roles of the epididymis and prostasomes in the attainment of fertilizing
capacity by stallion sperm. Animal Reproduction Science 107: 237-248.
Spalding, J. F. 1942. The nature and formation of the spermatophore and
sperm plug in Carcinus maenas. Quarterly Journal of Microscopical
Science 83: 399-422.
Tarlatzis, B. C., E. M. Kolibianakis, J. Bontis, M. Tousiou, S. Lagos, and
S. Mantalenakis. 1995. Effect of pentoxifylline on human sperm
motility and fertilizing capacity. Systems Biology in Reproductive
Medicine 34: 33-42.
Uma, K., and T. Subramoniam. 1979. Histochemical characteristics of
spermatophore layers of Scylla serrata (Forskal) (Decapoda: Portunidae). Journal of Invertebrate Reproduction 1: 31-40.
Wang, C. Z., Z. G. Xu, J. W. Zhuo, C. H. Xiao, and H. Wang. 1999.
Analysis on influence factor and activity of human sperm acrosin.
Journal of Norman Bethune University of Medical Science 22: 583-585.
Wang, Q., Y. L. Zhao, W. Lai, and N. S. Du. 2000. Ultrastructural Study
on Spermatophore Formation in Eriocheir sinensis H. Milne Edwards.
Journal of East China Normal University (Natural science) 3: 98-103.
Ying, Y., P. H. Chow, and W-S. O. 1998. Effects of male accessory sex
glands on deoxyribonucleic acid synthesis in the first cell cycle of
golden hamster embryos. Biology of Reproduction 58: 659-663.
RECEIVED: 26 May 2009.
ACCEPTED: 20 October 2009.