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- Original Article -
Assessment of released acrosin activity as a measurement of
the sperm acrosome reaction
Rui-Zhi Liu1, Wan-Li Na2, Hong-Guo
Zhang3, Zhi-Yong Lin4, Bai-Gong
Xue1, Zong-Ge Xu1
1Department of Cell Biology, School of Basic Medical Sciences, Jilin University, Institute for Reproductive Medicine of
Jilin Province, Changchun 130021, China
2Department of Urology, The China-Japan Union Hospital, Jilin University, Changchun 130021, China
3Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China
4Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
Abstract
Aim: To develop a method for assessing sperm function by measuring released acrosin activity during the acrosome
reaction (AR). Methods: Human semen samples were obtained from 24 healthy donors with proven fertility after
3_7 days of sexual abstinence. After collection, samples were liquefied for 30 min at room temperature. Standard
semen parameters were evaluated according to World Health Organization (WHO) criteria. Calcium ionophore
A23187 and progesterone (P4) were used to stimulate the sperm to undergo AR. After treatment, sperm were
incubated with the supravital dye Hoechst33258, fixed in a glutaraldehyde-phosphate-buffered saline solution, and
the acrosomal status was determined by fluorescence microscopy with fluorescein isothiocyanate-labeled
Pisum sativum agglutinin (FITC-PSA). The percentage of sperm undergoing AR (AR%) was compared to sperm acrosin
activities as assessed by spectrocolorimetry. The correlation between AR% and acrosin activity was determined by
statistical analysis. Results: The AR% and released acrosin activity were both markedly increased with A23187
and P4 stimulation. Sperm motility and viability were significantly higher after stimulation with P4 versus
stimulation with A23187 (P < 0.001). There was a significant positive correlation between released acrosin activity and
AR% determined by FITC-PSA staining (r = 0.916,
P < 0.001).
Conclusion: Spectrocolorimetric measurement
of released acrosin activity might serve as a reasonable alternative method to evaluate
AR. (Asian J Androl 2008 Mar; 10: 236_242)
Keywords: human sperm; released acrosin activity; sperm function
Correspondence to: Prof. Rui-Zhi Liu, PhD, Department of Cell Biology, School of Basic Medical Sciences, Jilin University, 126 Xin-min
Avenue, Changchun 130021, China.
Tel: +86-431-8888-3399 Fax: +86-431-8873-8877
E-mail: lrz420@126.com
Received 2006-12-10 Accepted 2007-05-20
DOI: 10.1111/j.1745-7262.2008.00312.x
1 Introduction
The acrosome reaction (AR) is an essential step for
the fertilization of ova by spermatozoa. The AR can
occur only in capacitated spermatozoa, and is a calcium
dependent, exocytotic event, resulting in the release of
hydrolytic enzymes. The AR facilitates the penetration
of the zona pellucida by spermatozoa and the subsequent fusion of the sperm plasma membrane with the
oocyte's oolemma. Reference studies have shown that
an abnormal AR can be one of the causes of unexplained
infertility [1].
Recently, the AR assay has become an important method for evaluating sperm function and investigating
human reproduction. Since Barros
et al. [2] verified the AR by electron microscopy, a variety of methods
for detecting the AR have been introduced using triple
or double staining or staining of acrosome with
fluorescein-isothiocyanate Pisum sativum agglutinin
(FITC-PSA). The most frequently used techniques for
evaluating the AR are based on light microscopy, in which
the acrosomal content is labeled either by a triple
staining technique or with fluorescent lectins, such as
FITC-PSA. However, these methods are either complex or
have difficulty in sperm sample conservation. Flow
cytometric analysis using monoclonal antibodies against
the spermatozoa membrane cofactor protein CD46 has
also been introduced to assess the AR. However, the
limited availability of these antibodies and their high cost
precludes their routine application. Electron
microscopy is the "gold standard" method for assessing the
AR [3]. However, the equipment is expensive and the
process is complex.
Because the acrosome of human spermatozoa is small, it is the difficult to detect the AR. Moreover, all
methods developed so far have some limitations,
therefore they cannot be used widely in clinical practice. All
these methods detect only acrosome-reacted spermatozoa,
and do not assess released acrosin activity, which is
characteristic of the AR. For example, when AR occurs,
there are differences in the quantity and activity of
released acrosin between large head sperm and small head
sperm. As a result, sperm AR is also different. Accurate
assessment of the level of acrosome-reacted sperm in a
sperm population is of great importance for both basic
research of human reproduction and the clinical
evaluation of male fertility. Therefore, it is important to
develop a technique for detecting the sperm AR function to
elucidate the mechanism of the AR and fertilization.
The purpose of this work was to develop a method
for assessing human sperm AR function by detecting
released acrosin activity during the sperm AR. AR is
required to obtain released acrosin. Physiologically, the
AR in human spermatozoa takes place only after
their binding to the zona pellucida. However, gaining zona
pellucida is not easy. Progesterone (P4) is used more
frequently as a near-physiological agent. However, it
is debatable whether P4 can promote the acrosome
reaction at physiological concentrations.
In the current study, several comparative analyses
were carried out to clarify whether released acrosin
activity of sperm AR can assess sperm function or not.
2 Materials and methods
2.1 Collection and analysis of semen sample
Human semen samples were collected from 24 healthy donors (age 26_34 years). All donors had proven
fertility and samples were taken after sexual abstinence
for 3_7 days. Semen samples from infertile patients were
provided by the First Hospital, Jilin University. Samples
were liquefied for 30 min at 37ºC. Completely liquefied
samples were analyzed for standard semen parameters
according to the World Health Organization (WHO)
criteria [4]. A simplified discontinuous Percoll gradient
centrifugation was applied to collect motile sperm. The sperm
viability was analyzed using eosin-Y staining.
In addition, semen samples were collected from 39
infertile subjects, including oligozoospermic (11),
asthe-nozoospermic (15) and teratozoospermic (13) semen.
Percentage of acrosome-reacted sperm and AR released
acrosin activity were detected.
2.2 Sperm capacitation
The concentration of the motile spermatozoa was
adjusted to 10 × 106/mL. The sperm suspension was
then incubated for 4 h under an atmosphere of 5%
CO2 at 37ºC.
2.3 Measure of AR released acrosin activity
P4 (Sigma, St. Louis, MO, USA) and calcium ionophore A23187 (Sigma, St. Louis, MO, USA) were
dissolved in DMSO (Sigma, St. Louis, MO, USA). Stock
solutions of P4 and A23187 were kept at _20ºC.
Working solutions were prepared by diluting the thawed stock
solutions 1:10 in BWW 30 min before adding them to the
sperm suspension. Capacitated sperm were treated with
10 µmol/L A23187, or with concentrations of P4
ranging from 1 µg/mL_40 µg/mL. Tubes were loosely
capped and incubated at 37ºC for 30 min. The
supernatant was collected after centrifugation in five
different Eppendof tubes, then sperm acrosin activities were
analyzed by spectroco-lorimety according to the method of
Cui et al. [5].
2.4 Acrosomal staining with FITC-PSA
After treatment with A23187 or P4, the sperm
solution was centrifuged (5 min). Glutaraldehyde-
phosphate-buffered saline (PBS) solution (1 mL, 3%) was added to
the sediment for fixation (10 min). Then, spermatozoa
were washed twice in PBS solution. Sperm suspensions
(10 µL) were smeared onto glass slides and air-dried.
Staining of acrosome with FITC-PSA was performed according to Mendoza
et al. [6]. The FITC-PSA solution was washed out by dipping the slides into PBS
solution.
2.5 Quality control
All experiments included a positive control. To avoid
a false measurement because of dead sperm or sperm
with degenerative acrosomal loss, double-staining of
sperm was performed using FITC-PSA and Hochest33258.
Every treated sperm sample was smeared onto two slides.
By means of a fluorescence microscope, at least 200
spermatozoa were evaluated blindly according to the
fluorescence pattern of their acrosomes. Two investigators
evaluated each slide, and the error between their data
was less than 15%. The average of two independent
values was recorded as the AR.
2.6 Statistical analysis
SPSS 11.5 software (SPSS, Chicago, IL, USA) was
used for the statistical calculations. The values reported
in the text and tables are mean ± SD
(range). The significance of the difference between the treatments was
assessed using unpaired t-test. Differences were
considered to be statistically significant when
P < 0.05.
3 Results
3.1 Undetected acrosomal contents revealed
After glutaraldehyde-PBS fixation agglutinin
(FIT-CPSA) Staining, undetected acrosomal contents were
revealed (Figure 1).
3.2 Comparison of the percentage of acrosome-reacted
sperm and AR-released acrosin activity after stimulation
with calcium ionosphore A23187 and P4
The percentage of acrosome-reacted sperm and
AR-released acrosin activity were both markedly increased
(P < 0.05), when compared to control (capacitation 4 h)
(Table 1).
3.3 Effect of P4 concentration on the percentage of
acrosome-reacted sperm, sperm motility and viability
At final concentrations of P4 between 10 µg/mL and
40 µg/mL, the acrosome reaction of the sperm was
markedly increased. The differences were statistically
significant when compared to the control group
(P < 0.05). At a final P4 concentration of
1 µg/mL, the difference was not statistically significant
(P > 0.05). There was no difference in the percentage of acrosome reacted sperm when
compared to the A23187 positive control
(P >0.05). There was also no difference in sperm motility and viability when
compared with control (P > 0.05). However, the
differences of sperm motility and viability were statistically
significant when compared to the A23187 control group
(P < 0.001) (Table 2).
3.4 Relationship of the percentage of acrosome-reacted
sperm and released acrosin activity, when using P4 at
different concentrations to induce AR
At concentration of 10 µg/mL, the percentage of
acrosome reacted sperm and released acrosin activity
were both markedly increased when compared to doses
of 1 µg/mL and 5 µg/mL
(P < 0.05) (Tables 2 and 3).
In addition, Spearman correlation coefficients were
calculated for the values obtained by labeling with
FITC-PSA and released acrosin activity during the AR. There
was a significant positive correlation between released
acrosin activity during the AR and the percentage of
acrosome-reacted sperm (r = 0.916,
P < 0.001).
3.5 Comparison of the percentage of acrosome-reacted
sperm and released acrosin activity of infertile group with
those of the fertile group
The percentage of acrosome-reacted sperm and released acrosin activity in the oligozoospermic,
asthenozoospermic and teratozoospermic groups were all lower
than those of the fertile group
(P < 0.05). The percentage of acrosome-reacted sperm and released acrosin
activity in the teratozoospermic group was the lowest of
the three infertile groups. However, there was no
difference between the result for the oligozoospermic,
asthenozoospermic or teratozoospermic groups
(P > 0.05) (Table 4).
Spearman correlation coefficients were calculated for
the values obtained by labeling with FITC-PSA and
released acrosin activity during the AR. There was a
significant positive correlation between released acrosin
activity during the AR and the percentage of acrosome-
reacted sperm (r = 0.931,
P < 0.001).
4 Discussion
Physiologically, the AR in human spermatozoa takes
place only after they bind to the zona pellucida
glycoprotein (ZP3). However, because of the paucity of available
human ova, the ZP protein is not often used in AR assays.
A suitable stimulus is necessary for routine AR assay.
The morphological changes of the human sperm acrosome after treatment with calcium ionophore A23187
are similar to those occurring during spontaneous or
physiologically-induced AR [7].
However, the spontaneous AR, as well as the AR
induced by P4, phorbol myristate ester and follicular fluid,
is usually partial. In contrast, the AR induced by A23187
is total (i.e. it includes both partial and complete)
[8]. P4 can also be used as a stimulus. Avalos-Rodriguez
et al. [9] described one phenotypic difference in a study of
rabbit sperm. After AR induction with P4, the localization
of phosphatidylserine was changed and the Annexin-V
binding sites were found only in the acrosomal region, but
with a higher number of binding sites in the equatorial
area. In contrast, after AR induction with A23187,
phosphatidylserine translocation, although predominant
over the acrosomal region, was also observed in the
post-acrosomal region. However, it is still debatable whether
P4 at physiological concentrations promotes AR. In the
present study, we used P4 as a near-physiological
inducer to induce AR.
From Figure 1, after acrosome stained with
FITC-PSA, fluorescently-labeled acrosome indicates whether
sperm structure is intact or not. If acrosome is not intact,
it indicates that sperm develops AR. Therefore,
percentage of acrosome-reacted sperm labeled with FITC-PSA
can evaluate sperm AR. The released acrosin activity
during AR and the percentage of acrosome-reacted sperm
were then evaluated. By comparison, we want to know
whether the released acrosin activity during AR can
determine sperm AR or not.
Acrosin is an acrosomal protease synthesized as a
pro-enzyme and activated into beta-acrosin during the
AR. It is presumed to be involved in the recognition and
binding of the sperm to the zona pellucida of the ovum
and in sperm penetration through the zona pellucida. In
2004, Lax et al. [10] described an acrosin activity assay
for the evaluation of the mammalian sperm acrosome
reaction. Acrosin is a key enzyme in fertilization and its
activity directly affects the fertilization rate.
Spermatozoa lacking acrosin protein
(Acr_) show a delayed fertilization. Acr
_ sperm have a selective disadvantage when they compete with
Acr+ sperm [11]. Langlois
et al. [12] conclude that the sperm acrosin assay could help to
predict sperm fertilizing capacity in in
vitro fertilization independent of sperm morphology. Chaudhury
et al. [13] reported that total acrosin activity might be
considered as a sensitive biochemical marker for the clinical
evaluation of unexplained infertility in men. Therefore,
acrosin activity during the AR can reflect the function of
the AR.
The percentage of acrosome reacted sperm and released acrosin activity were both markedly increased,
when either A23187 or P4 were used to induce AR (Table 1). This indicates that A23187 and P4 can induce
human sperm AR, as agreed by Katsuki
et al. [14], and by Bronson
et al. [15].
Our results showed that incubation of spermatozoa
with 10 µg/mL_40 µg/mL P4 for 30 min induced the sperm
AR (Table 2). The differences were statistically
significant when compared to control. No significant
differences were found between the 10 µg/mL,
20 µg/mL or 40 µg/mL P4-treated group. Some sperm plasma
membrane receptors are involved in the P4-initiated AR. If
the concentration of P4 is too low, some receptor sites
in the heads of the sperm are not active. Therefore, not
many cells get stimulated. However, when the
concentration of P4 reaches a certain level, the sites of all
receptors are activated. At this time, adding more P4 to
the sperm suspension cannot further increase activity of
P4-receptor.
We found that sperm motility and viability were both
markedly decreased with increased A23187 concentration
(P < 0.05). When using P4 as the AR inducer, there
was no significant difference in capacitated sperm
motility and viability. However, significant differences were
found between P4 and A23187
(P < 0.001). This finding implies that P4 would be a near-physiological AR
stimulus. Our data are consistent with the principle that
there is a clear difference between acrosome reactions
induced by P4 and A23287, as reported in the mouse by
Brucker et al. [16].
According to Bronson et al. [15], P4 promotes an
acrosome reaction within capacitated spermatozoa. Shoeb
et al. [17] indicate that P4 induces the formation of
horizontal microdomains within the exofacial surfaces of sperm
membranes, leading to progressive and independent
alterations in molecular dynamics. Pietrobon
et al. [18] report that the P4-induced AR is driven by activation of
G-proteins, which in turn activate PLA2 and PLC
simultaneously, which finally promote acrosomal exocytosis. Therefore, P4-induced AR would be a
near-physiological process.
After induction of the AR by P4, the percentage of
acrosome reacted sperm and released acrosin activity
were both markedly increased (Table 3). There was a
significant positive correlation between released acrosin
activity during the AR and the percentage of acrosome
reacted sperm (r = 0.916,
P < 0.001). This finding indicates that released acrosin activity during the AR is
consistent with the values obtained by labeling with
FITC-PSA, and suggests that released acrosin activity can
indirectly reflect the acrosomal status. The results confirm
the finding by Köhn et al. [3] that there is a positive
correlation between FITC-PSA labeling and studies using
transmission electron microscopy.
We compared the percentage of acrosome reacted sperm and released acrosin activity in fertile and infertile
men (Table 4). The finding that the acrosin activity of
asthenozoospermic men was lower than that of the
fertile group is consistent with the conclusion by Nakagawa
et al. [19]. Previous work has shown that spermatozoa
from asthenozoospermic might owe their poor motility
and their inability to properly capacitate and develop
hyperactivation to impair tyrosine phosphorylation of
critical proteins caused by decreased membrane fluidity
[20]. Because noncapacitated sperm do not undergo AR,
the percentage of acrosome-reacted sperm declines.
Released acrosin activity is low too. In addition, the
percentage of acrosome-reacted sperm and released acrosin
activity of the teratozoospermic group was the lowest
among the three infertile groups. Previous work has
reported that a defective sperm-zona pellucida
interaction was present in 64% of teratozoospermic infertile
men: 31% had defective sperm-zona pellucida binding,
and 33% low zona pellucida induced acrosome reaction
(ZPIAR) [21]. This finding showed that abnormal acrosin
activity might be one cause of teratozoospermic infertility.
There was a significant positive correlation between
released acrosin activity during the AR and the
percentage of acrosome reacted sperm (r = 0.931,
P < 0.001). This finding suggests that the released acrosin activity
during the AR is consistent with the values obtained by
labeling with FITC-PSA. In previous studies, most of
the different methods assess only the acrosomal status
without assessing released acrosin activity. Using these
methods, one has to make the assumption that the 200
sperm counted are representative of the entire sample.
Soderlund et al. [22] report that the evaluation of the
acrosome index would not accurately predict fertilization.
Our study suggests that measuring the released acrosin
activity during the AR can assess AR activity
independent of the percentage of acrosome reacted sperm.
In conclusion, detecting the sperm AR is an important
method for the assessment of sperm function and basic
research of human reproduction. Measuring the released
acrosin activity during the AR can determine sperm AR
function. The procedure is an alternative method for
evaluating true AR in whole sperm populations. This method is
an objective technique for accurate assessment of AR
function and, therefore, the observer's error that is
inherent in light microscopic methods is avoided.
Acknowledgment
We would like to thank Professor F. William Orr for
his critical reading of the manuscript. This work is
supported by a grant (No. 20030433) from the Department
of Science and Technology of Jilin Province, China.
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