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J Biol Chem, Vol. 273, Issue 17, 10470-10474, April 24, 1998
From the Institute of Applied Biochemistry and Tsukuba Advanced
Research Alliance, University of Tsukuba, and the National Institute
for Advanced Interdisciplinary Research, Tsukuba Science City, Ibaraki
305-8572, the Using homologous recombination, we have
previously produced male mice carrying a disruptive mutation
(Acr The acrosome reaction of sperm, a fusion (vesiculation) event
between the overlying plasma and outer acrosomal membranes, occurs
following the binding of sperm to the zona pellucida
(ZP),1 an extracellular
glycoprotein matrix surrounding the egg. This exocytotic reaction is
required for fertilization, because only acrosome-reacted sperm are
capable of penetrating ZP and of fusing with the egg plasma membrane
(for review see Ref. 1). The acrosomal components, including hydrolytic
enzymes, are released by the acrosome reaction and then interact
initially with ZP to facilitate the sperm penetration of the
glycoprotein matrix.
Acrosin, an endoprotease with a trypsin-like substrate specificity, is
localized in the acrosomal matrix as an enzymatically inactive zymogen,
proacrosin, that is then converted into the active form as a
consequence of the acrosome reaction (2-4). The physiological role of
acrosin in fertilization has long been believed to be the limited
proteolysis of the ZP, thus enabling the sperm to penetrate the ZP.
Using homologous recombination, we have successfully produced male mice
carrying a disruptive mutation in the acrosin gene (Acr) and
found that the mouse sperm lacking the acrosin protease activity
(Acr To elucidate the role of acrosin in fertilization, we have examined the
involvement of acrosin in the acrosome reaction of sperm, using
Acr+/+, Acr+/ Materials--
Monoclonal antibodies against a mouse sperm
protein (termed mAb OBF13) and an intra-acrosomal 155-kDa mouse protein
(mAb MC101) were prepared as described previously (7, 8). Anti-mouse sp56 monoclonal antibody 7C5 (mAb sp56) was originally purchased from
QED Biologicals (La Jolla, CA) by Dr. George L. Gerton and was kindly
provided by him. Rabbit anti-recombinant mouse PH2O antiserum was a
gift of Dr. Paul Primakoff. p-Aminobenzamidine and calcium
ionophore A23187 were purchased from Sigma and Dojindo Laboratories
(Kumamoto, Japan), respectively. Acr+/+,
Acr+/ Measurement of Sperm Binding to ZP--
Fresh cauda epididymal
sperm from male mice (2-3 months old) were dispersed in a 0.2-ml drop
of modified Krebs-Ringer bicarbonate solution (TYH medium) containing
glucose, sodium pyruvate, bovine albumin, and antibiotics (9) at
37 °C under 5% CO2 in air until the medium became
turbid (usually 15 min). The sperm were then capacitated by incubation
at 37 °C under 5% CO2 in air for 90 min. Female B6C3F1
mice (2-3 months old, Japan SLC, Inc., Shizuoka, Japan) were
superovulated following intraperitoneal injections of pregnant mare
serum gonadotropin and human chorionic gonadotropin at a 48-h interval.
Eggs associated with cumulus cells were recovered approximately 15 h after human chorionic gonadotropin injection and placed under warm
mineral oil in a plastic Petri dish containing TYH medium (0.2 ml),
treated at room temperature for 5 min with hyaluronidase (Sigma Type
I-S, 150 units/ml) to remove the cumulus cells, and washed with TYH
medium. The eight cumulus-free eggs and two 2-cell embryos in TYH
medium (5 µl) were incubated with 5 µl of the capacitated sperm
suspension (2,000 sperm) at 37 °C for 30 min, transferred to a
100-µl drop of fresh TYH medium, and washed by pipetting (10-15
times). After fixation with 4% paraformaldehyde, the number of sperm
bound to the egg ZP was counted using a Leica DMIRBE phytomicroscope.
The 2-cell embryos were used as an internal negative control for
nonspecific binding (10), and the number of the bound sperm per 2-cell
embryo was less than 4 under the above conditions.
Calcium Ionophore-induced Acrosome Reaction--
Capacitated
cauda epididymal sperm (4 × 106 sperm/ml) in 0.2 ml
of TYH medium were induced to undergo acrosome reaction by addition of
calcium ionophore A23187 at a final concentration of 5 µg/ml followed
by incubation at 37 °C under 5% CO2 in air. An aliquot
(50 µl each) was taken 3, 15, or 30 min after the incubation, transferred into a 1.5-ml microcentrifuge tube, and then centrifuged at
3,000 rpm for 10 min. The sperm pellets were resuspended in 50 µl of
phosphate-buffered saline (PBS).
ZP-induced Acrosome Reaction--
Solubilized ZP was prepared
from the superovulated eggs of 2-month-old female ICR mice by the
already described method (10) with a minor modification. Briefly, the
cumulus-free, ZP-intact eggs (400-500) were incubated for 20 min in a
bovine serum albumin-free acidified TYH medium (pH 2.2) at a
concentration of 10 eggs/µl. After centrifugation at 13,000 rpm for
10 min, the supernatant containing the solubilized ZP (20 µl each)
was neutralized with 16 µl of 62.5 mM Hepes/TYH medium
(pH 7.7) containing 8 mg/ml bovine serum albumin to give a final pH of
approximately 7.4. To the 36-µl solution, 4 µl of capacitated sperm
suspension (1.5 × 107 sperm/ml) were added, and the
mixture was then incubated for 60 min at 37 °C under 5%
CO2 in air.
Immunocytochemical Analysis of Sperm--
Sperm suspensions were
placed onto glass slides that had been coated with VECTABOND (Vector
laboratories, Burlingame, CA), treated with PBS containing 4%
paraformaldehyde or with PBS alone on ice for 30 min, and washed three
times with PBS. For indirect immunofluorescent staining, the slides
were incubated with primary antibodies diluted in PBS containing 5%
fetal bovine serum overnight, washed with PBS, and treated with
fluorescein isothiocyanate-conjugated goat anti-mouse IgA + IgG + IgM
(Cappel, Durham, NC) for 4 h. After washing with PBS, the slides
were observed using a Leitz DMRXE fluoromicroscope.
Acrosin Accelerates the Dispersal of Sperm Acrosomal Proteins
during Acrosome Reaction*
,, Research Institute for Microbial Diseases,
Osaka University, Yamadaoka 3-1, Suita, Osaka 565-0871, and the
§ Department of Anatomy, Miyazaki Medical College,
Miyazaki 889-1692, Japan
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
/) in the acrosin gene. Although
Acr/ mouse sperm lacking the acrosin
protease activity still penetrated the zona pellucida and fertilized
the egg, the mutant sperm exhibited a delay in penetration of the zona
pellucida solely at the early stages after insemination. To further
elucidate the role of acrosin in fertilization, we have examined the
involvement of acrosin in the acrosome reaction of sperm using the
Acr/ mutant mice. When the ability of sperm
to adhere (attach) and bind to the zona pellucida of cumulus-free eggs
was assessed in vitro, no significant difference was
observed among Acr+/+,
Acr+/, and Acr/
mouse sperm. Immunocytochemical analysis demonstrated that the release
of several acrosomal proteins from the acrosome of
Acr/ mouse sperm was significantly delayed
during the calcium ionophore- and solubilized zona pellucida-induced
acrosome reaction, despite normal membrane vesiculation. These data
indicate that the delayed sperm penetration of the zona pellucida in
the Acr/ mouse results from the altered
rate of protein dispersal from the acrosome and provide the first
evidence that the major role of acrosin is to accelerate the dispersal
of acrosomal components during acrosome reaction.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
/) still penetrate ZP and normally
fertilize the egg (5). These data provide evidence that acrosin is not
essential for sperm penetration of the ZP. However, as compared with
Acr+/+ and Acr+/ mice,
Acr/ mouse sperm showed a delay in sperm
penetration of the ZP solely at the early stages after insemination
(5). A recent report using separate lines of
Acr/ mice (6) has confirmed that sperm
lacking acrosin exhibit the delayed fertilization. Thus, these results
imply that acrosin plays an important role prior to the sperm
penetration of ZP, possibly at the time of the acrosome reaction,
although the participation of acrosin in the ZP hydrolysis cannot be
ruled out completely.
, and
Acr/ male mice. Immunocytochemical analysis
indicated that the release of several acrosomal proteins from the
acrosome of Acr/ mouse sperm during the
acrosome reaction was significantly delayed. Thus, acrosin likely
accelerates the dispersal of proteins from the sperm acrosome.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, and Acr/
male mice were obtained by mating between
Acr+/ males and females, as described
previously (5).
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
|---|
As described previously (5), a remarkable delay in the sperm
penetration of ZP was observed in Acr/ mouse
on in vitro fertilization within 60 min after insemination. To examine whether the delay is caused by a reduced ability of Acr/ mouse sperm for adhesion (attachment)
or binding to ZP, the cumulus-free, ZP-intact eggs were incubated with
capacitated sperm. No significant difference of the ability to adhere
to ZP was apparently observed among Acr+/+,
Acr+/, and Acr/
mouse sperm at 15 and 30 min after insemination. Moreover, the sperm
binding to ZP at 30 min after the incubation were found to be 28.5 ± 9.7, 38.2 ± 9.2, and 26.7 ± 7.7 (means of sperm
numbers/egg ± S.D.) in Acr+/+,
Acr+/, and Acr/
mice, respectively. These data indicate that
Acr/ mouse sperm still possess a normal
capacity for adhesion and binding to ZP and imply that the delay in the
sperm penetration of ZP in Acr/ mouse is
implicated in a physiological event occurring after the sperm bind
ZP.
Capacitated Acr+/+ and
Acr/ mouse sperm were treated with calcium
ionophore A23187, and the time course of protein release from the
acrosome was monitored by indirect immunofluorescent staining using
two monoclonal antibodies against mouse acrosomal proteins (mAb sp56
(12, 13) and mAb MC101 (8); Fig. 1). mAb
OBF13 (7), which immunoreacts both with a protein located at the acrosome cap region of capacitated sperm and with the same protein redistributed over the entire sperm head after acrosome reaction (14),
was also used as a control. The immunofluorescent staining patterns of
mAb sp56 and mAb MC101 disappeared from the
Acr+/+ sperm acrosome as the incubation time
elapsed, whereas the signals still remained in noticeable numbers of
the acrosome of Acr/ mouse sperm even at 30 min after addition of the ionophore. As expected, mAb OBF13 initially
immunostained the acrosomal cap region, and the signals quickly spread
over the sperm head. There was no difference of the immunostaining
pattern in mAb OBF13 between Acr+/+ and
Acr/ mouse sperm. Thus, these observations
clearly demonstrate that the dispersal of the antigenic proteins
recognized by mAb sp56 and mAb MC101 is delayed only in
Acr/ mouse sperm, despite apparently normal
membrane vesiculation during the acrosome reaction.
|
/To assess the acrosomal status more clearly, capacitated sperm with or
without ionophore treatment were stained by the ABC method (11) using
four different antibodies, including polyclonal anti-PH2O antibody
(Fig. 2). As found in indirect
immunostaining, some of the stained signals in
Acr+/+ and Acr/ mouse
sperm were lost from the acrosome by the ionophore treatment (data not
shown for Acr+/ mouse sperm). The sperm were
divided into two groups by the following criteria: (i) sperm that still
contained the antigens recognized by the antibodies in the acrosome, in
spite of the fact that they were acrosome-intact, and initiated the
acrosome reaction or had already acrosome-reacted and (ii) sperm that
contained no signal of the antigens in the acrosome. When anti-PH2O
antibody was used, several regions of sperm were stained; the acrosome
and sperm head were strongly and weakly stained, respectively. This
result may be due to the fact that PH2O is present in the sperm as two forms: a soluble form locating in the acrosome, and a membrane-anchored form on the plasma and inner acrosomal membranes (15, 16). Although the
sperm tail was also stained by anti-PH2O antibody, the signal may be
nonspecific.
|
As shown in Fig. 3, the sperm acrosome
immunostained by four antibodies following ionophore treatment was
quantified according to the above criteria. The number of the acrosome
immunostained rapidly decreased in Acr+/+ and
Acr+/ mouse sperm with the passage of time,
and approximately 90% of the sperm did not show any signal in the
acrosome after the 30-min ionophore treatment. However, the
disappearance of the signals in Acr/ sperm
acrosome was obviously delayed when mAb sp56, mAb MC101, and anti-PH2O
antibody were used. In the case of mAb OBF13, no significant difference
in the pattern of the signal disappearance was found among
Acr+/+, Acr+/, and
Acr/ mouse sperm. These results confirm the
delay of the dispersal of the acrosomal proteins in
Acr/ mouse sperm during the
ionophore-induced acrosome reaction. The number of the acrosome stained
by the four antibodies was estimated to be 79-86% of total
Acr+/+, Acr+/, or
Acr/ mouse sperm without the ionophore
treatment. The reduced numbers may result from the possibility that
14-21% of the sperm have undergone the spontaneous acrosome reaction
during capacitation. Moreover, almost 40% of
Acr+/+ and Acr+/ mouse
sperm without the ionophore treatment exhibited no signal in the
acrosome stained by anti-PH2O antibody. The reason for the unusual
observation is not clear at the present time.
|
3.
p-Aminobenzamidine, a competitive inhibitor for trypsin and
acrosin, has been reported to inhibit not the acrosome reaction itself,
but the dispersal of the acrosomal matrix and sperm penetration of ZP
(17-20). When the release of proteins, which were recognized by four
antibodies, from the acrosome of Acr+/+ mouse
sperm treated with ionophore was examined in the presence of 1 mM p-aminobenzamidine, the patterns of the
signal disappearance were similar to those in
Acr/ mouse sperm in the absence of the
acrosin inhibitor (Figs. 3 and 4). These
data verify the delayed release of several proteins from the
Acr/ sperm acrosome during ionophore-induced
acrosome reaction (Fig. 3).
|
Because calcium ionophore A23187 is not a physiological inducer of
acrosome reaction, we examined protein release from sperm acrosome
following solubilized ZP treatment (Table
I). Approximately 19-25% of
Acr+/+ mouse sperm lost the signals stained by
mAb sp56, mAb MC101, and mAb OBF13 from the acrosome at 60 min after
addition of the solubilized ZP. However, the number of the unstained
sperm was still 7-8% in Acr/ mouse when
mAb sp56 and mAb MC101 were used. Thus, the
Acr/ mouse sperm possess the reduced ability
for the protein dispersal from the acrosome during the ZP-induced
acrosome reaction.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
|---|
This paper demonstrates that the dispersal of acrosomal proteins
from the acrosome is delayed in Acr/ mouse
sperm during either calcium ionophore-induced or solubilized ZP-induced
acrosome reaction (Fig. 3 and Table I). These data are consistent with
the fact that only Acr/ mouse sperm show a
delayed sperm penetration of ZP at the early stages of in
vitro fertilization (5). Therefore, it may be concluded that
acrosin plays a major role in acceleration of the dispersal of
acrosomal proteins in vivo and that the decreased ability of
Acr/ mouse sperm to disperse acrosomal
proteins is responsible for the delayed sperm penetration of ZP.
However, the patterns of the signal disappearance from the acrosome in
Acr/ mouse sperm were distinguished from one
another by the antibody used (Fig. 3). In particular, approximately
50% of Acr/ mouse sperm still had the mAb
sp56-positive signal in the acrosome even at 30 min after the ionophore
treatment. This differential dispersal of the acrosomal proteins from
the acrosome may be correlated with the localization and associated
state of each of the proteins within the acrosomal matrix, as reported
previously (13, 17, 21, 22), if the protein release from the acrosome
during the ionophore-induced acrosome reaction totally reflects the
in vivo phenomenon.
In guinea pig, the sperm acrosome can be ultrastructurally separated into three domains (M1, M2, and M3) by electron density (23). Proacrosin is preferentially localized in the most electron-dense domains (M2 and M3) presumably in a state firmly associated with other matrix proteins (17, 21, 22, 24, 25), whereas some acrosomal proteins, including soluble PH2O and autoantigen 1, have been reported to associate weakly with the matrix (22, 26-28). Thus, acrosin, which is converted from proacrosin by autoactivation during the acrosome reaction, probably prefers to hydrolyze a core protein(s) of a stable acrosomal matrix, such as AM50 (25, 29) and AM67 (13) in guinea pig sperm, so that the acrosomal proteins, including the partially digested core proteins, would be readily released. Moreover, it is still reasonable to consider the possibility that acrosin possesses a selectivity for each of the acrosomal proteins in the matrix dispersal. If so, we can speculate that acrosin may act to destroy the physiological function of other acrosomal proteins and/or to give a function to latent forms of the proteins by proteolysis as well as to hydrolyze the core proteins forming the acrosomal matrix during acrosome reaction.
Our data provide the first evidence that the role of acrosin is to
accelerate the dispersal of acrosomal components during acrosome
reaction. However, we should not consider that the protein dispersal is
modulated solely by acrosin, because the acrosomal proteins are
released with a time delay, and their dispersal is not completely
blocked in the absence of acrosin (Fig. 3 and Table I) or in the
presence of p-aminobenzamidine (Fig. 4). Moreover, we have
found that Acr/ mouse sperm are incapable of
fertilizing the egg in the presence of
p-aminobenzamidine.2
Therefore, a protease(s) sensitive to the inhibitor must be present in
the sperm to enable it to penetrate the ZP. This protease may compensate for the insufficient function(s) of the
Acr/ mouse sperm due to the lack of acrosin.
Thus, to elucidate the sperm function in fertilization, the
identification and characterization of the novel sperm protease(s)
remains to be accomplished.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. P. Primakoff and G. L. Gerton for kind gifts of antibodies and critical reading of this manuscript.
| |
FOOTNOTES |
|---|
* This work was partly supported by grants from the Ministry of Education, Science, Sports, and Culture in Japan (to T. B., M. O., and K. T.) and by the Tsukuba Advanced Research Alliance Sakabe/Shoun Project and National Institute for Advanced Interdisciplinary Research Taira Project (to T. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Inst. of Applied Biochemistry, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan. Tel./Fax: 81-298-53-6632; E-mail: acroman{at}sakura.cc.tsukuba.ac.jp.
1 The abbreviations used are: ZP, zona pellucida; ABC, avidin-biotin peroxidase complex; PBS, phosphate-buffered saline; mAb, monoclonal antibody.
2 K. Yamagata, K. Murayama, N. Kohno, S. Kashiwabara, and T. Baba, submitted for publication.
| |
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D. Baba, S.-i. Kashiwabara, A. Honda, K. Yamagata, Q. Wu, M. Ikawa, M. Okabe, and T. Baba Mouse Sperm Lacking Cell Surface Hyaluronidase PH-20 Can Pass through the Layer of Cumulus Cells and Fertilize the Egg J. Biol. Chem., August 9, 2002; 277(33): 30310 - 30314. [Abstract] [Full Text] [PDF]
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A. Honda, K. Yamagata, S. Sugiura, K. Watanabe, and T. Baba A Mouse Serine Protease TESP5 Is Selectively Included into Lipid Rafts of Sperm Membrane Presumably as a Glycosylphosphatidylinositol-anchored Protein J. Biol. Chem., May 3, 2002; 277(19): 16976 - 16984. [Abstract] [Full Text] [PDF]
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 K. Nayernia, I. M. Adham, R. Shamsadin, C. Muller, U. Sancken, and W. Engel Proacrosin-deficient mice and zona pellucida modifications in an experimental model of multifactorial infertility Mol. Hum. Reprod., May 1, 2002; 8(5): 434 - 440. [Abstract] [Full Text] [PDF]
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S. Metayer, F. Dacheux, J.-L. Dacheux, and J.-L. Gatti Comparison, Characterization, and Identification of Proteases and Protease Inhibitors in Epididymal Fluids of Domestic Mammals. Matrix Metalloproteinases Are Major Fluid Gelatinases Biol Reprod, May 1, 2002; 66(5): 1219 - 1229. [Abstract] [Full Text]
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 E. Howes, J. C. Pascall, W. Engel, and R. Jones Interactions between mouse ZP2 glycoprotein and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during fertilization J. Cell Sci., March 13, 2002; 114(22): 4127 - 4136. [Abstract] [Full Text] [PDF]
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H. Sawada, N. Sakai, Y. Abe, E. Tanaka, Y. Takahashi, J. Fujino, E. Kodama, S. Takizawa, and H. Yokosawa Extracellular ubiquitination and proteasome-mediated degradation of the ascidian sperm receptor PNAS, January 24, 2002; (2002) 32389499. [Abstract] [Full Text] [PDF]
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 D. J. Miller, X. Shi, and H. Burkin Molecular Basis of Mammalian Gamete Binding Recent Prog. Horm. Res., January 1, 2002; 57(1): 37 - 73. [Abstract] [Full Text] [PDF]
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 K. J. Mengerink and V. D. Vacquier Glycobiology of sperm-egg interactions in deuterostomes Glycobiology, April 1, 2001; 11(4): 37R - 43R. [Abstract] [Full Text] [PDF]
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K.-S. Kim, M. C. Cha, and G. L. Gerton Mouse Sperm Protein sp56 Is a Component of the Acrosomal Matrix Biol Reprod, January 1, 2001; 64(1): 36 - 43. [Abstract] [Full Text]
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K.-S. Kim, J. A. Foster, and G. L. Gerton Differential Release of Guinea Pig Sperm Acrosomal Components During Exocytosis Biol Reprod, January 1, 2001; 64(1): 148 - 156. [Abstract] [Full Text]
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R.D. Moreno and C. Barros A Basic 18-Amino Acid Peptide Contains the Polysulfate-Binding Domain Responsible for Activation of the Boar Proacrosin/Acrosin System Biol Reprod, June 1, 2000; 62(6): 1536 - 1542. [Abstract] [Full Text]
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L. I. Furlong, U. Hellman, A. Krimer, J. G. Tezón, E. H. Charreau, and M. H. Vazquez-Levin Expression of Human Proacrosin in Escherichia coli and Binding to Zona Pellucida Biol Reprod, March 1, 2000; 62(3): 606 - 615. [Abstract] [Full Text]
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K. Ohmura, N. Kohno, Y. Kobayashi, K. Yamagata, S. Sato, S.-i. Kashiwabara, and T. Baba A Homologue of Pancreatic Trypsin Is Localized in the Acrosome of Mammalian Sperm and Is Released During Acrosome Reaction J. Biol. Chem., October 8, 1999; 274(41): 29426 - 29432. [Abstract] [Full Text] [PDF]
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J. D. Hooper, D. L. Nicol, J. L. Dickinson, H. J. Eyre, A. L. Scarman, J. F. Normyle, M. A. Stuttgen, M. L. Douglas, K. A. Lakoski Loveland, G. R. Sutherland, et al. Testisin, a New Human Serine Proteinase Expressed by Premeiotic Testicular Germ Cells and Lost in Testicular Germ Cell Tumors Cancer Res., July 1, 1999; 59(13): 3199 - 3205. [Abstract] [Full Text] [PDF]
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 V. D. Vacquier Evolution of Gamete Recognition Proteins Science, September 25, 1998; 281(5385): 1995 - 1998. [Abstract] [Full Text]
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E. Kodama, T. Baba, H. Yokosawa, and H. Sawada cDNA Cloning and Functional Analysis of Ascidian Sperm Proacrosin J. Biol. Chem., June 29, 2001; 276(27): 24594 - 24600. [Abstract] [Full Text] [PDF]
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H. Sawada, N. Sakai, Y. Abe, E. Tanaka, Y. Takahashi, J. Fujino, E. Kodama, S. Takizawa, and H. Yokosawa Extracellular ubiquitination and proteasome-mediated degradation of the ascidian sperm receptor PNAS, February 5, 2002; 99(3): 1223 - 1228. [Abstract] [Full Text] [PDF]
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