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Ultrastructural analysis of the acrosome reaction in a population of single guinea pig sperm.

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THE ANATOMICAL RECORD 229:186-194 (19911
Ultrastructural Analysis of the Acrosome Reaction
in a Population of Single Guinea Pig Sperm
SEAN P. FLAHERTY A N D GARY E. OLSON
Department of Cell B~ology,School of MedLcLne, VunderbLlt Universit.y,
Nashville, Tennessee 37232
ABSTRACT
Cauda epididymal guinea pig spermatozoa are arranged in
rouleaux, with the sperm heads stacked one on top of the other; the plasma membranes over the apical segment of the acrosomes of adjacent sperm are linked and
form non-fusigenic “junctional” zones. A complex structural and temporal sequence of membrane fusions occurs during the acrosome reaction of guinea pig
sperm in rouleaux. In this study, we have devised a procedure for dispersing the
rouleaux and isolating a population of single, motile guinea pig sperm, and have
investigated the ultrastructural features of the acrosome reaction in single sperm
to determine if the pattern of membrane fusions is different from sperm in
rouleaux. The rouleaux were dispersed using trypsin, and damaged cells were
removed by passing the sperm suspension through a glass bead column; a population of 70-904 motile, acrosome-intact, single sperm was obtained. Sperm were
then induced to undergo lysolecithin-mediated, “synchronous” acrosome reactions,
and processed for transmission electron microscopy. The acrosome reaction involved a complex sequence of membrane fusions between the plasma membrane
(PM) and outer acrosomal membrane (OAM). On the convex surface of the apical
segment, sheets of hybrid membrane and parallel arrays of hybrid membrane
tubules formed; filaments were associated with the luminal surface of the residual
OAM in these regions. Hybrid membrane vesicles were produced on the concave
surface of the apical segment, but fusion was delayed relative to the convex surface. In the principal segment, branching arrays of hybrid membrane tubules
formed and later vesiculated. Hence, in single guinea pig sperm, the sequence of
membrane fusions is similar to sperm in rouleaux except that fusion occurs in
regions of the apical segment which form the non-fusigenic PM “junctional” zones
in rouleaux. The results suggest that, regardless of whether the acrosome reaction
in vivo occurs before or after rouleaux dispersion, it will involve a complex sequence of membrane fusions which is determined by the structural properties of
the OAM and PM.
During the mammalian sperm acrosome reaction,
the plasma membrane and outer acrosomal membrane
fuse at multiple sites over the apical and principal segments of the acrosome to form a fenestrated hybrid
membrane complex (Barros et al., 1967; Bedford and
Cooper, 1978; Russell e t al., 1979). Dispersion of the
acrosomal matrix releases hydrolytic enzymes which
facilitate penetration of sperm through the cumulus
oophorus and zona pellucida surrounding the egg (Talbot, 1985; Yanagimachi, 1988). Fusion does not occur
in the equatorial segment of the acrosome, and this
region is involved in the initial fusion between the
sperm plasma membrane and the oolemma (Bedford et
al., 1979; Yanagimachi, 1988).
The guinea pig is a n excellent model system for
studying the acrosome reaction because the acrosome
is large and easily visualized by light microscopy,
methods a r e available for inducing “synchronous” acrosome reactions in a population of guinea pig sperm
(Yanagimachi and Usui, 1974; Fleming and Yanagimachi, 1981), and it is possible to isolate membranes
c
1991 WILEY-I,ISS. INC
from both unreacted and reacted sperm (Primakoff et
al., 1980; Olson et al., 1987).
Cauda epididymal guinea pig sperm are arranged in
rouleaux with the sperm heads stacked one on top of
the other. In rouleaux, the plasma membranes over the
apical segment of adjacent sperm are linked by cross
bridges and form “junctional” zones (Friend and Fawcett, 1974; Flaherty and Olson, 1988). Guinea pig
sperm undergo the acrosome reaction in vitro while in
rouleaux, and the rouleaux only disperse as a result of
the acrosome reaction (Yanagimachi and Usui, 1974;
Green, 1978a; Flaherty and Olson, 1988). We recently
described a complex structural and temporal pattern of
membrane fusions which occurs during the acrosome
Received November 13, 1989: accepted J u n e 19, 1990.
Sean P. Flaherty’s current address is Department of Obstetrics and
Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, Woodville. South Australia 5011.
GUINEA PIG SPERM ACROSOME: REACTION
TABLE 1. Composition of HmT, mT, and CmT’
Component
NaCl
KCl
CaCl,
NaHCO,
Sodium HEPES
D-glucose
Sodium lactate
Sodium pyruvate
Oxalacetic acid
Phenol red
Penicillin G
Streptomycin sulfate
Bovine serum albumin
DH (in air)
HmT
(mM)
111.76
2.70
4.00
21.00
5.56
10.00
1.00
0.10
0.001%
50 pgiml
50 pgiml
0.1 mgiml
7.5
mT
(mM)
111.76
2.70
-
25.07
CmT
(mM)
105.76
2.70
4.00
25.07
-
-
5.56
10.00
1.00
0.10
0.001%
50 pgiml
50 pgiml
3 mgiml
8.0-8.5
5.56
10.00
1.00
0.10
0.001%
50 pgiml
50 pgiml
3 mgiml
8.0-8.5
‘Based on Fleming and Yanagimachi (1981).
reaction of guinea pig sperm in rouleaux. Specific domains of the plasma membrane and outer acrosomal
membrane, including the plasma membrane “junctional” zones, a r e non-fusigenic, whilst membrane-associated cytoskeletal elements impart a directional
component to membrane fusion in other domains (Flaherty and Olson, 1988).
It is presently unclear 1)whether guinea pig sperm
rouleaux disperse in vivo before or after sperm undergo
the acrosome reaction (Martan and Shepherd, 1972;
Yanagimachi and Mahi, 1976), and 2) whether the
same complex sequence of membrane fusions occurs
during the acrosome reaction of single sperm as we
have described for sperm in rouleaux (Flaherty and
Olson, 1988). Hence, in this study we have developed a
method for dispersing guinea pig sperm rouleaux and
inducing the acrosome reaction in a population of motile, single sperm. Furthermore, we have examined the
morphological features of the acrosome reaction in single guinea pig sperm by transmission electron microscopy to determine if the pattern of membrane fusions is
the same a s when sperm are in rouleaux.
MATERIALS AND METHODS
Culture Media
A modified Tyrode’s medium (mT) was used for all
incubations (Fleming and Yanagimachi, 1981). Three
media were used: Ca2’-deficient HEPES-buffered mT
(HmT), Ca2’-deficient mT (mT), and 2X Ca2 mT
(CmT). The composition of the media is given in Table
1. Culture media were prepared with analytical grade
reagents and Milli Q water, and contained 0.1 mgiml
(HmT) or 3 mgiml (mT and CmT) bovine serum albumin (fatty acid free fraction V; Sigma Chemical Co., St.
Louis, MO). A 2 mgiml stock solution of lysolecithin
(Type I, from egg yolk; Sigma) was prepared in mT and
diluted to 80 pgiml in mT immediately before use.
+
Preparation of a Population of Single Guinea Pig Sperm
Adult male guinea pigs of proven fertility were killed
by a n overdose of sodium pentobarbitone (Sigma), and
sperm were flushed from the distal regions of the cauda
epididymis by retrograde infusion of warm HmT. The
sperm concentration was then adjusted to 20 x lo7
spermiml.
187
Sperm were diluted to a final concentration of 2 x
in HmT containing 0.2% trypsin (Type
IX; Sigma) and incubated in glass conical flasks a t
36°C in a waterbath with constant agitation (50 cycles/
min). Dispersion of the rouleaux was monitored by
phase contrast or Nomarski microscopy, and when only
single sperm were present, 1 ml of HmT containing 40
mgiml of egg white trypsin inhibitor (Type 111-0;
Sigma) was added to each flask.
The dispersed sperm suspensions were then passed
through glass bead columns to remove dead and damaged sperm (Lui et al., 1979). The columns were made
from disposable polypropylene chromatography columns (Biorad, Richmond, VA), and contained a single 5
mm diameter glass bead a t the bottom and a packed
volume of 1.5-1.8 ml of glass beads (150-212 pm diameter; Sigma). The glass beads were treated with 1N
HC1 and rinsed extensively with distilled water before
use. The glass bead columns were pre-equilibrated
with HmT before sperm were loaded. The sperm which
passed through the column were concentrated by gentle centrifugation (300g, 10 minutes), and the sperm
concentration was then adjusted to 7-10 x lo7 sperm/
ml.
lo7 spermiml
Induction of the Acrosome Reaction
Guinea pig spermatozoa were induced to undergo a
“synchronous” lysolecithin-mediated acrosome reaction (Fleming and Yanagimachi, 1981; Flaherty and
Olson, 1988). Sperm were diluted to a concentration of
7-10 x lo6 spermiml in mT containing 80 pgiml lysolecithin, and incubated at 36°C for 60-90 minutes with
constant agitation (50 cyclesimin). To maintain a high
percentage of viable cells, the sperm were then passed
through another glass bead column which had been
equilibrated with mT medium. An equal volume of
CmT medium (containing4 mM CaCl,) was then added
to aliquots of the sperm suspension, and they were incubated a t 36°C with agitation. The occurrence of the
acrosome reaction was assessed by phase contrast microscopy and transmission electron microscopy.
Transmission Electron Microscopy
Before and after rouleaux dispersion, and at various
times after the addition of calcium (30 seconds, 40-45
seconds, and 1, 1.25,1.5,2, and 3 minutes), sperm were
processed for thin section electron microscopy as previously described (Flaherty and Olson, 1988). Sperm
pellets were fixed in 2.5% glutaraldehyde in cacodylate
buffer, then some samples were transferred to fresh
AS
CB
cv
cx
ES
F
JZ
M
OAM
PM
PS
S
T
V
A bbreuiations
apical segment
plasma membrane cross bridges
concave surface
convex surface
equatorial segment
filaments
“junctional” zone
acrosomal matrix
outer acrosomal membrane
plasma membrane
principal segment
hybrid membrane sheet
hybrid membrane tubules
hybrid membrane vesicles
188
S.P. FLAHERTY AND G.E. OLSON
Flg. 1. Cauda epididymal guinea pig spermatozoa a r e arranged i n
rouleaux, with t h e sperm heads stacked one on top of t h e other. A few
single sperm are evident (arrows) Nomarski. x 470.
Flg. 2. T h e plasma membranes tPMi over the apical segment tAS) of
sperm in rouleaux are linked to form “junctional” zones tJZi. These
zones a r e absent from t h e principal segment (PSI The insert shows a
J Z after tannic acid fixation. Cross bridges tCBl link t h e plasma membranes 1P M ) of adjacent sperm, while bridging elements (arrows)link
the PM a n d outer acrosomal membrane ( O A M )on t h e concave surface
of the apical segment. x 13,000; x 125,000 iinsert).
Flg. 3. About 90% of t h e isolated single guinea pig sperm retain
their acrosomes. Nomarski. x 470.
Flg. 4. Transmission electron micrograph of the apical segment ( A S )
of a single sperm. On the concave surface tCV) of the apical segment,
the plasma membrane ( P M ) is still closely apposed to the outer acrosomal membrane (OAMI. Tannic acid fixation (insert) reveals t h a t
the bridging elements (arrows) between the PM and OAM are
present. CX, convex surface. x 24,000; x 150,000 (insert)
G U I N E A PIG SPERM ACIIOSOME REACTION
Fig. 5. The initial points of fusion (large arrows) between the PM
and OAM are on the concave surface (CV) of the apical segment near
its tip, and on the convex surface (CX) of the apical segment. Fusion
is delayed on the remainder of the concave surface (small arrows).
Cavitation of the acrosomal matrix has commenced (stars). x 38,500.
189
Fig. 6. A specific matrix component (Mi is closely apposed to the
OAM along the concave margin of the apical segment. The insert
shows the matrix component (Mi, as well as the PM-OAM bridging
elements (arrows).S, hybrid membrane sheet. Tannic acid. x 33,000;
x 82,000 (Insert).
190
Figs. 7-10
GUINEA PIG SPERM ACROSOME REACTION
fixative containing 2% tannic acid for 1 hour. The pellets were postfixed in osmium tetroxide, dehydrated in
a graded ethanol series which included en bloc staining
with uranyl acetate, then embedded in Embed 812
(Polysciences, Warrington, PA). Ultrathin sections
were examined in a Hitachi H-600 (Hitachi Ltd, Tokyo,
Japan) electron microscope.
RESULTS AND DISCUSSION
Cauda epididymal guinea pig sperm are arranged in
rouleaux, and the plasma membranes over the apical
segment of adjacent sperm are linked by periodic cross
bridges to form “junctional” zones (Friend and Fawcett,
1974; Flaherty and Olson, 1988). Our recent study of
the guinea pig sperm acrosome reaction revealed a
complex sequence of membrane fusions, and demonstrated 1) that non-fusigenic domains, including the
plasma membrane PM “junctional” zones, exist in the
PM and outer acrosomal membrane (OAM), and 2 ) the
filaments associated with the OAM impart a directional component to the membrane fusion process (Flaherty and Olson, 1988). In the present study, we have
examined the ultrastructural features of the acrosome
reaction in a population of single guinea pig sperm to
clarify whether this complex pattern of membrane fusions is defined by the arrangement of sperm in
rouleaux, or by the inherent properties of the PM and
OAM.
191
motile, acrosome-intact single sperm were isolated
with an overall sperm recovery of about 50% (Fig. 3).
Transmission electron microscopy revealed that the
ultrastructural features of most sperm were unchanged
by the isolation procedure, although in some there was
a slight indentation on the concave surface near the tip
of the apical segment. After tannic acid fixation of single sperm, slender bridging elements were observed
linking the closely apposed PM and OAM on the concave, but not convex, surface of the apical segment.
Remnants of the cross bridges which link sperm in
rouleaux were observed on the external surface of the
PM over the apical segment (Fig. 4). The cross bridges
may represent overlapping elements of the glycocalyx
(Friend and Fawcett, 1974; Friend, 1984; Flaherty and
Olson, 19881, and their sensitivity to trypsin and pronase implies that they have a polypeptide component
although the involvement of carbohydrates in this interaction has not been elucidated.
Ultrastructural Features of the Acrosome Reaction in
Single Sperm
Single sperm were incubated in mT containing lysolecithin (lysophosphatidylcholine), followed by the addition of calcium, to induce synchronous acrosome reactions (Fleming and Yanagimachi, 1981; Flaherty
and Olson, 1988). Sperm motility was maintained a t
>80%, and 60-80% of the motile sperm underwent a n
acrosome reaction within 10 minutes of adding calIsolation of Single Guinea Pig Sperm
cium. The acrosome reaction commenced in some
Greater than 90% of cauda epididymal guinea pig sperm about 20-30 seconds after adding calcium, and
spermatozoa were arranged in rouleaux, with up to 20 was underway in most sperm by 2 minutes. The reacsperm heads stacked one on top of the other (Fig. 1). tion appeared to be slightly less synchronous than for
The plasma membranes over the apical segment of ad- sperm in rouleaux, and this may be a damaging effect
jacent sperm were linked by cross bridges to form of trypsin treatment. Most of the acrosome-reacted
“junctional” zones, and slender bridging elements sperm exhibited a hyperactivated pattern of motility,
linked the PM and OAM on the concave surface of the although no attempt was made to correlate the onset of
apical segment in these zones. The bridging elements hyperactivation with the acrosome reaction.
were superimposed over the cross bridges (Fig. 2).
Sperm were fixed a t various times after the addition
Guinea pig sperm rouleaux were dispersed by incu- of calcium and examined by electron microscopy to asbation in HmT containing trypsin for 80-140 minutes. certain if the pattern of membrane fusions % different
The time and efficacy of dispersal was dependent on the from sperm which acrosome react while in rouleaux.
concentration and activity of the trypsin, a s well a s the There was a complex structural and temporal sequence
vigor of agitation. Increasing the trypsin concentration of membrane fusions between the PM and OAM. The
from 0.2 to 0.5%, or using 0.1% pronase, hastened initial points of fusion were on the concave surface of
rouleaux dispersal, but also resulted in poor motility the apical segment near its tip, and on the convex surand considerable head-tail cleavage. After trypsin face of the apical segment (Figs. 5, 6). On the convex
treatment, 50-70% of the sperm were motile and 80- surface of the apical segment, limited fusion created
90% were acrosome-intact. In order to retain a popula- large sheets of hybrid membrane and parallel arrays of
tion of highly motile, acrosome-intact sperm for studies hybrid membrane tubules (Figs. 7-10). Parallel arrays
of the acrosome reaction, sperm suspensions were then of filaments were adherent to the luminal surface of
passed through glass bead columns to remove dead and the residual OAM in the sheets and tubules, a s dedamaged sperm. Glass bead columns have been used to scribed by Olson et al. (1987) and Flaherty and Olson
remove dead sperm from suspensions of mouse (Mc- (1988). Remnants of the PM cross bridges which link
Grath et al., 19771, hamster (Lui et al., 19791, and hu- sperm in rouleaux were also evident on the external
man sperm (Daya et al., 1987). Populations of 70-90%
surface of the residual PM in the sheets and tubules,
Figs 7, 8. Transverse sections cut through the apical segment of
sperm a t early (Fig. 7) and later (Fig. 8)stages of the acrosome reaction. On the convex surface (CXI, fusion between the PM and OAM
produces large sheets of hybrid membrane (S)
and parallel arrays of
hybrid membrane tubules (TI. Fusion on the concave surface tCV1 is
delayed relative to the convex surface, which relates to persistence of
the matrix layer (M) associated with the OAM. Hybrid membrane
vesicles ( V ) with an indentation (arrow)on the PM side form on the
concave surface. The insert in Fig. 8 shows the oblique orientation of
the OAM filaments to the PM-PM cross bridges in a hybrid membrane
sheet. ~ 2 3 , 0 0 0(71; ~ 3 2 , 0 0 0(81; ~ 4 1 , 0 0 0(Fig. 8 insert).
Flgs. 9. 10. Parallel filaments tF1 are adherent to the luminal surface of the residual OAM in the hybrid membrane sheets IS)and
tubules IT) on the convex surface of the apical segment. Remnants of
the PM-PM cross bridges between sperm in rouleaux are also present
(arrow in Fig. 9). ~ 8 0 , 0 0 0(9); ~ 5 8 , 0 0 0(10).
192
S.1’. FLAHEKTY A N D G.E. OLSON
Flg. 11. When fusion h a s occurred throughout the apical segment,
cavitation of t h e acrosomal matrix (arrow) and membrane fusion
(double arrows) spread to t h e principal segment IPS). Note t h e hybrid
membrane tubules (TI, sheets (S),
and vesicles tVI in the apical segment ( A S ) . x 25,500.
Flg. 12. The equatorial segment remains unfused after completion
of the acrosome reaction; the PM and OAM are confluent along its
anterior border (arrows). x 55,000.
Fig. 13. In the principal segment, fusion between the PM and OAM
creates randomly-oriented arrays of branching hybrid membrane tubules (Ti; filaments a r e not evident on t h e luminal surface of t h e
residual OAM in these tubules. The tubules later form vesicles ( V I
with a n indentation on t h e residual PM side. The posterior border of
the equatorial segment tES1 is demarcated by finger-like projections
iarrowi. S,hybrid membrane sheet from apical segment. x 28,000.
GUINEA
r m SPERM ACROSOME
and the cross bridges were arranged in parallel rows a t
a n oblique angle to the filaments on the luminal surface of the OAM (Figs. 8-10).
Fusion on the concave surface of the apical segment
was delayed relative to the convex surface, and this
related to the persistence of a component of the acrosoma1 matrix which was closely associated with the OAM
and clearly distinguishable after tannic acid fixation
(Figs. 5-8). Once initiated, however, membrane fusion
along the concave surface of the apical segment resulted in the formation of hybrid membrane vesicles
which were indented on the PM surface (Figs. 8, 11).
Previous studies have described zones of differing solubility and electron density in the matrix of the apical
segment of the guinea pig sperm acrosome (Fawcett
and Hollenberg, 1963; Friend and Fawcett, 1974;
Green, 1978b; Huang et al., 19851, and the close apposition of this matrix component to the OAM suggests a
direct interaction between matrix and OAM components (Olson et al., 1988).
Membrane fusion in the principal segment was delayed relative to the apical segment, and was preceded
by swelling of the acrosomal matrix and undulation of
the OAM and PM (Fig. 11).Fusion produced random
arrays of branching hybrid membrane tubules, which
later vesiculated to form hybrid membrane vesicles
with a n indentation on the PM side. Filaments were
not observed on the luminal surface of the residual
OAM in these tubules (Figs. 11, 13). The equatorial
segment remained intact after completion of the acrosome reaction (Fig. 12), in line with its postulated
role in sperm-egg fusion (Bedford et al., 1979).
Hence, with the exception of the non-fusigenic “junctional zones,” the pattern of fusions between the PM
and OAM during the acrosome reaction is the same in
single guinea pig sperm and sperm in rouleaux (Flaherty and Olson, 1988), and therefore the various
structural manifestations of fusion in different regions
of the acrosome are not merely due to the arrangement
of sperm in rouleaux, but are due to inherent properties
of the PM and OAM and the membrane-associated cytoskeletal assemblies (Olson et al., 1989). Once the
“junctional zones” have been disrupted by trypsin
treatment, these domains in the apical segment undergo membrane fusion in single sperm even though
the PM-OAM bridging elements remain. Hence, the
inhibition of fusion in the “junctional” zones of sperm
in rouleaux is due to the presence of the PM-PM cross
bridges between sperm, rather than the PM-OAM
bridging elements which remain after rouleaux dispersal. As yet we have been unable to determine the
exact fate of the PM-OAM bridging elements, since
they are present during the early stages of the acrosome reaction prior to membrane fusion on the concave surface of the apical segment, but are not observed
within the resulting hybrid membrane tubules.
Function of Sperm Rouleaux
The function of sperm rouleaux is currently unclear.
This cell adhesion phenomenon has only been described in cauda epididymal sperm from the guinea pig
(Fawcett and Hollenberg, 1963), flying squirrel (Martan and Hruban, 19701, and naked-tail armadillo
(Heath e t al., 1987). In contrast, loris sperm are arranged in rouleaux during passage through the caput
REACTION
193
epididymis, but the rouleaux disperse later during
epididymal transit and do not reform (Phillips and Bedford, 1987). Whereas guinea pig sperm rouleaux disperse in vitro a s a result of the acrosome reaction, it is
unclear whether the rouleaux disperse in vivo before,
or as a result of, the acrosome reaction (Martan and
Shepherd, 1972; Yanagimachi and Mahi, 1976). Tung
et al. (1980) postulated that rouleaux preserve sperm
viability and prevent premature acrosome reactions.
The results of this study indicate that single guinea pig
spermatozoa remain viable and can be induced to undergo a morphologically normal acrosome reaction, so
rouleaux may not be involved in preserving sperm viability.
ACKNOWLEDGMENTS
The authors thank Ryuzu Yanagimachi for helpful
discussions, Matt Makinson for photographic assistance, and the staff of the Electron Microscope Unit at
The Queen Elizabeth Hospital. Supported by NIH
Grant HD-20419, NIH Center Grant HD-05797, and a
grant from the Mellon Foundation.
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