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.
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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
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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.
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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).
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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.
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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).
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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
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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. LITERATURE CITED Barros, C., J.M. Bedford, L.E. Franklin, and C.R. Austin 1967 Membrane vesiculation as a feature of the mammalian acrosome reaction. J. Cell. Biol., 34tCl-C5. Bedford, J.M. and G.W. Cooper 1978 Membrane fusion events in the fertilization of vertebrate eggs. In: Membrane Fusion. G. Poste and G.L. Nicolson, eds. Elsevier-North Holland, Amsterdam, pp. 65-125. Bedford, J.M., H.D.M. Moore, and L.E. Franklin 1979 Significance of the equatorial segment of the acrosome of the spermatozoon in eutherian mammals. Exp. Cell Res., 119t119-126. Daya, S., R.B.L. Gwatkin, and H. Bissessar 1987 Separation of motile human spermatozoa by means of a glass bead column. Gamete Res., 17t375-380. Fawcett, D.W. and R.D. Hollenberg 1963 Changes in the acrosome of guinea pig spermatozoa during passage through the epididymis. Z. Zellforsch. Mikrosk. Anat., 60t276-292. Flaherty, S.P. and G.E. Olson 1988 Membrane domains in guinea pig sperm and their role in the membrane fusion events of the guinea pig sperm acrosome reaction. Anat. Rec., 220t267-280. Fleming, A.D. and R. Yanagimachi 1981 Effects of various lipids on the acrosome reaction and fertilizing capacity of guinea pig spermatozoa with special reference to the possible involvement of lysophospholipids in the acrosome reaction. Gamete Res., 4t253273. Friend, D.S. 1984 Membrane organization and differentiation in the guinea pig spermatozoon. In: Ultrastructure of Reproduction. J. Van Blerkom and P.M. Motta, eds. Martinus Nijhoff, Boston, pp. 75-85. Friend, D.S. and D.W. Fawcett 1974 Membrane differentiations in freeze-fractured mammalian sperm. J. Cell Biol., 63t641-664. Green, D.P.L. 1978a The induction of the acrosome reaction in guineapig sperm by the divalent metal cation ionophore A23187. J. Cell Sci., 32t137-151. Green, D.P.L. 197813 The osmotic properties of the acrosome of guineapig sperm. J. Cell Sci., 32t165-176. Heath, E., N Schaeffer, D.A. Meritt, and J . A . Jeyendran 1987 Rouleaux formation by spermatozoa in the naked-tailed armadillo. Cahassous unicinctus. J. Reprod. Fertil., 7.9t153-158. Huang, T.T.F., D. Hardy, H. Yanagimachi, C. Teuscher, K. Tung, G. Wild, and R Yanagimachi 1985 pH and proteinase control of acrosomal content stasis and release during the guinea pig acrosome reaction. Biol. Reprod., 32.451-462. Lui, C.W., R.J. Mrsny, and S.Meizel 1979 Procedures for obtaining high percentages of viable in vitro capacitated hamster sperm. Gamete Res., 2.207-211. Martan, J. and Z. Hruban 1970 Unusual spermatozoan formations in the epididymis of the flying squirrel (Glaucomys volans). J . Reprod. Fertil., 21t167-170. Martan, J. and B.A. Shepherd 1972 Spermatozoa in rouleaux in the female guinea pig genital tract. Anat. Rec., 175t625-630.
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McGrath, J., N. Hillman, a n d M. Nadijcka 1977 Separation of dead and live mouse spermatozoa. Dev. Biol., 61:114-117. Olson, G.E., V.P. Winfrey, and G.R. Davenport 1988 Characterization of matrix domains of the hamster acrosome. Biol. Reprod.. 39: 1145-1158. Olson, G.E., V.P. Winfrey, and S.P. Flaherty 1989 Membranecytoskeleton interactions in the sperm acrosome. In: Gamete Physiology. R.H. Asch, J.P. Balmaceda and I. Johnston, eds. Serono Symposia, USA, Norwell, pp. 109-118. Olson, G.E., V.P. Winfrey, M.A. Winer, and G.R. Davenport 1987 The outer acrosomal membrane of guinea pig spermatozoa: Isolation and structural characterization. Gamete Res., 17:77-94. Phillips, D.M. a n d J.M. Bedford 1987 Sperm-sperm associations in t h e loris epididymis. Gamete Res., 18:17-25. Primakoff, P., D.G. Myles, a n d A.R. Bellve 1980 Biochemical analysis of the released products of the mammalian sperm acrosome reaction. Dev. Biol., 98:417-428. Russell, L.. R.N. Peterson, and M. Freund 1979 Direct evidence for
formation of hybrid vesicles by fusion of plasma and outer acrosoma1 membranes during t h e acrosome reaction in boar spermatozoa. J. Exp. Zool., 208:41-56. Talbot, P. 1985 Sperm penetration through oocyte investments in mammals. Am. J. Anat., 174:331-346. Tung, K.S.K., A. Okada, and R. Yanagimachi 1980 Sperm autoantigens and fertilization. I. Effects of antisperm antibodies on rouleaux formation, viability, and acrosome reaction of guinea pig spermatozoa. Biol. Reprod., 23:877-886. Yanagimachi, R. 1988 Mammalian fertilization. In: The Physiology of Reproduction. E. Knobil and J.D. Neill, eds. Raven Press, New York, Vol. 1, pp. 135-185. Yanagimachi, R. and C.A. Mahi 1976 The sperm acrosome reaction and fertilization in the guinea pig: A study in vivo. J. Reprod. Fertil., 46:49-54. Yanagimachi, R. and N. Usui 1974 Calcium dependence of the acrosome reaction and activation of guinea pig spermatozoa. Exp. Cell Res., 89:161-174.
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
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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).
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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
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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. LITERATURE CITED Barros, C., J.M. Bedford, L.E. Franklin, and C.R. Austin 1967 Membrane vesiculation as a feature of the mammalian acrosome reaction. J. Cell. Biol., 34tCl-C5. Bedford, J.M. and G.W. Cooper 1978 Membrane fusion events in the fertilization of vertebrate eggs. In: Membrane Fusion. G. Poste and G.L. Nicolson, eds. Elsevier-North Holland, Amsterdam, pp. 65-125. Bedford, J.M., H.D.M. Moore, and L.E. Franklin 1979 Significance of the equatorial segment of the acrosome of the spermatozoon in eutherian mammals. Exp. Cell Res., 119t119-126. Daya, S., R.B.L. Gwatkin, and H. Bissessar 1987 Separation of motile human spermatozoa by means of a glass bead column. Gamete Res., 17t375-380. Fawcett, D.W. and R.D. Hollenberg 1963 Changes in the acrosome of guinea pig spermatozoa during passage through the epididymis. Z. Zellforsch. Mikrosk. Anat., 60t276-292. Flaherty, S.P. and G.E. Olson 1988 Membrane domains in guinea pig sperm and their role in the membrane fusion events of the guinea pig sperm acrosome reaction. Anat. Rec., 220t267-280. Fleming, A.D. and R. Yanagimachi 1981 Effects of various lipids on the acrosome reaction and fertilizing capacity of guinea pig spermatozoa with special reference to the possible involvement of lysophospholipids in the acrosome reaction. Gamete Res., 4t253273. Friend, D.S. 1984 Membrane organization and differentiation in the guinea pig spermatozoon. In: Ultrastructure of Reproduction. J. Van Blerkom and P.M. Motta, eds. Martinus Nijhoff, Boston, pp. 75-85. Friend, D.S. and D.W. Fawcett 1974 Membrane differentiations in freeze-fractured mammalian sperm. J. Cell Biol., 63t641-664. Green, D.P.L. 1978a The induction of the acrosome reaction in guineapig sperm by the divalent metal cation ionophore A23187. J. Cell Sci., 32t137-151. Green, D.P.L. 197813 The osmotic properties of the acrosome of guineapig sperm. J. Cell Sci., 32t165-176. Heath, E., N Schaeffer, D.A. Meritt, and J . A . Jeyendran 1987 Rouleaux formation by spermatozoa in the naked-tailed armadillo. Cahassous unicinctus. J. Reprod. Fertil., 7.9t153-158. Huang, T.T.F., D. Hardy, H. Yanagimachi, C. Teuscher, K. Tung, G. Wild, and R Yanagimachi 1985 pH and proteinase control of acrosomal content stasis and release during the guinea pig acrosome reaction. Biol. Reprod., 32.451-462. Lui, C.W., R.J. Mrsny, and S.Meizel 1979 Procedures for obtaining high percentages of viable in vitro capacitated hamster sperm. Gamete Res., 2.207-211. Martan, J. and Z. Hruban 1970 Unusual spermatozoan formations in the epididymis of the flying squirrel (Glaucomys volans). J . Reprod. Fertil., 21t167-170. Martan, J. and B.A. Shepherd 1972 Spermatozoa in rouleaux in the female guinea pig genital tract. Anat. Rec., 175t625-630.
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McGrath, J., N. Hillman, a n d M. Nadijcka 1977 Separation of dead and live mouse spermatozoa. Dev. Biol., 61:114-117. Olson, G.E., V.P. Winfrey, and G.R. Davenport 1988 Characterization of matrix domains of the hamster acrosome. Biol. Reprod.. 39: 1145-1158. Olson, G.E., V.P. Winfrey, and S.P. Flaherty 1989 Membranecytoskeleton interactions in the sperm acrosome. In: Gamete Physiology. R.H. Asch, J.P. Balmaceda and I. Johnston, eds. Serono Symposia, USA, Norwell, pp. 109-118. Olson, G.E., V.P. Winfrey, M.A. Winer, and G.R. Davenport 1987 The outer acrosomal membrane of guinea pig spermatozoa: Isolation and structural characterization. Gamete Res., 17:77-94. Phillips, D.M. a n d J.M. Bedford 1987 Sperm-sperm associations in t h e loris epididymis. Gamete Res., 18:17-25. Primakoff, P., D.G. Myles, a n d A.R. Bellve 1980 Biochemical analysis of the released products of the mammalian sperm acrosome reaction. Dev. Biol., 98:417-428. Russell, L.. R.N. Peterson, and M. Freund 1979 Direct evidence for
formation of hybrid vesicles by fusion of plasma and outer acrosoma1 membranes during t h e acrosome reaction in boar spermatozoa. J. Exp. Zool., 208:41-56. Talbot, P. 1985 Sperm penetration through oocyte investments in mammals. Am. J. Anat., 174:331-346. Tung, K.S.K., A. Okada, and R. Yanagimachi 1980 Sperm autoantigens and fertilization. I. Effects of antisperm antibodies on rouleaux formation, viability, and acrosome reaction of guinea pig spermatozoa. Biol. Reprod., 23:877-886. Yanagimachi, R. 1988 Mammalian fertilization. In: The Physiology of Reproduction. E. Knobil and J.D. Neill, eds. Raven Press, New York, Vol. 1, pp. 135-185. Yanagimachi, R. and C.A. Mahi 1976 The sperm acrosome reaction and fertilization in the guinea pig: A study in vivo. J. Reprod. Fertil., 46:49-54. Yanagimachi, R. and N. Usui 1974 Calcium dependence of the acrosome reaction and activation of guinea pig spermatozoa. Exp. Cell Res., 89:161-174.
