Morphology and Kinetics of the Hamster Sperm Acrosome Reaction PRUDENCE TALBOT AND L. E. FRANKLIN Department of Biology, University of Houston, Houston, Texas 77004

ABSTRACT The morphology and kinetics of the normal acrosome reaction were examined in vitro using hamster sperm incubated in detoxified sera. The reaction involved either swelling and elevation or crenulation and fragmentation of the acrosomal cap. Swelling and elevation occurred during both normal and degenerative reactions, as reported by others. Crenulation with subsequent fragmentation of the cap was observed during normal reactions. Early crenulation of the acrosome could be induced by cold shock (5T, 25 minutes),but this did not decrease the incubation time required (at 37°C) for completion of the normal reaction. In appropriate sera, the occurrence of normal and degenerative acrosome reactions in motile sperm was significantly separated in time to study the reactions independently. The duration of the normal reaction, i.e., the time between the first morphological change in the acrosome (initiation)until the actual detachment of the cap (termination) was estimated to be 20 minutes. Saline dilution of these sera delayed initiation of the reaction and increased the duration of the reaction once it had started. Data from cold-shock and serum dilution experiments indicate that the mechanisms which govern the initiation and termination of the normal reaction are independently variable, and further suggest that initiation involves a change in membrane permeability and that termination includes membrane vesiculation. Mammalian sperm detach the acrosomal cap as a consequence of cell death (degenerative acrosome reaction) or as a physiological concomitant of fertilization (normal acrosome reaction). The degenerative acrosome reaction is characterized at the ultrastructural level by random changes in the plasma and outer acrosomal membrane (Saacke and Almquist, '64; Saacke and Marshall, '68; Bedford, '70;Franklin et al., '70; Yanagimachi and Noda, '70a; Jones, '73). In rats, hamsters, and rabbits, the normal acrosome reaction involves vesiculation of the plasma and outer acrosomal membrane forward from the equatorial segment (Piko and Tyler, '64; Barros et al., '67; Bedford, '68, '70; Franklin et al., '70; Yanagimachi and Noda, '70b). Most knowledge regarding the normal acrosome reaction has come from either static examination of dead sperm at the ultrastructural level or from studies in which the end point of the reaction was observed J. EXP. ZOOL., 198: 163-176

i.e., the number of reacted sperm was monitored. To understand the mechanism and physiological significance of the normal reaction, it is necessary to know what changes take place in sperm during the reaction. Our purpose was to conduct a time sequence study of Ziuing hamster sperm which w e r e in t h e process of undergoing normal acrosome reactions. Specific aspects of the reaction examined for hamster sperm were: (1)morphological changes occurring in the acrosomal cap during reaction and experimental induction of these changes by cold shock; (2) the rates at which sperm begin and complete the normal reaction; and (3) the effect of serum dilution and of cold shock on these rates. MATERIALS AND METHODS

Collection and in vitro incubation of sperm Hamster sperm from the lacerated




cauda epididymidis were collected in normal saline. Sperm concentration was determined turbidimetrically. Heat pretreated (60°C for 60 minutes) blood sera human, calf, Rhesus monkey or baboon) which consistently supported occurrence of activated motility and acrosome reactions were used for incubation. Blood serum (50 or 100 pl) was dispensed into culture dishes (Falcon Plastics, catalogue no, 3001) containing liquid paraffin. In some experiments, the serum was diluted 1:1with normal saline. Sperm (5 or 10 111) were added to the serum droplets (final concentration = 2 x 107 sperm/ml) and incubated at 37°C in air for variable periods of time up to 12 hours. At half-hourly or hourly intervals during incubation, small samples of sperm were examined at x 400 under a vaseline supported cover slip with a Wild M-20 phase contrast microscope. Sperm undergoing acrosome reactions (morphologically altered acrosomal caps) and/or sperm which had completed normal acrosome reactions highly motile sperm without acrosomal caps, fig. 2) were scored. Sperm were monitored until it became evident that the population was dying as judged by a decrease in the quality and percentage of motility. Morphological changes in hamster sperm acrosomes during reaction were photographed using a Reichert Zetopan research microscope equipped with Anoptral contrast optics. Cold shock of sperm Influx of extracellular Ca2' initiates acrosome reactions in guinea pig and echinoid sperm (Summerset al., '76).To attempt induction of hamster sperm acrosome reactions, sperm were cold shocked, a treatment which is not detrimental to epididymal hamster sperm (Morita and Chang, '70) and which increases the permeability of sperm membranes to numerous agents including Ca2+(e.g., Mann, '64; Quinn and White, '66; Purse1 et al., '70; Harrison and White, '72; Darin-Bennett et al., '73; Karagiannidis, '76).Droplets (50pl) of heat-pretreated blood serum were

placed in tissue culture dishes and cooled to 4-5°C; control droplets were equilibrated to 37°C. Sperm suspensions which had been prepared at 24°C were inoculated into the droplets (final concentration = 2 x 1O7/ml) and incubated under liquid paraffin in an air atmosphere. After ten or 30 minutes, sperm samples were placed under vaseline supported cover slips and the condition of the acrosome was assessed with phase contrast microscopy. To determine the effect of cold shock on the kinetics of the normal reaction, sperm were cold-shocked (4-5"C) for 25 minutes in heat-pretreated serum as described above, transferred to 37"C,and incubated an additional five hours. Sperm were sampled at hourly intervals and the percentages of motile reacting and reacted sperm were scored. Scanning electron microscopy of sperm Sperm undergoing normal acrosome reactions in serum and cold-shocked sperm were prepared for scanning electron microscopy in the following manner. Sperm suspensions were cold-shocked in serum or normal saline as described above, warmed to room temperature for 10 minutes, fixed in one-half strength glutaraldehyde-paraformaldehyde (Karnovsky, '65) buffered with 0.1 M cacodylate (pH 7.51, and pelleted by centrifugation. To selectively examine acrosome reacting sperm which were motile at the time of fixation,hamster ova were placed in serum droplets containing reacting sperm. After five to ten minutes, ova were removed, washed once in normal saline, and fixed as described above. Virtually all sperm adhering to the zona pellucida surface after the normal saline wash showed good motility. Following a rinse in cacodylate buffer, both the pellet of cold-shocked sperm and the ova with attached reacting sperm were post-fixed in 1%osmium tetroxide, dehydrated in a graded acetone series, enclosed in small bags made from Ross lens tissue, critically point dried (Samdri PVT-31, mounted on aluminum pedestals, coated



with carbon (100 N and gold (200 k , and adhered to the glass slide or cover slip. examined with a Cambridge S4-10 scanTo attempt induction of the reaction, ning electron microscope. sperm were cold-shocked (5°C for 25 minutes). Upon warming to room temperaRESULTS ture on a microscope slide, 65.8% & 4.8% Morphology of the acrosome reaction (mean of 5 experiments rt SEMI of the The acrosomal cap of epididymal ham- total sperm population was motile, and ster sperm is smooth-surfaced, reflective, 96.8%& 6.1%of the motile sperm formed and sickle-shaped (fig. 1). In reacted sperm crenulated acrosomes. These sperm were the acrosomal cap is absent (fig. 2). Motile indistinguishable from hamster sperm unsperm with morphologically altered acro- dergoing normal reactions by crenulation soma1 caps were interpreted to be in the of the acrosome. Sequential stages in crenprocess of undergoing acrosome reaction. ulation as it occurred in cold shocked Examination of intermediate reaction sperm are shown in figures 13 to 17. The tip stages revealed that two morphologically of the acrosome underwent progressive distinct modes of reaction occur. The bending in the direction of the concave acrosomal cap either became swollen and surface of the acrosome (figs. 14-16), reelevated prior to coming off or became sulting in formation of a blunt, non-reflective acrosome (fig. 17).This crenulation secrenulated and blunt at the tip. The reaction sequence for swelling and quence occurred in about 45 to 60 seconds elevation (figs. 3-71 was observed during for individual sperm. For better resolution of the topographiboth normal and degenerative acrosome reaction (the basis for distinguishing be- cal changes taking place during crenulatween these reactions will be explained tion, cold-shocked and normally reacting later). In order to present clearest micro- sperm were examined with scanning elecgraphs, immotile or poorly motile sperm tron microscopy. In the normally reacting were photographed during degeneration. sperm fixed on a zona pellucida surface (fig. Swelling occurred initially along the an- 191, the folded tip of the acrosome is not terior margin of the acrosome (fig. 3). The visible as such, suggesting that the plasma cap then elevated (figs. 4-61, and even- membrane has fused in the region of this tually the acrosomal “ghost” detached fold. A second fold occurs parallel to the completely from the sperm (fig. 7). Some- margin of the equatorial segment (arrow). times swollen acrosomes appeared scal- Topographically, cold-shocked sperm (fig. loped or rippled during normal reactions 18) were similar to those undergoing normal reaction, although in some cold(figs. 8, 9). Sperm with crenulated acrosomes (figs. shocked sperm the folded tip of the 10-12) were usually highly motile and un- acrosome was visible. In both sperm, it apdergoing normal acrosome reactions. Dur- pears that the plasma membrane is disconing crenulation, the tip of the acrosome tinuous at the interface between the equafolded under the concave surface of the torial segment and the acrosomal cap. In sperm populations forming crenulated cap, giving the acrosome a blunt appearance (fig. 10). The folded tip of the acrosomes, elevation of the cap was not obacrosome can be seen in figure 11, which served and there was a conspicuous abwas taken at a different plane of focus. The sence of bean pod shaped “ghosts” in the crenulated acrosome lost its reflectivity incubation medium at four hours. To deter(compare figs. 1and 101, and the surface of mine how crenulated acrosomes were dethe cap appeared slightly corrugated (fig. tached, sperm were incubated on a micro12). Sperm in figures 10 and 12 show a dif- scope slide at 37°C and the normal reacference in the degree of crenulation. Cren- tion was followed in individual sperm. In ulated acrosomes were sticky and often several instances, highly motile sperm with



crenulated acrosomes were observed shaking off small fragments of acrosomal cap. The event was virtually instantaneous. In a given serum batch, both sequences (swelling and crenulation) of acrosome reaction were usually observed, although one sequence was more prevalent, i.e., the majority of reacting sperm had either crendated or swollen acrosomes. Although the more prevalent sequence was often predictable based on experience with individual serum batches, we have noted that sera which often produced reaction by crenulation sometimes produced reaction by swelling and vice versa. Thus the existence of two morphologically distinct sequences of reaction can not be directly attributed to variations in serum composition among species. All serum batches used for the above morphological observations consistently supported the occurrence of a high percentage of acrosome reactions and activation of motility and have been routinely used in our laboratory for in vitro fertilization of hamster ova.

Kinetics of the acrosome reaction The percentage of sperm undergoing acrosome reactions (i.e., sperm in stages shown in figs. 3-12) was assessed as a function of time. The distribution of motiEe reacting sperm was bimodal (fig. 20). Sperm in the first peak of figure 20 were undergoing normal reactions and exhibited activated motility. Sperm in the second peak were undergoing degenerative reactions and exhibited poor motility. The position of the second peak varied with respect to time. In sera which did not sustain good sperm viability, two peaks were not clearly resolved. In some sera, sperm viability was unusually good, and the second peak occurred later in time. The data in figure 20 do not preclude the possibility that some motile sperm degenerated during the first four hours of incubation or that some normal reactions occurred during the second peak. However, based on the quality of the motility of the majority of the sperm, these events would be rare in the population. The main point demonstrated by this ex-

periment is that in selected sera, motile hamster sperm undergoing normal and degenerative reactions can be separated on the basis of time, and thus these reactions can be studied independently. An illustration of the conversion of normally reacting sperm into normally reacted sperm is shown in figure 21. Reacting sperm which were present between 2.1 and 3.7 hours reached a maximum percentage of 2.6 hours. The percentage of reacted sperm increased rapidly between 2.5 and 3.5 hours, reaching a maximum by four hours. The time required for a sperm to undergo reaction, i.e., the time interval between the first detectable morphological change in the acrosomal cap and the completion of the reaction as indicated by loss of the cap, was estimated. In order to make this estimation, it is useful to define the following points in figure 21: (1) the maximum percentage of reacting sperm, ARmax; (2) the time, T, required to reach half this percentage; and (3) the time, T', at which the reaction curve reached a percentage equivalent to the reacting curve at T. The time for the reaction to occur, A , is estimated by the interval T' - T. The duration of the reaction was 20.6 5 2.1 minutes (mean 2 S.E.M. of five experiments). In several experiments, exceptions to the reaction sequence shown in figure 21 were observed. In one instance, sperm started to undergo normal reactions at the usual time but did not complete the reaction, i.e., detach the acrosomal cap, by six to seven hours of incubation; reasons for this are not known. In the second case, very few reacting sperm were observed between two and four hours, although normally reacted sperm did appear at the usual time during incubation. This may be because swelling of the cap is sometimes subtle and difficult to detect at our routine magnification ( x 400). Specific serum components are required for in vitro occurrence of normal acrosome reactions of hamster sperm (Bavister and Morton, '74). The kinetics of the reaction were found to be influenced by the con-



centration of these serum components. Five pairs of experiments were performed in which sperm were incubated in serum or serum diluted 1:l with normal saline and the percentages of reacting and reacted sperm were monitored during time. Since the maximum percentage of reacted sperm varied among experiments, data were normalized to 100% reaction before being combined. Dilution of serum delayed the appearance of reacted sperm by 36 to 40 minutes (fig. 22). This delay is the combined results of: (1) a 28 minute postponement in the initiation of the reaction, T, and (2) a ten minute increase in the duration of the reaction, A . This indicates that there are at least two processes which are involved in the reaction and both are influenced by the concentration of serum components. The idea that the reaction involves two separate processes is reinforced by observations of cold-shocked sperm (fig. 23). Cold shocked sperm underwent immediate initiation of reaction (T, 12 minutes). Induction of this morphological change was not sufficient to cause completion of the reaction, and the duration of the intermediate stages ( A ) was long (about four hours). After sufficient incubation at 37"C, cold shocked sperm underwent normal reactions and activation of motility. The slight delay in the appearance of reacted, cold-shocked sperm is attributable to the time required for the incubation mixture to reach 37°C. DISCUSSION

Features of the morphology and kinetics of the hamster sperm acrosome reaction have been examined during in vitro incubation in detoxified serum. Two morphologically distinct modes of acrosome reaction were observed. These involved either swelling and elevation or crenulation and fragmentation of the acrosoma1 cap. Others have reported that hamster sperm undergo both degenerative (Austin and Bishop, '58a; Barros, '68; Franklin et al., '70; Yanagimachi and Noda, '70a) and normal (Austin and Bishop, '58b;

Yanagimachi and Chang, '64; Yanagimachi and Noda, '70b) acrosome reactions by swelling and elevation, and our results are in agreement. The occurrence of the normal reaction by crenulation of the cap in living hamster sperm has not been previously documented, although other authors may have observed equivalent changes (Yanagimachi, '69; Yanagimachi and Noda, '72). The mechanistic and functional significance of the two modes of reaction is not yet known. Variations in serum osmolarity were probably not responsible for causing the differences since: (1) both types of morphological change occurred during normal reaction in a given experiment (although one or the other mode was more prevalent); (2)osmolarities of all sera used were similar (278-306mOsm); and (3) the more prevalent mode of reaction was not affected by increasing serum osmolarity (unpublished data). Crenulation of the acrosome could be induced experimentally by cold shock. The crenulated acrosomes of cold-shocked and normally reacting sperm were indistinguishable with phase contrast and scanning electron microscopy, although this is not proof that the mechanism governing crenulation is the same in both cases. The fertilizing capacity of hamster sperm is decreased by cold shock, but this is not because sperm fail to undergo acrosome reactions or activation of motility (Talbot and Franklin, in preparation). Our morphological observations show that there are at least two identifiable phases in the normal reaction. These are initiation of the reaction (at time TI as evidenced by swelling and/or crenulation of the acrosomal cap and termination of the reaction (at time T' = T A 1 as evidenced by loss of the cap. The duration ( A 1 of the normal reaction for hamster sperm in serum was calculated to be about 20 minutes. Yanagimachi and Noda ('70b) observed part of the reaction sequence in several hamster sperm and estimated that it occurred in five to ten minutes. The time for occurrence of guinea pig sperm reactions was three to six minutes under in-




cubation conditions which promote synchronous reaction (Yanagimachi and Usui, '74)* The results with diluted serum and cold shock treatment show that the mechanisms which govern the initiation and termination of the reaction are independently variable. Evidence for this is: (1)dilution of serum affected the values of T and T' by different amounts; (2) the value of T was greatly decreased by cold shock, while T' was unaffected or increased; and (3) in some experiments (for unknown reasons) sperm began undergoing reactions at the usual value of T, but had not completed reactions even after six to seven hours of incubation, i.e., T' was abnormally long or non-existent . The first process, initiation of the reaction, is influenced by the concentration of serum components and probably involves an increase in the permeability of the outer acrosomal and/or periacrosomal plasma membrane. Evidence for a permeability change is: (1) swelling of the cap indicates increased permeability to at least water; and (2) cold shock, which induces initiation, causes profound permeability changes in sperm (e.g.,Mann, '64; Quinn and White, '66; Purse1 et al., '70; Harrison and White, '72; Darin-Bennett et al., '73). Our preliminary transmission electron micrographs and the more detailed observations of Jones ('75) indicate that vesiculation of the outer acrosomal and periacrosomal plasma membrane does not occur as an immediate consequence of cold shock. Although equivalent observations have not yet been made for normally reacting sperm these data suggest that initiation of the reaction does not include vesiculative breakdown of the acrosomal cap as described by Barros et al., ('67). The second process, termination of the reaction, is affected by the concentration of serum components, requires 2.5 to 4.5 hours of incubation even when initiation is decreased by cold shock, and does not necessarily take place as a consequence of initiation. Vesiculation between the outer acrosomal and plasma membrane may be

necessary for normal detachment of the acrosomal cap, and the events which lead up to and result in vesiculation with subsequent shedding of the cap may constitute the processes which were measured by A in these experiments. In summary, two morphologically distinct sequences of normal acrosome reaction occur in living hamster sperm during in vitro incubation in serum. The physiological significance of the reaction by swelling and elevation of the acrosomal cap versus crenulation and fragmentation of the cap is not yet known. Because the occurrence of normal and degenerative reactions is separated in time for selected sera, these reactions can be studied independently. Observations on the normal reaction indicate that: (1)the reaction can be initiated by cold shock; (2) initiation and termination of the reaction are independently variable processes; and (3) the duration of the reaction during standard incubation conditions is about 20 minutes. ACKNOWLEDGMENTS

This work was supported by Grant HD 07346 from the National Institutes of Health. We are indebted to R. J. Talbot and B. L. Hylander for their interest in and suggestions regarding this manuscript and to R. Keith and J. Randall for their assistance in the use of the Cambridge S4-10 scanning electron microscope. LITERATURE CITED Austin, C. R., and M. W. H. Bishop 1958a Some features of the acrosome and perforatorium in mammalian spermatozoa. Proc. Roy. Soc. Ser. B., 149: 234-240. -1958b Role of the rodent acrosome and perforatorium in fertilization. Proc. Roy. Soc. Ser. B., 149: 241-248. Barros, C. 1968 An in uitro study of sperm capacitation, the sperm acrosome reaction and fertilization in the golden hamster. Dissertation, Tulane University. 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., 34: C1-c5. Bavister, B. D., and D. B. Morton 1974 Separation of human serum components capable of inducing the


acrosome reaction in hamster spermatozoa. J. Reprod. Fert., 40: 495-498. Bedford, J. M. 1968 Ultrastructural changes in the sperm head during fertilization in the rabbit. Am. J. Anat., 123: 329-358. 1970 Sperm capacitation and fertilization in mammals. Biol. Reprod. Suppl., 2: 128-158. Darin-Bennett, A., A. Paulos and I. G. White 1973 The effect of cold shock and freeze-thawing on release of phospholipids by ram, bull and boar spermatozoa. Aust. J. Biol. Sci., 26:1409-1420. Franklin, L. E., C. Barros and E. N. Fussell 1970 The acrosomal region and the acrosome reaction in sperm of the golden hamster. Biol. Reprod., 3: 180-200. Harrison, R. A. P., and I. G. White 1972 Glycoiytic enzymes in the spermatozoa and cytoplasmic droplets of bull, boar and ram and their leakage after shock. J. Reprod. Fert., 30: 105-115. Jones, R. C. 1973 Changes occurring in the head of boar spermatozoa: vesiculation or vacuolation of the acrosome? J. Reprod. Fert., 33: 113-118. 1975 Fertility and infertility in mammals in relation to sperm structure. The Biology of the Male Gamete. J, G. Duckett and P. A. Racey, eds. Suppl. No. 1 Biol. J. Linnean Soc., 7: 343-365. Karagiannidis,A. 1976 The distribution of calcium in bovine spermatozoa and seminal plasma in relation to cold shock. J. Reprod. Fert., 46: 83-90. Karnovosky, M. J. 1965 A formaldyhyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol., 27: 137A-138A. Mann, T. 1964 The Biochemistry of Semen and of the Male Reproductive Tract. Metheun & Co., London, pp. 356-363. Morita, Z., and M. C. Chang 1970 The motility and aerobic metabolism of spermatozoa in laboratory animals with special reference to the effects of cold shock and the importance of calcium for the motility of hamster spermatozoa. Biol. Reprod., 3: 169-179


Piko, L., and A. Tyler 1964 Fine structural studies of sperm penetration in the rat. V. Int. Congr. Anim. Reprod. Artific. Insemin., Trento, 2: 372-377. Pursel, V. G., L. A. Johnson and R. J. Gerritis 1970 Distribution of glutamic oxalacetic transaminase and lactic dehydrogenase activities in boar semen after cold shock and freezing. Cryobiology, 7: 141-144. Quinn, P. G., and I. G. White 1966 The effect of cold shock and deep-freezing on the concentration of major cations in spermatozoa. J. Reprod. Fert., 12: 263-270. Saacke, R. G., and J. 0. Almquist 1964 Ultrastructure of bovine spermatozoa. I. The head of normal ejaculated sperm. Amer. J. Anat., 155: 143-162. Saacke, R. G., and C. E. Marshall 1968 Observations on the acrosomal cap of fixed and unfixed bovine spermatozoa. J. Reprod. Fert., 16: 511-514. Summers, R. G., P. Talbot, E. M. Keough, B. L. Hylander and L. E. Franklin 1976 Ionophore A23187 induces acrosome reactions in sea urchin and guinea pig spermatozoa. J. Exp. Zoo].,196: 381385. Yanagimachi, R. 1969 In vitro capacitation of hamster spermatozoa by follicular fluid. J. Reprod. Fertil., 18: 275-286. Yanagimachi, R., and M. C. Chang 1964 In uitro fertilization of golden hamster ova. J. Exp. Zool., 156: 361-376. Yanagimachi, R., and Y. D. Noda 1970a Fine structure of the hamster sperm head. Am. J. Anat., 128: 367-388. 1970b Ultrastructural changes in the hamster sperm head during fertilization. J. Ultrastruct. Res., 31: 465-485. ___ 1972 Scanning electron microscopy of golden hamster spermatozoa before and during fertilization. Experientia, 28: 69-72. Yanagimachi, R., and N. Usui 1974 Calcium dependence of the acrosome reaction and activation of guinea pig spermatozoa. Exp. Cell Res., 6: 161-174.


Morphological changes in the hamster sperm acrosome during reaction. x 3,000.

1 An epididymal hamster sperm with a morphologically normal acrosome. The surface of the acrosomal cap is smooth in contour and reflective. 2 A highly motile hamster sperm which has undergone a normal acrosome

reaction. The acrosomal cap has been completely detached. 3-7 Sequential stages in the acrosome reaction as it occurred by swelling and elevation of the acrosomal cap. These stages were observed in motile sperm undergoing both normal and degenerative reaction. 8,9 Normally reacting sperm with swollen acrosomes which are rippled (fig. 8) and partially fragmented (fig. 9). Photographed from highly motile sperm undergoing normal reactions.

10-12 Reacting sperm with crenulated acrosomes. These stages were usually seen during normal reactions. Focus in figure 10 is on the surface of the cap which is nonreflective (compare to fig. 1). Focus in figure 11 is on the folded tip of the cap. Figure 12 is a more advanced stage of crenulation (compare to fig. 10). The surface of the sperm in figure 12 is slightly corrugated.


HAMSTER SPERM ACROSOME REACTION Prudence Talhot and L. E. Franklin




13-17 Sequential stages in crenulation of the acrosome induced by cold shock of epididymal hamster sperm. Crenulation was easily studied and photographed in cold shocked suspensions as the change occurred with a high degree of synchrony. x 3,000. 18 Scanning electron micrograph of a cold shocked hamster sperm with a

crenulated acrosome. x 10,000. 19 Scanning electron micrograph of a hamster sperm on a zona pellucida surface. This sperm was undergoing a normal reaction at the time of fixation and had a crenulated acrosome. x 9,200.





23 The conversion of cold-shocked sperm into normally reacted sperm. The percentage of motile cold-shocked sperm with crenulated acrosomes (x-X) increases rapidly, but these sperm do not complete reactions ( 0 - 1 until three to five hours later, i.e., A is very long. Control sperm completed reactions ( 0 - - - - 0 ) earlier than cold-shocked sperm.

22 The effect of dilution of serum on the occurrence of hamster sperm acrosome reactions. The curve generated in diluted serum ( 0 - 0 ) appears 36 to 40 minutes later in time than the curve generated in undiluted serum (0-0). Each point is the mean of five observations. Data were normalized to 100%reaction before taking means. S.E.M. are not shown as values were smaller than points representing the means.

21 The conversion of normally reacting sperm ( x - x ) into normally reacted sperm ( 0 - 0 ). The duration of the reaction was estimated by A and found to be about 20 minutes (based on five experiments).

20 The percentage of motile sperm in the process of undergoing acrosome reactions (figs. 3-12) is shown as a function of time for a single experiment. Sperm in the first peak were undergoing normal reactions; the percentage of reacting sperm decreases by four to five hours as these sperm complete the normal reaction (also see fig. 21). In this experiment, about 50%of the motile sperm completed normal reaction by four hours. The remaining 50%had intact acrosomal caps, but underwent degenerative reactions (second peak) between six to ten hours. Motility of reacting sperm in the second peak was poor. The exact position of the second peak with respect to time was variable (see text).





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Morphology and kinetics of the hamster sperm acrosome reaction.

Morphology and Kinetics of the Hamster Sperm Acrosome Reaction PRUDENCE TALBOT AND L. E. FRANKLIN Department of Biology, University of Houston, Housto...
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