The Time and Location of the Acrosome Reaction during Sperm Transport in the Female Rabbit J. W. OVERSTREET AND G . W. COOPER Laboratory of Reproductive Biology, Department of Obstetrics and Gynecology, Cornell Uniuersity Medical College, New York, New York 10021
ABSTRACT Motile spermatozoa with lifted or absent acrosomal caps (“reacted acrosomes”) were observed by phase contrast microscopy exclusively in the oviduct of the rabbit reproductive tract during the first 16 hours post coiturn (p.c.1. They were first identified a t six hours p.c. in flushings from the lower isthmus of t h e oviduct and were present in both the lower and upper isthmus two hours later. Motile spermatozoa with reacted acrosomes appeared in the lower ampulla (the site of fertilization) a t ten hours p.c. Such spermatozoa were obtained from all regions of the oviduct at 12 and 16 hours p.c. but they always constituted a minority of the freely swimming spermatozoa recovered. The percentage of spermatozoa with activated motility was higher following the acrosome reaction, and their apparent swimming speeds were lower than those of intact spermatozoa. The role of ovulation in t h e induction of acrosome reactions was studied in females artificially inseminated per uuginum (AI), with or without the induction of ovulation, and in females mated either two hours before or two hours after ovulation. The proportion of motile spermatozoa with reacted acrosomes in the isthmus and ampulla of non-ovulatory females 12 hours after A1 was similar to their proportions in these regions 12 hours after mating or 12 hours after A1 with induced ovulation. Four hours after delayed matings, i.e., two or six hours after ovulation, motile spermatozoa were confined to the tuba1 isthmus and none had undergone the acrosome reaction. These findings indicate that the products of ovulation and the endocrine changes associated with ovulation in the rabbit need not be directly involved in the induction of the acrosome reaction in vivo. Direct observations have not been reported previously of t h e site and time of occurrence of the acrosome reaction of rabbit spermatozoa within the female reproductive tract. In part this has been due to the difficulty of visualizing alterations in the head membranes and acrosomal content of living spermatozoa of this species (Austin, ’63; Bedford, ’67). The sequence of membrane changes involved in the acrosome reaction of rabbit spermatozoa and the relationship of these events to the ovum and its vestments have been inferred from ultrastructural studies following natural mating and intratubal insemination (Bedford, ’67, ’68, ’721,and it is generally believed t h a t initiation of t h e acrosome reaction requires specific chemical and/or physical stimuli associated with the products of ovulation or the site of fertilization (see Meizel, ’78; Bedford and Cooper, ’78, for reviews). InforJ. EXP. ZOOL. (1979) 209: 97-104.
mation on t h e time and site of occurrence of the acrosome reaction during sperm transport and fertilization after natural mating is necessary for complete interpretation of the morphological and biochemical information now available on rabbit gamete interaction. In the course of a study of sperm motility in the rabbit oviduct (Overstreet and Cooper, ’751, we became able to recognize acrosomal alterations in motile spermatozoa with phase-contrast microscopy. In this communication we report the locations within t h e female tract in which motile spermatozoa were recovered with altered or missing acrosomes, as well as our visual assessments of the movement characteristics of rabbit spermatozoa following the acrosome reaction. ‘Present address: Department of Human Anatomy, Schml of Medicine. University of California, Davis, California 95616.
97
98
J. W. OVERSTREET AND G. W. COOPER
MATERIALS AND METHODS
Observations were made with phase contrast microscopy of the acrosomal morphology and movement characteristics of rabbit spermatozoa collected from t h e reproductive tracts of 61 virgin New Zealand White females. Forty-five were studied after a single natural mating and were assayed in groups of five a t 1, 15 and 90 minutes, and a t 4 , 6 , 8 , 10, 12 and 16 hours post coitum (p.c.1. Eight does were each artificially inseminated per vaginam (AI) with a single ejaculate (Adams, ’62) and were examined 12 hours later. Three of these animals were induced to ovulate by mounting and/or coitus with a vasectomized male immediately before AT. Five does received no ovulation inducing stimulus a t the time of AI. In a second group of eight does ovulation was induced by mating with a vasectomized male after which “delayed mating” was allowed with a fertile male either 8 hours (4 does) or 12 hours (4 does) after the sterile coitus. All of these animals were examined four hours after the fertile mating, i.e., either two or six hours after the onset of ovulation. Details of the numerical distribution of spermatozoa in the reproductive tracts of these 61 females are the subject of other communications (Overstreet and Cooper, ’78, ’79; Overstreet et al., ’78). Females were sacrificed by overdose of sodium pentobarbital (Nembutal, Abbott), and the reproductive tract was divided with hemostats into lower isthmus, upper isthmus, lower ampulla, upper ampulla, uterus, endocervix and vagina. All manipulations and observations were carried out a t 37°C in a controlled environment room. Each region of the oviduct was cannulated with a micropipette and its luminal contents were collected by flushing 10 p1 of a 5% mixture (v/v) of rabbit serum in Tyrode solution (pH 7.4) into the segment. When animals were examined after ovulation, ampullar segments were flushed a second time with 0.5 ml of 0.9% NaCl (w/v) to maximize ovum recovery. Each uterine horn, endocervix and the vagina were flushed with 1 ml of serum Tyrode. Five to ten microliters of these flushes were placed on a slide and covered with a coverslip (#l, 18 X 18 mm) (see Overstreet and Cooper, ’78, ’79, for details). Spermatozoa were observed with dry phasecontrast optics a t magnifications of 100400 x (Leitz, Ortholux). A spermatozoon was scored as motile if any flagellar undulations
were seen. Those motile spermatozoa with visible changes in the head membranes (Overstreet and Cooper, ’75) were classified as having undergone the acrosome reaction. The motility of spermatozoa was classified as progressive Le., space gaining movement) or non-progressive, and if progressive as linear, circular or activated (Cooper et al., ’79). Ova were recovered from the serum Tyrode and saline flushings and were mounted under Vaseline-paraffin supported coverslips. After fixation in acetic ethanol and staining with lacmoid (Chang, ’521, the number of spermatozoa associated with the ovum vestments was counted and sperm entry into the vitellus was assessed. RESULTS
With phase-contrast optics the acrosome of the rabbit spermatozoon appears as a phasedense crescent around the rostra1 margin of the head. By focusing up and down over the flattened head, the intact acrosome appears alternately as a phase-dense (dark) or refractile (bright) structure which ends a t the equatorial segment - a dark band girdling the middle of the head (Overstreet and Cooper, ’75). Alterations in the acrosome which can be appreciated visually always involve loss of its phase-density and refractility. Most motile spermatozoa with “reacted acrosomes” had no visible evidence of acrosomal membranes or content. A minority retained a cap of expanded membranes surrounding the acrosomal region which sometimes were crenated and adherent to the head. All of these states of acrosomal alteration were seen in motile spermatozoa sampled from each region of the oviduct, and were indistinguishable from those described previously for non-motile spermatozoa (Overstreet and Cooper, ’75). Approximately 10%of the spermatozoa recovered from the female tract were swimming too rapidly for an accurate assessment of acrosomal morphology. These spermatozoa were always classified as intact. Motile spermatozoa with reacted acrosomes were never observed in flushings from the vagina, endocervix or uterus of the animals in this study. Nor was any visible alteration apparent in the acrosomes of 1,198 motile oviductal spermatozoa examined between one minute and four hours p.c. Motile spermatozoa with reacted acrosomes were first seen in flushings from the isthmus in three of five does assayed six hours p.c. and in three of five
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ACROSOME REACTION DURING SPERM TRANSPORT IN RABBITS
does assayed eight hours p.c. (table 1). They were also found in the ampullae of females examined from 10 through 16 hours P.c., but always constituted a small percentage of the motile spermatozoa recovered (table 1). Four of five does assayed ten hours p.c. had ovulated and none of the 18 eggs recovered were fertilized (56%egg recovery, based on estimates of ovarian ovulation points). These eggs were completely invested by cumulus cells and 98%of the ampullar sperm were observed swimming freely in the medium, i.e., they were not associated with the granulosa cell egg masses. All does examined 12 and 16 hours px. had ovulated. Most of the granulosa cells had dispersed from the 36 eggs recovered 12 hours p.c. (90% egg recovery) and 53% were fertilized. Fifty-two percent of the ampullar spermatozoa sampled were associated with the eggs and their remaining vestments - the corona radiata cells and the zona pellucida. By 16 hours p.c. all of the 53 eggs (88%recovery) were completely denuded of cumulus and corona cells and 98%were fertilized. Ninety-two percent of the ampullar sperm from these oviducts were recovered in association with the zona pellucida of the eggs. It seems likely that many of these sperm had undergone an acrosome reaction. Since the data presented in table 1 refer only to free spermatozoa which were swimming slowly enough to be classified, those percentages must be a substantial underestimate of the incidence of the acrosome reaction in the rabbit ampulla a t 12 and 16 hours p.c. The patterns of motility displayed by acrosome reacted spermatozoa were similar to those of intact spermatozoa. However, following the acrosome reaction, the proportion of motile spermatozoa swimming in linear trajectories declined, while the incidence of activated motility (alternating episodes of forward progressive and vigorous non-progressive movement, see Cooper et al., '79) increased (table 2). The apparent swimming speeds of acrosome reacted spermatozoa were generally lower than those of intact spermatozoa in the same suspensions. In the tuba1 isthmus non-progressive motility (i.e., stationary spermatozoa with flagellar undulations) was more frequent after the acrosome reaction (table 2). Ampullar sperm with reacted acrosomes appeared to swim more slowly than intact spermatozoa, but 94% of acrosome reacted spermatozoa in the ampulla had progressive motility as compared with only 64%
o
o
o
m
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J. W. OVERSTREET AND G. W. COOPER
that were progressive in the lower isthmus (table 2). The experiments with delayed matings and those with artificial insemination (AI) of nonovulatory females provided evidence that neither ovulation-associated endocrine changes nor the products of ovulation are required for induction of the acrosome reaction in vivo. In the absence of ovulation only a few spermatozoa reach the tubal ampulla by 12 hours after A1 (Overstreet and Cooper, '79). Nevertheless, the proportion of motile, acrosome reacted spermatozoa in the isthmus and ampulla of these females did not differ from that found in ovulatory females 12 hours after natural mating or A1 (table 3). In delayed mating experiments, motile spermatozoa were recovered exclusively from the tubal isthmus four hours after fertile matings initiated 8 or 12 hours after sterile coitus (Overstreet and Cooper, '79). None of the 1,189 motile isthmic spermatozoa examined had undergone a n acrosome reaction, and all eggs recovered from the ampulla were unfertilized and remained in cumulus. DISCUSSION
We have observed that rabbit spermatozoa undergo visible acrosomal alterations which are compatible with sustained motility after they have resided for more than four hours in the female tract. Such spermatozoa were first seen six hours p.c. in the lower isthmus of the oviduct and were present in the ampulla after the onset of ovulation. Motile spermatozoa with acrosome reactions were not seen in the rabbit vagina, cervix or uterus during the first 16 hours P.c., but they were found in the uterus by 24 to 27 hours P.c., i.e., well after the completion of fertilization (Cooper and Overstreet, unpublished). These observations are consistent with the notion that completion of sperm capacitation must precede the "true" acrosome reaction (Bedford, '691, and that visible alterations of the head membranes of vigorously moving spermatozoa are physiologic acrosome reactions (Austin, '75). Motile, acrosome reacted spermatozoa have been recovered from the oviducts of several rodent species a t the time of fertilization (Austin and Bishop, '58; Yanagimachi and Mahi, '76) and their appearence in vitro is considered evidence that the incubation medium promotes capacitation and physiologic acrosome reactions (Meizel, '78). "True" acrosome reactions
ACROSOME REACTION DURING SPERM TRANSPORT IN RABBITS
101
TABLE 3
Proportion of motile oviductal spermatozoa with "reacted "acrosomes recovered 12 hours after a single natural mating (Group I ) or after artificial insemination (AIl of a single ejaculate. Group IIIfemales did not ovulate after AI. Group IIfemales were induced to ovulate prior to insemination by coitus with a vasectomized buck Median percentages of reacted motile spermatozoa per animal (range)' Region
Upper ampulla Lower ampulla Upper isthmus Lower isthmus
Group I (Natural mating)
Group I1 (Al, atenle coitus)
Group 111 (AI, no ovulation)
4
(0-33) 8 (0-21) 13 (0-25)
10 (5-24) 23 (22-38) 4 (0-57)
8 (2-28)
10 (0-33) 20 (0-67) 02 (0-3) 8
(1-2)
1 2
(0-12)
For each region sampled there were no significant differences among Groups I, 11, and 111 (Kruskal-WallisOne Way Analysia of Variance by Ranks, P > 0.10 in all cases). ' Median percentages (range)based on motile spermatozoa recovered from five females in Groupa I and 111, and three in Group 11. A total of 173 reacted motile spermatozoa were examined in Group I, 94 in Group I1 and 52 in Group 111. Mode
involve ordered membrane fusions and vesiculations between the plasmalemma and outer acrosomal membrane followed by loss of the acrosomal content. Only electron microscopy can distinguish this type of acrosome reaction from the uncoordinated, degenerative changes in the plasmalemma and outer acrosomal membrane, the so-called "false" acrosome reaction. Immotile rabbit spermatozoa with visibly altered acrosomes can be recovered from the female tract throughout the period of sperm transport, beginning as early as one minute p.c. (Overstreet and Cooper, '75; Overstreet et al., '78). These cells do not appear to be viable and it is probable that most have undergone false acrosome reactions. In view of the apparent requirement for enzyme mediated dispersal of the granulosa cell investment during the approach of spermatozoa to eggs (Bedford, '74), most investigators have assumed t h a t the acrosome reaction of the fertilizing spermatozoon must be induced a t or near the site of fertilization. Recently, it has been suggested that the integrity of the sperm plasmalemma may be critical for attachment to the zona pellucida prior to its penetration, thus necessitating initiation of the acrosome reaction a t or near the zona surface (Gwatkin et al., '76; Bedford and Cooper, '78). Both transmission (Yanagimachi and Noda, '70; Bedford, '72) and scanning electron micrographs (Gwatkin et al., '76) indicate that spermatozoa need not loose the vesiculated acrosomal cap until zona penetra-
tion has begun. There is as yet, however, no morphological or experimental evidence conclusively showing t h a t spermatozoa are infertile if they undergo a n acrosome reaction in the outer cumulus as suggested by Gwatkin et al. ('76). The granulosa cell investment of t h e rabbit egg is not essential for the induction of acrosome reactions, since eggs free of these cells have been fertilized in vivo (Harper, '70; Overstreet and Bedford, '74) and in vitro (Fraser et al., '711, and the treatment of denuded eggs with a variety of enzymes fails to limit rabbit sperm penetration through the zona pellucida in vivo (Overstreet and Bedford, '75). Our observations that motile spermatozoa with reacted acrosomes are present in the oviducts before ovulation and in the oviducts of non-ovulatory, estrous females after artificial insemination support t h e above experimental findings and suggest further that the specific environmental factors promoting t h e acrosome reaction in t h e oviduct are not controlled by ovulation-associated changes in circulating steroid levels or by any of the products of ovulation including follicular fluids. If initiation of the acrosome reaction in capacitated spermatozoa is accomplished by a specific stimulus, it appears that this may occur throughout the oviduct, and not a t the site of fertilization alone. Even if controlled release of acrosomal enzymes occurs within t h e cumulus mass in those spermatozoa first
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J. W. OVERSTREET AND G. W. COOPER
reaching the egg, the stimulus for this could still be distant from the egg. Depending on the time course of the biochemical and morphological events preceding release of the acrosome's enzyme content, initiation of the acrosome reaction could occur in the isthmus and not in the ampulla of the oviduct. Completion of the membrane fusion events of the rabbit sperm acrosome reaction in vitro requires a minimum of five to ten minutes (Singhas and Oliphant, '78) and it is likely that the initial biochemical events preceding visible morphological changes would also require some minutes (Meizel, '78). The final ascent of rabbit spermatozoa to the site of fertilization is synchronized with ovulation (Harper, '73; Overstreet et al., '78) and may require activation of sperm motility (Overstreet and Cooper, '79). Rodent spermatozoa which have undergone an acrosome reaction are usually activated (Yanagimachi, '701, and our observations suggest that a shift to activated motility may also accompany the acrosome reaction in rabbit spermatozoa (table 2). Since the passage of rabbit spermatozoa a t ovulation from the tubal isthmus to the ampulla takes place within 15 to 30 minutes (Harper, '73; Overstreet et al., '78), the acrosome reaction of ascending spermatozoa, if initiated in the lower isthmus, could reach completion in the vicinity of the eggs a t the ampullar isthmic junction. Most spermatozoa in the isthmus of the rabbit oviduct are located within 2 cm of the uterotubal junction (Overstreet et al., '781, and the majority appear closely associated with ciliated epithelial cells (Cooper and Overstreet, unpublished). The effect of these cell-cell interactions on the physiology of oviductal spermatozoa may include membrane changes resulting in activation of sperm motility and initiation of the biochemical events leading to the acrosome reaction. ACKNOWLEDGMENTS
We thank Doctor D. F. Katz for performing the statistical analysis of the data. This research was supported by NIH Grant HD09215 and a grant from the Ford Foundation. LITERATURE CITED Adams, C. E. 1962 Artificial insemination i n d e n t s . In: Semen of Animals and Artificial Insemination. J. P. Maule, ed. Commonwealth Agricultural Bureau, pp. 316-330. Austin, C. R. 1963 Acrosome loss from the rabbit spermatozoon in relation to entry into the egg. J. Reprod. Fertil., 6: 313-314.
1975 Membrane fusion events in fertilization. J. Reprod. Fertil., 44: 155-166. Austin, C. R., and M. W. H. Bishop 1958 Role of the rodent acrosome and perforatorium in fertilization. Proc. R. SOC. B., 14: 241-248. Bedford, J. M. 1967 Experimental requirement for capacitation and observations on ultrastructural changes in rabbit spermatozoa during fertilization. J. Reprod. Fertil. (Suppl.), 2: 35-48. 1968 Ultrastructural changes i n the sperm head during fertilization in t h e rabbit. Am. J. Anat., 123: 329-358. 1969 Morphological aspects of capacitation. Adv. Biosci., 4; 35-50. 1972 An electron microscopic study of sperm penetration into the rabbit egg after natural mating. Am. J. Anat., 133: 213-254. 1974 Mechanisms involved in penetration of spermatozoa through the vestments of the mammalian egg. In: Physiology and Genetics of Reproduction. Part B. E. M. Coutinho and F. Fuchs, eds. Plenum Publishing Corp., New York, pp. 55-68. 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 Press, Amsterdam, pp. 65-125. Chang, M. C. 1952 Fertilizability of rabbit ova and the effects of temperature in uitro on their subsequent fertilization and activation in uiuo. J. Exp. Zool., 121: 351-370. Cooper, G. W., J. W. Overstreet and D. F. Katz 1979 Sperm transport in the reproductive tract of the female rabbit. Sperm motility and patterns of movement at different levels of the tract. Gamete Res., in press. Fraser, L. R., P. V. Dandekar and R. A. Vaidya 1971 In uitro fertilization of tubal rabbit ova partially or totally denuded of follicular cells. Biol. Reprod., 4: 229-233. Gwatkin, R. B. L., H. W. Carter and H. Patterson 1976 Association of mammalian sperm with the cumulus cells and the zona pellucida studied by scanning electron microscopy. Proc. workshop SEM in Reproduction Biology, IIT Research Institute, Chicago, pp. 379-384. Harper, M. J. K. 1970 Factors influencing sperm penetration of rabbit eggs in uiuo. J. Exp. Zool., 173: 47-62. 1973 Stimulation of sperm movement from the isthmus to the site of fertilization in the rabbit oviduct. Biol. Reprod., 8: 369-377. Meizel, S. M. 1978 The mammalian sperm acrosome reaction, a biochemical approach. In: Development in Mammals. Vol. 3. M. H. Johnson, ed. Elsevier, North Holland Press, Amsterdam, pp. 1-64. Overstreet, J. W., and J. M. Bedford 1974 Comparison of the penetrability of the egg vestments i n follicular oocytes, unfertilized and fertilized ova of the rabbit. Dev. Biol., 41: 185-192. 1975 The penetrability of rabbit ova treated with enzymes or anti-progesterone antibody: Aprobe into the nature of a mammalian fertilizin. J. Reprod. Fertil., 44: 273-284. Overstreet, J. W., and G. W. Cooper 1975 Reduced sperm motility in the isthmus of the rabbit oviduct. Nature, 258: 718-719. 1978 Sperm transport in the reproductive tract of the female rabbit: I. The rapid transit phase of transport. Biol. Reprod., 19: 101-114. 1979 Effect of ovulation and sperm motility on the migration of rabbit spermatozoa to the site of fertilization. J. Reprod. Fertil., 55: 53-59. Overstreet, J. W., G . W. Cooper and D. F. Katz 1978 Sperm
ACROSOME REACTION DURING SPERM TRANSPORT IN RABBITS transport in th e reproductive tract of the female rabbit: 11. The sustained phase of transport. Biol. Reprod., 19: 115.132. Singhas, C. A., and G. Oliphant 1978 Ultrastructural observations of the time sequence of induction of acrosomal membrane alterations by ovarian follicular fluid. Fertil. Steril., 29: 194-203. Yanagimachi, R. 1970 The movement of golden hamster
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spermatozoa before and after capacitation. J. Reprod. Fertil., 23: 193-196. Yanagimachi, R., and C. A. Mahi 1976 Sperm acrosome reaction in the guinea pig: a study in uiuo. J. Reprod. Fertil., 46: 49-54. Yanagimachi, R., and Y. D. Noda 1970 Ultrastructural changes in the hamster sperm head during fertilization. J. Ultrastruc. Res., 31: 465-485.
ABSTRACT Motile spermatozoa with lifted or absent acrosomal caps (“reacted acrosomes”) were observed by phase contrast microscopy exclusively in the oviduct of the rabbit reproductive tract during the first 16 hours post coiturn (p.c.1. They were first identified a t six hours p.c. in flushings from the lower isthmus of t h e oviduct and were present in both the lower and upper isthmus two hours later. Motile spermatozoa with reacted acrosomes appeared in the lower ampulla (the site of fertilization) a t ten hours p.c. Such spermatozoa were obtained from all regions of the oviduct at 12 and 16 hours p.c. but they always constituted a minority of the freely swimming spermatozoa recovered. The percentage of spermatozoa with activated motility was higher following the acrosome reaction, and their apparent swimming speeds were lower than those of intact spermatozoa. The role of ovulation in t h e induction of acrosome reactions was studied in females artificially inseminated per uuginum (AI), with or without the induction of ovulation, and in females mated either two hours before or two hours after ovulation. The proportion of motile spermatozoa with reacted acrosomes in the isthmus and ampulla of non-ovulatory females 12 hours after A1 was similar to their proportions in these regions 12 hours after mating or 12 hours after A1 with induced ovulation. Four hours after delayed matings, i.e., two or six hours after ovulation, motile spermatozoa were confined to the tuba1 isthmus and none had undergone the acrosome reaction. These findings indicate that the products of ovulation and the endocrine changes associated with ovulation in the rabbit need not be directly involved in the induction of the acrosome reaction in vivo. Direct observations have not been reported previously of t h e site and time of occurrence of the acrosome reaction of rabbit spermatozoa within the female reproductive tract. In part this has been due to the difficulty of visualizing alterations in the head membranes and acrosomal content of living spermatozoa of this species (Austin, ’63; Bedford, ’67). The sequence of membrane changes involved in the acrosome reaction of rabbit spermatozoa and the relationship of these events to the ovum and its vestments have been inferred from ultrastructural studies following natural mating and intratubal insemination (Bedford, ’67, ’68, ’721,and it is generally believed t h a t initiation of t h e acrosome reaction requires specific chemical and/or physical stimuli associated with the products of ovulation or the site of fertilization (see Meizel, ’78; Bedford and Cooper, ’78, for reviews). InforJ. EXP. ZOOL. (1979) 209: 97-104.
mation on t h e time and site of occurrence of the acrosome reaction during sperm transport and fertilization after natural mating is necessary for complete interpretation of the morphological and biochemical information now available on rabbit gamete interaction. In the course of a study of sperm motility in the rabbit oviduct (Overstreet and Cooper, ’751, we became able to recognize acrosomal alterations in motile spermatozoa with phase-contrast microscopy. In this communication we report the locations within t h e female tract in which motile spermatozoa were recovered with altered or missing acrosomes, as well as our visual assessments of the movement characteristics of rabbit spermatozoa following the acrosome reaction. ‘Present address: Department of Human Anatomy, Schml of Medicine. University of California, Davis, California 95616.
97
98
J. W. OVERSTREET AND G. W. COOPER
MATERIALS AND METHODS
Observations were made with phase contrast microscopy of the acrosomal morphology and movement characteristics of rabbit spermatozoa collected from t h e reproductive tracts of 61 virgin New Zealand White females. Forty-five were studied after a single natural mating and were assayed in groups of five a t 1, 15 and 90 minutes, and a t 4 , 6 , 8 , 10, 12 and 16 hours post coitum (p.c.1. Eight does were each artificially inseminated per vaginam (AI) with a single ejaculate (Adams, ’62) and were examined 12 hours later. Three of these animals were induced to ovulate by mounting and/or coitus with a vasectomized male immediately before AT. Five does received no ovulation inducing stimulus a t the time of AI. In a second group of eight does ovulation was induced by mating with a vasectomized male after which “delayed mating” was allowed with a fertile male either 8 hours (4 does) or 12 hours (4 does) after the sterile coitus. All of these animals were examined four hours after the fertile mating, i.e., either two or six hours after the onset of ovulation. Details of the numerical distribution of spermatozoa in the reproductive tracts of these 61 females are the subject of other communications (Overstreet and Cooper, ’78, ’79; Overstreet et al., ’78). Females were sacrificed by overdose of sodium pentobarbital (Nembutal, Abbott), and the reproductive tract was divided with hemostats into lower isthmus, upper isthmus, lower ampulla, upper ampulla, uterus, endocervix and vagina. All manipulations and observations were carried out a t 37°C in a controlled environment room. Each region of the oviduct was cannulated with a micropipette and its luminal contents were collected by flushing 10 p1 of a 5% mixture (v/v) of rabbit serum in Tyrode solution (pH 7.4) into the segment. When animals were examined after ovulation, ampullar segments were flushed a second time with 0.5 ml of 0.9% NaCl (w/v) to maximize ovum recovery. Each uterine horn, endocervix and the vagina were flushed with 1 ml of serum Tyrode. Five to ten microliters of these flushes were placed on a slide and covered with a coverslip (#l, 18 X 18 mm) (see Overstreet and Cooper, ’78, ’79, for details). Spermatozoa were observed with dry phasecontrast optics a t magnifications of 100400 x (Leitz, Ortholux). A spermatozoon was scored as motile if any flagellar undulations
were seen. Those motile spermatozoa with visible changes in the head membranes (Overstreet and Cooper, ’75) were classified as having undergone the acrosome reaction. The motility of spermatozoa was classified as progressive Le., space gaining movement) or non-progressive, and if progressive as linear, circular or activated (Cooper et al., ’79). Ova were recovered from the serum Tyrode and saline flushings and were mounted under Vaseline-paraffin supported coverslips. After fixation in acetic ethanol and staining with lacmoid (Chang, ’521, the number of spermatozoa associated with the ovum vestments was counted and sperm entry into the vitellus was assessed. RESULTS
With phase-contrast optics the acrosome of the rabbit spermatozoon appears as a phasedense crescent around the rostra1 margin of the head. By focusing up and down over the flattened head, the intact acrosome appears alternately as a phase-dense (dark) or refractile (bright) structure which ends a t the equatorial segment - a dark band girdling the middle of the head (Overstreet and Cooper, ’75). Alterations in the acrosome which can be appreciated visually always involve loss of its phase-density and refractility. Most motile spermatozoa with “reacted acrosomes” had no visible evidence of acrosomal membranes or content. A minority retained a cap of expanded membranes surrounding the acrosomal region which sometimes were crenated and adherent to the head. All of these states of acrosomal alteration were seen in motile spermatozoa sampled from each region of the oviduct, and were indistinguishable from those described previously for non-motile spermatozoa (Overstreet and Cooper, ’75). Approximately 10%of the spermatozoa recovered from the female tract were swimming too rapidly for an accurate assessment of acrosomal morphology. These spermatozoa were always classified as intact. Motile spermatozoa with reacted acrosomes were never observed in flushings from the vagina, endocervix or uterus of the animals in this study. Nor was any visible alteration apparent in the acrosomes of 1,198 motile oviductal spermatozoa examined between one minute and four hours p.c. Motile spermatozoa with reacted acrosomes were first seen in flushings from the isthmus in three of five does assayed six hours p.c. and in three of five
99
ACROSOME REACTION DURING SPERM TRANSPORT IN RABBITS
does assayed eight hours p.c. (table 1). They were also found in the ampullae of females examined from 10 through 16 hours P.c., but always constituted a small percentage of the motile spermatozoa recovered (table 1). Four of five does assayed ten hours p.c. had ovulated and none of the 18 eggs recovered were fertilized (56%egg recovery, based on estimates of ovarian ovulation points). These eggs were completely invested by cumulus cells and 98%of the ampullar sperm were observed swimming freely in the medium, i.e., they were not associated with the granulosa cell egg masses. All does examined 12 and 16 hours px. had ovulated. Most of the granulosa cells had dispersed from the 36 eggs recovered 12 hours p.c. (90% egg recovery) and 53% were fertilized. Fifty-two percent of the ampullar spermatozoa sampled were associated with the eggs and their remaining vestments - the corona radiata cells and the zona pellucida. By 16 hours p.c. all of the 53 eggs (88%recovery) were completely denuded of cumulus and corona cells and 98%were fertilized. Ninety-two percent of the ampullar sperm from these oviducts were recovered in association with the zona pellucida of the eggs. It seems likely that many of these sperm had undergone an acrosome reaction. Since the data presented in table 1 refer only to free spermatozoa which were swimming slowly enough to be classified, those percentages must be a substantial underestimate of the incidence of the acrosome reaction in the rabbit ampulla a t 12 and 16 hours p.c. The patterns of motility displayed by acrosome reacted spermatozoa were similar to those of intact spermatozoa. However, following the acrosome reaction, the proportion of motile spermatozoa swimming in linear trajectories declined, while the incidence of activated motility (alternating episodes of forward progressive and vigorous non-progressive movement, see Cooper et al., '79) increased (table 2). The apparent swimming speeds of acrosome reacted spermatozoa were generally lower than those of intact spermatozoa in the same suspensions. In the tuba1 isthmus non-progressive motility (i.e., stationary spermatozoa with flagellar undulations) was more frequent after the acrosome reaction (table 2). Ampullar sperm with reacted acrosomes appeared to swim more slowly than intact spermatozoa, but 94% of acrosome reacted spermatozoa in the ampulla had progressive motility as compared with only 64%
o
o
o
m
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J. W. OVERSTREET AND G. W. COOPER
that were progressive in the lower isthmus (table 2). The experiments with delayed matings and those with artificial insemination (AI) of nonovulatory females provided evidence that neither ovulation-associated endocrine changes nor the products of ovulation are required for induction of the acrosome reaction in vivo. In the absence of ovulation only a few spermatozoa reach the tubal ampulla by 12 hours after A1 (Overstreet and Cooper, '79). Nevertheless, the proportion of motile, acrosome reacted spermatozoa in the isthmus and ampulla of these females did not differ from that found in ovulatory females 12 hours after natural mating or A1 (table 3). In delayed mating experiments, motile spermatozoa were recovered exclusively from the tubal isthmus four hours after fertile matings initiated 8 or 12 hours after sterile coitus (Overstreet and Cooper, '79). None of the 1,189 motile isthmic spermatozoa examined had undergone a n acrosome reaction, and all eggs recovered from the ampulla were unfertilized and remained in cumulus. DISCUSSION
We have observed that rabbit spermatozoa undergo visible acrosomal alterations which are compatible with sustained motility after they have resided for more than four hours in the female tract. Such spermatozoa were first seen six hours p.c. in the lower isthmus of the oviduct and were present in the ampulla after the onset of ovulation. Motile spermatozoa with acrosome reactions were not seen in the rabbit vagina, cervix or uterus during the first 16 hours P.c., but they were found in the uterus by 24 to 27 hours P.c., i.e., well after the completion of fertilization (Cooper and Overstreet, unpublished). These observations are consistent with the notion that completion of sperm capacitation must precede the "true" acrosome reaction (Bedford, '691, and that visible alterations of the head membranes of vigorously moving spermatozoa are physiologic acrosome reactions (Austin, '75). Motile, acrosome reacted spermatozoa have been recovered from the oviducts of several rodent species a t the time of fertilization (Austin and Bishop, '58; Yanagimachi and Mahi, '76) and their appearence in vitro is considered evidence that the incubation medium promotes capacitation and physiologic acrosome reactions (Meizel, '78). "True" acrosome reactions
ACROSOME REACTION DURING SPERM TRANSPORT IN RABBITS
101
TABLE 3
Proportion of motile oviductal spermatozoa with "reacted "acrosomes recovered 12 hours after a single natural mating (Group I ) or after artificial insemination (AIl of a single ejaculate. Group IIIfemales did not ovulate after AI. Group IIfemales were induced to ovulate prior to insemination by coitus with a vasectomized buck Median percentages of reacted motile spermatozoa per animal (range)' Region
Upper ampulla Lower ampulla Upper isthmus Lower isthmus
Group I (Natural mating)
Group I1 (Al, atenle coitus)
Group 111 (AI, no ovulation)
4
(0-33) 8 (0-21) 13 (0-25)
10 (5-24) 23 (22-38) 4 (0-57)
8 (2-28)
10 (0-33) 20 (0-67) 02 (0-3) 8
(1-2)
1 2
(0-12)
For each region sampled there were no significant differences among Groups I, 11, and 111 (Kruskal-WallisOne Way Analysia of Variance by Ranks, P > 0.10 in all cases). ' Median percentages (range)based on motile spermatozoa recovered from five females in Groupa I and 111, and three in Group 11. A total of 173 reacted motile spermatozoa were examined in Group I, 94 in Group I1 and 52 in Group 111. Mode
involve ordered membrane fusions and vesiculations between the plasmalemma and outer acrosomal membrane followed by loss of the acrosomal content. Only electron microscopy can distinguish this type of acrosome reaction from the uncoordinated, degenerative changes in the plasmalemma and outer acrosomal membrane, the so-called "false" acrosome reaction. Immotile rabbit spermatozoa with visibly altered acrosomes can be recovered from the female tract throughout the period of sperm transport, beginning as early as one minute p.c. (Overstreet and Cooper, '75; Overstreet et al., '78). These cells do not appear to be viable and it is probable that most have undergone false acrosome reactions. In view of the apparent requirement for enzyme mediated dispersal of the granulosa cell investment during the approach of spermatozoa to eggs (Bedford, '74), most investigators have assumed t h a t the acrosome reaction of the fertilizing spermatozoon must be induced a t or near the site of fertilization. Recently, it has been suggested that the integrity of the sperm plasmalemma may be critical for attachment to the zona pellucida prior to its penetration, thus necessitating initiation of the acrosome reaction a t or near the zona surface (Gwatkin et al., '76; Bedford and Cooper, '78). Both transmission (Yanagimachi and Noda, '70; Bedford, '72) and scanning electron micrographs (Gwatkin et al., '76) indicate that spermatozoa need not loose the vesiculated acrosomal cap until zona penetra-
tion has begun. There is as yet, however, no morphological or experimental evidence conclusively showing t h a t spermatozoa are infertile if they undergo a n acrosome reaction in the outer cumulus as suggested by Gwatkin et al. ('76). The granulosa cell investment of t h e rabbit egg is not essential for the induction of acrosome reactions, since eggs free of these cells have been fertilized in vivo (Harper, '70; Overstreet and Bedford, '74) and in vitro (Fraser et al., '711, and the treatment of denuded eggs with a variety of enzymes fails to limit rabbit sperm penetration through the zona pellucida in vivo (Overstreet and Bedford, '75). Our observations that motile spermatozoa with reacted acrosomes are present in the oviducts before ovulation and in the oviducts of non-ovulatory, estrous females after artificial insemination support t h e above experimental findings and suggest further that the specific environmental factors promoting t h e acrosome reaction in t h e oviduct are not controlled by ovulation-associated changes in circulating steroid levels or by any of the products of ovulation including follicular fluids. If initiation of the acrosome reaction in capacitated spermatozoa is accomplished by a specific stimulus, it appears that this may occur throughout the oviduct, and not a t the site of fertilization alone. Even if controlled release of acrosomal enzymes occurs within t h e cumulus mass in those spermatozoa first
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J. W. OVERSTREET AND G. W. COOPER
reaching the egg, the stimulus for this could still be distant from the egg. Depending on the time course of the biochemical and morphological events preceding release of the acrosome's enzyme content, initiation of the acrosome reaction could occur in the isthmus and not in the ampulla of the oviduct. Completion of the membrane fusion events of the rabbit sperm acrosome reaction in vitro requires a minimum of five to ten minutes (Singhas and Oliphant, '78) and it is likely that the initial biochemical events preceding visible morphological changes would also require some minutes (Meizel, '78). The final ascent of rabbit spermatozoa to the site of fertilization is synchronized with ovulation (Harper, '73; Overstreet et al., '78) and may require activation of sperm motility (Overstreet and Cooper, '79). Rodent spermatozoa which have undergone an acrosome reaction are usually activated (Yanagimachi, '701, and our observations suggest that a shift to activated motility may also accompany the acrosome reaction in rabbit spermatozoa (table 2). Since the passage of rabbit spermatozoa a t ovulation from the tubal isthmus to the ampulla takes place within 15 to 30 minutes (Harper, '73; Overstreet et al., '78), the acrosome reaction of ascending spermatozoa, if initiated in the lower isthmus, could reach completion in the vicinity of the eggs a t the ampullar isthmic junction. Most spermatozoa in the isthmus of the rabbit oviduct are located within 2 cm of the uterotubal junction (Overstreet et al., '781, and the majority appear closely associated with ciliated epithelial cells (Cooper and Overstreet, unpublished). The effect of these cell-cell interactions on the physiology of oviductal spermatozoa may include membrane changes resulting in activation of sperm motility and initiation of the biochemical events leading to the acrosome reaction. ACKNOWLEDGMENTS
We thank Doctor D. F. Katz for performing the statistical analysis of the data. This research was supported by NIH Grant HD09215 and a grant from the Ford Foundation. LITERATURE CITED Adams, C. E. 1962 Artificial insemination i n d e n t s . In: Semen of Animals and Artificial Insemination. J. P. Maule, ed. Commonwealth Agricultural Bureau, pp. 316-330. Austin, C. R. 1963 Acrosome loss from the rabbit spermatozoon in relation to entry into the egg. J. Reprod. Fertil., 6: 313-314.
1975 Membrane fusion events in fertilization. J. Reprod. Fertil., 44: 155-166. Austin, C. R., and M. W. H. Bishop 1958 Role of the rodent acrosome and perforatorium in fertilization. Proc. R. SOC. B., 14: 241-248. Bedford, J. M. 1967 Experimental requirement for capacitation and observations on ultrastructural changes in rabbit spermatozoa during fertilization. J. Reprod. Fertil. (Suppl.), 2: 35-48. 1968 Ultrastructural changes i n the sperm head during fertilization in t h e rabbit. Am. J. Anat., 123: 329-358. 1969 Morphological aspects of capacitation. Adv. Biosci., 4; 35-50. 1972 An electron microscopic study of sperm penetration into the rabbit egg after natural mating. Am. J. Anat., 133: 213-254. 1974 Mechanisms involved in penetration of spermatozoa through the vestments of the mammalian egg. In: Physiology and Genetics of Reproduction. Part B. E. M. Coutinho and F. Fuchs, eds. Plenum Publishing Corp., New York, pp. 55-68. 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 Press, Amsterdam, pp. 65-125. Chang, M. C. 1952 Fertilizability of rabbit ova and the effects of temperature in uitro on their subsequent fertilization and activation in uiuo. J. Exp. Zool., 121: 351-370. Cooper, G. W., J. W. Overstreet and D. F. Katz 1979 Sperm transport in the reproductive tract of the female rabbit. Sperm motility and patterns of movement at different levels of the tract. Gamete Res., in press. Fraser, L. R., P. V. Dandekar and R. A. Vaidya 1971 In uitro fertilization of tubal rabbit ova partially or totally denuded of follicular cells. Biol. Reprod., 4: 229-233. Gwatkin, R. B. L., H. W. Carter and H. Patterson 1976 Association of mammalian sperm with the cumulus cells and the zona pellucida studied by scanning electron microscopy. Proc. workshop SEM in Reproduction Biology, IIT Research Institute, Chicago, pp. 379-384. Harper, M. J. K. 1970 Factors influencing sperm penetration of rabbit eggs in uiuo. J. Exp. Zool., 173: 47-62. 1973 Stimulation of sperm movement from the isthmus to the site of fertilization in the rabbit oviduct. Biol. Reprod., 8: 369-377. Meizel, S. M. 1978 The mammalian sperm acrosome reaction, a biochemical approach. In: Development in Mammals. Vol. 3. M. H. Johnson, ed. Elsevier, North Holland Press, Amsterdam, pp. 1-64. Overstreet, J. W., and J. M. Bedford 1974 Comparison of the penetrability of the egg vestments i n follicular oocytes, unfertilized and fertilized ova of the rabbit. Dev. Biol., 41: 185-192. 1975 The penetrability of rabbit ova treated with enzymes or anti-progesterone antibody: Aprobe into the nature of a mammalian fertilizin. J. Reprod. Fertil., 44: 273-284. Overstreet, J. W., and G. W. Cooper 1975 Reduced sperm motility in the isthmus of the rabbit oviduct. Nature, 258: 718-719. 1978 Sperm transport in the reproductive tract of the female rabbit: I. The rapid transit phase of transport. Biol. Reprod., 19: 101-114. 1979 Effect of ovulation and sperm motility on the migration of rabbit spermatozoa to the site of fertilization. J. Reprod. Fertil., 55: 53-59. Overstreet, J. W., G . W. Cooper and D. F. Katz 1978 Sperm
ACROSOME REACTION DURING SPERM TRANSPORT IN RABBITS transport in th e reproductive tract of the female rabbit: 11. The sustained phase of transport. Biol. Reprod., 19: 115.132. Singhas, C. A., and G. Oliphant 1978 Ultrastructural observations of the time sequence of induction of acrosomal membrane alterations by ovarian follicular fluid. Fertil. Steril., 29: 194-203. Yanagimachi, R. 1970 The movement of golden hamster
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spermatozoa before and after capacitation. J. Reprod. Fertil., 23: 193-196. Yanagimachi, R., and C. A. Mahi 1976 Sperm acrosome reaction in the guinea pig: a study in uiuo. J. Reprod. Fertil., 46: 49-54. Yanagimachi, R., and Y. D. Noda 1970 Ultrastructural changes in the hamster sperm head during fertilization. J. Ultrastruc. Res., 31: 465-485.
