ULTRASTRUCTURAL OBSERVATIONS OF THE TIME SEQUENCE OF INDUCTION OF ACROSOMAL MEMBRANE ALTERATIONS BY OVARIAN FOLLICULAR FLUID*

Transcription

1 FERTILITY AND STERILITY Copyright ~ 1978 The American Fertility Society Vol. 29, No.2, February 1978 Printed in U.SA. ULTRASTRUCTURAL OBSERVATIONS OF THE TIME SEQUENCE OF INDUCTION OF ACROSOMAL MEMBRANE ALTERATIONS BY OVARIAN FOLLICULAR FLUID* CHARLES A. SINGHAS, PH.D.t GENE OLIPHANT, PH.D. Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Virginia School of Medicine, Charlottesville, Virginia Ultrastructural studies were made of rabbit sperm pretreated with high ionic strength medium and fixed after 0, 5, 10, 15, and 30 minutes of incubation in bovine follicular fluid (BFF) or control medium. Membrane changes associated with loss of the acrosomal contents were divided into three stages: membrane fusion; a vesiculation stage where approximately 50% of the contents of the acrosome were lost; and completion, where the outer acrosomal membrane and adjacent plasma membrane were lost. The rate of each step appeared to be independent of the subsequent phase, since no steady-state concentration was seen for any of the stages during the reaction. After 5 minutes' exposure to BFF, multiple fusions and vesiculations had occurred which involved the plasma and outer acrosomal membranes. After 10,15, and 30 minutes of incubation, increasing numbers of sperm showed fusion and the formation of vesicles. By 30 minutes, sperm showed vesicle formation and an additional 30% showed complete loss of vesicles and the acrosomal contents. The inner acrosomal membrane, nuclear membrane, and membranes posterior to the equatorial segment were unaffected by this treatment, as were all membranes after 30 minutes in control medium. The sequence of events indicated by these observations suggests that the BFF-induced acrosome reaction may be similar to that occurring in vivo to sperm in juxtaposition to ova. Sperm capacitation l 2 and the subsequent acrosome reaction 3 are required for successful fertilization of mammalian ova. In the rabbit, sperm must reside in the environment of the female reproductive tract for 6 to 12 hours' in order to become fully capacitated and must then initiate acrosomal breakdown in juxtaposition to the egg. Although the capacitating environment of the female tract apparently fails to alter the ultrastructural features of the spermatozoon,4. 5 it has been implicated in the removal or alteration of sperm-bound seminal and/or epididymal plasma components,6.7 suggesting that capacitation, through the removal of spermbound components, may promote membrane destabilization.5-7. Received July 5, 1977; revised August 31, 1977; accepted September 1, *Supported by United States Public Health Service Grant ROl-HD tto whom reprint requests should be addressed. 194 After capacitation, sperm undergo what has. been termed the acrosome reaction as they approach the outer investments of the egg. Ultrastructural studies have shown that during the acrosome reaction the plasma membrane and the outer acrosomal membrane covering the anterior aspect of the head undergo an orderly breakdown resulting in the formation of vesicles and fenestrations. 3 Membrane fenestration allows the release of lytic enzymes which apparently facilitate penetration of the zona pellucida and entry of the sperm into the ovum. Although Bedford4. 8 found no gross structural changes in rabbit sperm during capacitation, when these sperm were placed in fresh follicular fluid, acrosomal disruption occurred within 30 minutes. A similar effect when culture medium was used alone was not observed, indicating that follicular fluid contained sperm acrosome reaction-inducing activity (ARIA). Recently, Brackett and Oliphant 9 successfully capacitated '"

2 Vol. 29, No.2 TIME SEQUENCE OF ACROSOMAL CHANGES 195 rabbit sperm by pretreatment in a defined medium of high ionic strength. When sperm pretreated in this manner were incubated for 2 hours in vitro in the presence of bovine follicular fluid, the acrosome reaction readily occurred, whereas sperm not pretreated in high ionic strength medium underwent the acrosome reaction only very slowly.7 Sperm treated with high ionic strength medium but not with follicular fluid failed to shed the acrosomal caps. Since the in vitro incubation of sperm in high ionic strength medium appears to institute changes similar to capacitation in vivo, and since sperm capacitated in high ionic strength rapidly undergo an acrosome reaction, an ultrastructural examination of the time sequence of sperm pretreated in high ionic strength medium and incubated in the presence of follicular fluid was undertaken. This paper describes the sequence of events occurring to the plasma and outer acrosomal membranes using an in vitro system to determine the ultrastructural similarity or difference of the in vivo rabbit acrosome reaction described by others.lo MATERIALS AND METHODS Bovine ovaries were obtained from an abattoir and placed on ice. Within 2 to 3lh hours after removal of the ovaries, follicular fluid was aspirated from mature follicles, pooled, divided into 0.5-ml aliquots, and frozen at -20 C until use. Sperm Cultures. Ejaculated sperm were obtained from New Zealand White bucks of proven fertility by means of an artificial vagina. Individual ejaculates were examined for highly motile sperm, pooled, and placed in a constanttemperature bath at 37 C. Concentrations of sperm were determined by using a hemocytometer and adjusted with Cross-Brinster ll medium to a final concentration of 10 8 spermlml. High ionic strength (HIS) medium was prepared by adding 12 mg ofnaci to 1.0 ml ofcross-brinster medium. Sperm were preincubated in high ionic strength medium by mixing 1.0 ml of the sperm preparation with 1.0 ml of HIS medium to produce an environment of 380 mosm. After the 5-minute preincubation period at 37 C, a volume of 100ILI of the sperm mixture was transferred to the final reaction mixture consisting of 200 ILl of Cross-Brinster medium (255 to 270 mosm, ph 7.4) and 200 ILl of bovine follicular fluid. Control incubations consisted of replacing the ARIA with an equal volume of Cross-Brinster medium. Both experimental and control cultures were covered with sterile paraffin oil and maintained at 37 C. Aliquots of 0.1 ml were taken from both groups at 0, 5, 10, 15, and 30 minutes of incubation, gently transferred into size 00 Beem capsules containing fixative, and prepared for electron microscopy. At the termination of the experiment all cultures were examined for the presence of motile sperm; only those cultures having at least 90% highly motile sperm showing progressive movement were used for ultrastructural studies. Clumping was not observed when spermatozoa were mixed with follicular fluid. Electron Microscopy. Samples were fixed in 3% gluteraldehyde in M sodium phosphate buffer (ph 7.4) for 2 hours at room temperature and centrifuged at 600 x g for 20 minutes; the fixative was then carefully pipetted off. The samples were rinsed in phosphate buffer containing 10% (w/v) sucrose (420 mosm) and then postfixed for 2 hours in buffered 1% osmium tetroxide. 12 After washing for 30 minutes in cold 0.9% saline, the samples were dehydrated by using a graded series of acetone and embedded in Epon Thin sections were cut on a Reichert OM-12 ultramicrotome, stained with uranyl acetate and lead citrate, and examined on a Hitachi HU-lle-1 electron microscope operated at 75 kv. RESULTS Sperm exposed to either high ionic strength,medium alone or preincubated in high ionic strength medium followed by a 30-minute incubation in Cross-Brinster's medium were morphologically similar to freshly ejaculated sperm (Figs. 1 to 5). Figures 1 and 2 show the characteristic organization of the head region of an ejaculated rabbit spermatozoon. The acrosome is bound by an inner and outer acrosomal membrane extending from the equatorial segment anteriorly to enclose the acrosomal contents. The plasma membrane is loosely applied over the head except at the anterior margin, where it lies in close apposition to the outer acrosomal membrane. As controls, sperm incubated either in HIS medium for 5 minutes (Fig. 3) or after HIS pretreatment followed by 30 minutes of incubation in Cross-Brinster's medium (Fig. 4) were similar in appearance to normal ejaculated sperm and to sperm incubated for 30 minutes '" in Cross- Brinster's medium alone (Fig. 5). Breaks in the

3 196 SINGHAS AND OLIPHANT February 1978

4 Vol. 29, No.2 TIME SEQUENCE OF ACROSOMAL CHANGES 197 plasma membrane were occasionally observed but did not appear to be involved in loss of the acrosomal contents or to interfere substantially with the results seen in control media preparations. Exposure of HIS-pretreated sperm to media containing ovarian follicular fluid rapidly initiated changes in the acrosome region ofthe sperm. Table 1 shows the number of sperm undergoing acrosomal changes at each time interval (0, 5, 10, 15, 30 minutes) expressed in terms of the nature of the acrosomal change, i.e., fusion, vesicle formation, completion of the reaction as indicated by shedding of the acrosomal cap, or no change. Fusion involving the plasma and outer acrosomal membranes of HIS-pretreated sperm occurred after 5 minutes' exposure to the medium containing follicular fluid. Typically, the points of fusion between these two membranes were first observed over the anterior aspect of the head, where both membranes are closely apposed (Fig. 6), rather than posteriorly where the plasma membrane is more loosely applied. The precise nature of this fusion remains unclear; whether the plasma membrane breaks prior to fusing with the outer acrosomal membrane or whether both membranes fuse and break simultaneously remains unanswered in the present investigation. It appears, however, that fusion, defined as the physical contact between the two membrane systems, occurs prior to vesiculation. Vesicles formed from plasma and outer acrosomal membrane material were visible in some sections as early as 5 minutes after the start of incubation. The vesicles themselves seemed to involve a larger area of the membranes than the early points of fusion (Fig. 7). This observation suggests the possibility that fusion points may form, break down, and re-form, resulting in the appearance of vesicles larger than would normally be expected from the distance between the early fusion points. Between 10 and 15 minutes in follicular fluid, progressive changes occurred involving fusion and vesiculation of the membranes overlying the acrosome in an increasing number of HISpretreated sperm. Remnants of membranous vesicles appeared in close proximity to the sperm head, and the acrosomal contents were progressively lost (Fig. 8). By 15 and 30 minutes, some sperm had completely shed the acrosome (Fig. 9). The inner acrosomal membrane remained intact, showing traces of electron-opaque acrosomal material bound to it, and the perforatorium was retained. By 30 minutes of incubation, 37/100 sperm counted showed varying degrees of vesicle formation, while an additional 30% had completely shed the acrosome. Acrosomal loss in samples treated for 30 minutes in medium containing follicular fluid appeared to involve only the vesiculation of the plasma and outer acrosomal membranes, with the inner membrane, including the perforatorium, being trained. Similarly, membranes posterior to the equatorial segment remained intact and did not appear to be extensively involved in fusion or vesiculation. Posterior remnants of the plasma membrane were secured about the equatorial segment by fusion to the outer acrosomal membrane. Occasional vesicles were observed posterior to the equatorial segment, but were probably derived from displacement during sample preparation. When the number of sperm at the fusion stage or having completed it was plotted as a function of time (Fig. 10, top) the plot showed a relatively linear increase-up to 87% of sperm reaching this stage in 30 minutes. This corresponds to a rate of 4 x 105 sperm attaining the fusion stage/minute. Likewise, Figure 10, bottom, shows the results for sperm attaining the vesiculation stage. The initial rate for attaining this phase was 3.5 x 10 5 sperm/minute in the last 20 minutes of incubation. The percentages of sperm at specific phases during each period are shown in Figure 11. FIG. 1. Longitudinal section of a freshly ejaculated rabbit spermatozoon. The acrosome and its associated membranes are intact, as is the plasma membrane. The nucleus is covered anteriorly by the acrosome (AR), which is secured by the inner (lam) and outer (OAM) acrosomal membranes. The plasma membrane (PM) is continuous over the surface of the sperm. Arrows indicate the equatorial segment (x26,000). FIG. 2. Cross-section through the anterior aspect of the head of a normal ejaculated spermatozoon showing the relationship between the inner acrosomal, outer acrosomal, and plasma membranes. The perforatorium (subacrosomal space) is indicated by arrows (x26,800). FIG. 3. Spermatozoon pretreated for 5 minutes in HIS medium prior to fixatior'. Sperm prepared after this treatment showed no evidence of membrane fusion and vesiculation, nor loss of acrosomal contents (x 12,900).

5 198 SINGHAS AND OLIPHANT February 1978 FIG. 4. Section through a spermatozoon pretreated for 5 minutes in HIS medium followed by incubation for 30 minutes in CroBB-Brinster medium (control). Plasma and outer acrosomal membranes remain intact and the acro80mal contents are retained (x 18,250). FIG. 5. Ejaculated rabbit spermatozoon pretreated for 30 minutes in CroBB-BrinBter medium (without HIS pretreatment). No evidence for acrosomal membrane alteration is observed (x25,000)..i

6 Vol. 29, No.2 TIME SEQUENCE OF ACROSOMAL CHANGES 199 Treatment(1 TABLE 1. Effect of HIS Treatment and Exposure to an ARIA on Ultrastructural Changes in the Rabbit Sperm Acrosome Acrosomal change b Total no. counted Intact Fusion V. It' Reaction eslcu a Ion completed HIS alone O min FF min FF min FF min FF min CB min EJ aff, Bovine follicular fluid (ARIA); CB, Cross-Brinster's medium; EJ, ejaculated sperm (incubated in seminal plasma). bnumber of sperm. Increasing percentages of sperm reached stages of fusion and vesiculation throughout the 30- minute period. In contrast, the completion of the acrosome reaction commenced at an initial rate of 2 x 105 sperm/minute and then rapidly leveled off to a rate of 0.5 x 10 5 sperm/minute. DISCUSSION No previous description has been made of the time sequence of the rabbit acrosome reaction induced in vitro under conditions approaching physiologic. The present work indicates (1) that sperm pretreated in hypertonic medium and then treated with ovarian follicular fluid readily undergo ultrastructural changes approaching those of the "true" acrosome reaction; (2) that the changes are initiated immediately upon introduction of the sperm into the follicular fluid milieu; (3) that pretreatment of sperm with a hypertonic medium similar to that previously shown to have the ability to capacitate rabbit spermatozoa has no observable effect on spermatozoon ultrastructure. It has been suggested that capacitation may promote destabilization of the sperm plasma membrane, outer acrosomal membrane, or both, and cause increased susceptibility for the sperm to undergo the acrosome reaction. Recently, successful in vitro capacitation of rabbit 9 and mouse spermatozoa has been achieved by using a defined medium of high ionic strength to pretreat the spermatozoa. 6 9 In these experiments, the time required for fertilization of ova was delayed for HIS-treated sperm. Furthermore, a radioimmunoassay for quanti tat ion of spermbound seminal plasma antigens indicated that these antigens were rapidly removed after HIS treatment of the sperm. Using the same medium, Koehler 14 observed a decrease of hemocyaninlabeled rabbit anti-rabbit sperm globulin in the plasma membrane over the acrosomal region after 30 minutes of incubation, and also concluded that capacitating incubations may remove sperm surface (glycocalyx) material. The work presented in this paper further indicates that treatment of sperm in high ionic strength medium does not by itself induce any obvious ultrastructural changes in the sperm. The follicular fluid induction of a physiologic rabbit sperm acrosome reaction is implied by the positive results obtained by in vitro fertilization of rabbit ova in the presence of ovarian follicular fluid.7 15 The requirement of follicular fluid in this process was suggested when ova denuded of cumulus and corona cells were readily fertilized in vitro in the presence of follicular fluid, but ova remained unpenetrated in the absence of follicular fluid. 7 This laboratory has further indicated by light microscopy that a rapid loss of the rabbit sperm acrosome occurs upon pretreatment of either ejaculated or epididymal sperm with a high ionic strength medium followed by treatment with ovarian follicular fluid. Rabbit sperm capacitated in high ionic strength medium also appear to undergo the acrosome reaction in vitro during exposure to follicular fluid from various species. 16 The nature of the acrosomal changes observed in the present work appears similar to the "true" acrosome reaction described by Bedford5 for sperm penetrating ova in vivo. Initially, fusion between the plasma and outer acrosomal membrane occurs in the region anterior to the equatorial segment, while the plasma membrane at the equatorial segment and more posteriorly remains unaffected. The present study indicates that the fusion process begins virtually immediately upon the introduction of the spermatozoa into the follicular fluid and proceeds in probably a linear manner for 30 minutes of incubation. This finding, however, indicates that the fusion process is not rate-limiting to vesiculation or acrosomal shedding, since the number of sperm at the fusion stage increases and does not assume a steady-state concentration. The rate of sperm attaining fusion after between 5 and 15 minutes of incubation is 4 x 105 sperm! minute. After 30 minutes of )ncubation 87% of the sperm have initiated the fusion phase of the reaction.

7 ) I > 200 SINGHAS AND OLIPHANT February 1978 FIG. 6. Tangential section of a sperm head showing early fusion and membrane breakage (arrows). Vesiculation has apparently not yet occurred. Some lytic enzymes from the acrosome may be released at this stage (x98,180). FIG. 7. Early vesiculation involving the plasma and outer acrosomal membranes. Sperm at this stage of the acrosome reaction were seen as early as 5 minutes after exposure to an ARIA following capacitation (x 112,000). j

8 Vol. 29, No.2 TIME SEQUENCE OF ACROSOMAL CHANGES 201,,~ ". FIG. 8. Thin sectiqn of a spermatozoon showing a late stage ofthe acrosome reaction. Prominent vesicles and almost complete loss of the acrosomal contents are visible, as is an intact inner acrosomal membrane (x27,600). FIG. 9. Terminal stage of the acrosome reaction in vitro involves the loss ofmembranoub vesicles and the acrosomal contents. The inner acrosomal membrane, however, remains continuous; remnants of the plasma and outer acrosomal membranes have fused about the equatorial -segment (arrow) (x25,750).

9 " 202 SINGHAS AND OLIPHANT February 1978 c.2 -u Fusion Ii: ~ II) e u c -0 f m.: c -c E... Vesicula Time (min) FIG. 10. Percentages of sperm attaining the phases of the acrosome reaction. Top, Fusion-indicates all sperm presently at or beyond the fusion stage; bottom, vesiculation-indicates all sperm presently at or beyond this phase. Experimental details are given under "Materials and Methods." " CD 40 Fusion 0 Vesicula. II " f c.2.. u II ~ 20 QI E 0 " 0 0 U C i E CD Co UJ & U) '#- 0 te- 20 Complete 30 Time (min) FIG. 11. Percentages of sperm. at the acrosome reaction phases. Only the numbers of sperm present at the various phases of the reaction at the times observed are indicated. Experimental details are given under "Materials and Methods." The formation of membrane-bound vesicles occurred concurrently with the formation of fenestrations and loss of the acrosomal filubstance. Again, this step does not limit the rate at which the reaction is completed, since the number of sperm at the vesicle stage increased continually throughout the 30-minute incubation. This process does show a lag period of several minutes (only 3% in the vesiculation stage in 5 minutes), reflecting the requirement for a certain amount of fusion to have occurred before a large loss of the acrosomal contents ensues. The vesiculation phase appears to proceed at a rate of 3.5 x 105 sperm/minute between 5 and 15 minutes of incubation, and decreases to 1.8 x 10 5 sperm! minute between 15 and 30 minutes of incubation. Finally, completion of the reaction as judged by the total loss of the outer acrosomal membrane and adjacent plasma membrane as well as the contents of the acrosome lagged behind the other phases of the reaction. The earliest time at which complete acrosomal loss occurred was between 5 and 10 minutes of incubation with an initial rate of 2 x 10 5 sperm showing acrosomal loss/ minute. This rate rapidly leveled off to 5 x 10 4 sperm showing acrosomal loss/minute. This is in reasonable agreement with the rate observed for rabbit sperm16 by light microscopic observation. After 1 hour under the same conditions used in this work, Oliphant et al.16 observed a rate of 4 x 10 4 sperm acrosome reaction completed! minute. This is, however, in marked contrast to the extremely rapid acrosome reaction of the guinea pig (approximately 6 x 105 sperm!minute) observed after preincubation for 24 hours in a calcium-free medium17 or the very slow reaction (only a "small number" in 8 hours) of human sperm, placed directly in follicular fluid. 1s Aside from the obvious morphologic differences in the various phases of the reaction, the rates at which they occur suggest reaction mechanisms. The fusion process is certainly the membrane phenomenon normally associated with the acrosome reaction in vivo; the stage described in this work as vesiculation may reflect the rate of release of the acrosomal contents, i.e., the dissociation of the material within the acrosome. The complete loss of the membranes likely represents the simple rate of dissociation of the vesicles away from the sperm surface. During these studies, occasionally ( < 1%) sperm undergoing the "false" acrosome reaction were observed. The "false" acrosome r.eaction did not involve fusion between the plasma membrane 1 I

10 Vol. 29, No.2 TIME SEQUENCE OF ACROSOMAL CHANGES 203 and outer acrosomal membrane, and vesicles formed by the breakdown of the plasma membrane surrounded the outer acrosomal membrane. The outer acrosomal membrane itself appeared to be lost through a process of random breakage. The cause of the "false" acrosome reaction in spermatozoa is unclear, but may reflect dead or dying sperm in the process of degeneration. A role of follicular fluid may be that of altering some of the properties of the plasma membrane and/or outer acrosomal membrane following removal of protein components of seminal or epididymal plasma during capacitation; these changes may involve a redistribution of membrane components or increased permeability of the membranes to calcium ions. It has been shown that the acrosome reaction in guinea pig17 and rabbit16 spermatozoa is dependent on the presence of calcium ions. Furthermore, the treatment of guinea pig sperm with detergents, such as Hyamine, resulted in an immediate acrosome reaction which was blocked when calcium was omitted from the medium.19 However, the mechanism by which the membranes are altered in these two systems is not resolved. Although follicular fluid is required in the rabbit system (the defined medium alone has little effect), only a very simple, defined medium is needed for the guinea pig acrosome reaction. It is envisioned that, after ovulation, follicular fluid, entrapped in the matrix of cumulus cells, is carried into the fallopian tube. There it may provide the stimulus to induce the acrosome reaction as the sperm comes in close proximity to the egg-cumulus cell mass. This work provides ultrastructural evidence that the sequence of alteration induced by follicular fluid in vitro is similar in many respects to the "true" in vivo induced acrosome reaction. REFERENCES 1. Austin C: Observations on the penetration of the sperm into the mammalian egg. Aust J Sci Res [Ser B] 4:581, Chang M: Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 168:697, Barros C, Bedford J, Franklin L, Austin C: Membrane vesiculation as a feature of the mammalian acrosome reaction. J Cell BioI 34:C1, Bedford J: Morphological aspects of capacitation. In Advances in Biosciences. A Shering Symposium on Mechanisms Involved in Contraception. New York, Pergamon Press, 1969, p Bedford J: Sperm capacitation and fertilization in mammals. BioI Reprod [Suppl] 2:128, Oliphant G, Brackett B: Immunological assessment of surface changes of rabbit sperm undergoing capacitation. BioI Reprod 9:404, Oliphant G: Removal of sperm-bound seminal plasma components as a prerequisite to induction of the rabbit acrosome reaction. Fertil Steril 27:28, Bedford J: Experimental requirement for capacitation and observations on ultrastructural changes in rabbit spermatozoa during fertilization. J Reprod Fertil [Suppl] 2:35, Brackett B, Oliphant G: Capacitation of rabbit spermatozoa in vitro. BioI Reprod 12:260, Hadek R: Submicroscopic changes in the penetrating spermatozoon of the rabbit. J Ultrastruct Res 8:161, Cross P, Brinster R: In vitro development of mouse oocytes. BioI Reprod 3:298, Millong G: Further observations on a phosphate buffer for osmium solutions in fixations. In Proceedings of the Congress of Electron Microscopy, Vol 2, Fifth Edition. New York, Academic Press, 1962, p Luft J: Improvements in epoxy resin embedding. J Biophys Biochem Cytol 9:409, Koehler J: Changes in anti-rabbit sperm antibody labelling patterns following "capacitation" (abstr 438). J Cell BioI 67:219a, Rosado A, Hicks J, Reyes A, Blanco I: Capacitation in vitro of rabbit spermatozoa with cyclic adenosine monophosphate and human follicular fluid. Fertil Steril 25:821, Oliphant G, Cabot C, Singhas C: The nature of the rabbit sperm acrosome reaction inducing activity from follicular fluid. J Reprod Fertil 50:245, Yanagimachi R, U sui N: Calcium dependence of the acrosome reaction and activation of guinea pig spermatozoa. Exp Cell Res 89:161, Roomans G, Afzelius R: Acrosome vesiculation in human sperm. J Submicrosc Cytol 7:61, Yanagimachi R: Acceleration of the acrosome reaction and activation of guinea pig spermatozoa by detergents and other reagents. BioI Reprod 13:519, 1975