A scanning electron microscopic study of rabbit spermatozoa in the female reproductive tract following coitus

Cell Tiss. Res. 163, 29--44 (1975) �9 by Springer-Verlag 1975

A Scanning Electron Microscopic Study of Rabbit Spermatozoa

in the Female Reproductive Tract Following Coitus *

Pietro Motta

Department of Anatomy, Faculty of Medicine, University of Rome, Rome, Italy

J o n a t h a n Van Blerkom

B. F. Stolinsky Laboratories, Department of Pediatrics, University of Colorado School of Medicine, Denver, Colorado 80220, U.S.A.

Received May 9, 1975

Summary. The surface morphology of rabbit spermatozoa, fixed in situ (female reproductive tract) and prepared for scanning electron microscopy by critical point drying, was studied for as many as 36 hours post coitum. The findings demonstrate that 1) spermatozoa in the reproductive tract following coitus exist as a heterogenous, morphological population and 2) with time, shifts within this population from one predominant morphology to another take place.

In the fresh ejaculate, most spermatozoa have intact surfaces free of membranous disruptions. With time, a process of labilization (denudation) of the membranes covering the acrosomal region occurs in a progressively larger proportion of spermatozoa. The labilization originates by a process of vesiculation and/or vacuolation and leads to the appearance of a series of small fenestrations or perforations of the surface membranes. The perforations coalesce, and gradually larger areas of the surface membranes are eroded such that by 15 hours post coitum, the outer acrosomal membrane, as well as other acrosomal areas, are to varying degrees, directly exposed to the uterine milieu.

Secretory granules, picked up by cilia and transferred to the spermatozoa become localized over the acrosomal region shortly after coitus. The possible significance of these time-dependent, morphological events with the phenomena of capacitation and the "true" and "false" acrosome reactions are discussed.

Key words: Spermatozoa --- Capacitation - - Uterus - - Acrosome Reaction - - Scanning electron microscopy.

Introduct ion

Following ejaculation, the spermatozoa of several mammals are required to reside in the female reproductive t rac t for a period of t ime before they are com- pe tent to penet ra te and fertilize an egg. This process, described independent ly by Aus t in (1951) and Chang (1951), has been termed capacitation by Aus t in (1952). As a process, capaci ta t ion is thought to proceed and to be dis t inct from changes occurring at the acrosomal region of the spermatozoon when in the immedia te v ic in i ty of an ovulated egg (Barros et al., 1967; Aust in, 1968; Bedford, 1970; Zamboni , 1971; Bemste in and Teichman, 1972; Soupart , 1972; Williams, 1972).

Send o//print requests to: Dr. J. Van Blerkom, B.F. Stolinsky Laboratories, Department of Pediatrics, University of Colorado School of Medicine, Denver, Colorado 80220 U.S.A.

* This investigation was partially supported by NIH grant HD-04274 and by a special fund of the Italian Foreign Office and was made in the Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado. We are indebted to Professor Keith R. Porter for his generosity in making laboratory facilities available.

30 P. Motta and J. Van Blerkom

These acrosomal changes, collectively termed the acrosome reaction, are thought to be initiated by factors present in the egg and/or corona cells and to consist primarily of a vesiculation of the apposing acrosomal and plasma membranes, with the consequent liberation of acrosomal enzymes (Austin, 1968; Barros et al., 1967; Bedford, 1968). The acrosome reaction is a necessary prerequisite to spermatozoa penetration and egg fertilization.

The term "fa lse" acrosome reaction was introduced by Bedford (1970) to describe the changes in the acrosome that are usually seen in testicular, epididymal, ejaculated, and capacitated spermatozoa as a feature of degenerative breakdown. I t has been suggested tha t the " fa lse" acrosome reaction is quite different from the " t r u e " acrosome reaction which takes place only around the ova (Bedford, 1970). Other investigators consider both of these processes to be morphologically indistinguishable (Zamboni, 1971; 1972).

Numerous studies by transmission electron microscopy (TEM) of isolated spermatozoa have failed to detect obvious differences between capacitated and uncapacitated spermatozoa insofar as general morphology and "proport ion of organelles" is concerned (Bedford, 1969, 1970; Zaneveld et al., 1971; McRorie and Williams, 1974). Recent studies by scanning electron microscopy (SEM) of mature spermatozoa, obtained primarily by centrifugation and prepared either by air drying (Fujita et al., 1970; Yanagimachi and Nods, 1972), freeze drying (Zaneveld et al., 1971), or critical point drying (Lung and Bahr, 1972), have appeared in the literature. In relating capacitation with images of sperm obtained by SEM, the results of Zaneveld et al. (1971) indicate tha t no "visible differences can be detected between capacitated and ejaculated rabbit spermatozoa."

Capacitation is a time-dependent phenomenon which takes place in the female reproductive tract. Therefore, in order to relate possibly subtle morphological events occurring on the surface of spermatozoa with the process of capacitation, we thought it essential to examine a large population of ejaculated spermatozoa while they were both undisturbed and situated in their natural loca t ion- - the female reproductive tract. Specimens were prepared for SEM by critical point drying, because this procedure, in comparison to other preparative techniques, subjects fragile material to a minimum of disruptive surface tension forces (Porter et al., 1972) and produces images of superior detail and preservation (Motta et al.,

1975).

Materials and Methods Capacitated rabbit spermatozoa can be recovered from the reproductive tract after as few

as 4 to 6 hours (Soupart, 1972) or as many as 18 hours post coitum (Bedford, 1970; Seitz et al., 1970). The most successful rates of in vitro fertilization have been obtained with spermatozoa recovered from the reproductive tract between 16 and 18 hours post coitum (Seitz et at., 1970). In the present study, the surface morphology of spermatozoa, fixed in situ, was examined at the following times after coitus: 1, 10, and 30 minutes, and 31/2, 9, 15, 24, and 36 hours.

The genital tracts of 16 mature, Dutch-Belted rabbits were placed in Karnovsky's formaldehyde-glutaraldehyde fixative (Karnovsky, 1965) at the times post coitum indicated above. Different portions of the reproductive tract (vagina, uterus, oviduct, ovary) were then excised and immersed in fresh fixative for a period of approximately 24 hours (Motta and Porter, 1974; Motta and Van Blerkom, 1975). After fixation, the tissues were gently rinsed in 0.1 M sodium cacodylate (pH 7.4) for 11/2 hours, cut into small blocks, dehydrated through a graded series of acetone, and finally critical point dried in liquid C02 (Porter et al., 1972). The dried

S.E.M. of Rabbit Spermatozoa Following Coitus 31

samples were mounted on aluminum studs by means of conductive silver paint and then coated with a thin layer of carbon and gold in a high vacuum evaporator (DV-502, Denton Vacuum) under continuous rotation and with the appropriate tilt of the stud. All specimens were examined and photographed in a JEOL Model 35 scanning electron microscope operated at 25 kV. Approximately 2 000 spermatozoa were examined in this study, and the surface features of about 600 were recorded photographically.

Results

Sur/ace Morphology o/ Spermatozoa in the Vagina at 1, 10, and 30 Minutes post eoitum

Several distinct morphologies were displayed by spermatozoa in the fresh ejaculate (1 minute post eoitum). Typically, the paddle-shaped head of most sperm was quite smooth and free of granules, membranous disruptions, and/or irregularities (Fig. 1). At 10 minutes and 30 minutes post coitum, some spermatozoa were covered to varying degrees with a granular material that was distributed over both head and tail regions in an apparently random fashion (Fig. 2). (I t is also evident from Fig. 2 that spermatozoa in the same cluster are devoid of granules.) Occasionally, spermatozoa which exhibited a disruption, possibly a vacuolation and/or vesiculation of membranes covering the acrosome were observed. Fig. 3 illustrates an example of a spermatozoon in which extensive disruption has taken place. For the first few hours following copulation, spermatozoa of this type were comparatively rare.

Sur/ace Morphology o/Spermatozoa in the Uterus at 31/2 Hours post coitum Spermatozoa observed in the uterus at 31/2 hours post coitum were generally

found in association with ciliated cells (Fig. 4). This association was evident not only in the endocervix (where ciliated cells are more abundant) but also in areas of the uterus containing a reduced population of ciliated cells. The most typical relationship between spermatozoa and ciliated cells resulted in the spermatozoon head being oriented towards the ceils (Fig. 4); in many cases, the entire head region was masked by cilia such that at low magnification, only spermatozoa tails were easily distinguishable.

At higher magnification, it was apparent that the same granular material present on the head region of most spermatozoa was also present on the cilia (Figs. 5-7). A thin filamentous material (Figs. 5-7) could be recognized on the acrosomal surface. In some areas this filamentous substance seemed to inter- connect adjacent granules (Fig. 5) and may represent a residual cell coat ("glyco- calix"). Observations of numerous interactions between spermatozoa and cilia suggested that the granular material was originally localized to the ciliated cells and, through the beating and brushing action of the cilia, was gradually transferred to and distributed over the head region of the spermatozoa (Figs. 6, 7). Many spermatozoa, especially in regions of the uterus containing few ciliated cells, were nevertheless covered with granules (in the head region), suggesting an earlier encounter with ciliated cells.

The outer membrane of most spermatozoa examined at this t ime was intact and free of disruptions (Fig. 6). However, some spermatozoa did contain regions in which a relatively mild disruption (perforation) of the outer membrane had

32 :P. Motta and J. Van Blerkom


occurred (Figs. 8, 9). Characteristically, this disruption was limited to the membranes overlying the acrosomal region. Fig. 9 illustrates the appearance of a spermatozoon with an intact plasma membrane (the predominant type) as well as a spermatozoon in which a slight degree of disruption (perforation) has taken place. Spermatozoa with extensive membrane disruption (as presented in Fig. 3) were observed infrequently.

Sur/aee Morphology o/Spermatozoa in the Uterus and on the Ovary at 9 Hours post coitum

The surface morphology of spermatozoa in the uterus at 9 hours post coitum is shown in Figs. 10-13. Whereas the plasma membrane of many spermatozoa was still intact (Fig. 13) in a greater proportion of the spermatozoa than observed previously, the membranes overlying the acrosomal region showed varying degrees of disruption. Fig. 10 illustrates a comparat ively mild level of disruption, whereas Figs. 11 and 12 demonstrate the results of more extensive perforation. Typically, areas of disruption (fenestration) were restricted to the surface membranes covering the acrosomal region and appeared to arise from the coalescence of small fenestrations. The small fenestrations seemed to originate primarily at the equatorial segment through a process of vesiculation of the plasma membrane, and with time, these fenestrations coalesced and gradually eroded the plasma membrane and possibly, the outer acrosomal membrane as well (covering the acrosomal cap) (Figs. 10-12). The erosion of the surface membranes is apparently limited to the acrosomal region since no signs of disruption were observed in areas posterior to the acrosome or on the membranous coverings of the tail (Figs. 10-13).

A small but significant percent of the sperm observed at this t ime had undisrupted surfaces (Fig. 13). However, all spermatozoa in this category which were examined contained surface granules restricted to the acrosomal region (Fig. 13). At higher magnification, these particular spermatozoa did have a slight perforation of the plasma membrane, but only in areas directly under the granules (Fig. 13). The association of spermatozoa with ciliated ceils described for 31/2 hours post coitum remained unchanged at 9 hours post coitum.

A large number of spermatozoa in proximity to ovulated eggs, in association with corona cells, or in regions near the infundibulum were observed at 9 hours post eoitum (Motta and Van Blerkom, 1975). Spermatozoa in contact with corona cells were presumably involved in the process of fertilization. The surface

Fig. 1. In the fresh ejaculate, most spermatozoa examined had a smooth surface free of membranous disruptions (vagina, 1 minute post coitum). • 9300

Fig. 2. Between 10 and 30 minutes post coitum, some spermatozoa were covered to varying degrees with a granular material that was distributed over both head and tail regions in an apparently random fashion. In addition, many spermatozoa were completely devoid of granules

(vagina, 10 minutes post coitum). • 3700

Fig. 3. Occasionally, spermatozoa that exhibited a disruption of the surface membranes covering the acrosomal area were observed. This micrograph illustrates an example of a spermatozoon in which extensive disruption had taken place (arrows). Spermatozoa of this type

were comparatively rare at this time (30 minutes post coitum). • 7200

3 Cell Tiss. Res.



Figs. 6 and 7. These micrographs illustrate the association between cilia and spermatozoa in the uterus at 31/2 hours post coitum. At higher magnifications, it appeared likely that the granules were transferred to the acrosomal region of the head by the brushing and beating action of the cilia (arrows). A thin filamentous material (residual glycocalyx ?) appeared

randomly distributed over the head's surface. X15000; x20000

Figs. 8 and 9. Although most spermatozoa have intact, undisrupted surfaces at 31/2 hours post coitum, some spermatozoa did contain regions in which a relatively mild disruption of the surface membranes overlying the acrosomal area had taken place(*). Cilia (C). Fig. 9 demonstrates the appearance of a spermatozzoon in which a slight degree of perforation had occurred (*) as well as a spermatozoon with an intact surface (the predominant type)

x 8000; x 6000

Fig. 4. At 31/2 hours post coitum, most spermatozoa examined in the uterus were in association with ciliated cells of the endometrium. Cilia (C) ; Secretory Cells (S). X 4200

Fig. 5. Numerous granules were observed on the plasma membrane covering the acrosomal cap (A). These granules appeared to be transferred from cilia surrounding the spermatozoa (C). The post acrosomal region of the head (arrows) and the entire tail section were not covered with granules. Thin strands of a filamentous material (residual glycocalyx ?) were also evident

in the granulated region of the acrosome, x 12800


Figs. 10--13. The typical appearance of spermatozoa in the uterus at 9 hours post coitum is shown in this series of micrographs. In a greater proportion of spermatozoa than observed previously, the membranes overlying the acrosomal area showed varying degrees of disruption. Fig. 10 illustrates a comparatively mild level of disruption whereas figures 11 and 12 demonstrate the results of more extensive perforation (arrows). Spermatozoa in which the overlying membranes had eroded have their outer acrosomal membrane and discrete zones of the acrosome directly exposed to the uterine milieu (arrows, *). A small, but significant percent of the spermatozoa observed at this time had intact surfaces (Fig. 13). In all cases, the tail (T) and post acrosomal regions (PA) were free of disruption. • • 11500, •

• 10000

characteristics of these spermatozoa were identical to the features of sperm tha t were still in the uterus at 9 hours post coitum (illustrated in Figs. 10-13). The surface membranes overlying the acrosomal region displayed several morphologies from being undis tu rbed (but granulated) to expressing varying degrees of denuda t ion ; no one surface morphology predominated.


Sur/ace Morphology o/Spermatozoa in the Uterus and on the Ovary at 15 Hours Post Coitum

In most spermatozoa observed at 15 hours post coitum, the surface membranes covering the acrosome were extensively eroded (Figs. 14, 15). This erosion resulted from the coalescence of small fenestrations that seemed to originate in the equatorial segment. As a consequence of this process, large areas of the surface membranes appeared to be degenerating (Fig. 14) and/or exfoliating (Fig. 15). In spermatozoa in which advanced erosion of the plasma membrane had taken place, the underlying acrosomal membrane, and possibly the acrosome itself, were directly exposed to the uterine milieu (Fig. 14). Many spermatozoa were still observed in contact with ciliated cells, and both the head region of the spermatozoa and the cilia were coated with granules (Fig. 16). Spermatozoa with comparatively intact membranes were rarely encountered at this time (Fig. 17). Occasionally, some uterine spermatozoa were associated with leukocytes (Fig. 18) or were in close proximity to them (Fig. 19); most of these spermatozoa were degenerating.

Numerous spermatozoa were found on the ovary (especially around the stigma of ovulated follicles) at 15 hours post coitum. Whereas some of these spermatozoa were in advanced stages of complete degeneration (Fig. 20), most displayed an extensive erosion of the membranes overlying the acrosomal area (Fig. 21). Since rabbits ovulate approximately 12 hours post coitum (Austin and Braden, 1954), it is unlikely that these spermatozoa were directly involved in fertilization.

Sur/ace Morphology o/Spermatozoa in the Uterus and Oviduct at 24 Hours Post Coitum

In the majority of remove spermatozoa examined at 24 hours post coitum, the perforation of the membranes covering the acrosomc and the denudation of the acrosomal cap were complete (Figs. 22, 23). Only rarely were spermatozoa with relatively undisturbed surfaces observed (Fig. 24). Where ciliated cells were present, spermatozoa still showed a strict association with these elements (Fig. 24).

Surface Morphology o/ Spermatozoa in the Uterus and Oviduct at 36 Hours Post Coitum

Compared to the population of spermatozoa previously available for study, only a reduced number of spermatozoa were observed at 36 hours post coitum. Generally, these spermatozoa were in advanced stages of complete degeneration (Fig. 25), with only a very small portion having the vesiculated-granulated appearance typical of spermatozoa between 15 and 24 hours post coitum.

Discussion

The results obtained in the present study, derived from a detailed examination of approximately 2000 spermatozoa, indicate that several surface morphologies are displayed by spermatozoa both in the fresh ejaculate and during the first 36 hours post eoitum. Due to the enormous number of spermatozoa present in the reproductive tract following coitus, a statistical analysis of the different surface morphologies was not undertaken. However, because a relatively sizeable population of spermatozoa was examined, it is felt that the impressions obtained


Figs. 14--17. This series of micrographs illustrates the appearance of spermatozoa in the uterus at 15 hours post coitum. In most spermatozoa examined at this time, the membranes covering the acrosomal region were extensively eroded owing to the coalescence of small fenestrations that possibly originated in the equatorial segment (Fig. 14, arrow ES). As a consequence of this process, large areas of the plasma membrane were degenerating (Fig. 14) and/or exfoliating (arrow, Fig. 15). Spermatozoa were still observed in contact with ciliated cells containing granules (G, arrows, Fig. 16). Spermatozoa with comparatively intact surfaces

were rarely encountered at this time (Fig. 17). x9000; • • •

f rom such an examina t ion are both signif icant and represen ta t ive of the popula t ion of spermatozoa presen t in the female reproduc t ive t r a c t for as m a n y as 36 hours post coitum.

I n the fresh e jacula te (up to 10 minutes post coitum), near ly every spe rmatozoon examined had a re la t ive ly smooth surface free of membranous disrupt ions . I t should be noted, however, t h a t even in the fresh e jacula te , some spermatozoa dis- p l a y e d vary ing degrees of per fora t ion of the surface membranes over lying the


Figs. 18-21. At 15 hours post coitum, some spermatozoa were in association with (Fig. 18) or in close proximity to leukocytes (L) (Fig. 19). Of the numerous spermatozoa observed on the ovary at this time, some were in advanced stages of complete breakdown (Fig. 20) while most displayed extensive erosion of the membranes covering the acrosomal area (Fig. 21). Spermato-

tozoa (S). x4800; x1300; x3000; x4000

acrosomal area; spermatozoa with surface disruptions of this type were compara- t ively rare. By 30 minutes post coitum, most spermatozoa were covered with a granular material located over both head and tail regions. The granules appeared to originate from secretory cells and to be transferred to spermatozoa through the beating and brushing action of cilia. Ciliated cells were quite abundan t in the cervical canal and lower port ion of the uterus, but became less numerous toward the uterotubal junction. By 31/2 hours post coitum, surface granules appeared to be restricted to the acrosomal surface.

Wi th increasing intervals of t ime following ejaculation (between 31/2 and 24 hours), a progressively larger port ion of the spermatozoa observed displayed disruptions and irregularities due to the presence of granules, perforations, and pits localized to the membranes covering the acrosomal region. For as m a n y as


Figs. 22--25. At 24 hours post coitum, almost every spermatozoon examined (in the uterus) displayed an advanced erosion of the membranes covering the acrosomal area (Fig. 22). While some spermatozoa appeared to be related to ciliated cells (C). Others appeared to be seated on the surface of secretory cells (S) (Figs, 23, 24). In Fig. 23, large droplets on the surface of the spermatozoa are Amilar to secretory droplets on the surface of surrounding cells (arrows). Only rarely were spermatozoa with relatively intact surfaces encountered (Fig. 24; note accumulation of granules and/or vesicles at the equatorial segment ES). By 36 hours post coitu~n, almost every spermatozoon encountered was in an advanced stage of complete degeneration (Fig. 25). Head, (H), Post Acrosomal Region (PA), Tail (T). • 4400;

• • )<4400

24 hours, the post-acrosomal region of the head, and the entire tail section of almost every spermatozoon examined were free of disruptions and granules. Between 24 and 36 hours post coitum, m a n y spermatozoa were observed in close proximi ty to, or in association with (sometimes engulfed by), leukocytes, Most spermatozoa


in this situation were clearly degenerating. However, even as late as 36 hours post coitum, an occasional spermatozoon was found in which only a partial erosion of the membranes overlying the acrosomal area had taken place.

Collectively, these observations suggest that a gradual (time-dependent) labilization and denudation of the membranous coverings of the acrosomal region occur in a progressively larger proportion of spermatozoa during post-coital residence in the reproductive tract. The initial labilization of the surface membrane(s) usually originates at the equatorial segment as a series of small fenestrations or perforations. With time, these fenestrations coalesce, and gradually, larger and larger areas of membrane are eroded. By 15 hours post coitum, in most spermatozoa examined, the outer acrosomal membrane, and possibly some areas of the acrosome itself are, to varying degrees, directly exposed to the uterine milieu.

I t is of interest to note that by SEM many of the events leading to the erosion of the surface membranes observed resemble morphological changes described by TEM and termed the "acrosome reaction" (Barros et al., 1967; Austin, 1968; Bedford, 1968, 1970; Zamboni, 1971, 1972). As mentioned previously, the acre- some reaction is believed to occur only when spermatozoa are in close proximity to a recently ovulated egg, and to consist primarily of a coalescence (at various points) of the outer acrosomal membrane with the overlying plasma membrane through a process of vesiculation (Bedford, 1970; Zamboni, 1971) and/or vacu- elation (Jones, 1973; Roomans and Afzelius, 1975). A very similar pattern of degeneration and loss of the plasma and acrosomal membranes, if occurring in regions of the reproductive tract removed from the immediate vicinity of an egg, has been referred to as the "false" acrosome reaction (Bedford, 1970). By TEM, some investigators have been unable to detect morphological differences between the "false" and " t rue" acrosome reactions (Zamboni, 1971, 1972). The present SEM observations demonstrate that alterations of surface membranes (possibly both plasma and acrosomal membranes) occur in a very similar pattern in spermatozoa present in the vagina, uterus, or oviduct, as well as in spermatozoa in the immediate vicinity of an egg during fertilization. These findings suggest that the so*called "false" and " t rue" acrosome reactions are morphologically indistinguishable and consequently may be different stages of the same general phenomenon (i.e. related to a cytolytic process of both acrosomal and plasma membranes).

Since the requirement of spermatozoa to be "capaci ta ted" was first described (Austin, 1951; Chang, 1951), numerous biochemical and electron microscopic studies have at tempted to define what factors or events are involved in this process (for a review, see McRorie and Williams, 1974). At present, it is believed that the acquisition of fertilizing capability (capacitation) is a time-dependent phenomenon which takes place in the uterus and/or oviduct and may require as many as 16 hours to complete (Bedford, 1968; Bernstein and Teichman, 1972; Soupart, 1972). In order to be able to detect subtle changes on the surface of spermatozoa during the interval of time required for capacitation, it was thought essential to examine spermatozoa while they were in their natural location following coitus--the female reproductive tract.

The results of this study demonstrate that with increasing periods of time following copulation, the surfaces of progressively larger numbers of spermatozoa appears granulated and provided with a heterogenous population of


vesicles and perforations. Ultimately, this disruptive process leads to the erosion of the membranes overlying the acrosomal area and possibly results in the direct exposure of the acrosome to the oviductal and/or uterine milieu. Although from the present study it would be premature to conclude a functional correspondence between membranous disruptions and capacitation, it is nevertheless an intriguing possibility that such a relationship exists. In this respect, capacitation and the acrosome reaction might be different stages of the same phenomenon consisting of (1) a gradual labilization and perforation of the membranes covering the acrosomal region (capacitation) and (2) a progressive exposure of the acrosome to the oviductal and/or uterine milieu. The observation that membranous disruptions are specifically limited to the acrosomal segment may be related to the different properties (morphological, cytochcmical and immunological) of the periacrosomal portion of the plasmalemma from those of the postacrosomal region (Gordon et al., 1975). Furthermore, the specific disruption of the periacrosomal membranes tends to support the hypothesis that the fragility of the acrosomal area could be related to the lytic activity of acrosomal enzymes. This view seems to be in agreement with the idea that the acrosome is a modified from of a lysosome and that the fertilizable state of spermatozoa ("capacitation" and "acrosome reaction") may involve a process of labilization similar to that known for lysosomcs.

An additional finding from this study indicates that spermatozoa have a curious tendency to come into close contact with ciliated cells, not only in regions of the genital tract where these cells are abundant, but also in areas where these cells are less numerous. Recent studies by phase-contrast microscopy and cinemato- graphy demonstrate that cilia in the cervix of the rabbit beat toward the vagina, and such movement may facilitate the release and distribution of secretory granules originating from cells adjacent to the ciliated cells (Kanagana and Hafcz, 1973). I t has also been shown that ciliated cells of the rabbit uterus and oviduct may function to distribute secretory granules produced in those regions of the genital tract (Motta and Andrews, 1975). The present observations indicate that small granules adhering to the surface of cilia are transferred to spermatozoa through the brushing and beating action of the cilia. These granules eventually become localized to the region of the plasma membrane overlying the acrosomal cap. At present, it is unknown whether these granules contribute to the increasing labilization and fragility of the surface membranes that occurs during the length of time required for capacitation. I t may be only coincidental that membranous perforations are restricted to regions of the spermatozoon head over which these granules are localized. Further studies are required to establish whether the granules are simply mucins or mucopolysaecharides (as a number of histochemical studies have suggested; for references see Lawn, 1974) or whether they may contain lytic substances that could possibly function to labilize the acrosomal surface. Such a process could result in the progressive removal of the surface glycoprotein coat (Gordon et al., 1975) and ultimately, the perforation (by vesiculation and/or vacuolation) of the plasmalemma and outer acrosomal membranes.

Finally, the existence of "decapacitation factor(s)" (Chang, 1957; Bedford and Chang, 1962), and the observation that the action of such a factor(s) is reversible, has been used to support the idea that no structural changes are expressed in spermatozoa as a "concomitant of capacitation" (Bedford, 1970).


Resul ts ob ta ined in the present s t u d y d e m o n s t r a t e t h a t af ter as m a n y as 24 hours of residence in the reproduc t ive t rac t , spe rmatozoa with e i ther an in tac t or pa r t i a l l y eroded surface membrane(s) are encountered. Spermatozoa of this t y p e are much more obvious in specimens examined a t 9 and 15 hours post coitum t h a n a t 24 hours. The " r e c a p a c i t a t i o n " of " d e c a p a c i t a t e d " spermatozoa could involve a resumption of the erosion process in those spermatozoa in which only pa r t i a l d i s rupt ion of the membrane(s) over ly ing the acrosomal region had occurred, or the in i t ia t ion of the erosion process in spermatozoa t h a t were in t ac t upon t r e a t m e n t with decapac i t a t ion factor(s).

I t is i m p o r t a n t to re-emphasize t h a t by SEM, spe rmatozoa in the reproduc t ive t r ac t following coitus exis t as a heterogenous, morphologica l popula t ion , and t ha t wi th t ime, shifts wi th in this popu la t ion from one p r e d o m i n a n t surface morpho logy to ano ther t ake place. This observa t ion is of pr ime concern when a t t e m p t i n g to reconcile differences in the in t e rp re t a t ion of sperm fine s t ruc ture when images of sperm ob ta ined b y SEM are compared with those ob ta ined by TEM. An approach in which the same mate r i a l is examined by both TEM and SEM should provide a clearer unde r s t and ing of the cellular basis of physiological events occurr ing dur ing the ear ly pos t coi tal period.

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Documents

A scanning electron microscopic study of rabbit spermatozoa in the female reproductive tract following coitus