33
J. Anat. (1978), 127, 1, pp. 33-41 With 8 figures Printed in Great Britain
The nature and occurrence of the acrosome reaction in spermatozoa of the domestic pig, Sus scrofa DANIEL SZOLLOSI AND R. H. F. HUNTER*
Station de Physiologie Animale, 78350 Jouy-en-Josas and Laboratoire d'Histologie d'Embryologie, Universite Paris-Sud, Kremlin-Bicetre and * School of Agriculture, University of Edinburgh, Scotland
(Accepted 1 August 1977) INTRODUCTION
Extensive vesiculation between the plasma membrane and the outer acrosomal membrane has been described in mammalian spermatozoa and termed the acrosome reaction (Barros et al. 1967; Bedford, 1967, 1968, 1972; Piko, 1969; Franklin, Barros & Fussell, 1970; Yanagimachi & Noda, 1970). This membranous reaction may be initiated as the spermatozoa start their passage between the cells of the cumulus oophorus (Austin, 1960), and is generally considered to be well advanced by the time they arrive at the surface of the zona pellucida. The acrosomal contents, of which hyaluronidase is a major component, would be released as soon as apertures were formed in the membranes surrounding the heads of viable spermatozoa. More recently, however, it has become evident that much hyaluronidase can be released from hamster sperm in vitro before initiation of membrane vesiculation is detected (Talbot & Franklin, 1974a, b). Interpretation of a normally developing acrosome reaction is made more difficult if degenerative changes have arisen during sperm passage in the female reproductive tract, or during prolonged periods of in vitro incubation, or even after exposure of spermatozoa to solutions of detergents of various strengths, for the pattern of membrane vesiculation appears morphologically to be nearly identical (Bedford, 1970; Flechon, 1970; Wooding, 1975; Yanagimachi, 1975). More recently, the swelling of the acrosome has been described as the initial stage in the acrosome reaction in the golden hamster (Talbot & Franklin, 1976). In the present study we report that, after the experimental manipulation of surgical insemination directly into the Fallopian tube of the domestic pig, the unbound contents of the acrosome seemed to have dispersed following an apparent swelling, while the plasma membrane and outer acrosomal membrane were still intact. Moreover, in this experimental situation, the acrosome reaction of many of the penetrating spermatozoa is able to take place on the surface of the zona pellucida. MATERIALS AND METHODS
Mature pigs were inseminated, with 0 05 ml of freshly collected ejaculate containing 2-21-2-74 x 108 cells per ml of seminal plasma, directly into the lower isthmus of the Fallopian tube after surgical intervention as described elsewhere (Hunter, 1973). These conditions of insemination lead to polyspermic penetration of the eggs * Address for correspondence (R.H.F.H.): School of Agriculture, University of Edinburgh, West Mains Road, Edinburgh EH9 3JG.
3
ANA
I27
34
D. SZOLLOSI AND R. H. F. HUNTER
(Hunter, 1973, 1976; Szollosi & Hunter, 1973), and hence are well suited to increase the number of spermatozoa around and within the zona pellucida. Eggs were recovered within 2 hours of ovulation by flushing the tubes with Eagle's medium, and, after a brief wash in the medium, were fixed in 1-5 % glutaraldehyde in either Dulbecco's phosphate buffered saline (PBS) or 0O15 M cacodylate buffer, at pH 7-3. Following dehydration in a graded ethyl alcohol series, the eggs were embedded in Spurr's medium and sections prepared as described previously (Szollosi & Hunter, 1973). RESULTS
In each preparation there were large numbers of spermatozoa in the vicinity of the egg, and many of these spermatozoa were probably affixed to it in a very weak gel of hyaluronic acid and other glycoproteins, although morphologically this gel was not easily demonstrable. Several of the spermatozoa were barely in contact with the zona pellucida, whereas others had entered the less compact peripheral portion of the zona (Szollosi & Hunter, 1973), while a smaller but variable proportion of the cell was passing through the more compact major portion of the zona. A total sperm count in the environs of each of the eggs sectioned was not attempted, and full serial sections, which this kind of a study would require, were not obtained. In the case of those oocytes in which spermatozoa were loosely associated with the egg surface, no perivitelline spermatozoa were observed, although the eggs had clearly been penetrated. By contrast, in those eggs in which the number of perivitelline spermatozoa was high, few loosely associated spermatozoa were found on the zona surface. Most of the spermatozoa studied had in any event already undergone the acrosome reaction, either on the zona surface or within its width. It should be emphasized that the oocytes were usually denuded of cells of the cumulus oophorus at the time of fixation, which probably reflects the conditions of insemination employed, and influences the following interpretation of events. The spermatozoa differed characteristically in each of the regions described; morphological analysis of each region allows the reconstitution of successive stages in the acrosome reaction. The approaching spermatozoa, which were loosely associated with the egg surface, and were often separated by several microns from the most peripheral portion of the recognizable zona, demonstrated an intact acrosome, fully comparable with those in either cauda epididymal or freshly ejaculated spermatozoa (Fig. 1). In most cases the cell membrane was not very closely applied to the acrosome, being separated from it by a space of varying dimensions. This separation may be artefactual, as suggested by fixation experiments on ejaculated boar spermatozoa (Jones, 1973). A few spermatozoa showed some dispersion of the acrosomal contents, which may be reflected in a slight swelling of the acrosome, and in these instances the contents had become less dense overall (Fig. 2). Moreover, within the acrosome of such spermatozoa, regions with different densities of contents were clearly visible, suggesting a different internal organization or compaction of acrosomal substance. On occasions the plasma membrane was absent, most likely a preparatory artefact. Spermatozoa making contact with the surface of the zona were of two kinds. In the first, the acrosome was intact even though slightly swollen, but the bulk of the acrosomal material was lacking (Fig. 3). Nonetheless, along the inner surface of the outer acrosomal membrane a slight density was sometimes visible, and a few membranous elements were found within the lumen of the acrosome. In the second,
35
The acrosome reaction in boar spermatozoa
"IS
I V.
A
/
is
(.
l
I
1
~~~~~~~~~~2
Fig. 1. The spermatozoon, which is in loose association with the egg surface, is fully ccmparable morphologically with those in the cauda epididymidis. Note the swelling of the membrane around the sperm head. x 32000. Fig. 2. The acrosome of this sperm near the surface of the zona pellucida is slightly swollen. The acrosomal contents are seen to have differing densities in different regions. x 58000. 3-2
36
D. SZOLLOSI AND R. H. F. HUNTER
W.\
I~~~~~~~~~I
I/I
?;
;A
I
.t
II
A.
3
Fig. 3. The spermatozoon at the surface of the zona shows a slight swelling. Note that the acrosomal substance is lacking even though the acrosomal vesiculation has not taken place. The inner surface of the outer acrosomal membrane is coated with a dense material. x 29000.
The acrosome reaction in boar spermatozoa
37
..
V~~~~~~~~~~~~
Fig. 4. Vesiculation of the acrosomal membrane is initiated apically. Near the equatorial segment the acrosome is still intact. x 24000.
Fig. 5. Membrane vesiculation has been completed in this specimen. Most vesicles are of small size, although one large vesicle is seen near the apex. x 25000.
the acrosomal substance was also lacking. Vesiculation had been initiated rostrally in such spermatozoa, while the dense nucleus was being enveloped by vesicles (Fig. 4). At the equatorial segment, portions of the still intact plasma membrane and outer acrosomal membrane were found. In spermatozoa on the surface and within the loose part of the zona, the membrane vesiculation between plasma membrane and outer acrosomal membrane had been completed. The vesicles which surrounded the inner acrosomal membrane and the nucleus were of varying dimensions, but were usually small, although on occasions one to several large vesicles remained (Fig. 5). Also, two rows of vesicles frequently lay on the surface of the zona, most probably indicating loci where the vesiculated membranes were left behind by the actively moving spermatozoon (Fig. 6). On the surface of eggs fixed at a longer time interval after ovulation, spermatozoa were
>.):s,'!i.;_teAU rw,;li:^.te'-*sS 38
,
J. Anat. (1978), 127, 1, pp. 33-41 With 8 figures Printed in Great Britain
The nature and occurrence of the acrosome reaction in spermatozoa of the domestic pig, Sus scrofa DANIEL SZOLLOSI AND R. H. F. HUNTER*
Station de Physiologie Animale, 78350 Jouy-en-Josas and Laboratoire d'Histologie d'Embryologie, Universite Paris-Sud, Kremlin-Bicetre and * School of Agriculture, University of Edinburgh, Scotland
(Accepted 1 August 1977) INTRODUCTION
Extensive vesiculation between the plasma membrane and the outer acrosomal membrane has been described in mammalian spermatozoa and termed the acrosome reaction (Barros et al. 1967; Bedford, 1967, 1968, 1972; Piko, 1969; Franklin, Barros & Fussell, 1970; Yanagimachi & Noda, 1970). This membranous reaction may be initiated as the spermatozoa start their passage between the cells of the cumulus oophorus (Austin, 1960), and is generally considered to be well advanced by the time they arrive at the surface of the zona pellucida. The acrosomal contents, of which hyaluronidase is a major component, would be released as soon as apertures were formed in the membranes surrounding the heads of viable spermatozoa. More recently, however, it has become evident that much hyaluronidase can be released from hamster sperm in vitro before initiation of membrane vesiculation is detected (Talbot & Franklin, 1974a, b). Interpretation of a normally developing acrosome reaction is made more difficult if degenerative changes have arisen during sperm passage in the female reproductive tract, or during prolonged periods of in vitro incubation, or even after exposure of spermatozoa to solutions of detergents of various strengths, for the pattern of membrane vesiculation appears morphologically to be nearly identical (Bedford, 1970; Flechon, 1970; Wooding, 1975; Yanagimachi, 1975). More recently, the swelling of the acrosome has been described as the initial stage in the acrosome reaction in the golden hamster (Talbot & Franklin, 1976). In the present study we report that, after the experimental manipulation of surgical insemination directly into the Fallopian tube of the domestic pig, the unbound contents of the acrosome seemed to have dispersed following an apparent swelling, while the plasma membrane and outer acrosomal membrane were still intact. Moreover, in this experimental situation, the acrosome reaction of many of the penetrating spermatozoa is able to take place on the surface of the zona pellucida. MATERIALS AND METHODS
Mature pigs were inseminated, with 0 05 ml of freshly collected ejaculate containing 2-21-2-74 x 108 cells per ml of seminal plasma, directly into the lower isthmus of the Fallopian tube after surgical intervention as described elsewhere (Hunter, 1973). These conditions of insemination lead to polyspermic penetration of the eggs * Address for correspondence (R.H.F.H.): School of Agriculture, University of Edinburgh, West Mains Road, Edinburgh EH9 3JG.
3
ANA
I27
34
D. SZOLLOSI AND R. H. F. HUNTER
(Hunter, 1973, 1976; Szollosi & Hunter, 1973), and hence are well suited to increase the number of spermatozoa around and within the zona pellucida. Eggs were recovered within 2 hours of ovulation by flushing the tubes with Eagle's medium, and, after a brief wash in the medium, were fixed in 1-5 % glutaraldehyde in either Dulbecco's phosphate buffered saline (PBS) or 0O15 M cacodylate buffer, at pH 7-3. Following dehydration in a graded ethyl alcohol series, the eggs were embedded in Spurr's medium and sections prepared as described previously (Szollosi & Hunter, 1973). RESULTS
In each preparation there were large numbers of spermatozoa in the vicinity of the egg, and many of these spermatozoa were probably affixed to it in a very weak gel of hyaluronic acid and other glycoproteins, although morphologically this gel was not easily demonstrable. Several of the spermatozoa were barely in contact with the zona pellucida, whereas others had entered the less compact peripheral portion of the zona (Szollosi & Hunter, 1973), while a smaller but variable proportion of the cell was passing through the more compact major portion of the zona. A total sperm count in the environs of each of the eggs sectioned was not attempted, and full serial sections, which this kind of a study would require, were not obtained. In the case of those oocytes in which spermatozoa were loosely associated with the egg surface, no perivitelline spermatozoa were observed, although the eggs had clearly been penetrated. By contrast, in those eggs in which the number of perivitelline spermatozoa was high, few loosely associated spermatozoa were found on the zona surface. Most of the spermatozoa studied had in any event already undergone the acrosome reaction, either on the zona surface or within its width. It should be emphasized that the oocytes were usually denuded of cells of the cumulus oophorus at the time of fixation, which probably reflects the conditions of insemination employed, and influences the following interpretation of events. The spermatozoa differed characteristically in each of the regions described; morphological analysis of each region allows the reconstitution of successive stages in the acrosome reaction. The approaching spermatozoa, which were loosely associated with the egg surface, and were often separated by several microns from the most peripheral portion of the recognizable zona, demonstrated an intact acrosome, fully comparable with those in either cauda epididymal or freshly ejaculated spermatozoa (Fig. 1). In most cases the cell membrane was not very closely applied to the acrosome, being separated from it by a space of varying dimensions. This separation may be artefactual, as suggested by fixation experiments on ejaculated boar spermatozoa (Jones, 1973). A few spermatozoa showed some dispersion of the acrosomal contents, which may be reflected in a slight swelling of the acrosome, and in these instances the contents had become less dense overall (Fig. 2). Moreover, within the acrosome of such spermatozoa, regions with different densities of contents were clearly visible, suggesting a different internal organization or compaction of acrosomal substance. On occasions the plasma membrane was absent, most likely a preparatory artefact. Spermatozoa making contact with the surface of the zona were of two kinds. In the first, the acrosome was intact even though slightly swollen, but the bulk of the acrosomal material was lacking (Fig. 3). Nonetheless, along the inner surface of the outer acrosomal membrane a slight density was sometimes visible, and a few membranous elements were found within the lumen of the acrosome. In the second,
35
The acrosome reaction in boar spermatozoa
"IS
I V.
A
/
is
(.
l
I
1
~~~~~~~~~~2
Fig. 1. The spermatozoon, which is in loose association with the egg surface, is fully ccmparable morphologically with those in the cauda epididymidis. Note the swelling of the membrane around the sperm head. x 32000. Fig. 2. The acrosome of this sperm near the surface of the zona pellucida is slightly swollen. The acrosomal contents are seen to have differing densities in different regions. x 58000. 3-2
36
D. SZOLLOSI AND R. H. F. HUNTER
W.\
I~~~~~~~~~I
I/I
?;
;A
I
.t
II
A.
3
Fig. 3. The spermatozoon at the surface of the zona shows a slight swelling. Note that the acrosomal substance is lacking even though the acrosomal vesiculation has not taken place. The inner surface of the outer acrosomal membrane is coated with a dense material. x 29000.
The acrosome reaction in boar spermatozoa
37
..
V~~~~~~~~~~~~
Fig. 4. Vesiculation of the acrosomal membrane is initiated apically. Near the equatorial segment the acrosome is still intact. x 24000.
Fig. 5. Membrane vesiculation has been completed in this specimen. Most vesicles are of small size, although one large vesicle is seen near the apex. x 25000.
the acrosomal substance was also lacking. Vesiculation had been initiated rostrally in such spermatozoa, while the dense nucleus was being enveloped by vesicles (Fig. 4). At the equatorial segment, portions of the still intact plasma membrane and outer acrosomal membrane were found. In spermatozoa on the surface and within the loose part of the zona, the membrane vesiculation between plasma membrane and outer acrosomal membrane had been completed. The vesicles which surrounded the inner acrosomal membrane and the nucleus were of varying dimensions, but were usually small, although on occasions one to several large vesicles remained (Fig. 5). Also, two rows of vesicles frequently lay on the surface of the zona, most probably indicating loci where the vesiculated membranes were left behind by the actively moving spermatozoon (Fig. 6). On the surface of eggs fixed at a longer time interval after ovulation, spermatozoa were
>.):s,'!i.;_teAU rw,;li:^.te'-*sS 38
,
