MOLECULAR REPRODUCTION AND DEVELOPMENT 27:152-158 (1990)
Acrosome Reaction of Stallion Spermatozoa Evaluated With Monoclonal Antibody and Zona-Free Hamster Eggs J. ZHANG,' M.S. BOYLE,' C.A. SMITH,^ AND H.D.M. MOO RE^ 'Thorough Breeders' Association Equine Fertility Unit, Mertoun Paddocks, Newmarket, Suffolk, and 2MRCIAFRC Comparative Physiology Research Group, Institute of Zoology, Zoological Society of London, London, England The acrosome of the stallion ABSTRACT spermatozoon was visualized by indirect immunofluorescence with monoclonal antibody ( 18.6) which recognized an integral acrosomal membrane component. Localization was confirmed by electron microscopy using peroxidase labelled antibody. In fresh semen samples (n = 19), 73.9 ? 9.1% of the spermatozoa from five fertile stallions displayed a uniform bright fluorescence over their acrosome region. In two semen samples from an infertile stallion only 28% and 35% of spermatozoa showed the same pattern of fluorescence. Spermatozoa from fertile stallions incubated for up to 12 hours in TALP medium maintained motility and exhibited a significant progressive loss of acrosomes as detected by immunofluorescence. Alternatively, a similar loss of acrosomes could be induced with calcium ionophore A231 87 over a 90 minute incubation. Ultrastructural observations and incubation with zona-free hamster eggs indicated that only with ionophore treatment was immunofluorescent acrosome loss correlated with a physiological acrosome reaction, while prolonged sperm incubation led to degenerative membrane changes. It was concluded that, if carefully validated, immunofluorescent localization of the acrosome of stallion sperm with monoclonal antibody could be used to monitor the acrosome reaction. Furthermore, definitive acrosome visualization would be valuable in assessing semen quality.
Key Words: Stallion spermatozoa, Acrosome reaction, Monoclonal antibody, Zona-free hamster eggs
INTRODUCTION The sperm acrosome reaction is a prerequisite for fertilization in mammals (Moore and Bedford, 1983; Yanagimachi, 1988). Objective methods of assessing the morphology and function Of the acroSome are therefore essential for evaluating the fertilizing capacity of spermatozoa. In the stallion, the kinetics of the ac-
0 1990 WILEY-LISS, INC.
rosome reaction have been poorly documented due both to the difficulties of visualizing the status of the apical acrosome under bright or phase contrast microscopy (Voss et al., 1981) and to the lack of a reliable method for inducing capacitation and the acrosome reaction in vitro. Monoclonal antibodies in conjunction with indirect immunofluorescence have been used to evaluate acrosomal status in the human and laboratory animals (Ellis et al., 1985; Wolf et al., 1985; Moore et al., 1987) and recently to assess the integrity of the plasma membrane of stallion spermatozoa (Blach et al., 1988). In the present investigation a monoclonal antibody (18.6) which recognizes a n integral membrane component of the sperm acrosomal membranes of all eutherian mammals so far tested (Ellis et al., 1985) has been used to visualize the acrosome of stallion spermatozoa during incubation in vitro and following induction of the acrosome reaction with calcium ionophore A23187. A correlation between immunofluorescent staining of spermatozoa and true induction of the acrosome reaction was investigated using ultrastructural observations (see Moore and Bedford, 1983) and sperm penetration of zona-free hamster eggs (see Yanagimachi, 1988).
MATERIALS AND METHODS Semen Samples Samples of ejaculated semen were obtained from five normal, fertile pony stallions and from one infertile stallion by means of a n artificial vagina. The infertile stallion had failed to produce any foals in two seasons a t stud, although conventional semen analysis and clinical investigation had revealed no abnormalities. Indirect Immunofluorescence Visualization of acrosomes was carried out using monoclonal antibody 18.6 (Ellis et al., 1985), originally
Received October 30, 1989; accepted January 22, 1990. Address reprint requests to Dr. J. Zhang, Thorough Breeders' Association Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk, CB89BH, United Kingdom.
STALLION SPERM ACROSOME REACTION generated in mice immunized with washed hamster spermatozoa. This antibody cross-reacts with spermatozoa from all eutherian mammals so far tested and binds to the anterior acrosome. Immunofluorescent localization was carried out on methanol-fixed spermatozoa using a similar protocol to that for human spermatozoa (Moore et al., 1987). Semen from the fertile stallions and from the one infertile stallion was washed twice by centrifugation a t 320g for 5 minutes in TALP medium (Graham e t al., 1986) and then diluted to a concentration of 10-20 x lo6 spermiml. A smear made from a drop of this washed sperm suspension was air dried and fixed in absolute methanol for 10 seconds. Hybridoma supernatant containing 18.6 antibody (lop11 was spread onto the smear. After 10 minutes' incubation a t 37"C, the smear was gently rinsed with PBS and spread with FITC-antimouse IgG (heavy and light chain, Miles Research, diluted 1 5 0 with PBS). After a further 10 minutes' incubation, the drop was rinsed again and cells were examined under oil a t x 500 with a Nikon microscope equipped with epifluorescent optics. In each experiment 150 spermatozoa were examined.
Induction of the Acrosome Reaction After Incubation or After Addition of Calcium Ionophore Washed semen samples from fertile stallions were diluted to 100-200 x lo6 spermiml and incubated in TALP medium a t 37.5"C in 5% CO,. After 6, 9, and 12 hours of incubation, a drop of sperm suspension was removed and diluted a further ten times for evaluation of sperm motility and to assess acrosome morphology using indirect immunofluorescence. At 6 and 12 hours of incubation, aliquots were also recovered for the zonafree hamster egg penetration test (see below). Acrosome reactions were also induced using calcium ionophore A23187 as described by Shams-Borhan and Harrison (1981). Semen was washed twice by centrifugation a t 320g for 5 minutes in TALP medium supplemented with 0.1% polyvinyl alcohol (PVA), and then diluted to a concentration of 10-15 x lo6 spermiml. The acrosome reaction was initiated by addition of A23187 from a stock solution in DMSO a t a concentration of 0.1 pM. The spermatozoa were then incubated a t 375°C in a culture tube and 50 pl aliquots removed a t 0, 10, 30, 60, 90, and 120 minutes for evaluation of the acrosome reaction by indirect immunofluorescence and electron microscopy (see below). A total of 19 semen samples from five fertile stallions were incubated in medium with and without ionophore. For each time point 150 sperm were assessed by immunofluorescence. Stallion Sperm Penetration of Zona-Free Hamster Eggs The zona-free hamster egg penetration test was carried out using aliquots of washed sperm suspension
153
that had been incubated alone for 6 and 12 hours or had been treated with calcium ionophore A23187 for 10 minutes as described above. These samples were diluted with TALP medium containing 0.3% bovine serum albumin (Sigma, fraction V) to give final concentrations of 0.5 x lo6 spermiml (sperm incubated alone for 6 or 12 hours) and 5 x lo6 spermiml (A23187 treated sperm). Aliquots (50 p1) of these diluted suspensions were then added to 50 pl of TALP medium (under oil) containing 10 zona-free hamster eggs prepared using the method described by Moore and Hartman (1984). Spermiegg preparations were incubated a t 375°C under oil in 5% CO, for 3 hours. Oocytes were then removed and carefully rinsed in culture medium before being fixed in alcoho1:acetic acid (3:l) and stained with lacmoid for assessment of sperm penetration or fixed in 1% paraformaldehyde for indirect immunofluorescence (Moore et al., 1987).
Electron Microscopy After being exposed to calcium ionophore A23187 for periods of 10-120 minutes, or after prolonged incubation for 6 to 12 hours, spermatozoa were also fixed and processed for electron microscopy (Shams-Borhan and Harrison, 1981). Ultrastructural localization of 18.6 monoclonal antibody binding was carried out using the peroxidase-antiperoxidase(PAP) techniques described by Smith et al. (1986). RESULTS Immunofluorescent Localization of the Acrosome With Monoclonal Antibody On methanol-fixed stallion spermatozoa, monoclonal antibody 18.6 was visualized by indirect immunofluorescence binding over the apical acrosome region (Fig. 1).Bright even fluorescence over the acrosome (Fig. 9.1% of spermatozoa (19 l a ) was displayed by 73.9 samples) from fertile stallions. In contrast, for the two semen samples collected from the infertile stallion, only 28% and 35% of spermatozoa exhibited a similar pattern of fluorescence, while 60% and 55%, respectively, showed a patchy distribution of fluorescence (Fig. lc,d).
*
The Effects of Incubation and Calcium Ionophore on Acrosome Immunofluorescence During prolonged incubation for up to 12 hours a t 37.5"C, the proportion of progressively motile spermatozoa (from fertile stallions) remained constant (60 k 10%). However, the mean proportion of spermatozoa displaying either patchy fluorescence (Fig. Id) or loss of fluorescence (Fig. l e ) over the acrosome region increased significantly with time (Fig. 2) so that by 12 hours spermatozoa with bright acrosome fluorescence represented about 32.9 12.6% of the sample. Interstallion variation was less than 8%. When diluted semen samples were treated with the calcium ionophore A23187, the proportion of sperma-
*
154
ZHANG ET AL.
Fig. 1. Micrographs of stallion spermatozoa displaying various forms of fluorescence over their acrosome region after incubation in TALP medium. a: Even bright fluorescence. b Fading fluorescence.
c,d: Patchy distribution of fluorescence. e: Total loss of fluorescence. f: Spermatozoa treated with A23187 displayed even bright fluorescence or loss of fluorescence (arrows). x 1,000.
-
1OOF v)
jg
Stall,""
60-
H"
m z
d -o
40-
:z$ 5
20 -
1
100-
Fluorescence
80-
e
STALLION SPERM ACROSOME REACTION
Total loss
0: 3 k t l o "
80 60 -
40 -
Evedbrrght 20 -
R
Incubation
Time
Ihl
Fig. 2. Histogram of the proportion of stallion spermatozoa displaying various degrees of fluorescence during incubation at 37.5"C in TALP medium. Means of 19 samples.
155
tozoa displaying faded (Fig. l b ) or absent fluorescence (Fig. I f ) over the acrosome increased substantially within 30 minutes of the addition of 0.1 pM A23187 and reached a maximum by 90 minutes. By 120 minutes only 29.2 i 6.3% of spermatozoa had bright acrosomal fluorescence. The time course for this change in the acrosome varied between horses as shown for three stallions in Figure 3.
Zona-Free Hamster Egg Test Spermatozoa incubated for 6 and 12 hours remained motile and bound avidly to zona-free hamster eggs but failed to fuse with the oolemma. By contrast, spermatozoa treated with A23187 ionophore for 10 minutes consistently fused with zona-free hamster eggs (Table 1)as confirmed by the presence of one or more decondensing sperm heads in the vitellus (Fig. 4a). At high magnification this decondensation of sperm nuclear chromatin was often seen to be irregular (Fig. 4b). Indirect immunofluorescent localization with 18.6 monoclonal antibody revealed that many of the spermatozoa bound to the surface of zona-free hamster eggs had a n acrosome present (Fig. 4c).
Time atter the addition of L123187 Imin.1
Fig. 3. Histogram of the changes in fluorescence for spermatozoa from three stallions during incubation in the presence of A23187.
TABLE 1. Penetration of Zona-Free Hamster Eggs by Stallion Sperm Treated With Ionophore A23187* Stallion A (2)" B (3) c (3) c (1) Total
No. eggs penetrated/ No. eggs inseminated 17/30 19/30 17/25 15/25 681110 (62%)
No. sperm heads/ penetrated egg
Ultrastructure of the Acrosome 2.1 3.1 In sperm samples incubated for 6 and 12 hours, cells 2.2 undergoing a n acrosome reaction were not observed. 1.5 Spermatozoa with vesiculated or absent acrosomal 2.2 membranes were present but these features were accompanied by degenerative changes to membrane over '"ee text for details. the equatorial segment and post-acrosomal region. aNo. of semen samples. However, in all the sperm samples incubated with A23187, true acrosome-reacted spermatozoa could be identified by fusion and vesiculation of plasma and microscopy was greater in those samples incubated for outer acrosomal membrane over the apical sperm head; the longer time periods, although a quantitative analthe specific fusion of plasma membrane and outer ac- ysis was not performed. At the ultrastructural level 18.6, monoclonal antirosomal membrane a t the apical border of the equatorial segment and a contiguous plasma membrane over body was shown with immunoperoxidase labelling to the remaining sperm head (Fig. 5a,b). The number of bind to a n intracellular antigen associated mainly with acrosome-reacted spermatozoa observed by electron the outer acrosomal membranes (Fig. 6a,b), although
156
ZHANG ET AL. Fig. 4. Micrographs of zona-free hamster oocytes co-incubated with stallion spermatozoa. a: Oocyte with many hound spermatozoa and containing two decondensed sperm heads with associated tails. Lacmoid stain. x 300. b A swollen head at higher magnification. X 790. c: Zona-free hamster oocyte stained with 18.6 antibody showing fluorescent sperm hound to the oolemma.
some label was present on the inner acrosomal membrane of disrupted cells.
DISCUSSION A means of inducing and subsequently monitoring the acrosome reaction in equids would be of considerable benefit for the development of in vitro fertilization techniques in these animals. But difficulties of visualizing reliably the compact horse sperm acrosome either by light microscopy alone or in combination with histochemical staining techniques (nigrosin and eosin: Dott and Foster, 1972; triple stain: Talbot and Chacon, 1981) has retarded progress. The objective of the present study was to attempt to induce the acrosomereaction in stallion spermatozoa and to investigate whether acrosomal status can be monitored with specific monoclonal antibody. Immunofluorescent localization made the acrosomes of stallion spermatozoa clearly visible after fixation in methanol. This was because the sperm membranes had become more permeable, allowing antibody to gain access to the internally located antigen (see Moore et al., 1987). Although a t the ultrastructural level some antibody binding (identified by immunoperoxidase staining) was present on the inner acrosomal membrane of damaged spermatozoa (Fig. 61, this was not apparent by immunofluorescence. Hence, bright, even stain represented the full complement of acrosomal content and membranes, while a loss of fluorescence (of various degrees) denoted a breakdown of the acrosome. The clear difference in the pattern of fluorescence between semen samples from fertile stallions and the two samples from a n infertile stallion indicated a difference in acrosome morphology which had not been identified by conventional semen analysis. A lack of spermatozoa with intact acrosomes a t ejaculation may contribute to male infertility and definitive visualization of acrosome morphology could provide a rapid diagnosis of ejaculate quality; a similar conclusion was reached by Blach and co-workers (1988), who also assessed membrane integrity in stallion spermatozoa with a monoclonal antibody. Since only one infertile animal was tested, it is not yet possible to say how useful this test would be for predicting infertility in stallions. Although prolonged incubation of diluted spermatozoa for 6-12 hours did not appreciably alter sperm motility, the fraction of spermatozoa with intact acrosomes a s detected by bright fluorescence decreased markedly. Indirect evidence would suggest that this change was probably due to a degenerative process rather than induction of the acrosome reaction. Spermatozoa observed by electron microscopy often exhibited a general breakdown in their membranes, but spe-
STALLION SPERM ACROSOME REACTION
157
Fig. 5. Electron micrographs of a stallion sperm head after treatment with A23187. a: Vesiculation o f plasma and outer acrosomal membranes typical of the acrosome reaction (arrow indicates anterior border o f the equatorial segment). x 10,000. b: Higher power of the equatorial region showing intact plasma membrane. x 30,000.
Fig. 6. Electron micrograph of immunolocalization of monoclonal antibody binding using peroxidaseantiperoxidase. a: Reaction product is present over the acrosomal region. x 12,000. b Control spermatozoa without first antibody. x 12,000.
cific membrane fusion associated with a physiological acrosome reaction (Fig. 5) was not seen. Moreover, spermatozoa incubated for 6 or 12 hours failed to fuse and penetrate zona-free hamster eggs. A normal acrosome reaction would appear to be essential €or gamete fusion in mammals (Yanagimachi, 1988). Since
sperm viability was maintained during spermiegg incubations, a n absence of sperm penetration was indicative of a failure to induce a true acrosome reaction. In contrast, the decline in immunofluorescence over the acrosomes of spermatozoa treated with A23187 calcium ionophore (Fig. 3) was probably associated with a
158
ZHANG ET AL.
true rather than a false acrosome reaction. In other species, treatment of spermatozoa with A23187 provides a way of producing a synchronous acrosome-reacted population (Shams-Borhan and Harrison, 1981; Topfer-Peterson et al., 1988). With stallion spermatozoa, the fading of fluorescence over the acrosome over a relatively short time span (60-90 minutes to reach maximum) was reminiscent of similar changes to fluorescence seen in human spermatozoa during in vitro culture, leading to a spontaneous acrosome reaction (Moore et al., 1987). Of more significance, stallion spermatozoa treated with A23187 displayed physiological acrosome reactions under the electron microscope and penetrated zona-free hamster eggs. With respect to the latter, a n unusual feature was the binding to the egg membrane of sperm with uniform bright fluorescence over the apical head. In other species, only acrosome reacted spermatozoa normally attach to the oolemma (Talbot and Chacon, 1982; Moore et al., 1987). In conclusion, we have demonstrated that the acrosome reaction of stallion spermatozoa could be induced by a short incubation with calcium ionophore A23187 and that this process was inversely correlated with immunofluorescent visualization of the acrosome with specific monoclonal antibody. Degenerative changes to the acrosome associated with prolonged incubation also altered immunofluorescent staining patterns. Therefore care must be taken that under different conditions such antibody localization techniques reflect the kinetics of the true acrosome reaction. The ability to assess rapidly the acrosome status of stallion spermatozoa will aid in the development of in vitro fertilization techniques. Furthermore, i t should prove useful for testing semen quality at ejaculation and after cryopreservation.
ACKNOWLEDGMENTS Work at the Equine Fertility Unit was supported by the Thoroughbred Breeder’s Association and the Horserace Betting Levy Board. The work was also supported in part by a programme grant to H.D.M.M. from
the Medical Research Council and Agricultural and Food Research Council.
REFERENCES Blach EL, Amann RP, Bowen RA, Sawyer HR, Hermenet MJ (1988) Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion spermatozoa. Gamete Res 21233-241. Dott HM, Foster GC (1972) A technique for studying the morphology of mammalian spermatozoa which are eosinophilic in a differential live/dead stain. J Reprod Fertil 29443-445. Ellis DH, Hartman TD, Moore HDM (1985) Maturation and function of the hamster spermatozoa probed with monoclonal antibodies. J Reprod Immunol 7:299-314. Graham JK, Foote RH, Parrish JJ (1986) Effect of dilauroylphosphatidylcholine on the acrosome reaction and subsequent penetration of hull spermatozoa into zona-free hamster eggs. Biol Reprod 35413-424. Moore HDM, Bedford JM (1983): The interaction of mammalian gametes in the female. In JF Hartmann (ed):“Mechanism and Control of Animal Fertilization.” New York: Academic Press, pp 435-497. Moore HDM, Hartman TD (1984) Localization by monoclonal antibodies of various surface antigens on hamster spermatozoa and effect of antibody on fertilization in uitro. J Reprod Fertil70:175-183. Moore HDM, Smith CA, Hartman TD, Bye AP (1987) Visualization and characterization of the acrosome reaction of human spermatozoa by immunolocalization with monoclonal antibody. Gamete Res 17245-259. Shams-Borhan G, Harrison RAP (1981) Production, characterization and use of ionophore-induced calcium dependent acrosome reaction in ram spermatozoa. Gamete Res 4:417-432. Smith CA, Hartman TD, Moore HDM (1986) A determinant of Mr 34000 expressed by hamster epididymal epithelium binds specifically to spermatozoa in co-culture. J Reprod Fertil 78:337-345. Talbot P, Chacon RS (1981) A triple-stain technique for evaluating normal acrosome reactions of human sperm. J Exp Zoo1 215201208. Talbot P, Chacon RS (1982) Ultrastructural observations on binding and membrane fusion between human sperm and zona pellucidafree hamster oocytes. Fertil Steril 37:240-248. Topfer-Peterson E, Friess AE, Schill WB (1988) The acrosome reaction in boar spermatozoa. Human Reprod 3:319-326. Voss JL, Pickett BW, Squires EL (1981) Stallion spermatozoa1 morphology and motility and their relationship to fertility. J Am Vet Med Assoc 178287-289. Wolf DP, Boldt J , Byrd W, Bechtol KB (1985) Acrosomal status evaluation in human ejaculate sperm with monoclonal antibodies. Biol Reprod 32:1157-1 162. Yanagimachi R (1988): Mammalian fertilization. In E Knobil and J Neil (eds): “Physiology of Reproduction.“ New York: Raven Press, pp 135-185.
Acrosome Reaction of Stallion Spermatozoa Evaluated With Monoclonal Antibody and Zona-Free Hamster Eggs J. ZHANG,' M.S. BOYLE,' C.A. SMITH,^ AND H.D.M. MOO RE^ 'Thorough Breeders' Association Equine Fertility Unit, Mertoun Paddocks, Newmarket, Suffolk, and 2MRCIAFRC Comparative Physiology Research Group, Institute of Zoology, Zoological Society of London, London, England The acrosome of the stallion ABSTRACT spermatozoon was visualized by indirect immunofluorescence with monoclonal antibody ( 18.6) which recognized an integral acrosomal membrane component. Localization was confirmed by electron microscopy using peroxidase labelled antibody. In fresh semen samples (n = 19), 73.9 ? 9.1% of the spermatozoa from five fertile stallions displayed a uniform bright fluorescence over their acrosome region. In two semen samples from an infertile stallion only 28% and 35% of spermatozoa showed the same pattern of fluorescence. Spermatozoa from fertile stallions incubated for up to 12 hours in TALP medium maintained motility and exhibited a significant progressive loss of acrosomes as detected by immunofluorescence. Alternatively, a similar loss of acrosomes could be induced with calcium ionophore A231 87 over a 90 minute incubation. Ultrastructural observations and incubation with zona-free hamster eggs indicated that only with ionophore treatment was immunofluorescent acrosome loss correlated with a physiological acrosome reaction, while prolonged sperm incubation led to degenerative membrane changes. It was concluded that, if carefully validated, immunofluorescent localization of the acrosome of stallion sperm with monoclonal antibody could be used to monitor the acrosome reaction. Furthermore, definitive acrosome visualization would be valuable in assessing semen quality.
Key Words: Stallion spermatozoa, Acrosome reaction, Monoclonal antibody, Zona-free hamster eggs
INTRODUCTION The sperm acrosome reaction is a prerequisite for fertilization in mammals (Moore and Bedford, 1983; Yanagimachi, 1988). Objective methods of assessing the morphology and function Of the acroSome are therefore essential for evaluating the fertilizing capacity of spermatozoa. In the stallion, the kinetics of the ac-
0 1990 WILEY-LISS, INC.
rosome reaction have been poorly documented due both to the difficulties of visualizing the status of the apical acrosome under bright or phase contrast microscopy (Voss et al., 1981) and to the lack of a reliable method for inducing capacitation and the acrosome reaction in vitro. Monoclonal antibodies in conjunction with indirect immunofluorescence have been used to evaluate acrosomal status in the human and laboratory animals (Ellis et al., 1985; Wolf et al., 1985; Moore et al., 1987) and recently to assess the integrity of the plasma membrane of stallion spermatozoa (Blach et al., 1988). In the present investigation a monoclonal antibody (18.6) which recognizes a n integral membrane component of the sperm acrosomal membranes of all eutherian mammals so far tested (Ellis et al., 1985) has been used to visualize the acrosome of stallion spermatozoa during incubation in vitro and following induction of the acrosome reaction with calcium ionophore A23187. A correlation between immunofluorescent staining of spermatozoa and true induction of the acrosome reaction was investigated using ultrastructural observations (see Moore and Bedford, 1983) and sperm penetration of zona-free hamster eggs (see Yanagimachi, 1988).
MATERIALS AND METHODS Semen Samples Samples of ejaculated semen were obtained from five normal, fertile pony stallions and from one infertile stallion by means of a n artificial vagina. The infertile stallion had failed to produce any foals in two seasons a t stud, although conventional semen analysis and clinical investigation had revealed no abnormalities. Indirect Immunofluorescence Visualization of acrosomes was carried out using monoclonal antibody 18.6 (Ellis et al., 1985), originally
Received October 30, 1989; accepted January 22, 1990. Address reprint requests to Dr. J. Zhang, Thorough Breeders' Association Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk, CB89BH, United Kingdom.
STALLION SPERM ACROSOME REACTION generated in mice immunized with washed hamster spermatozoa. This antibody cross-reacts with spermatozoa from all eutherian mammals so far tested and binds to the anterior acrosome. Immunofluorescent localization was carried out on methanol-fixed spermatozoa using a similar protocol to that for human spermatozoa (Moore et al., 1987). Semen from the fertile stallions and from the one infertile stallion was washed twice by centrifugation a t 320g for 5 minutes in TALP medium (Graham e t al., 1986) and then diluted to a concentration of 10-20 x lo6 spermiml. A smear made from a drop of this washed sperm suspension was air dried and fixed in absolute methanol for 10 seconds. Hybridoma supernatant containing 18.6 antibody (lop11 was spread onto the smear. After 10 minutes' incubation a t 37"C, the smear was gently rinsed with PBS and spread with FITC-antimouse IgG (heavy and light chain, Miles Research, diluted 1 5 0 with PBS). After a further 10 minutes' incubation, the drop was rinsed again and cells were examined under oil a t x 500 with a Nikon microscope equipped with epifluorescent optics. In each experiment 150 spermatozoa were examined.
Induction of the Acrosome Reaction After Incubation or After Addition of Calcium Ionophore Washed semen samples from fertile stallions were diluted to 100-200 x lo6 spermiml and incubated in TALP medium a t 37.5"C in 5% CO,. After 6, 9, and 12 hours of incubation, a drop of sperm suspension was removed and diluted a further ten times for evaluation of sperm motility and to assess acrosome morphology using indirect immunofluorescence. At 6 and 12 hours of incubation, aliquots were also recovered for the zonafree hamster egg penetration test (see below). Acrosome reactions were also induced using calcium ionophore A23187 as described by Shams-Borhan and Harrison (1981). Semen was washed twice by centrifugation a t 320g for 5 minutes in TALP medium supplemented with 0.1% polyvinyl alcohol (PVA), and then diluted to a concentration of 10-15 x lo6 spermiml. The acrosome reaction was initiated by addition of A23187 from a stock solution in DMSO a t a concentration of 0.1 pM. The spermatozoa were then incubated a t 375°C in a culture tube and 50 pl aliquots removed a t 0, 10, 30, 60, 90, and 120 minutes for evaluation of the acrosome reaction by indirect immunofluorescence and electron microscopy (see below). A total of 19 semen samples from five fertile stallions were incubated in medium with and without ionophore. For each time point 150 sperm were assessed by immunofluorescence. Stallion Sperm Penetration of Zona-Free Hamster Eggs The zona-free hamster egg penetration test was carried out using aliquots of washed sperm suspension
153
that had been incubated alone for 6 and 12 hours or had been treated with calcium ionophore A23187 for 10 minutes as described above. These samples were diluted with TALP medium containing 0.3% bovine serum albumin (Sigma, fraction V) to give final concentrations of 0.5 x lo6 spermiml (sperm incubated alone for 6 or 12 hours) and 5 x lo6 spermiml (A23187 treated sperm). Aliquots (50 p1) of these diluted suspensions were then added to 50 pl of TALP medium (under oil) containing 10 zona-free hamster eggs prepared using the method described by Moore and Hartman (1984). Spermiegg preparations were incubated a t 375°C under oil in 5% CO, for 3 hours. Oocytes were then removed and carefully rinsed in culture medium before being fixed in alcoho1:acetic acid (3:l) and stained with lacmoid for assessment of sperm penetration or fixed in 1% paraformaldehyde for indirect immunofluorescence (Moore et al., 1987).
Electron Microscopy After being exposed to calcium ionophore A23187 for periods of 10-120 minutes, or after prolonged incubation for 6 to 12 hours, spermatozoa were also fixed and processed for electron microscopy (Shams-Borhan and Harrison, 1981). Ultrastructural localization of 18.6 monoclonal antibody binding was carried out using the peroxidase-antiperoxidase(PAP) techniques described by Smith et al. (1986). RESULTS Immunofluorescent Localization of the Acrosome With Monoclonal Antibody On methanol-fixed stallion spermatozoa, monoclonal antibody 18.6 was visualized by indirect immunofluorescence binding over the apical acrosome region (Fig. 1).Bright even fluorescence over the acrosome (Fig. 9.1% of spermatozoa (19 l a ) was displayed by 73.9 samples) from fertile stallions. In contrast, for the two semen samples collected from the infertile stallion, only 28% and 35% of spermatozoa exhibited a similar pattern of fluorescence, while 60% and 55%, respectively, showed a patchy distribution of fluorescence (Fig. lc,d).
*
The Effects of Incubation and Calcium Ionophore on Acrosome Immunofluorescence During prolonged incubation for up to 12 hours a t 37.5"C, the proportion of progressively motile spermatozoa (from fertile stallions) remained constant (60 k 10%). However, the mean proportion of spermatozoa displaying either patchy fluorescence (Fig. Id) or loss of fluorescence (Fig. l e ) over the acrosome region increased significantly with time (Fig. 2) so that by 12 hours spermatozoa with bright acrosome fluorescence represented about 32.9 12.6% of the sample. Interstallion variation was less than 8%. When diluted semen samples were treated with the calcium ionophore A23187, the proportion of sperma-
*
154
ZHANG ET AL.
Fig. 1. Micrographs of stallion spermatozoa displaying various forms of fluorescence over their acrosome region after incubation in TALP medium. a: Even bright fluorescence. b Fading fluorescence.
c,d: Patchy distribution of fluorescence. e: Total loss of fluorescence. f: Spermatozoa treated with A23187 displayed even bright fluorescence or loss of fluorescence (arrows). x 1,000.
-
1OOF v)
jg
Stall,""
60-
H"
m z
d -o
40-
:z$ 5
20 -
1
100-
Fluorescence
80-
e
STALLION SPERM ACROSOME REACTION
Total loss
0: 3 k t l o "
80 60 -
40 -
Evedbrrght 20 -
R
Incubation
Time
Ihl
Fig. 2. Histogram of the proportion of stallion spermatozoa displaying various degrees of fluorescence during incubation at 37.5"C in TALP medium. Means of 19 samples.
155
tozoa displaying faded (Fig. l b ) or absent fluorescence (Fig. I f ) over the acrosome increased substantially within 30 minutes of the addition of 0.1 pM A23187 and reached a maximum by 90 minutes. By 120 minutes only 29.2 i 6.3% of spermatozoa had bright acrosomal fluorescence. The time course for this change in the acrosome varied between horses as shown for three stallions in Figure 3.
Zona-Free Hamster Egg Test Spermatozoa incubated for 6 and 12 hours remained motile and bound avidly to zona-free hamster eggs but failed to fuse with the oolemma. By contrast, spermatozoa treated with A23187 ionophore for 10 minutes consistently fused with zona-free hamster eggs (Table 1)as confirmed by the presence of one or more decondensing sperm heads in the vitellus (Fig. 4a). At high magnification this decondensation of sperm nuclear chromatin was often seen to be irregular (Fig. 4b). Indirect immunofluorescent localization with 18.6 monoclonal antibody revealed that many of the spermatozoa bound to the surface of zona-free hamster eggs had a n acrosome present (Fig. 4c).
Time atter the addition of L123187 Imin.1
Fig. 3. Histogram of the changes in fluorescence for spermatozoa from three stallions during incubation in the presence of A23187.
TABLE 1. Penetration of Zona-Free Hamster Eggs by Stallion Sperm Treated With Ionophore A23187* Stallion A (2)" B (3) c (3) c (1) Total
No. eggs penetrated/ No. eggs inseminated 17/30 19/30 17/25 15/25 681110 (62%)
No. sperm heads/ penetrated egg
Ultrastructure of the Acrosome 2.1 3.1 In sperm samples incubated for 6 and 12 hours, cells 2.2 undergoing a n acrosome reaction were not observed. 1.5 Spermatozoa with vesiculated or absent acrosomal 2.2 membranes were present but these features were accompanied by degenerative changes to membrane over '"ee text for details. the equatorial segment and post-acrosomal region. aNo. of semen samples. However, in all the sperm samples incubated with A23187, true acrosome-reacted spermatozoa could be identified by fusion and vesiculation of plasma and microscopy was greater in those samples incubated for outer acrosomal membrane over the apical sperm head; the longer time periods, although a quantitative analthe specific fusion of plasma membrane and outer ac- ysis was not performed. At the ultrastructural level 18.6, monoclonal antirosomal membrane a t the apical border of the equatorial segment and a contiguous plasma membrane over body was shown with immunoperoxidase labelling to the remaining sperm head (Fig. 5a,b). The number of bind to a n intracellular antigen associated mainly with acrosome-reacted spermatozoa observed by electron the outer acrosomal membranes (Fig. 6a,b), although
156
ZHANG ET AL. Fig. 4. Micrographs of zona-free hamster oocytes co-incubated with stallion spermatozoa. a: Oocyte with many hound spermatozoa and containing two decondensed sperm heads with associated tails. Lacmoid stain. x 300. b A swollen head at higher magnification. X 790. c: Zona-free hamster oocyte stained with 18.6 antibody showing fluorescent sperm hound to the oolemma.
some label was present on the inner acrosomal membrane of disrupted cells.
DISCUSSION A means of inducing and subsequently monitoring the acrosome reaction in equids would be of considerable benefit for the development of in vitro fertilization techniques in these animals. But difficulties of visualizing reliably the compact horse sperm acrosome either by light microscopy alone or in combination with histochemical staining techniques (nigrosin and eosin: Dott and Foster, 1972; triple stain: Talbot and Chacon, 1981) has retarded progress. The objective of the present study was to attempt to induce the acrosomereaction in stallion spermatozoa and to investigate whether acrosomal status can be monitored with specific monoclonal antibody. Immunofluorescent localization made the acrosomes of stallion spermatozoa clearly visible after fixation in methanol. This was because the sperm membranes had become more permeable, allowing antibody to gain access to the internally located antigen (see Moore et al., 1987). Although a t the ultrastructural level some antibody binding (identified by immunoperoxidase staining) was present on the inner acrosomal membrane of damaged spermatozoa (Fig. 61, this was not apparent by immunofluorescence. Hence, bright, even stain represented the full complement of acrosomal content and membranes, while a loss of fluorescence (of various degrees) denoted a breakdown of the acrosome. The clear difference in the pattern of fluorescence between semen samples from fertile stallions and the two samples from a n infertile stallion indicated a difference in acrosome morphology which had not been identified by conventional semen analysis. A lack of spermatozoa with intact acrosomes a t ejaculation may contribute to male infertility and definitive visualization of acrosome morphology could provide a rapid diagnosis of ejaculate quality; a similar conclusion was reached by Blach and co-workers (1988), who also assessed membrane integrity in stallion spermatozoa with a monoclonal antibody. Since only one infertile animal was tested, it is not yet possible to say how useful this test would be for predicting infertility in stallions. Although prolonged incubation of diluted spermatozoa for 6-12 hours did not appreciably alter sperm motility, the fraction of spermatozoa with intact acrosomes a s detected by bright fluorescence decreased markedly. Indirect evidence would suggest that this change was probably due to a degenerative process rather than induction of the acrosome reaction. Spermatozoa observed by electron microscopy often exhibited a general breakdown in their membranes, but spe-
STALLION SPERM ACROSOME REACTION
157
Fig. 5. Electron micrographs of a stallion sperm head after treatment with A23187. a: Vesiculation o f plasma and outer acrosomal membranes typical of the acrosome reaction (arrow indicates anterior border o f the equatorial segment). x 10,000. b: Higher power of the equatorial region showing intact plasma membrane. x 30,000.
Fig. 6. Electron micrograph of immunolocalization of monoclonal antibody binding using peroxidaseantiperoxidase. a: Reaction product is present over the acrosomal region. x 12,000. b Control spermatozoa without first antibody. x 12,000.
cific membrane fusion associated with a physiological acrosome reaction (Fig. 5) was not seen. Moreover, spermatozoa incubated for 6 or 12 hours failed to fuse and penetrate zona-free hamster eggs. A normal acrosome reaction would appear to be essential €or gamete fusion in mammals (Yanagimachi, 1988). Since
sperm viability was maintained during spermiegg incubations, a n absence of sperm penetration was indicative of a failure to induce a true acrosome reaction. In contrast, the decline in immunofluorescence over the acrosomes of spermatozoa treated with A23187 calcium ionophore (Fig. 3) was probably associated with a
158
ZHANG ET AL.
true rather than a false acrosome reaction. In other species, treatment of spermatozoa with A23187 provides a way of producing a synchronous acrosome-reacted population (Shams-Borhan and Harrison, 1981; Topfer-Peterson et al., 1988). With stallion spermatozoa, the fading of fluorescence over the acrosome over a relatively short time span (60-90 minutes to reach maximum) was reminiscent of similar changes to fluorescence seen in human spermatozoa during in vitro culture, leading to a spontaneous acrosome reaction (Moore et al., 1987). Of more significance, stallion spermatozoa treated with A23187 displayed physiological acrosome reactions under the electron microscope and penetrated zona-free hamster eggs. With respect to the latter, a n unusual feature was the binding to the egg membrane of sperm with uniform bright fluorescence over the apical head. In other species, only acrosome reacted spermatozoa normally attach to the oolemma (Talbot and Chacon, 1982; Moore et al., 1987). In conclusion, we have demonstrated that the acrosome reaction of stallion spermatozoa could be induced by a short incubation with calcium ionophore A23187 and that this process was inversely correlated with immunofluorescent visualization of the acrosome with specific monoclonal antibody. Degenerative changes to the acrosome associated with prolonged incubation also altered immunofluorescent staining patterns. Therefore care must be taken that under different conditions such antibody localization techniques reflect the kinetics of the true acrosome reaction. The ability to assess rapidly the acrosome status of stallion spermatozoa will aid in the development of in vitro fertilization techniques. Furthermore, i t should prove useful for testing semen quality at ejaculation and after cryopreservation.
ACKNOWLEDGMENTS Work at the Equine Fertility Unit was supported by the Thoroughbred Breeder’s Association and the Horserace Betting Levy Board. The work was also supported in part by a programme grant to H.D.M.M. from
the Medical Research Council and Agricultural and Food Research Council.
REFERENCES Blach EL, Amann RP, Bowen RA, Sawyer HR, Hermenet MJ (1988) Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion spermatozoa. Gamete Res 21233-241. Dott HM, Foster GC (1972) A technique for studying the morphology of mammalian spermatozoa which are eosinophilic in a differential live/dead stain. J Reprod Fertil 29443-445. Ellis DH, Hartman TD, Moore HDM (1985) Maturation and function of the hamster spermatozoa probed with monoclonal antibodies. J Reprod Immunol 7:299-314. Graham JK, Foote RH, Parrish JJ (1986) Effect of dilauroylphosphatidylcholine on the acrosome reaction and subsequent penetration of hull spermatozoa into zona-free hamster eggs. Biol Reprod 35413-424. Moore HDM, Bedford JM (1983): The interaction of mammalian gametes in the female. In JF Hartmann (ed):“Mechanism and Control of Animal Fertilization.” New York: Academic Press, pp 435-497. Moore HDM, Hartman TD (1984) Localization by monoclonal antibodies of various surface antigens on hamster spermatozoa and effect of antibody on fertilization in uitro. J Reprod Fertil70:175-183. Moore HDM, Smith CA, Hartman TD, Bye AP (1987) Visualization and characterization of the acrosome reaction of human spermatozoa by immunolocalization with monoclonal antibody. Gamete Res 17245-259. Shams-Borhan G, Harrison RAP (1981) Production, characterization and use of ionophore-induced calcium dependent acrosome reaction in ram spermatozoa. Gamete Res 4:417-432. Smith CA, Hartman TD, Moore HDM (1986) A determinant of Mr 34000 expressed by hamster epididymal epithelium binds specifically to spermatozoa in co-culture. J Reprod Fertil 78:337-345. Talbot P, Chacon RS (1981) A triple-stain technique for evaluating normal acrosome reactions of human sperm. J Exp Zoo1 215201208. Talbot P, Chacon RS (1982) Ultrastructural observations on binding and membrane fusion between human sperm and zona pellucidafree hamster oocytes. Fertil Steril 37:240-248. Topfer-Peterson E, Friess AE, Schill WB (1988) The acrosome reaction in boar spermatozoa. Human Reprod 3:319-326. Voss JL, Pickett BW, Squires EL (1981) Stallion spermatozoa1 morphology and motility and their relationship to fertility. J Am Vet Med Assoc 178287-289. Wolf DP, Boldt J , Byrd W, Bechtol KB (1985) Acrosomal status evaluation in human ejaculate sperm with monoclonal antibodies. Biol Reprod 32:1157-1 162. Yanagimachi R (1988): Mammalian fertilization. In E Knobil and J Neil (eds): “Physiology of Reproduction.“ New York: Raven Press, pp 135-185.
