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Simultaneous detection of the acrosomal status and viability of incubated ram spermatozoa using fluorescent markers
REAt!isiION SCIENCE
ELSEVIER
Animal Reproduction
Science 46 (1997) 89-96
Simultaneous detection of the acrosomal status and viability of incubated ram spermatozoa using fluorescent markers S. Sukardi, M.R. Curry, P.F. Watson Depurtment
*
of Veterinary Basic Sciences, Royal Veterinnry College, Royul College Street, London NW1 OTU, UK Accepted 25 June 1996
Abstract Incubation of diluted ram spermatozoa at 39°C results in a high percentage of acrosome reactions, but previously we have not been able to demonstrate the viability of these cells. Detection of the viability and stages of acrosomal exocytosis, either spontaneous or induced, was carried out using fluorescent probes. Propidium iodide (PI) was used to determine cell viability and, simultaneously, FITC-Pisum satiuum lectin (FITC-PSA) was used to assess acrosomal status by staining glycoproteins in the acrosome of permeabilised spermatozoa. Diluted ram semen was incubated for 6 hours at 39°C. At 2 hourly intervals, samples were taken and examined for evidence of a spontaneous acrosome reaction. In addition, calcium ionophore A23187 was used to induce the acrosome reaction and samples were examined at 10 minute intervals. PI was added and then washed out by filtration. Smears were made and air-dried, permeabilised with absolute ethanol and then stained with FITC-PSA. The slides were later viewed under the fluorescence microscope with a peak excitation wavelength of 488 nm. With this combination of two fluorescent probes using a single excitation wavelength, both the cell viability and the acrosomal status could be simultaneously and easily visualized. Results showed four categories of staining: PI-ve/PSA + ve (Live and acrosome-intact), PI + ve/PSA + ve (dead and acrosome-intact), PI - ve/PSA - ve (live and acrosome-reacted) and PI + ve/PSA - ve (dead and acrosome-degenerated). About 75% spermatozoa that were acrosome-reacted were still viable after 4h incubation in the absence of ionophore, and approximately 90% spermatozoa were acrosome-reacted and still viable after 30min incubation in the presence of ionophore. Keywords: Ram spermatozoa; Acrosome tinin; Viability
* Corresponding
Propidium
author. Tel: M_(O)17 l-468 5235. Fax: 44-(0)171-388
0378-4320/97/$17.00 PII
reaction; Incubation;
SO378-4320(96)01599-O
Copyright
iodide; FITC-pisum
sativum a&-
1027. E-mail: [email protected]
0 1997 Elsevier Science B.V. All rights reserved.
S. Sukardi et al./Animal Reproduction Science 46 (1997) 89-96
90
1. Introduction The acrosome reaction is an exocytotic reaction involving multiple sites of fusion
between the outer acrosomal membrane and the overlying plasma membrane with release of acrosomal enzymes required for fertilization (reviewed by Yanagimachi, 1994). The acrosome reaction occurs in response to natural inducers, i.e., the zona pellucida, oviductal fluids (Yanagimachi, 1994), but the reaction can also be artificially induced by a variety of substances, calcium ionophore A23187 being one of the most common (Jamil and White, 1981; Shams-Borhan and Harrison, 1981; Flechon et al., 1986; Pampiglione et al., 1993; Troup et al., 1994). Fresh ram spermatozoa will spontaneously undergo the acrosome reaction when they are diluted and incubated at 39°C over a period of 4h in the absence of any inducing agent (Watson et al., 1991). When artificially induced with calcium ionophore, up to 90% of spermatozoa undergo acrosome reactions within 30min (Watson et al., 1991). The membrane fusion that occurs in the spontaneous acrosome reaction is observed morphologically to resemble a true acrosome reaction and to differ from the ionophoreinduced reaction in terms of vesicle size and distribution (Watson et al., 1992). Spermatozoa spontaneously undergoing the acrosome reaction retained motility (Watson et al., 1991) but the limiting membrane integrity of these acrosome-reacting spermatozoa is important because it is recognised that degenerating sperm can also show similar acrosomal changes (Bedford, 1970; Mendoza et al., 1992). As the ram sperm acrosome is smaller than that of the hamster and guinea pig, the acrosome cannot be directly visualized wihout a specific stain. Fluorescein-conjugated plant lectins like fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITCPSA) from the edible pea has been used as a selective acrosomal staining of the spermatozoa of human (Cross et al., 1986; Mendoza et al., 1992; Tesarik et al., 1993), stallion (Farlin et al., 1992; Casey et al., 1993) and monkey (Cross et al., 1989). This lectin shows an affinity for terminal alpha-D-glucosyl and alpha-D-mannosyl residues of glycoproteins (Trowbridge, 1974) and binds specifically to the sugar alpha-mannoside which is found in the acrosomal contents (Cross et al., 1986). Sperm viability was measured with propidium iodide (PI), a nucleic acid-specific fluorophore which stains dead spermatozoa and is relatively impermeant (Garner et al., 1986), i.e. it is excluded by cells with intact limiting membranes. As PI and PSA have the same peak excitation wavelength of 488nm (Centola et al., 1990), simultaneous detection of the viability and acrosomal status using fluorescence microscopy is easily carried out. Combination of these fluorophores has been reported in earlier work with human spermatozoa using fluorescence microscopy (Centola et al., 1990) and flow cytometry (Miyazaki et al., 1990; Henley et al., 1994). This is the first study with ram spermatozoa. 2. Materials and methods Fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), propidium iodide (PI) and all chemicals used in the experiment were of analytical grade and purchased from Sigma Chemical Co. Ltd (Poole, Dorset, UK)
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Semen was collected from trained Friesland rams housed at the Royal Veterinary College using the artificial vagina. Individual ejaculates were collected and diluted with HEPES Glucose buffer (139mM NaCl, 2.5 mM KCl, 10 mM glucose, 20mM HEPES and 3.0mM CaCl,, pH 7.4, Shams-Borhan and Harrison, 1981). The final concentration of spermatozoa used for the study was 10’ spermatozoaml- ’. 2.1. Induced acrosome reaction Diluted fresh ram spermatozoa were induced to undergo the acrosome reaction with calcium ionophore A23 187 at 39°C as described by Watson et al. (1991). Briefly, calcium ionophore A23187 dissolved in dimethyl sulphoxide was added to a diluted suspension of spermatozoa (10’ spermatozoa ml- ’) at a final concentration of 1 pM A23187 and 1% DMSO (Shams-Borhan and Harrison, 1981) and incubation at 39°C commenced. Subsamples were taken for fluorescent labelling after 13 min and 28 min after ionophore addition. 2.2. Spontaneous acrosome reaction For the spontaneous acrosome reaction, diluted fresh ram spermatozoa were incubated for 6 hours without ionophore and subsamples taken for fluorescent labelling at 2, 4 and 6h (Watson et al., 1991). 2.3. Fluorescent staining Propidium iodide (PI), at 10 pgml- ’ final concentration, was added to the sample for 2 min. Excess PI was removed by diluting the suspension ten-fold with fresh HEPES buffer as follows. The whole suspension (1 ml) was drawn into a lOm1 plastic syringe, followed by 9ml of fresh HEPES buffer, thus mixing and diluting the suspension. A millipore filter (0.45 km - Millipore UK Ltd, Watford, Hertfordshire, UK) was fixed to the syringe and the mixture was carefully filtered to the original volume. Cells deposited on the filter membrane were recovered by gently flushing back and forth across the filter several times with the remaining 1 ml suspension. Approximately 85% of the total were recovered using this technique. A 20 p,l of supension was then smeared on a clean glass slide and air-dried. Three replicate slides were made for each subsample. Spermatozoa were permeabilised by flooding the slides with 100% ethanol for 5 min. Excess ethanol was removed by washing slides with Phosphate buffer saline (PBS - 160mM NaCl, 8mM Na,HPO,, 2mM NaH,P0,.2H,O, pH 7.4). The slides were then flooded with FITC-PSA at 40 p_gml-’ in HEPES buffer and kept in the dark for 10 min. The slides were then agitated in deionized water to remove excess FITC-PSA and drained. A drop of p-phenylenediamine (0.1% in 9: 1 Glycero1:PB.S) was placed on the slide to enhance fluorescence, a coverslip added and the edges sealed with nail varnish. Slides were viewed within 2 h under an Olympus BH-2 microscope equipped
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Science 46 (1997) 89-96
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pisum sativum agglutinin (green)
m propidium iodide (red) 0
no fluorescence
Fig. 1. Categories of acrosomal status and sperm viability detected with Pisum satiuum agglutinin (PSA) and propidium iodide (PI). AIL - Acrosome intact, live sperm, AID - acrosome intact, dead sperm, ARL acrosome reacted live sperm, ARD - acrosome reacted dead sperm.
with standard FITC filter set at 400X magnification. A total of 200 cells were counted for each slide.
3. Results Spermatozoa were allocated to one of four groups on the basis of their PSA and PI staining patterns (Fig. 1): 1. PSA positive and PI negative - Acrosome intact live (AIL) 2. PSA positive and PI positive - Acrosome intact dead (AID)
0
10
20
30
Time abler addition of ionophore (min) Fig. 2. Percentage viability and acrosomal status of fresh ram spermatozoa during the ionophore-induced acrosome reaction. AIL - Acrosome intact, live sperm, AID - acrosome intact, dead sperm, ARL acrosome reacted live sperm, ARD - acrosome reacted dead sperm.
S. Sukardi
et ul./Animal
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0
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Time of incubation (h) Fig. 3. Percentage viability and acrosomal status of fresh ram spermatozoa during the spontaneous acrosome reaction. AIL - Acrosome intact, live sperm, AID - acrosome intact, dead sperm, ARL acrosome reacted live sperm, ARD - acrosome reacted dead sperm.
3. PSA negative and PI negative - Acrosome reacted live CARL) 4. PSA negative and PI positive - Acrosome degenerate dead CARD) Various staining patterns and intensity were observed. Cells which retained staining of the equatorial segment were considered fully acrosome-reacted. They were grouped together with cells totally devoid of PSA staining in the head region. Cells that showed a patchy fluorescence were grouped as acrosome-intact since the majority of the acrosoma1 contents was still in situ, although membrane fusion had probably commenced. In the presence of ionophore, the proportion of live acrosome-reacted spermatozoa steadily increased to approximately 90% of the total over a period of 30min. The live acrosome-intact cells decreased in number correspondingly over the same period (Fig. 2). The numbers of dead cells did not change significantly. However, in the absence of ionophore, there was a steady increase in cells which were acrosome-reacted while remaining viable reaching about 75% of the total after 4 h incubation (Fig. 3). By 6 h, there was a dramatic increase in dead acrosome-reacted cells, indicating the loss of barrier function of the plasma membrane in a high proportion of these cells. These cells were totally devoid of PSA staining.
4. Discussion The PSA staining patterns observed with ram spermatozoa corresponded to the patterns previously reported for human spermatozoa (Cross et al., 1986; Centola et al., 1990) but with additional weak staining of the midpiece. As PSA binds to the acrosomal contents, the progress of the acrosome reaction is indicated by the intensity and
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distribution of fluorescence over the acrosomal region. The acrosome reaction in ram spermatozoa commences just anterior to the equatorial segment and proceeds in an arborizing fashion towards the apical ridge (Flechon, 1985; Watson and Piummer, 1985). Therefore, as the reaction progresses, more acrosomal contents will be lost and, thercfore, less fluorescence will be seen. Staining only in the equatorial segment is also characteristic of a cell that has only recently completed its acrosome reaction whereas cells devoid of staining in this region have fully completed the acrosome reaction some time previously (Tesarik et al., 1993). The use of FITC-PSA enables easy visualization of acrosomes in mammalian species in which other means are more problematic. Pisum satiuum agglutinin has been widely used in mammalian spermatozoa to detect acrosomal status as it gives essentially the same staining patterns as an antiserum to the acrosomal enzyme acrosin (Cross et al., 1986). In our experience, the lectin was particularly useful in showing the various stages of the acrosome reaction by the degree and distribution of staining. The viability of the cells was readily detected with PI which was chosen because both PI and FITC-PSA are excited at the same wavelength of 488nm (Centola et al., 1990) making it simpler to detect the staining pattern under the fluorescence microscope. Propidium iodide in the external medium must be removed or diluted before permeabilising viable cells for PSA staining. Repeated centrifugation serves to stress the cell and can cause damage to intact acrosomes thus distorting the numbers of cells in various classes. Moreover, PI is only loosely bound to the nucleic acid and may be removed by washing procedures (Riedy et al., 1991). The method of gently removing excess PI from the solution by filtering through a syringe filter appeared not to stress the cell membranes and proved satisfactory to reduce the concentration. We found that the amount of PI remaining bound to the nucleic acid was quite sufficient for counting dead cells. The use of the filter also reduced the remaining concentration of the ionophore and seminal plasma. The recovery of cells was sufficient and did not apparently grossly distort the distribution of staining patterns. Fresh ram spermatozoa will spontaneously undergo the acrosome reaction when incubated at 39°C for up to 4h in buffer (Watson et al., 1991). Our results confirmed that the majority of these cells were indeed both acrosome-reacted and viable. We have previously reported that such cells are also motile (Watson et al., 1991) However the population of acrosome-reacted viable cells declined with further incubation indicating the cells have only a limited life span in culture after completing the acrosome reaction, a finding which supports other studies of acrosome-reacted spermatozoa (Chang and Hunter, 1975; Hunter, 1989). Results from this study suggest that cells remain viable for no more than 2-4h after the acrosome reaction occurs. With ionophore, 90% were acrosome-reacted and still viable after 30min incubation, suggesting that the ionophoreinduced acrosome reaction occurs in viable cells and that 1 p,M A23187 is compatible with survival in the short-term. The results obtained support our recent work that the spontaneous acrosome reaction of ram spermatozoa resembles a true acrosome reaction (Watson et al., 1992) and is not a degenerative change occurring in dead cells. We suggest it can be used as a model of the physiological acrosome reaction which occurs in the vicinity of the oocyte at fertilization.
S. Sukardi et al./Animal Reproduction Science 46 (1997) 89-96
9.5
Acknowledgements The authors wish to thank Mr. John Millar and Ms. Angie Poole for technical help. S. Sukardi is a recipient of a Malaysian Federal Government scholarship and is attached to Universiti Pertanian Malaysia.
References Bedford, J.M., 1970. Sperm capacitation and fertilization in mammals. Biol. Reprod. 2, suppl. 2: 128-158. Casey, P.J., Hillman, R.B., Robertson, K.R., Yudin, A.I., Liu, I.K. and Drobnis, E.Z., 1993. Validation of an acrosomal stain for equine sperm that differentiates between living and dead sperm. J. Androl., 14: 289-297. Centola, G.M., Mattox, J.H., Burde, S. and Leary, J.F., 1990. Assessment of the viability and acrosome status of fresh and frozen-thawed human spermatozoa using single wavelength fluorescence microscopy. Mol. Reprod. Dev., 27: 130-135. Chang, M.C. and Hunter, R.H.F., 1975. Capacitation of mammalian sperm: biological and experimental aspects. In: D.W. Hamilton and R.O. Greep (Eds), Handbook of Physiology, Section 7: Endocrinology, Vol. V. American Physiological Society: Washington D.C., pp: 339-351. Cross, N., Morales, P., Overstreet, J. and Hanson, F., 1986. Two simple methods for detecting acrosome-reacted human sperm. Gamete Res., 15: 213-226. Cross, N.L., Morales, P., Fukuda, M. and Behboodi, E., 1989. Determining acrosomal status of the cynomolgus monkey (Macaca jbscicularis) sperm by fluorescence microscopy. Am. J. Primat., 17: 157-163. Farlin, M.E., Jasko, D.J., Graham, J.K. and Squires, E.L., 1992. Assessment of Pisum satiuum agglutinin in identifying acrosomal damage in stallion spermatozoa. Mol. Reprod. Dev., 32: 23-27. Flechon, J.E., 1985. Sperm surface changes during the acrosome reaction as observed by freeze-fracture. Am. J. Anat., 174: 239-248. Flechon, J.E., Harrison, R.A., Flechon, B. and Escaig, J., 1986. Membrane fusion events in the Ca*+/ionophoreinduced acrosome reaction of ram spermatozoa. J. Cell Sci., 81: 43-63. Garner, D.L., Pinkel, D., Johnson, L.A. and Pace, M.M., 1986. Assessment of spermatozoa1 function using dual fluorescent staining and flow cytometric analyses. Biol. Reprod., 34: 127- 138. Henley, N., Baron, C. and Roberts, K.D., 1994. Flow cytometric evaluation of the acrosome reaction of human spermatozoa: a new method using a photoactivated supravital stain. Int. J. Androl., 17: 78-84. Hunter, R.H.F., 1989. Ovarian programming of gamete progression and maturation in the female genital tract. 2001. J. Linn. Sot., 95: 117- 124. Jamil. K. and White, I.G.. 1981. Induction of acrosomal reaction in sperm with ionophore A23187 and calcium. Arch. Androl., 7: 283-292. Mendoza, C., Carreras, A., Moos, J. and Tesarik, J., 1992. Distinction between true acrosome reaction and degenerative acrosome loss by a one-step staining method using Pisum satiuum agglutinin. J. Reprod. Fert., 95: 755-763. Miyazaki, R., Fukuda, M., Takeuchi, H., Itoh, S. and Takada. M., 1990. Flow cytometry to evaluate acrosome-reacted sperm. Arch. Androl., 25: 243-25 1. Pampiglione, J.S., Tan, S.L. and Campbell, S., 1993. The use of the stimulated acrosome reaction test as a test of fertilizing ability in human spermatozoa. Fe&l. Steril., 59: 1280- 1284. Riedy, MC., Muirhead, K.A., Jensen, C.P. and Stewart, CC., 1991. Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations. Cytometry, 12: 1x3- 139. Shams-Borhan G. and Harrison, R., 1981. Production, characterization, and use of ionophore-induced, calcium-dependent acrosome reaction in ram spermatozoa. Gamete Res., 4: 407-432. Tesarik, J., Mendoza, C. and Carreras, A., 1993. Fast acrosome reaction measure: a highly sensitive method for evaluating stimulus-induced acrosome reaction. Fertil. Steril., 59: 424-430.
%
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Troup, S.A., Lieberman, B.A. and Watson, P.L., 1994. The acrosome reaction to ionophore challenge test assay reproducibility, effect of sexual abstinence and results for fertile men. Human Reprod., 9: 2079-2083. Trowbridge, I.S., 1974. Isolation and chemical characterization of a mitogenic lectin from Pisum satiuum. J. Biol. Chem., 249: 6004-6012. Watson, P.F. and Plummer, J.M., 1986. Relationship between calcium binding sites and membrane fusion during the acrosome reaction induced by ionophore in ram spermatozoa. J. Exp. Zool., 238: 113-l 18. Watson, P.F., Jones, P.S. and Plummer, J.M., 1991. A quantitative comparison of the spontaneous and ionophore-induced acrosome reaction in ejaculated ram spermatozoa: the effects of temperature, time and individual. Anim. Reprod. Sci., 24: 93-108. Watson, P.F., Phtmmer, J.M. and Jones, P.S., 1992. The ionophore-induced acrosome reaction differs structurally from the spontaneous acrosome reaction. 1. Exp. Zool., 264: 231-235. Yanagimachi, R., 1994. Mammalian fertilization. In, E. Knobil and J. Neil1 (Eds). The Physiology of Reproduction. Vol. 1. Raven press Ltd. New York. pp. 189-317.
ELSEVIER
Animal Reproduction
Science 46 (1997) 89-96
Simultaneous detection of the acrosomal status and viability of incubated ram spermatozoa using fluorescent markers S. Sukardi, M.R. Curry, P.F. Watson Depurtment
*
of Veterinary Basic Sciences, Royal Veterinnry College, Royul College Street, London NW1 OTU, UK Accepted 25 June 1996
Abstract Incubation of diluted ram spermatozoa at 39°C results in a high percentage of acrosome reactions, but previously we have not been able to demonstrate the viability of these cells. Detection of the viability and stages of acrosomal exocytosis, either spontaneous or induced, was carried out using fluorescent probes. Propidium iodide (PI) was used to determine cell viability and, simultaneously, FITC-Pisum satiuum lectin (FITC-PSA) was used to assess acrosomal status by staining glycoproteins in the acrosome of permeabilised spermatozoa. Diluted ram semen was incubated for 6 hours at 39°C. At 2 hourly intervals, samples were taken and examined for evidence of a spontaneous acrosome reaction. In addition, calcium ionophore A23187 was used to induce the acrosome reaction and samples were examined at 10 minute intervals. PI was added and then washed out by filtration. Smears were made and air-dried, permeabilised with absolute ethanol and then stained with FITC-PSA. The slides were later viewed under the fluorescence microscope with a peak excitation wavelength of 488 nm. With this combination of two fluorescent probes using a single excitation wavelength, both the cell viability and the acrosomal status could be simultaneously and easily visualized. Results showed four categories of staining: PI-ve/PSA + ve (Live and acrosome-intact), PI + ve/PSA + ve (dead and acrosome-intact), PI - ve/PSA - ve (live and acrosome-reacted) and PI + ve/PSA - ve (dead and acrosome-degenerated). About 75% spermatozoa that were acrosome-reacted were still viable after 4h incubation in the absence of ionophore, and approximately 90% spermatozoa were acrosome-reacted and still viable after 30min incubation in the presence of ionophore. Keywords: Ram spermatozoa; Acrosome tinin; Viability
* Corresponding
Propidium
author. Tel: M_(O)17 l-468 5235. Fax: 44-(0)171-388
0378-4320/97/$17.00 PII
reaction; Incubation;
SO378-4320(96)01599-O
Copyright
iodide; FITC-pisum
sativum a&-
1027. E-mail: [email protected]
0 1997 Elsevier Science B.V. All rights reserved.
S. Sukardi et al./Animal Reproduction Science 46 (1997) 89-96
90
1. Introduction The acrosome reaction is an exocytotic reaction involving multiple sites of fusion
between the outer acrosomal membrane and the overlying plasma membrane with release of acrosomal enzymes required for fertilization (reviewed by Yanagimachi, 1994). The acrosome reaction occurs in response to natural inducers, i.e., the zona pellucida, oviductal fluids (Yanagimachi, 1994), but the reaction can also be artificially induced by a variety of substances, calcium ionophore A23187 being one of the most common (Jamil and White, 1981; Shams-Borhan and Harrison, 1981; Flechon et al., 1986; Pampiglione et al., 1993; Troup et al., 1994). Fresh ram spermatozoa will spontaneously undergo the acrosome reaction when they are diluted and incubated at 39°C over a period of 4h in the absence of any inducing agent (Watson et al., 1991). When artificially induced with calcium ionophore, up to 90% of spermatozoa undergo acrosome reactions within 30min (Watson et al., 1991). The membrane fusion that occurs in the spontaneous acrosome reaction is observed morphologically to resemble a true acrosome reaction and to differ from the ionophoreinduced reaction in terms of vesicle size and distribution (Watson et al., 1992). Spermatozoa spontaneously undergoing the acrosome reaction retained motility (Watson et al., 1991) but the limiting membrane integrity of these acrosome-reacting spermatozoa is important because it is recognised that degenerating sperm can also show similar acrosomal changes (Bedford, 1970; Mendoza et al., 1992). As the ram sperm acrosome is smaller than that of the hamster and guinea pig, the acrosome cannot be directly visualized wihout a specific stain. Fluorescein-conjugated plant lectins like fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITCPSA) from the edible pea has been used as a selective acrosomal staining of the spermatozoa of human (Cross et al., 1986; Mendoza et al., 1992; Tesarik et al., 1993), stallion (Farlin et al., 1992; Casey et al., 1993) and monkey (Cross et al., 1989). This lectin shows an affinity for terminal alpha-D-glucosyl and alpha-D-mannosyl residues of glycoproteins (Trowbridge, 1974) and binds specifically to the sugar alpha-mannoside which is found in the acrosomal contents (Cross et al., 1986). Sperm viability was measured with propidium iodide (PI), a nucleic acid-specific fluorophore which stains dead spermatozoa and is relatively impermeant (Garner et al., 1986), i.e. it is excluded by cells with intact limiting membranes. As PI and PSA have the same peak excitation wavelength of 488nm (Centola et al., 1990), simultaneous detection of the viability and acrosomal status using fluorescence microscopy is easily carried out. Combination of these fluorophores has been reported in earlier work with human spermatozoa using fluorescence microscopy (Centola et al., 1990) and flow cytometry (Miyazaki et al., 1990; Henley et al., 1994). This is the first study with ram spermatozoa. 2. Materials and methods Fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), propidium iodide (PI) and all chemicals used in the experiment were of analytical grade and purchased from Sigma Chemical Co. Ltd (Poole, Dorset, UK)
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Semen was collected from trained Friesland rams housed at the Royal Veterinary College using the artificial vagina. Individual ejaculates were collected and diluted with HEPES Glucose buffer (139mM NaCl, 2.5 mM KCl, 10 mM glucose, 20mM HEPES and 3.0mM CaCl,, pH 7.4, Shams-Borhan and Harrison, 1981). The final concentration of spermatozoa used for the study was 10’ spermatozoaml- ’. 2.1. Induced acrosome reaction Diluted fresh ram spermatozoa were induced to undergo the acrosome reaction with calcium ionophore A23 187 at 39°C as described by Watson et al. (1991). Briefly, calcium ionophore A23187 dissolved in dimethyl sulphoxide was added to a diluted suspension of spermatozoa (10’ spermatozoa ml- ’) at a final concentration of 1 pM A23187 and 1% DMSO (Shams-Borhan and Harrison, 1981) and incubation at 39°C commenced. Subsamples were taken for fluorescent labelling after 13 min and 28 min after ionophore addition. 2.2. Spontaneous acrosome reaction For the spontaneous acrosome reaction, diluted fresh ram spermatozoa were incubated for 6 hours without ionophore and subsamples taken for fluorescent labelling at 2, 4 and 6h (Watson et al., 1991). 2.3. Fluorescent staining Propidium iodide (PI), at 10 pgml- ’ final concentration, was added to the sample for 2 min. Excess PI was removed by diluting the suspension ten-fold with fresh HEPES buffer as follows. The whole suspension (1 ml) was drawn into a lOm1 plastic syringe, followed by 9ml of fresh HEPES buffer, thus mixing and diluting the suspension. A millipore filter (0.45 km - Millipore UK Ltd, Watford, Hertfordshire, UK) was fixed to the syringe and the mixture was carefully filtered to the original volume. Cells deposited on the filter membrane were recovered by gently flushing back and forth across the filter several times with the remaining 1 ml suspension. Approximately 85% of the total were recovered using this technique. A 20 p,l of supension was then smeared on a clean glass slide and air-dried. Three replicate slides were made for each subsample. Spermatozoa were permeabilised by flooding the slides with 100% ethanol for 5 min. Excess ethanol was removed by washing slides with Phosphate buffer saline (PBS - 160mM NaCl, 8mM Na,HPO,, 2mM NaH,P0,.2H,O, pH 7.4). The slides were then flooded with FITC-PSA at 40 p_gml-’ in HEPES buffer and kept in the dark for 10 min. The slides were then agitated in deionized water to remove excess FITC-PSA and drained. A drop of p-phenylenediamine (0.1% in 9: 1 Glycero1:PB.S) was placed on the slide to enhance fluorescence, a coverslip added and the edges sealed with nail varnish. Slides were viewed within 2 h under an Olympus BH-2 microscope equipped
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pisum sativum agglutinin (green)
m propidium iodide (red) 0
no fluorescence
Fig. 1. Categories of acrosomal status and sperm viability detected with Pisum satiuum agglutinin (PSA) and propidium iodide (PI). AIL - Acrosome intact, live sperm, AID - acrosome intact, dead sperm, ARL acrosome reacted live sperm, ARD - acrosome reacted dead sperm.
with standard FITC filter set at 400X magnification. A total of 200 cells were counted for each slide.
3. Results Spermatozoa were allocated to one of four groups on the basis of their PSA and PI staining patterns (Fig. 1): 1. PSA positive and PI negative - Acrosome intact live (AIL) 2. PSA positive and PI positive - Acrosome intact dead (AID)
0
10
20
30
Time abler addition of ionophore (min) Fig. 2. Percentage viability and acrosomal status of fresh ram spermatozoa during the ionophore-induced acrosome reaction. AIL - Acrosome intact, live sperm, AID - acrosome intact, dead sperm, ARL acrosome reacted live sperm, ARD - acrosome reacted dead sperm.
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et ul./Animal
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0
2
4
6
Time of incubation (h) Fig. 3. Percentage viability and acrosomal status of fresh ram spermatozoa during the spontaneous acrosome reaction. AIL - Acrosome intact, live sperm, AID - acrosome intact, dead sperm, ARL acrosome reacted live sperm, ARD - acrosome reacted dead sperm.
3. PSA negative and PI negative - Acrosome reacted live CARL) 4. PSA negative and PI positive - Acrosome degenerate dead CARD) Various staining patterns and intensity were observed. Cells which retained staining of the equatorial segment were considered fully acrosome-reacted. They were grouped together with cells totally devoid of PSA staining in the head region. Cells that showed a patchy fluorescence were grouped as acrosome-intact since the majority of the acrosoma1 contents was still in situ, although membrane fusion had probably commenced. In the presence of ionophore, the proportion of live acrosome-reacted spermatozoa steadily increased to approximately 90% of the total over a period of 30min. The live acrosome-intact cells decreased in number correspondingly over the same period (Fig. 2). The numbers of dead cells did not change significantly. However, in the absence of ionophore, there was a steady increase in cells which were acrosome-reacted while remaining viable reaching about 75% of the total after 4 h incubation (Fig. 3). By 6 h, there was a dramatic increase in dead acrosome-reacted cells, indicating the loss of barrier function of the plasma membrane in a high proportion of these cells. These cells were totally devoid of PSA staining.
4. Discussion The PSA staining patterns observed with ram spermatozoa corresponded to the patterns previously reported for human spermatozoa (Cross et al., 1986; Centola et al., 1990) but with additional weak staining of the midpiece. As PSA binds to the acrosomal contents, the progress of the acrosome reaction is indicated by the intensity and
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distribution of fluorescence over the acrosomal region. The acrosome reaction in ram spermatozoa commences just anterior to the equatorial segment and proceeds in an arborizing fashion towards the apical ridge (Flechon, 1985; Watson and Piummer, 1985). Therefore, as the reaction progresses, more acrosomal contents will be lost and, thercfore, less fluorescence will be seen. Staining only in the equatorial segment is also characteristic of a cell that has only recently completed its acrosome reaction whereas cells devoid of staining in this region have fully completed the acrosome reaction some time previously (Tesarik et al., 1993). The use of FITC-PSA enables easy visualization of acrosomes in mammalian species in which other means are more problematic. Pisum satiuum agglutinin has been widely used in mammalian spermatozoa to detect acrosomal status as it gives essentially the same staining patterns as an antiserum to the acrosomal enzyme acrosin (Cross et al., 1986). In our experience, the lectin was particularly useful in showing the various stages of the acrosome reaction by the degree and distribution of staining. The viability of the cells was readily detected with PI which was chosen because both PI and FITC-PSA are excited at the same wavelength of 488nm (Centola et al., 1990) making it simpler to detect the staining pattern under the fluorescence microscope. Propidium iodide in the external medium must be removed or diluted before permeabilising viable cells for PSA staining. Repeated centrifugation serves to stress the cell and can cause damage to intact acrosomes thus distorting the numbers of cells in various classes. Moreover, PI is only loosely bound to the nucleic acid and may be removed by washing procedures (Riedy et al., 1991). The method of gently removing excess PI from the solution by filtering through a syringe filter appeared not to stress the cell membranes and proved satisfactory to reduce the concentration. We found that the amount of PI remaining bound to the nucleic acid was quite sufficient for counting dead cells. The use of the filter also reduced the remaining concentration of the ionophore and seminal plasma. The recovery of cells was sufficient and did not apparently grossly distort the distribution of staining patterns. Fresh ram spermatozoa will spontaneously undergo the acrosome reaction when incubated at 39°C for up to 4h in buffer (Watson et al., 1991). Our results confirmed that the majority of these cells were indeed both acrosome-reacted and viable. We have previously reported that such cells are also motile (Watson et al., 1991) However the population of acrosome-reacted viable cells declined with further incubation indicating the cells have only a limited life span in culture after completing the acrosome reaction, a finding which supports other studies of acrosome-reacted spermatozoa (Chang and Hunter, 1975; Hunter, 1989). Results from this study suggest that cells remain viable for no more than 2-4h after the acrosome reaction occurs. With ionophore, 90% were acrosome-reacted and still viable after 30min incubation, suggesting that the ionophoreinduced acrosome reaction occurs in viable cells and that 1 p,M A23187 is compatible with survival in the short-term. The results obtained support our recent work that the spontaneous acrosome reaction of ram spermatozoa resembles a true acrosome reaction (Watson et al., 1992) and is not a degenerative change occurring in dead cells. We suggest it can be used as a model of the physiological acrosome reaction which occurs in the vicinity of the oocyte at fertilization.
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9.5
Acknowledgements The authors wish to thank Mr. John Millar and Ms. Angie Poole for technical help. S. Sukardi is a recipient of a Malaysian Federal Government scholarship and is attached to Universiti Pertanian Malaysia.
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