ANDROLOCIA
24, 155- 159 ( 1992)
ACCEPTED: JANUARY 22, 1992
Loss of acid phosphatase from rat spermatozoa as a method for assessing the acrosome reaction Z. Salzberger, L. M. Lewin and R. Shalgi'
I
Key words. Spermatozoa - acid phosphatase - acrosome
Summary. A method is presented for evaluating the extent of the acrosome reaction by measuring the release of acrosomal acid phosphatase from rat spermatozoa during incubation under capacitating conditions. Treatment of spermatozoa with lysophosphatidylcholine or Triton X- 100 released the acid phosphatase from the sperm cell. Using this enzymatic method we could not detect an alteration in enzyme activity following 5 h incubation under capacitating conditions. The effect of in vitro capacitation for 5 h in the absence or presence of heparin or ionophore A23187 was studied. Incubation in the presence of heparin (lOpgml-') caused a 32% increase in enzyme activity. After exposure of the spermatozoa to ionophore A23187 (0.5 p ~ )16% increase of enzyme activity could be detected.
Introduction
The acrosomes of spermatozoa are modified lysosomes containing enzymes with an acid pH optimum (Eddy, 1988). At least some of these may play a role in passage of the spermatozoa through the egg investments. Before sperm can enter the zona pellucida they must undergo the acrosome reaction, during which the outer acrosomal membrane fuses with the overlying plasma membrane, releasing the contents of the acrosomal matrix. In this work we have studied the loss of the acrosomal enzyme, acid phosphatase, from epididymal and ejaculated rat spermatozoa after incubation under capacitating conditions or after
- acrosome
reaction - rat.
treatment with various agents known to affect acrosomal membranes. Materials and methods
Animals Wistar-derived rats of the Tel Aviv University colony were maintained at 21-23 "C, supplied with food and water ad libitum, and provided, daily, with 14 h of artificial light. Sexually mature male and female rats were at least 3 months old. Immature females for in vitro fertilization were 23-26 d old at the day of hormone induction.
Epididymal spermatozoa Isolation of epididymal spermatozoa was carried out as previously described (Shalgi et al., 1989b). Epididymal tissue from caput, corpus, and cauda regions was cleared of blood and spermatozoa were obtained by cutting the tube in rat fertilization media (RFMm) (Shalgi, 1991), allowing the luminal contents to disperse, and separating the spermatozoa away from tissue fragments. Spermatozoa were collected 'from the vas deferens by flushing the vas with RFMm, using a 21 G needle. The sperm suspensions were centrifuged at 600 x g for 10 min to sediment the spermatozoa, which were washed twice more by centrifugation and resuspension in fresh RFMm. Sperm concentration was evaluated using a haemocytometer and the final suspension was adjusted to 2 x 107 rn1-1.
'Department of Embryology and Teratology and 'Department of Chemical Pathology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.
Ejaculated spermatozoa and fertilization in vitro
Correspondence: Dr Ruth Shalgi, Department of Embryology and Teratology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978, Israel.
Spermatozoa were collected from uteri of mature cycling rats soon after mating (Shalgi et al., 1981) and sperm concentration was adjusted to 4-
156
Z. SALZBERGER ET A L .
7 x lo5 ml-' by dilution with RFMm. Aliquots of sperm suspension (100 p1 or 200 pl) were incubated under heavy paraffin oil (BDH- U K ) at 37 "C for 5 h in an atmosphere of 5% CO, in air to allow capacitation (Shalgi et al., 1983). Immature female rats (23-25 d old) were induced to superovulate by injection of pregnant mare serum gonadotrophin (15 I.U., PMSG Folligon, Intervet Laboratories, Angers, France) followed 48 h later by human chorionic gonadotrophin (15 I.U., hCG Sigma Ltd, St Louis, MO, USA). Egg masses were collected from the ampullae of the oviducts 18-20 h after hCG injection and cumulus cells were dispersed by exposure to hyaluronidase (0.1yo; type IV from Sigma Ltd). The eggs were washed thoroughly in RFMm, pooled and then allocated randomly to droplets of sperm suspensions. Sperm-egg suspensions were incubated overnight and fertilization assessed the following day using interference contrast microscopy. Ova were considered fertilized when a spermatozoon flagellum was observed inside the vitellus (Shalgi & Kraicer, 1978).
Induction of acrosome reaction heparin
Ionophore A-23187 or
To investigate the effect of treatment of spermatozoa with inducers of capacitation, caudal or ejaculated spermatozoa were incubated for 30 min in medium containing 0.5 or 5.0 p~ of Ionophore A-23187. As control we added an equivalent volume of the solvent (DMSO). Alternatively, spermatozoa were incubated for 5 h in medium containing 10 pg ml-' of heparin (Fluka, 130668) and suspensions were washed twice as described above.
Extraction of acid phosphatase Spermatozoa were centrifuged (2000 x g for 2 min), supernatant discarded, and the pellet resuspended again in medium, to give a concentration of 2 x lo7 ml-'. The spermatozoa were washed twice by centrifugation. For extraction of acrosomal enzymes, spermatozoa were resuspended in 1% of Triton X-100, at a final concentration of 2 x lo7 cells ml-I, frozen using dry ice and acetone, and thawed in warm water. The procedure was repeated three times. Alternatively, spermatozoa were incubated for 30 min at 37 "C in the presence of L-a-lysophosphatidyl choline (LPC) (Byrd & Wolf, 1986) (final concentration of 0.227 mg ml- I ) . Following either treatment, sperm suspensions were centrifuged in 2000 x g for 15 min.
Measurement of Acid phosphatase Acid phosphatase activity was measured using the spectrophotometric technique described in Sigma diagnostics manual (Procedure No. 104). For this purpose 0.125 ml of extract or medium was added to 0.125 ml of paranitrophenyl phosphate (4 mg ml-' in 0.125 ml of 0 . 0 9 ~citrate buffer p H 4.8). The reaction was conducted at 37 "C for 30 min, was stopped with 0.25 ml of 0.5M NaOH, and the product measured at 410 nm.
Chemicals Unless otherwise indicated, all chemicals were purchased from Sigma Co. Ltd. St. Louis, USA.
Results I t is well established that the male reproductive tract secretions contain acid phosphatase. The number of washes needed to rid the spermatozoa of any acid phosphatase originating from the male accessory glands was determined by measuring enzyme activity in the supernatant fluid after washes. I t was demonstrated that the second wash contained only 5% of the enzyme activity in the first supernatant. T h e third wash results were not different from the second. Thus, to minimize damaging spermatozoa by centrifugation, we limited ourselves to two washes. Preliminary experiments demonstrated that measurable acid phosphatase activity could be extracted from lo7 washed sperm cells using either Triton X-100 or LPC. The two methods for extraction of acrosomal enzymes, released identical amounts of enzyme. The LPC method is easier to perform, and therefore in all further experiments we used this method. Enzyme activity was measured in room temperature (27°C) and in 37°C. The reaction was linear with time, with higher activity at 37°C (Fig. 1). The activity of the enzyme was recorded in 1 0 m I U per lo6 spermatozoa. The method was applied to investigate the amount of acid phosphatase activity of spermatozoa obtained from the caput and cauda epididymis and from the vas deferens. T h e results of 7 experiments demonstrated enzyme activity of 20.9 mIU lo7 spermatozoa- ' in cauda spermatozoa. This result represents a decrease of 41% in enzyme activity during passage from caput to cauda epididymis with only a small additional, insignificant decrease during passage to the vas deferens (54%). ANDROLOGIA 24, 155-159 (1992)
ACIDPHOSPHATASE
FROM RAT SPERMATOZOA
157
401 -
/
-8 35
/
p ._ 30
./
/
25
$ 20 c .-
i T
15
lo 5 0
I -
Control
I. (L.)
I. (H.)
Heparin
Treatment group
Figure 2. Increase in acid phosphatase release after various treatments (meanfstandard error). The change is expressed as yo difference. Results were collected from 13 different experiments. Significant difference by the Mann-Whitney-Wilcoxon Test, is marked as different letters above the histogram. I ( H ) - Ionophore high concentration; 5 p ~I(L); Ionophore low concentration, 0.5 p ~ .
observed in release of acrosomal acid phosphatase (Fig. 2).
Discussion
I
TIME
(min 1
I
Figure 1. Acid phosphatase activity as measured at different temperatures. -027°C; - 0 37°C.
In seven of 13 experiments, sperm samples were also used for in vitro fertilization of oocytes, as described in ‘Material and methods’. Results of these experiments yielded an average of 89.5% fertilization (51 out of 57). These results confirm that spermatozoa incubated under these conditions were capacitated and capable of fertilizing oocytes. Acid phosphatase activity was assayed in spermatozoa at the beginning of incubation and following 5 h of incubation under capacitation conditions, during which time the content of enzyme in the spermatozoa remained relatively constant (Fig. 2). Ionophore was applied at two different concentrations. Incubating spermatozoa in medium containing 0.5 ~ L Mof ionophore (low concentration) allowed acrosome reaction to occur to a small but significant extent, leaving enzyme activity remaining in the sperm cells (Fig. 2). When the ionophore was supplied a t ten fold that concentration (high concentration) enzyme release was not significantly different from the control (P>0.47 by Mann-Whitney Test). When spermatozoa were capacitated for 5 h in the presence of 10 pg ml-’ heparin, an increase of 32% was ANDROLOGIA 24, 155-159 (1992)
There are a number of reasons why acrosomal status is an important parameter of sperm fertilizing potential. I n some cases spermatozoa are infertile because the acrosome is missing or damaged. The ability of spermatozoa to respond to physiological inducers and undergo the acrosome reaction can serve as a measure of the extent to which they have undergone capacitation. I n IVF, the status of the acrosome is important not only for evaluating damage to spermatozoa. I n cases of male infertility which are treated by getting spermatozoa around the physiological barriers presented by the cumulus oophorus and the zona pellucida it may be desirable to utilize acrosomereacted spermatozoa. Occurrence of sperm acrosome reactions can be assayed by a number of different methods. In species with large acrosomes, which are easily observed in the light microscope, the breakdown of the acrosome has been assessed using different stains or by phase microscopy (for review see Cross & Meizel, 1989). I n other species such as the rat (Shalgi et al., 1989a) only electron microscopy can resolve the acrosomal status. Even in those cases where the acrosome reaction can be assayed microscopically by either of these methods or by specific lectin or antibody binding, these methods require counting numerous sperm cells for statistically significant results to be obtained. The method presented in this study,
158
Z. SALZBERCER ET AL.
which measures the amount of acrosomal acid phosphatase remaining in cells, presents a different approach especially suitable for the rat, a species with small acrosomes which are not easily visualized by light microscopy. The results allow estimation of the amount of acrosomal enzyme which has been lost from the cells during the acrosomal reaction. Agents such as Triton X-100 and lysolecithin (LPC), which destroy the sperm membranes, released all of the acid phosphatase from the cells. Other groups have used a number of methods to expose the acrosomal enzymes. A calmodulin antagonist (Nagae & Srivastava., 1986), follicular fluid (Yudin et al., 1988), Ionophore A23187 (Tesarik, 1985) and sulphated glycoconjugates and heparin (Meizel & Turner, 1986; Parrish et al., 1989) reportedly do this. Acrosome reaction was also assayed in the ram by release of acrosin (Ben-Av et al., 1988). On the other hand, incubation of rat spermatozoa under capacitating conditions did not lead to significant release of acid phosphatase from these cells. This is consistent with the hypothesis that acrosomal enzyme release occurs when the spermatozoa undergo the acrosome reaction after binding to the zona pellucida (Wassarman, 1988). Lewin et al. (1982), reported leakage of no more than 4-774 of hyaluronidase of rat spermatozoa upon incubation under capacitating conditions for 24h. In human spermatozoa Mack et al. (1983) reported no loss of acrosomal enzymes except for hyaluronidase after 8 h of incubation under capacitating conditions. In other species, the proportion of spermatozoa which have undergone the acrosome reaction spontaneously by the time suspensions are highly fertile is considerably higher (mouse 30-35y0, [Fraser, 19811 Hamster 10-65y0, depending on the medium used [Yanagimachi, 198I]). Some of these apparent acrosome reactions may be non-physiological, reflecting death of some cells with subsequent breakdown of their membranes. It is interesting to note that only 10% of human spermatozoa lost their acrosome after 24 h under capacitating conditions (Mallet et al., 1985). It has been proposed that fertilizing spermatozoa undergo an acrosome reaction induced by glycosaminoglycans either in the follicular fluid, in the extracellular matrix between the cumulus cells (Lenz et al., 1983a; Cross & Meizel, 1989), or on the surface of the zona pellucida (Wassarman, 1988). Other studies indicated that glycosaminoglycan components of the proteoglycans present in the follicular fluid, can stimulate the acrosome reaction of spermatozoa in rabbits (Lenz et al., 198313) boar (Reyes et al., 1984) and human (Suarez et al., 1986). In those studies, the
molecules in question were incubated together with spermatozoa for a prolonged period of time before acrosome reaction was assayed, so the molecules could have been involved in capacitation as well as in the acrosome reaction. Nevertheless, a study by Meizel & Turner (1986) has shown that late addition of glycosaminoglycans can induce the acrosome reaction in hamster spermatozoa previously capacitated in uitro, suggesting that the possibility that the active component of human follicular fluid is a proteoglycan or glycosaminoglycan, must also be considered. Proteoglycans (Chondroitin sulphate, glycosaminoglycans) have been reported to enhance the acrosome reaction in bovine and rabbit spermatozoa as determined by light and EM (Lenz et al., 1983a), but the nature of this effect has not been fully investigated as yet. On the other hand heparin, another glycosaminoglycan, was found to bind to sperm heads in a typical receptorligand interaction (Handrow et al., 1984) and heparin binding proteins were reported by Miller & Ax (1990). In the studies reported here exposure to heparin led to a significant increase in enzyme released. The calcium ion is also reportedly required for the acrosome reaction but not for capacitation (Yanagimachi, 1988). The cation ionophore A-23187 in the presence of Ca2+, can induce capacitated spermatozoa to undergo the acrosome reaction (Talbot et al., 1976; Aitken et al., 1984; Tesarik, 1985; Morales et al., 1989). The results reported here confirmed that more acid phosphatase was released from spermatozoa that had been incubated with the calcium ionophore A-23187. The procedure reported here provides a simple, quantitative assay of release of acid phosphatase from sperm acrosomes, a method which can be used conveniently to assess the acrosome reaction in rat spermatozoa, where microscopic visualization of the acrosome is difficult. The simplicity and quantitative nature of this method should make it useful for evaluating the acrosome reaction in other species as well as in the rat.
References Aitken RJ, Ross A, Hargreave T, Richardson D, Best F ( 1984) Analysis of human sperm function following exposure to the ionophore A23187. J Androl 5:321-329. Ben-Av P, Rubinstein S, Breitbart H (1988) Induction of acrosomal reaction and calcium uptake in ram spermatozoa by ionophore. Biochem Biophys Acta 939:2 14-222. Byrd W, Wolf DP (1986) Acrosomal status in fresh and capacitated human ejaculated sperm. Biol Reprod 34~859-869. Cross NL, Meizel S (1989) Methods for evaluating the ANDROLOGIA 24, 155-159 (1992)
Acm
acrosomal status of mammalian sperm. Biol Reprod 41 1635-641. Eddy EM (1988) The spermatozoon. In: The Physiology of Reproduction. Knobil E, Neill JD, Ewing LL, Greenwald GS, Market C, Pfaff W. (eds) Raven Press, Ltd., New York, pp 27-68. Fraser LR (1981) Dibutyryl cyclic AMP decreases capacitation time in uitro in mouse spermatozoa. J Reprod Fertil 62:63-72. Handrow RR, Boehm SK, Lenz RW, Robinson JA, Ax RL (1984) Specific binding of the glycosaminoglycan 3Hheparin to bull, monkey, and rabbit spermatozoa in vitro. J Androl 5:51-63. Lenz RW, Ball GD, Lohse JK, First NL, Ax R L (1983a) Chondroitin sulphate facilitates an acrosome reaction in bovine spermatozoa as evidenced by light microscopy, electron microscopy and in uitro fertilization. Biol Reprod 28~683-690. Lenz RW, Bellin ME, Ax RL (1983b) Rabbit spermatozoa undergo an acrosome reaction in the presence of glycosaminoglycans. Gamete Res 8: 11-19. Lewin LM, Nevo Z, Gabsu A, Weissenberg R (1982) The role of sperm bound hyaluronidase in the dispersal of the cumulus oophorus surrounding the rat ova. Int J Androl 5:37-44. Mack S, Bhattacharyya AK, Joyce C, Van Der Ven H, Zaneveld LJD ( 1983) Acrosomal enzymes of human spermatozoa before and after in uitro capacitation. Biol Reprod 28: 1032- 1042. Mallett PJ, Stock CE, Fraser RL (1985) Acrosome loss in human sperm incubated in vitro under capacitating conditions. Int J Androl 8:357-364. Meizel S, Turner K O ( 1986) Glycosaminoglycans stimulate the acrosome reaction of previously capacitated hamster sperm. J Exp Zool 237:137-139. Miller DJ, Ax RL (1990), Carbohydrates and fertilization in animals. Mol Reprod Dev 26:184-198. Morales P, Cross NL, Oversteet JW, Hanson FW (1989) Acrosome intact and acrosome reacted human sperm can initiate binding to the zona pellucida. Dev Biol 133~385-392. Nagae T, Srivastava PN (1986) Induction of the acrosome reaction in guinea pig spermatozoa by calmodulin antagonist W-7. Gamete Res 14:197-208. Parrish JJ, Susko-Parrish JL, Handrow RR, Ax RL, First NL (1989) Effect of sulfated glycoconjugates on capacitation and the acrosome reaction of bovine and hamster spermatozoa, Gamete Res 24:403-413.
ANDROLOGIA 24, 155-159 (1992)
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159
Reyes R, Carranco A, Hernandez 0, Rosado A, Merchant H, Delgado NM (1984) Glycosaminoglycan sulfate as acrosomal reaction-inducing factor of follicular fluid. Arch Androl 12:203-209. Shalgi R (1991) Fertilization in the rat. In: A Comparative Overview of Mammalian Fertilization. Dunbar BS, O’Rand MG (eds) Plenum Press, pp 245-255. Shalgi R, Kraicer PF (1978) Timing of sperm transport, sperm penetration and cleavage in the rat. J Exp Zool 2041353-360. Shalgi R, Kaplan R, Nebel L, Kraicer PF (1981) The male factor in fertilization of rat eggs in uitro. J Exp Zool 21 7:399-402. Shalgi R, Kaplan R, Nebel L (1983) The capacitation rate of rat sperm in vitro. In: The Sperm Cell. Andre J (ed) Martinus Nijhoff, The Hague, pp 47-50. Shalgi R, Phillips DM, Jones R (1989a) Status of the rat acrosome during sperm-zona pellucida interactions. Gamete Res 22:l-13. Shalgi R, Seligman J, Kosower NS (1989b) Dynamics of the thiol status of rat spermatozoa during maturation: Analysis with the fluorescent labeling agent monobromobimane. Biol Reprod 40:1037-1045. Suarez SS, Wolf DP, Meizel S (1986) Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid. Gamete Res 14:107-121. Talbot P, Summers RG, Hylander BL, Keough EM, Franklin LE (1976) The role of calcium in acrosome reaction. An analysis using ionophore A23187. J Exp Zool 198~383-392. Tesarik J ( 1985) Comparison of acrosome reaction-inducing activities of human cumulus oophorus, follicular fluid and ionophore A23187 in human sperm populations of proven fertilizing ability in vitro. J Reprod Fert 74:383-388. Wassarman PM (1988) Zona pellucida glycoproteins. Ann Rev Biochem 57:415-442. Yanagimachi R (1981) Mechanisms of fertilization in mammals. In: Fertilization and Embryonic Development In Vitro. Mastroiani L, Biggers LD (eds) Plenum Publishing Corporation, New York, pp 81b.182. Yanagimachi R (1988) Mammalian fertifization. In: The Physiology of Reproduction, Knobil E, Neill JD, Ewing LL, Greenwald GS, Market C, Pfaff W. (eds) Raven Press, Ltd, New York, pp 85-135. Yudin AI, Gottleib W, Meizel S (1988) Ultrastructural studies of the early events of the human sperm acrosome reaction as initiated by human follicular fluid. Gamete Res 20: 1 1-24.
24, 155- 159 ( 1992)
ACCEPTED: JANUARY 22, 1992
Loss of acid phosphatase from rat spermatozoa as a method for assessing the acrosome reaction Z. Salzberger, L. M. Lewin and R. Shalgi'
I
Key words. Spermatozoa - acid phosphatase - acrosome
Summary. A method is presented for evaluating the extent of the acrosome reaction by measuring the release of acrosomal acid phosphatase from rat spermatozoa during incubation under capacitating conditions. Treatment of spermatozoa with lysophosphatidylcholine or Triton X- 100 released the acid phosphatase from the sperm cell. Using this enzymatic method we could not detect an alteration in enzyme activity following 5 h incubation under capacitating conditions. The effect of in vitro capacitation for 5 h in the absence or presence of heparin or ionophore A23187 was studied. Incubation in the presence of heparin (lOpgml-') caused a 32% increase in enzyme activity. After exposure of the spermatozoa to ionophore A23187 (0.5 p ~ )16% increase of enzyme activity could be detected.
Introduction
The acrosomes of spermatozoa are modified lysosomes containing enzymes with an acid pH optimum (Eddy, 1988). At least some of these may play a role in passage of the spermatozoa through the egg investments. Before sperm can enter the zona pellucida they must undergo the acrosome reaction, during which the outer acrosomal membrane fuses with the overlying plasma membrane, releasing the contents of the acrosomal matrix. In this work we have studied the loss of the acrosomal enzyme, acid phosphatase, from epididymal and ejaculated rat spermatozoa after incubation under capacitating conditions or after
- acrosome
reaction - rat.
treatment with various agents known to affect acrosomal membranes. Materials and methods
Animals Wistar-derived rats of the Tel Aviv University colony were maintained at 21-23 "C, supplied with food and water ad libitum, and provided, daily, with 14 h of artificial light. Sexually mature male and female rats were at least 3 months old. Immature females for in vitro fertilization were 23-26 d old at the day of hormone induction.
Epididymal spermatozoa Isolation of epididymal spermatozoa was carried out as previously described (Shalgi et al., 1989b). Epididymal tissue from caput, corpus, and cauda regions was cleared of blood and spermatozoa were obtained by cutting the tube in rat fertilization media (RFMm) (Shalgi, 1991), allowing the luminal contents to disperse, and separating the spermatozoa away from tissue fragments. Spermatozoa were collected 'from the vas deferens by flushing the vas with RFMm, using a 21 G needle. The sperm suspensions were centrifuged at 600 x g for 10 min to sediment the spermatozoa, which were washed twice more by centrifugation and resuspension in fresh RFMm. Sperm concentration was evaluated using a haemocytometer and the final suspension was adjusted to 2 x 107 rn1-1.
'Department of Embryology and Teratology and 'Department of Chemical Pathology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.
Ejaculated spermatozoa and fertilization in vitro
Correspondence: Dr Ruth Shalgi, Department of Embryology and Teratology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978, Israel.
Spermatozoa were collected from uteri of mature cycling rats soon after mating (Shalgi et al., 1981) and sperm concentration was adjusted to 4-
156
Z. SALZBERGER ET A L .
7 x lo5 ml-' by dilution with RFMm. Aliquots of sperm suspension (100 p1 or 200 pl) were incubated under heavy paraffin oil (BDH- U K ) at 37 "C for 5 h in an atmosphere of 5% CO, in air to allow capacitation (Shalgi et al., 1983). Immature female rats (23-25 d old) were induced to superovulate by injection of pregnant mare serum gonadotrophin (15 I.U., PMSG Folligon, Intervet Laboratories, Angers, France) followed 48 h later by human chorionic gonadotrophin (15 I.U., hCG Sigma Ltd, St Louis, MO, USA). Egg masses were collected from the ampullae of the oviducts 18-20 h after hCG injection and cumulus cells were dispersed by exposure to hyaluronidase (0.1yo; type IV from Sigma Ltd). The eggs were washed thoroughly in RFMm, pooled and then allocated randomly to droplets of sperm suspensions. Sperm-egg suspensions were incubated overnight and fertilization assessed the following day using interference contrast microscopy. Ova were considered fertilized when a spermatozoon flagellum was observed inside the vitellus (Shalgi & Kraicer, 1978).
Induction of acrosome reaction heparin
Ionophore A-23187 or
To investigate the effect of treatment of spermatozoa with inducers of capacitation, caudal or ejaculated spermatozoa were incubated for 30 min in medium containing 0.5 or 5.0 p~ of Ionophore A-23187. As control we added an equivalent volume of the solvent (DMSO). Alternatively, spermatozoa were incubated for 5 h in medium containing 10 pg ml-' of heparin (Fluka, 130668) and suspensions were washed twice as described above.
Extraction of acid phosphatase Spermatozoa were centrifuged (2000 x g for 2 min), supernatant discarded, and the pellet resuspended again in medium, to give a concentration of 2 x lo7 ml-'. The spermatozoa were washed twice by centrifugation. For extraction of acrosomal enzymes, spermatozoa were resuspended in 1% of Triton X-100, at a final concentration of 2 x lo7 cells ml-I, frozen using dry ice and acetone, and thawed in warm water. The procedure was repeated three times. Alternatively, spermatozoa were incubated for 30 min at 37 "C in the presence of L-a-lysophosphatidyl choline (LPC) (Byrd & Wolf, 1986) (final concentration of 0.227 mg ml- I ) . Following either treatment, sperm suspensions were centrifuged in 2000 x g for 15 min.
Measurement of Acid phosphatase Acid phosphatase activity was measured using the spectrophotometric technique described in Sigma diagnostics manual (Procedure No. 104). For this purpose 0.125 ml of extract or medium was added to 0.125 ml of paranitrophenyl phosphate (4 mg ml-' in 0.125 ml of 0 . 0 9 ~citrate buffer p H 4.8). The reaction was conducted at 37 "C for 30 min, was stopped with 0.25 ml of 0.5M NaOH, and the product measured at 410 nm.
Chemicals Unless otherwise indicated, all chemicals were purchased from Sigma Co. Ltd. St. Louis, USA.
Results I t is well established that the male reproductive tract secretions contain acid phosphatase. The number of washes needed to rid the spermatozoa of any acid phosphatase originating from the male accessory glands was determined by measuring enzyme activity in the supernatant fluid after washes. I t was demonstrated that the second wash contained only 5% of the enzyme activity in the first supernatant. T h e third wash results were not different from the second. Thus, to minimize damaging spermatozoa by centrifugation, we limited ourselves to two washes. Preliminary experiments demonstrated that measurable acid phosphatase activity could be extracted from lo7 washed sperm cells using either Triton X-100 or LPC. The two methods for extraction of acrosomal enzymes, released identical amounts of enzyme. The LPC method is easier to perform, and therefore in all further experiments we used this method. Enzyme activity was measured in room temperature (27°C) and in 37°C. The reaction was linear with time, with higher activity at 37°C (Fig. 1). The activity of the enzyme was recorded in 1 0 m I U per lo6 spermatozoa. The method was applied to investigate the amount of acid phosphatase activity of spermatozoa obtained from the caput and cauda epididymis and from the vas deferens. T h e results of 7 experiments demonstrated enzyme activity of 20.9 mIU lo7 spermatozoa- ' in cauda spermatozoa. This result represents a decrease of 41% in enzyme activity during passage from caput to cauda epididymis with only a small additional, insignificant decrease during passage to the vas deferens (54%). ANDROLOGIA 24, 155-159 (1992)
ACIDPHOSPHATASE
FROM RAT SPERMATOZOA
157
401 -
/
-8 35
/
p ._ 30
./
/
25
$ 20 c .-
i T
15
lo 5 0
I -
Control
I. (L.)
I. (H.)
Heparin
Treatment group
Figure 2. Increase in acid phosphatase release after various treatments (meanfstandard error). The change is expressed as yo difference. Results were collected from 13 different experiments. Significant difference by the Mann-Whitney-Wilcoxon Test, is marked as different letters above the histogram. I ( H ) - Ionophore high concentration; 5 p ~I(L); Ionophore low concentration, 0.5 p ~ .
observed in release of acrosomal acid phosphatase (Fig. 2).
Discussion
I
TIME
(min 1
I
Figure 1. Acid phosphatase activity as measured at different temperatures. -027°C; - 0 37°C.
In seven of 13 experiments, sperm samples were also used for in vitro fertilization of oocytes, as described in ‘Material and methods’. Results of these experiments yielded an average of 89.5% fertilization (51 out of 57). These results confirm that spermatozoa incubated under these conditions were capacitated and capable of fertilizing oocytes. Acid phosphatase activity was assayed in spermatozoa at the beginning of incubation and following 5 h of incubation under capacitation conditions, during which time the content of enzyme in the spermatozoa remained relatively constant (Fig. 2). Ionophore was applied at two different concentrations. Incubating spermatozoa in medium containing 0.5 ~ L Mof ionophore (low concentration) allowed acrosome reaction to occur to a small but significant extent, leaving enzyme activity remaining in the sperm cells (Fig. 2). When the ionophore was supplied a t ten fold that concentration (high concentration) enzyme release was not significantly different from the control (P>0.47 by Mann-Whitney Test). When spermatozoa were capacitated for 5 h in the presence of 10 pg ml-’ heparin, an increase of 32% was ANDROLOGIA 24, 155-159 (1992)
There are a number of reasons why acrosomal status is an important parameter of sperm fertilizing potential. I n some cases spermatozoa are infertile because the acrosome is missing or damaged. The ability of spermatozoa to respond to physiological inducers and undergo the acrosome reaction can serve as a measure of the extent to which they have undergone capacitation. I n IVF, the status of the acrosome is important not only for evaluating damage to spermatozoa. I n cases of male infertility which are treated by getting spermatozoa around the physiological barriers presented by the cumulus oophorus and the zona pellucida it may be desirable to utilize acrosomereacted spermatozoa. Occurrence of sperm acrosome reactions can be assayed by a number of different methods. In species with large acrosomes, which are easily observed in the light microscope, the breakdown of the acrosome has been assessed using different stains or by phase microscopy (for review see Cross & Meizel, 1989). I n other species such as the rat (Shalgi et al., 1989a) only electron microscopy can resolve the acrosomal status. Even in those cases where the acrosome reaction can be assayed microscopically by either of these methods or by specific lectin or antibody binding, these methods require counting numerous sperm cells for statistically significant results to be obtained. The method presented in this study,
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which measures the amount of acrosomal acid phosphatase remaining in cells, presents a different approach especially suitable for the rat, a species with small acrosomes which are not easily visualized by light microscopy. The results allow estimation of the amount of acrosomal enzyme which has been lost from the cells during the acrosomal reaction. Agents such as Triton X-100 and lysolecithin (LPC), which destroy the sperm membranes, released all of the acid phosphatase from the cells. Other groups have used a number of methods to expose the acrosomal enzymes. A calmodulin antagonist (Nagae & Srivastava., 1986), follicular fluid (Yudin et al., 1988), Ionophore A23187 (Tesarik, 1985) and sulphated glycoconjugates and heparin (Meizel & Turner, 1986; Parrish et al., 1989) reportedly do this. Acrosome reaction was also assayed in the ram by release of acrosin (Ben-Av et al., 1988). On the other hand, incubation of rat spermatozoa under capacitating conditions did not lead to significant release of acid phosphatase from these cells. This is consistent with the hypothesis that acrosomal enzyme release occurs when the spermatozoa undergo the acrosome reaction after binding to the zona pellucida (Wassarman, 1988). Lewin et al. (1982), reported leakage of no more than 4-774 of hyaluronidase of rat spermatozoa upon incubation under capacitating conditions for 24h. In human spermatozoa Mack et al. (1983) reported no loss of acrosomal enzymes except for hyaluronidase after 8 h of incubation under capacitating conditions. In other species, the proportion of spermatozoa which have undergone the acrosome reaction spontaneously by the time suspensions are highly fertile is considerably higher (mouse 30-35y0, [Fraser, 19811 Hamster 10-65y0, depending on the medium used [Yanagimachi, 198I]). Some of these apparent acrosome reactions may be non-physiological, reflecting death of some cells with subsequent breakdown of their membranes. It is interesting to note that only 10% of human spermatozoa lost their acrosome after 24 h under capacitating conditions (Mallet et al., 1985). It has been proposed that fertilizing spermatozoa undergo an acrosome reaction induced by glycosaminoglycans either in the follicular fluid, in the extracellular matrix between the cumulus cells (Lenz et al., 1983a; Cross & Meizel, 1989), or on the surface of the zona pellucida (Wassarman, 1988). Other studies indicated that glycosaminoglycan components of the proteoglycans present in the follicular fluid, can stimulate the acrosome reaction of spermatozoa in rabbits (Lenz et al., 198313) boar (Reyes et al., 1984) and human (Suarez et al., 1986). In those studies, the
molecules in question were incubated together with spermatozoa for a prolonged period of time before acrosome reaction was assayed, so the molecules could have been involved in capacitation as well as in the acrosome reaction. Nevertheless, a study by Meizel & Turner (1986) has shown that late addition of glycosaminoglycans can induce the acrosome reaction in hamster spermatozoa previously capacitated in uitro, suggesting that the possibility that the active component of human follicular fluid is a proteoglycan or glycosaminoglycan, must also be considered. Proteoglycans (Chondroitin sulphate, glycosaminoglycans) have been reported to enhance the acrosome reaction in bovine and rabbit spermatozoa as determined by light and EM (Lenz et al., 1983a), but the nature of this effect has not been fully investigated as yet. On the other hand heparin, another glycosaminoglycan, was found to bind to sperm heads in a typical receptorligand interaction (Handrow et al., 1984) and heparin binding proteins were reported by Miller & Ax (1990). In the studies reported here exposure to heparin led to a significant increase in enzyme released. The calcium ion is also reportedly required for the acrosome reaction but not for capacitation (Yanagimachi, 1988). The cation ionophore A-23187 in the presence of Ca2+, can induce capacitated spermatozoa to undergo the acrosome reaction (Talbot et al., 1976; Aitken et al., 1984; Tesarik, 1985; Morales et al., 1989). The results reported here confirmed that more acid phosphatase was released from spermatozoa that had been incubated with the calcium ionophore A-23187. The procedure reported here provides a simple, quantitative assay of release of acid phosphatase from sperm acrosomes, a method which can be used conveniently to assess the acrosome reaction in rat spermatozoa, where microscopic visualization of the acrosome is difficult. The simplicity and quantitative nature of this method should make it useful for evaluating the acrosome reaction in other species as well as in the rat.
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