MORPHOLOGIC CHARACTERISTICS OF THE CHEMICALLY INDUCED ACROSOME REACTION IN HUMAN SPERMATOZOA*

Transcription

1 FERTILITY AND STERILITY Copyright 1979 The American Fertility Society Vol. 32, No.1, July 1979 Printed in U.SA. MORPHOLOGIC CHARACTERISTICS OF THE CHEMICALLY INDUCED ACROSOME REACTION IN HUMAN SPERMATOZOA* LONNIE RUSSELL, PH.D.t RUDOLPH N. PETERSON, PH.D. MATTHEW FREUND, PH.D. Department of Physiology and School of Medicine, Southern Illinois University, Carbondale, Illinois The morphologic changes accompanying the acrosome reaction in human spermatozoa, as it is induced by the antibiotic A23187 and calcium ions, are described. The reaction is shown to be similar to that observed in other species when the reaction occurs spontaneously or is induced by physiologic fluids. The reaction in human spermatozoa differs from the chemically induced reaction in other species in that plasma membrane microfilaments, prominent in the boar, and tubular-like elements prominent in boar, rabbit, and monkey sperm, are not observed. Motility remains high when human spermatozoa are treated with A23187 and calcium and it is possible that these agents may be useful in the study of certain causes of infertility. Fertil SteriI32:87, The ability to induce the acrosome reaction in mammalian spermatozoa by various chemical means has provided a useful way for studying the membrane changes occurring during this reaction. Morphologic changes in boar and guinea pig spermatozoa, induced by the ionophore A23187 and calcium, have been recently described. I - 3 A description for human spermatozoa has yet to be reported, although such an analysis should be of particular interest in view of the potential practical value of acrosome-reacted human spermatozoa in the study of infertility. In this report the ultrastructural changes in human spermatozoa caused by the ionophore A23187 and calcium, as well as the effects of these agents on motility, are described. METHODS AND MATERIALS Semen was obtained from normal student donors, pooled, used within 2 hours of collection. The Received March 12, 1979; accepted March 29, * Supported by Grant HD from the National Institutes of Health. tto whom reprint requests should be addressed. 87 combined specimen was then washed free of seminal plasma by centrifugation at 1000 x g (two times) and resuspended in a Tris-based buffer of the following composition: tris(hydroxymethyl)aminomethane chloride, 0.02 M; sodium chloride, 0.12 M; potassium chloride, M; sodium pyruvate, M; and glucose, M. The ph was 7.4 at 37 C. Spermatozoal motility was rated subjectively at 37 C using a light microscope. The ratings were made by two trained observers who were unaware of particular treatments. Forward progression (rated on a scale of 1 to 10) and the percentage of motile cells were recorded. Samples were prepared for electron microscopy by fixation in 2% glutaraldehyde in cacodylate buffer (ph 7.0), washed and then postfixed in 2% osmium tetroxide-1.25% ferrocyanide solution,4 dehydrated, infiltrated with propylene oxide, and embedded in Epon. Silver and silver-gray appearing sections were stained with uranyl acetate and examined on a Phillips 201 electron microscope. The divalent cation ionophore A23187, which increases the permeability of membranes to calcium, was the gift of the Eli Lilly Pharmaceutical Co., Indianapolis, Ind. Other chemicals were pur-

2 88 RUSSELL ET AL. July 1979 TABLE 1. Effect of the Ionophore A23187 and Calcium on the Motility of Washed Human Spermatozoa Addition Time after addition A23187 o min 15 min 30 min 90 min 300 min motile cells (%)"., None 60 (4) 60 (5) 50 (4) 40 (4) 20 (2) A23187 (1 pm) 60 (4) 60 (6) 60 (5) 50 (5) mm Ca (4) 40 (3) 50 (3) 50 (3) mm Ca2+ 60 (4) 50 (4) 50 (4) 40 (3) mm Ca (4) 60 (6) 60 (5) 40 (4) A23187 (10 pm) 60 (4) 60 (6) 30 (3) 40 (4) mm Ca (4) 50 (4) 50 (4) 40 (3) mm Ca (4) 60 (6) 50 (4) 30 (2) mm Ca (4) 60 (5) 50 (4) 30 (3) 20 (2) -Sperm were diluted in buffer to give a final concentration of approximately 0.5 x 10 8 /ml. bnumbers in parentheses are ratings for forward progression (scale 1 to 10). chased from the Sigma Chemical Co., St. Louis, Mo., and the J. T. Baker Chemical Co., Phillipsburg, N. J. RESULTS Table 1 shows the effects of different concentrations of A23187 and calcium on the motility of washed suspensions of human spermatozoa. In this buffer, it was difficult to recognize any significant effects ofthese agents on motility. Even at the highest concentrations of the ionophore (10 pm) and calcium ions (3 mm), motility was not significantly different from that of controls. Moreover, exposure to these agents for as long as 5 hours did not abolish motility. This finding is in marked contrast to the effects of these agents on boar and guinea pig spermatozoa, where motility is rapidly abolished.!' 3 Rapid sperm motion and the small size of the head and acrosomal cap of human spermatozoa made it difficult to discern by light microscopy any morphologic changes induced by the chemical treatments. However, when spermatozoa were treated with the ionophore (10 pm) and calcium (1 mm) for 1 hour, electron microscopic analysis indicated that approximately 40% of the spermatozoa had undergone the acrosome reaction. The details of the ultrastructural changes occurring in these treated sperm are shown in Figures 1 and 2. Figure 1, band c, shows the pattern of vesiculation observed. Vesicles of different sizes form over the acrosomal region that extends caudally from the tip of the sperm head to the equatorial segment. In a small number of the spermatozoa, the contents of both the principal segment and equatorial segments have been dispersed (Fig. 1b), but in the majority of reacted sperm, vesiculation ceases at the equatorial segment, where the plasma membrane has fused with the outer acrosomal membrane (Fig. 2a). The fusion occurs typically in spermatozoa induced to undergo the acrosome reaction by physiologic buffers and fluids 5 and maintains continuity with the exposed inner acrosomal membrane that extends anteriorly into the area of vesiculation (Figs. 1c and 2a). The vesicular structures at high magnification are shown in Figure 2b. Those vesicles that remain close to the sperm head appear to be bound to the inner acrosomal membrane by a dense material. Similar material also appears to link some vesicles to each other. These lateral attachments, however, are not a prominent feature of the vesiculation reaction as they are in the boar, where vesicles are linked in beadlike fashion over the entire area of the principal segment. 1 DISCUSSION In previous reports it has been shown that the acrosome reaction in the boar, induced by the ionophore A23187 and calcium, is similar in its morphologic expression to that which occurs spontaneously or that which has been observed in other FIG. 1. a, Untreated human sperm showing the plasma membrane (pm) and the acrosomal sac which is formed by the outer (oa) and inner acrosomal (ia) membranes. Division of the acrosomal sac into principal (ps) and equatorial segments (es)is indicated, as well as is the postacrosomal region (par). b, A minority of sperm treated with the ionophore A23187 and Ca2+ show vesiculation (u) over the entire acrosomal region. The acrosomal contents of both the principal and equatorial regions have been dispersed. c, In most acrosome-reacted sperm the equatorial segment of the acrosome is unaffected. Here fusion of the plasma membrane (pm) with the outer acrosomal membrane (oa) of the equatorial segment is evident. Vesicles formed over the anterior tip of the head have been shed and the acrosomal contents dispersed. The inner acrosomal membrane (ia) remains intact (a, x51,000; b, x39,100; c, x63,600).

3 Vol. 32, No.1 CHEMICALLY INDUCED ACROSOME REACTION 89

4 90 RUSSELL ET AL. July 1979

5 Vol. 32, No.1 CHEMICALLY INDUCED ACROSOME REACTION 91 species when the reaction is induced by physiologic fluids or buffers.i-3 Several differences in the reaction between human and boar spermatozoa should be noted, however. Microfilament formation along the plasma membrane, which is a normal concomitant of the reaction in boar spermatozoa, was not observed in acrosome-reacted human spermatozoa. Tubular elements, which have been observed to occur spontaneously in chemically induced acrosome-reacted boar, rabbit, and monkey sperm,!. 6 were also not observed in human acrosome-reacted spermatozoa. The reason for these differences is not clear. Actin, however, has been demonstrated to be present in human spermatozoa, where it is concentrated in the postacrosomal region of the head piece. 7 It is possible that the conditions required for the polymerization of actin and tubulin may differ among these species. In other respects the morphologic changes in the acrosome reaction, as it is induced by the ionophore and calcium, appear to be similar among the species thus far examined. Characteristically, vesiculation occurs all along the principal segment of the head piece, and a fusion of membranes links the outer acrosomal and plasma membrane at the equatorial segment. Of particular interest is the observation that the antibiotic A23187 had very little effect on the motility of human spermatozoa in the buffer used in these experiments, even in the presence of high calcium concentrations. This finding contrasts with the effects of A23187 and calcium in the boar and guinea pig spermatozoa, L 3 which are rapidly rendered immotile. This may be related to the significant differences in energy metabolism among these species. Studies in this laboratory have shown that boar and guinea pig spermatozoa depend on the presence of oxygen for survival. 8, 9 The rapid uptake of calcium by mitochondria in the presence of A23187, which in the boar is accompanied by a large increase in oxygen uptake,8 very probably contributes to an uncoupling of oxidative phosphorylation. This may enhance the loss of cell viability. Human spermatozoa, on the other hand, survive equally well in aerobic and anaerobic environments and ordinarily consume com paratively small amounts of oxygen. 10 This smaller dependence on oxidative metabolism may account for the absence of significant effects of the ionophore and calcium on motility. The observation that A23187 and calcium have minor effects (if any) on motility and yet promote the acrosome reaction in human spermatozoa has practical significance. Rogers et al. ll have recently described a diagnostic technique for infertility based on the inability of spermatozoa from infertile males to fuse with zona-free hamster eggs. It is well known that the acrosome reaction is required for this fusion reaction l2 and therefore it is possible that the inability of spermatozoa to undergo this essential reaction may be involved in some cases of infertility. Since our observations show that human spermatozoa can be treated chemically to induce the acrosome reaction, without significant loss of motility, such chemical treatment of infertile spermatozoa may prove useful in determining certain causes of infertility. REFERENCES 1. Peterson RN, Russell L, Bundman D, Freund M: Presence of micro filaments and tubular structures in boar spermatozoa after chemically inducing to acrosome reaction. BioI Reprod 19:459, Russell L, Peterson R, Freund M: Direct evidence for formation of hybrid vesicles by fusion of plasma and outer acrosomal membranes during the acrosome reaction in boar spermatozoa. J Exp Zool. In press, Green DPL: The induction of the acrosome reaction in guinea-pig sperm by the divalent cation ionophore A J Cell Sci 32:137, Russell LD, Burguet S: Ultrastructure of Leydig cells as revealed by secondary tissue treatment with a ferrocyanide-osmium mixture. Tissue Cell 9:751, Barros C, BedfordJM, Franklin LE, Austin CR: Membrane vesiculation as a feature ofthe mammalian acrosome reaction. J Cell BioI 34:C1, Stambaugh R, Smith M: Microtubular structures in mammalian acrosomes. J Exp Zool 203:135, Clarke GN, Boyd RL, Muller HK: Actin like proteins in human sperm heads. In Immunological Influences on Human Fertility, Edited by B Boettcher. New York, Academic Press, 1977, p Peterson RN, Seyler D, Bundman D, Freund M: The effect of theophylline and dibutyryl cyclic AMP on the uptake of calcium and phosphate ions by boar and human spermatozoa. J Reprod Fertil 55:385, 1979 FIG. 2. a, Most reacted sperm treated with the ionophore A23187 and Ca 2 + show a typical acrosome reaction. The equatorial segment (es) remains intact, whereas the acrosomal contents ofthe anterior head region have dispersed and have exposed the inner acrosomal membrane (iam). A few vesicles (v) (formed by fusion of the plasma and outer acrosomal membrane) are indicated (arrows). Note the continuity of the plasma membrane with the outer acrosomal membrane in the unvesiculated region of the acrosome (arrowheads). b, High magnification micrograph showing the anterior aspect ofthe sperm head and the vesicles (v) produced after ionophore and Ca2+ treatment. Vesicles appear to be bound by dense material (arrowheads) to the inner acrosomal membrane (iam). The hybrid nature of the vesicles is not easily discerned, although the presumptive inner acrosomal membrane usually forms a "rigid" hemisphere whereas the plasma membrane follows a somewhat irregular course (a, x57,100; b, x 139,300).

6 92 RUSSELL ET AL. July Frenkel G, Peterson RN, Freund M: Oxidative and glycolytic metabolism of semen components by washed guineapig spermatozoa. Fertil SterH 28:144, Peterson RN, Freund M: An evaluation of the respiratory capacity of human spermatozoa. J Reprod Fertil 17:357, Rogers BJ, Uena M, Hambert H, Hale RW: Analysis of human spermatozoa by in"vitro fertilization of zona-free animal eggs. Squibb Prize Lecture, presented at the Thirty-Fifth Annual Meeting of The American Fertility Society, February 3 to 7, 1979, San Francisco 12. Yanagimachi R, Noda YD: Physiologic changes in the post nuclear cap regions of mammalian spermatozoa: a necessary preliminary to the membrane fusion between sperm and egg cells. J Ultrastruct Res 31:486, 1970