Acrosomal ATPase in starfish and bivalve mollusk spermatozoa

Printed in Sweden Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in my form reserved

Experimental

ACROSOMAL

Cell Research 82 (1973) 271-219

ATPASE IN STARFISH

AND

BIVALVE

MOLLUSK

SPERMATOZOA YORIKO

MABUCHI’

and ISSEI MABUCHP

‘Department of Biology, Ochanomizu University, Bunkyo-ku, Tokyo 112 and 2Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, and The Misaki Marine Biological Station, Miura-shi, Kanagawa, Japan

SUMMARY ATPase activity was found in acrosomes of starfish and bivalve mollusk spermatozoa, using a cytochemical method with electron microscopy.The activity waslocated in central material of the starfish acrosomeand in material lining the acrosomal membrane of the Mytilus acrosome, as well as in the basal part of the starfish acrosome.The ATPase activity in the former material was preferably activatedby Ca2+, while that in the starfish basalmaterial was preferably activated by Mg2+. Both types of activity persisted during and after the acrosome reaction. ATPase activity was also observed in the region of the axial filament complex of the flagella, in centriolesand in a basal matrix. ATPase in the acrosome a.lso hydrolysed other nucleoside triphosphates. However, there was no detectable phosphatase activity, Ad little pyrophosphat&e 0; 5’-nucleotidase activity. Evidence was obtained that adenylate kinase may be included in the acrosome. A possible role of the ATPase activity in the acrosdme reaction is discussed.

The acrosome reaction of starfish and Mytilus spermatozoa involves a rapidly occurring change in the contents of the acrosome [2, lo]. In the starfish, this consists in the centrifugal expulsion of material filling the center of the acrosome. In Mytilus, this is seen as an explosive eversion of material lining the acrosomal membrane. The swelling of this material, caused by hydration, has been suggested as the source of the force which induces this rapid change in the reacting MytiZus acrosome [lo]. In addition to such physical properties of the material, its chemical properties should also be considered in the attempt to explain this phenol Present address: Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, Hyakunincho, Shunjuku-ku, Tokyo 160, Japan. 2 Present address: Department of Biology, University of Tokyo, Komaba, Meguro-Ku, Tokyo 153, Japan. 19-731808

menon. The demonstrate components somes which

results reported strong ATPase of starfish and are the agents

MATERIALS

in this paper activity in the Mytilus acroof this change.

AND METHODS

Biological materials. Gametes of the starfishes, Asterina pectinifera and Asterias amurensis, and the bivalve mollusk, Mytilus edulis, were used. Spermatozoa were obtained by squeezing the gonads through nylon cloth, and suspended in-sea water containing 0.1 mM EDTA as described by Hagiwara et al. [6]. Egg-jelly solution was obtained by dissolving the jelly at pH 5.0 from a dense suspension of spawned eggs [2]. The pH was returned to 8.2 immediately. Induction of acrosome reaction. The acrosome reaction was induced by exposing spermatozoa to eggjelly solution in starfish, and by increasing the Ca2+ cont. of the sperm suspension in Mytilus [13]. Fixation of spermatozoa. Suspensions of Asterina and Asterias spermatozoa were fixed with 0.3 % glutaraldehyde in sea water for 30 min in an ice bath. Mytilus sperm were fixed with 0.1 % glutaraldehydesea water for 13 min. Exptl Cell Res 82 (1973)

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Y. Mabuchi & I. Mabuchi

Detection of ATPase and other enzyme activities. After fixation, sperm were washed twice by suspension in chilled 50 mM Tris-maleate buffer (pH 7.8), dialysed against the same buffer solution for 1 h at 4°C and incubated at 25°C for 20 min in Wachstein-Meisel (WM) medium [12] as modified by Marchesi & Palade [8] by reducing the concentration of lead nitrate from 2 mM to 0.2 mM. The incubation medium contained 4 mM substrate. 80 mM Tris-maleate (uH 7.8), 10 mM CaCI, or MgS’O,, and 0.2 mM Pb(NO& This medium was prepared just before use. Lead nitrate was added dropwise in the last stage to prevent precipitation. For the control, samples were incubated without either divalent cations or substrate. The substrates used were: ATP (Kohjin Co.), ITP (Sigma Chem. Co.), GTP (Yamasa Shoyu Co.), ADP (Kyowa Hal&o Kogyo Co.), AMP (Kohjin Co.), B-glycerophosphate (E. Merck AG), and inorganic pyrophosphate. Acid and alkaline phosphatase activities were also detected using /3-glycerophosphate as substrate, according to method of Gomori [4] modified by Barka [ 1] and Hugon & Borgers [7].

low concentrations of the fixative preserved cell structures sufficiently for the purposes of this experiment, although damage, probably incurred during washing out the fixative, was often observed in the midpieces. Glutaraldehyde routinely fails to fix a small granule of osmiophilic material located in the center of the acrosome (g, fig. 1; figs 2, 3, 4).

No deposit of lead phosphate was observed in any region of the spermatozoon in controls in which Ca2+, Mg2+ or ATP was omitted from the Wachstein-Meisel (WM) medium (fig. 2). After incubation in the standard WM medium using ATP as substrate, however, deposits of lead phosphate were observed Preparation for electron microscopy. The sperm were throughout the material, filling the center of post-fixed with 1 % OsO,, dehydrated with ethanol and embedded in stylene-n-butylmethacrylate. Sec- the acrosome (a material) in Asterina spermations were cut on a Porter-Blum microtome and tozoa (figs 3, 4). This indicates that the viewed with a Hitachi HS-7 electron microscope. ATPase in this region is strongly activated by either Ca2+ or Mg2+. Ca2+ seemed to be RESULTS more effective than Mg2+ at 10 mM, judging from the amount of lead phosphate deposited. After Dan & Hagiwara [2] and Niijima & On the contrary, no activity was observed in Dan [IO], the pertinent changes in the course of the acrosome reaction in starfish and the g region. Weak ATPase activity was also induced by Mytilus spermatozoa will be summarized as Ca2+ in the f material, which would be located follows. In the first step of the reaction, the at the base of the fibrous shaft of the acroperiacrosomal plasma membrane and underlying acrosomal trigger component are dis- somal process when the acrosome reacted rupted following stimulation by contact (fig. 3). The deposit of lead phosphate, howwith the egg-jelly (starfish) or egg-surface ever, was more conspicuous when the prepara(Mytilus). Then the rapid centrifugal retrac- tion was activated by Mg2+ at 10 mM (fig. 4). During the acrosome reaction, the enzyme tion followed by the outflowing (starfish) or the rapid eversion (Mytilus) of the acrosomal activity was still observed in the a material contents takes place (fig. 1, A-2 and B-2). In after it had flowed out around the nucleus the last step, the acrosomal process elongates (fig. 5). The activity in the f material remained in the same place, at the base of the process, (fig. 1, A-3, and B-3). after the reaction (fig. 5). None was found in ATPase activity in acrosome association with the fibrillar components of The very low concentrations of glutaralde- the acrosomal process shaft. A similar distribution of ATPase in the a hyde (0.1-0.3 %) used for fixation, resulted in satisfactory retention of ATPase activity and f materials was observed in the spermatoin the acrosomes, whereas higher concentra- zoa of Asterias (fig. 6). tions completely destroyed such activity. The In the case of Mytilus edulis, the precipiExptl

Cell Res 82 (1973)

Acrosomal A TPase

2 73

A-3

a

M

B-3

am

:y< ,; ;..I,. .7. .._/ I.. k ‘;:. fs :.: )).. \;...

1

a

Fig. I. Diagrammatic representation of acrosome reaction in starfish and Myths spermatozoa. (A) starfish. (B) Mytilus. (A-l, B-l) intact acrosomes. (A-2, B-2) acrosome opened, a material retracts (in starfish sperm) or everts (in Myths sperm), and fibrous shaft begins to elongate from f region (starfish sperm). (A-3, B-3) after the acrosome reaction. a material flows outside of the cell and the fibrous shaft elongates completely. To illustrate these sections, reports of Dan & Hagiwara [2] and Niijima & Dan [IO] were referred to. Terms, cr,f. andg were according the former. a, a material; am, acrosomal membrane; BM, basal matrix; Cd, distal centriole: Cp, proximal centriole; F, flagellum; f, f material; fs, fibrous shaft; g, granule; M, mitochondrion; N, nucleus: nm, nuclear membrane; pm, plasma membrane.

tates of lead phosphate were observed in the material lining the acrosomal membrane, which may correspond to the a material of the starfish acrosome (fig. 7). After the acrosome reaction had taken place, ATPase activity was still detectable in remnants of this material which persisted along the everted acrosomal membrane (fig. 8). Other nucleotidase activities in Asterina acrosome No significant difference in the amount of deposit of lead phosphate was observed in

either a or f materials when ATP was replaced by ITP, GTP or ADP in the presence of Ca2+ (fig. 9-C). When 5’-AMP was used as a substrate, most cells showed no deposit. although in 2-3 % of cells a weak 5’-nucleotidase activity was observed (100 %, of the cells showed a strong ATPase activity in the same series of experiments) in the a material. Phosphatase activities were never detected in the acrosomes (fig. IO), although a weak pyrophosphatase activity was observed in the a material in the presence of Mg*+. Therefore, a part of Mg2+-activated hydrolysis of Exptl Cell RES82 (1973)

Figs 2-5. ATPase activities in Asterina

pectinifera spermatozoa. x 35 000. Fig. 2, unreacted spermatozoon incubated in WM medium without divalent cations; fig. 3, unreacted spermatozoon incubated in WM medium containing 10 mM CaCI,. Deposits of lead phosphate are clearly seen in the a material (a) and flagellum (F). A few deposits are also seen in the f material (f); fig. 4, unreacted spermatozoon incubated in WM medium containing 10 mM MgSO,. Deposits of lead phosphate are seen in the a material (a) (but fewer than seen in fig. 3), f, material (j), flagella (F), centriole (C), plasma membrane (pm) and around acrosomal membrane (am); fig. 5, reacted spermatozoon incubated in WM medium containing 10 mM CaCI,. Deposits are abundant in the outflowing a material (a). They are also seen in the f material cf) and centriole (C); N, nucleus; M, mitochondrion; fs, fibrous shaft; g, granule. Exptl

Cell Res 82 (1973)

Figs 6-8. ATPase activities in Asterias amurensis and Myths edulis spermatozoa. 3 30 000. Fig. 6. unreacted Asterias amurensis spermatozoon incubated in WM medium containing 10 mM CaCI,. Deposits of lead phosphate are seen in the a material (a) and f material (f); fig. 7, unreacted Mytilus edulis spermatozoon incumated in WM medium containing 10 mM CaCl,. Deposits are seen in the a material (a) and flagella (F);fig. 8, reacted Myths edulis spermatozoon incubated in WM medium containing 10 mM CaCl,. Deposits are seen in the everting a material (a) and in the flagella (F); N, nucleus; M, mitochondrion; fs, fibrous shaft. Exptl Cell Res N? (i97.7)

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Y. Mabuchi & I. Mabuchi

Fig. 9. ATPase and ITPase activities in the centriolar region of Asterina pectinifera spermatozoa. x 60 000. (A) Transverse section of the proximal centriole. Incubated in Wachstein-Meisel medium containing ATP and CaCI,. Deposits are seen in the proximal centriole (Cp), basal matrix (BM), mitochondrion, and a material; (B) transverse section of the proximal centriole and oblique section of the distal centriole. Incubated in WM medium containing ATP and CaCl,. Deposits are seen in the two centrioles (Cp, Cd) and basal matrix (BM); (C) longitudinal section of the distal centriole. Incubated in WM medium containing ITP and CaCl,. Deposits are seen in the distal centriole (Cd), basal matrix (BM), flagellum (F), a material and f material.

[9] and Gordon & Barrnett [5]. The enzyme activity in the centrioles was located in the region of the microtubular triplets (fig. 9). ATPase in the flagella, centrioles and basal ATPase and other enzyme activities in matrix hydrolysed ITP and GTP as well as other region of Asterina spermatozoon ATP (fig. 9-C). Deposits were also observed ATPase activity was also observed in the in these three regions when ADP was used mitochondria (fig. 9-A), flagella (figs 3, 4, 7, as the substrate. The effects of Ca2+and Mg2+ 8), proximal and distal centrioles (figs 4, 5, on these ATPase activities seemed to be 9), basal matrix (fig. 9) and plasma and acro- similar. These results are summarized in somal membranes (fig. 4). Flagellar ATPase table 1. The mitochondrial and the membraneactivity was located in the region of the axial filament complex, as reported by Nagano bound ATPase activities were found to be

ATP may be due to the pyrophosphatase activity.

Exptl CelI Res 82 (19 73)

Acrosomal A TPase 271 Table I. Summary of enzywzqkctivities found in Asterina spermatozoa ATP Part of sperm cell a material f material Flagellum Centrioles Basal matrix

Ca*+ ,/

1

Mg2+ T + t - t ,- ~. by!

ITP Ca2+

GTP Ca2+

~- + I-

I _~ -~ L I-

-r 1 ~-

ADP CaY+

AMP Ca’,

b-G’ Ca’+

Mg”-

PPi

Mg’

-~--

All expts were performed in Wachstein-Meisel medium. f, strong activity always observed; , activity always observed; &, weak activity sometimes observed; ~, activity never observed. /SGP, ,!?-glycerophosphate: PPi, inorganic pyrophosphate.

relatively weak under the present experimental conditions so that they could not be detected in some spermatozoa (figs 3, 4, 5). However, a considerable amount of strong phosphatase activity (especially alkaline phosphatase activity) was observed in association with the mitochondria and the plasma and nuclear membranes (fig. IO).

major constituent of the a material is protein, on the basis of its removal from unfixed, thin sectioned acrosomes by pronase digestion (I’. Mabuchi, unpublished). This material seems to exist as radiational arrays of fila-

DISCUSSION Except for the observation made by Gordon & Barrnett [5] of ATPase activity associated with the acrosomal membrane of guinea pig spermatozoa, no evidence has been reported concerning the activity of this enzyme in the acrosome. However, the present work demonstrates ATPase activity in a centrally located component of starfish and Mytihs acrosomes, and in material of the starfish acrosome concerned with the formation of the axial fiber that supports the acrosomal process. Biochemical explanations for the various changes making up the acrosome reaction have not been proposed, except for a recent suggestion that the elongation of the starfish acrosomal process is due to the polymerization of an actin-like substance [ll]. The only information concerning the chemical constitution of these acrosomes is that a

Fig. 10. Phosphatase activity in Asterina

pectinifera

spermatozoa. x 35 000. Unreacted spermatozoon incubated in WM medium containing p-glycerophosphate as substrate and CaCI,. Deposits of lead phosphate are seen in the mitochondrion (M), plasma membrane (pm) and nuclear membrane (nm). while a and f materials and flagella (F) show no deposit. Exptl Cell Res 82 (1973)

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Y. Mabuchi & I. Mabuchi

mentous structures in the both starfish [6] and MytiZus ([lo] and S. Endo, unpublished) acrosomes when they are fixed with 0~0, only. (The best electron micrograph showing these elements is fig. 6 in [6]). The present investigation has added the information that ATPase activity is present in the a material of both starfish and Mytilus acrosomes. These observations form the bases of our assumption that the material(s) contiguous to the acrosomal membranes in starfish and Mytilus acrosomes have essentially the same role in the acrosome reaction. The consideration that ATPase generally provides cell organelles with energy for some kind of change, suggeststhat the ATPase in the a material may produce energy for the acrosome reaction. From the explanation of the course of the acrosome reaction, it is possible to consider that the ATPase in the a material is activated by the entrance of sea water in the first step of the reaction, which would be confirmed by the fact that this enzyme is strongly activated by Ca2+. The enzyme might generate the energy for the rapid eversion of the a material interacting with the filamentous structures. However, the possibility that the filamentous structures are artefacts due to fixation cannot be denied. The ATPase activity in the f material of the starfish acrosome seemedto be relatively weaker than that of the a material. The activity remained in that region after the acrosome reaction. Recently Tilney et al. found evidences that the fibrous shaft of the starfish acrosome consists of a bundle of F-actin filaments which extend from the f material when the reaction occurs [I 11. The ATPase in this region might play some role in the process of elongation of these filaments. The ATPase in the acrosome may have a low substrate specificity becauseit hydrolysed GTP and ITP as well as ATP. The hydrolysis of ADP indicates the existence of adenylate Exptl

Cd Res 82 (1973)

kinase in the acrosome. Furthermore, 5’nucleotidase and pyrophosphatase activities were found to some extent in the a material. The significance of these enzymes in the acrosome reaction or fertilization is not clear. In addition to that related to the acrosome, ATPase activity was also observed in the two centrioles. Transverse sections show that the accumulation of deposit describes a circle (fig. 9), the outer and inner diameters of which are 0.18 and 0.07 pm, respectively (average value from 5 good transverse sections). These dimensions correspond well with those of the circle of triplet microtubules of the same Asterina spermatozoon, which are about 0.19 and 0.10 ,um (calculated from fig. 17 in [3]). This means that an ATPase is located around the microtubular triplets of the centrioles. A “basal matrix”, which surrounds the two centrioles and extends around the base of the flagellum along the surface of the midpiece mitochondrion, has not been described before. Although no apparent structure was seen by the fixation procedure used here, ATPase activity was found in this region. The activity in the centrioles and in the basal matrix was similar to that in the flagellum with respect to substrate requirement and degree of activation by Mg2+ or Ca2+. This ATPase activity may play some role in flagellar movement. The authors are indebted to Dr Jean Clark Dan for her encouragement throughout the work, valuable criticism and review of the manuscript. They also thank Dr Hikoichi Sakai for his interest and valuable discussions.

REFERENCES 1. Barka, T, J histochem cytochem 12 (1964) 229. 2. Dan, J C & Hagiwara, Y, J ultrastruct res 18 (1967) 562. 3. Dan, J C & Sirakami, A, Develop growth differ 13 (1971) 37. 4. Gomori, G, Proc sot exptl biol med 42 (1939) 23. 5. Gordon, M & Barrnett, R J, Exptl cell res 48 (1967) 395.

Acrosomal ATPase 6. Hagiwara, Y, Dan, J C & Saito, A, J ultrastruct res 18 (1967) 551. 7. Hugon, J & Borgers, M, J histochem cytochem 14 (1966) 629. 8. Marchesi, V T & Palade, G E, J cell bio135 (1967) 385. 9. Nagano, T, J cell biol 25 (1965) 101. 10. Niijima, L & Dan, J, J cell biol 25 (1965) 249. 11. Tilney, L G, Hatano, S & Mooseker, M S, J cell biol 55 (1972) 261 a.

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12. Wachstein, M & Meisel, E, Am j clin pathol 27 (1957) 13. 13. Wada, S K, Collier, J R & Dan, J, Exptl cell res 10 (1956) 377.

Received December 12, 1972 Revised version received July 3, 1973

Exptl Cell Res 82 (1973)