Printed in Sweden Copyright 0 1975 by Academic Press, Inc. A/I rights of rrproduction in any form reserwd

Experimental

PHYSIOLOGICAL COMPONENT

Cell Research 90 (1975) 56-62

STUDIES RESPONSIBLE

IN SEA URCHIN II.

ON THE FOR

SPERM

SPERM-EGG

SURFACE BONDING

FERTILIZATION

Effect of Concanavalin A on the Fertilizing

Capacity of Sperm

K. AKETA Nagoya,

Biological Institute, Faculty of Science, 464, and Sugashima Marine Biological

Nagoya Station,

University, Toba, 517, Japan

SUMMARY Sperm of the sea urchin, Anthociduris crassispina loses its fertilizing capacity, without losing its motility, on prior exposure to both native and trypsin-digested, univalent Concanavalin A (Con A). Neither agglutination nor acrosome reaction is evoked by ConA treatment. Fluoresceinconjugated ConA binds to the apex of sperm head and to the midpiece. The observed effects of ConA are cancelled by methyl a-o-mannoside. ConA neither binds to sperm of Hemicentrotus pulcherrimus nor renders it infertile. Fertilizability of egg of both species is not reduced by Con A, though formation of the fertilization membrane and 1st cleavage are seriously affected. It is suggested that the species-specific polysaccharide component is situated on the apex of the sea urchin sperm head and constitutes the counterpart to the sperm-binding protein of the vitelline membrane of the egg which belongs to the same species.

In a preceding paper [I], this author presented some data in favour of a view that an unknown component is situated on the apex of sea urchin sperm head and bears complementary relationship to the sperm-binding protein on the vitelline membrane of the egg of the samespecies.The initial association between sperm and egg surfaces, sperm-egg bonding, may be interpreted in terms of interaction between these components. Although trypsin reduces the fertilizability of the seaurchin egg by disrupting the spermbinding site on the vitelline membrane [2], it does not depress but prolongs the fertilizing capacity of sea urchin sperm [3-61. This fact seems to imply that the factor functioning in sperm-egg bonding at the Exptl

Cell Res 90 (1975)

sperm surface is not of a proteinaceous nature, or, at least, does not have protein at its significant component. It was previously shown that carbohydrate moiety of sperm-binding protein from the egg surface does not seemto play an essential role in binding the sperm [7]. This is coincident with the work that sodium periodate rather improves the fertilizability of sea urchin egg [8, 91. On the other hand, polysaccharide may constitute the main component of the counterpart at the sperm surface to the sperm-binding protein of the egg surface. Presence of polysaccharide or glycolipid on the acrosomal region was reported in mammals [lo, 1l] and in sea urchin [12]. This possibility is studied in the present

Sperm wrfuce 100

1

component in fertilization. 0

0

II

57

Cl-0

--em_ ----L,,--

---*

b

0 0

15

30

45

GO

0

15

30

45

60

Fig. 1. Abscissa: duration of exposure to ConA (mm); ordinate: fertilization rate ( “6). O-O, Con A; O-O, ConA+mannoside; O---O, normal sea water. Effect of ConA on the fertilizing capacity of sperm. (a) Anthociduris crusispina; (6) Hemicentrorus pulcherrimus.

work with the aid of Concanavalin A (ConA), a phytoagglutinin obtained from jack bean, Canavalia ensiformis. This protein reacts with a variety of polysaccharides which have branched structures and a-D-glycopyranosyl, cc-D-mannopyranosyl, /?-D-fructofuranosyl, or a-D-arabinofuranosyl residuesoccupying nonreducing terminal positions (for references see [13]). MATERIAL

AND METHOD

Eggs of the sea urchins Hemicentrotus pulcherrimus and Anthocidaris (Heliocidaris) crassispina were obtained by pouring isotonic KC1 into the opened body cavity. Jellv coat surrounding the egg was removed by titrating the egg suspension to FH 5.2 with dilute HCI. Snermatozoa were obtained bv cutting ripe testes removed from dissected male. Sperm concentration was expressed by dilution from the original semen (‘dry’ sperm). Fertilizing capacity was estimated by counting cleaving eggs out of 300 specimens and expressed by percentage.

Preparation of univalent Con A Univalent ConA was prepared according to the method of Burger & Noonan [ 141. Ten mg Con A

(Pharmacia) was dissolved in 10 ml of 0.2 M phosphate buffer (pH 7.0) containing 10 mg trypsin (Difco) and incubated at 37°C for 5 h. Enzymic digestion of ConA was stopped by addition of 7 mg soy bean trypsin inhibitor (Sigma, Type I-S). As control, trypsin and inhibitor were added to the ConA solution at the same time and incubated as above.

Conjugation of jluorescein to ConA The method of Tkacz et al. [15] was employed with some modifications. One mg of fluorescein isothiocyanate (Baltimore Biol. Lab.) was dissolved in 1 ml of 0.1 M Na,HPO,. To this solution was added 12.5 mg of Con A (Boehringer) in 2 ml of 1 M NaCl. The reaction mixture, pH 8.3, was incubated at room temperature (23°C) for 3 h and then dialysed overnight at 4°C against 2 1of 1 M NaCl. After the small amount of precipitate which formed on dialysis was removed by centrifugation (15 000 rpm, 15 min, 4”C), the mixture was placed on a column (0.8 by 24 cm) of Sephadex G-75 (Pharmacia) previously equilibrated with 1 M NaCl. When the column was eluted with 1 M NaCl, a yellow peak appeared in the void volume. The bulk of the vellow material which remained on the upper third-of the column was eluted bv a solution of 0.1 M glucose in 1 M NaCl. The second peak, which con&ted of active, fluoresceinconjugated Con A and any remaining unmodified ConA, was pooled (ca 7 ml) and dialysed overnight at 4°C against 2 1 of normal sea water to remove the glucose. Exptl

Cell Res 90 (1975)

58

K. Aketa

Table 1. Effect of trypsin-digested, univalent ConA on the fertilizing capacity of Anthocidaris sperm (percentage fertilization of jellyless eggs) with 10 min pretreatment Sperm dilution

(1 m/ml)

Native Con A Univalent ConA

10-a

5 x 10-d

48 43

28 21

Fluorescence was observed under an Olympus fluorescence microscope Model FLM. For inspection of the acrosomal region of spermatozoa, the material was fixed for 10-30 min in 10 % formalin-sea water, passed through sea water and distilled water, and then observed under an electron microscope, JEM “Superscope”, at a magnification of x 4 000.

RESULTS Effect of native ConA on the fertilizing capacity of sperm Sperm diluted 200-2 OOO-foldwas mixed with an equal volume of sea water containing ConA at a concentration of 1 mg/ml, and, after given intervals of time, the aliquots were added to a given volume of egg suspension for insemination. Quite different results were obtained between the sperm of Anthocidaris and of Hemicentrotus with respect to sensitivity to ConA. As shown in fig. 1, in which 400-fold dilution sperm was used, Anthocidaris sperm gradually lost its fertilizing capacity, without losing its motility, by the treatment, whereas Hemicentrotus sperm remained fertilizable longer than in normal sea water. ConAtreated Anthocidaris sperm seldom became bound to the egg surface. Only when the suspension was denser (below 200-fold dilution) than that used in the above experiment, did Con A cause agglutination of head-tohead, head-to-tail or tail-to-tail Anthocidaris sperm. Agglutination of Hemicentrotus sperm was never observed. No acrosome reaction was evoked by ConA in either species. Exptl

Cell Res 90 (1975)

2. Anthociduris spermatozoa exposed to fluorescein-conjugated ConA. Apex of the acrosome (urrows) and midpiece fluoresce. :’ 400.

Fig.

None of the above effects of Con A to Anthocidaris sperm were observed in the presence of methyl a-D-mannoside (100 pmole/ml). Effect of univalent ConA on the fertilizing capacity of sperm To avoid the criticism that the observed effects of ConA result by virtue of bi- or multivalency of ConA, univalent Con A was prepared [14], and was examined with Anthocidaris sperm. Being different from native ConA, the digested one did not cause sperm agglutination, even in an extraordinarily dense suspension. However, it reduced the fertilizing capacity to a similar extent as did the native one (table 1). The experiment was

Sperm surface component in fertilization.

II

59

Fig. 3. Hemicentrotus eggs exposed to ConA (a) or ConA+mannoside (b) and fertilized. Many precipitates appear in the perivitelline space of ConA-exposed eggs whose membranes sink gradually afterwards.

performed following the procedure employed in the native Con A experiment except that the duration of exposure to ConA was restricted to 10 min. Binding of fluorescent ConA to sperm surface Sperm diluted 200-I OOO-fold was mixed with an equal volume of fluorescent Con A +sea water (ca 1 mg/ml) and incubated at room temperature (20°C) for 30 min. After being washed in two changes of filtered sea water, the sperm was fixed in 10 y0 formalin sea water. The specimens were dialysed against distilled water overnight and airdried in the cold. It was then mounted with a medium consisting of 9 vol glycerine and 1 vol 0.25 M Na,CO,-NaHCO,(pH 9.0). As

control 30 mg of methyl cr-D-mannoside was added to 1 ml of fluorescent Con A + sea water, and examined as above. Fluorescence was detected in pinpoint at the apex of the acrosomal region under the fluorescence microscope, though it was blurred in fig. 2 in the process of taking the photograph. The midpiece also fluoresced. The fluorescence was negligible in sperm treated with fluorescent Con A t- mannoside mixture. No binding of fluorescent Con A to Hemicentrotus sperm was detected. Effect of ConA on the egg To draw a comparison with the sensitivity of sperms to ConA, unfertilized eggs were also treated with Con A + sea water (final Exptl

Cell Res 90 (197.5)

60

K. Aketa

Fig. 4. Anthociduris eggs exposed to fluorescein-conjugated Egg surface fluoresces. x 200.

cont. 0.5 mg/ml) for more than 1.5 h. No apparent morphological change occurred. The jelly coat was not precipitated. The eggs with or deprived of jelly coat were washed with three changes of normal sea water and inseminated. Fertilization was not inhibited at all, but the elevation of the fertilization membrane was incomplete, both in Anthocidarb and Hemicentrotu eggs. In the former, the membrane formed was lower than the control from the beginning, and in the latter, the membrane elevated high at first and gradually sank to the egg surface. Such an incomplete elevation of the fertilization membrane appears to be caused by the precipitation of the component derived from the cortical granules broken up following fertilization (fig. 3). Cleavage was seriously blocked in Anthocidaris eggs, but nuclear division did take place. In the presence of methyl a-D-mannoside, the described effects of ConA were not observed. Exptl

Cell

Res 90 (197.5)

Con A. Formalin-fixed,

unsectioned specimen.

Fluorescent ConA bound to the surface, probably the vitelline membrane, of Anthocidaris egg (fig. 4). In Hemicentrotus egg, fluorescence could only be detected in the perivitelline space of fertilized egg. It must be caused by binding of fluorescent ConA to the substance derived from the cortical granules. DISCUSSION As described above, ConA binds to the apex of Anthocidaris sperm head and seriously interferes with the binding of sperm to the surface of the egg. On the other hand, this reagent does not bind to Hemicentrotus sperm and does not affect its fertilizing capacity. Such a difference in ConA sensitivity between these two speciesis of great interest from the aspect of speciesspecificity of sea urchin fertilization. Unpublished data of the present author show that wheat germ agglutinin affects the fertilizing capacity of

Sperm surface component in fertilization. sperm, not of Anthocidaris, but of Hemicentrotus. Thus, the effects of ConA and wheat germ agglutinin to these two species are reciprocal. These data seem to strongly suggest that a factor of polysaccharide in nature is located at the apical surface of sperm acrosome and plays the central role in establishing species specific bonding between sperm and egg. The isolation of the substance which appears to be this factor has recently been made with success and will be reported later. Edelman & Millette [l l] found that Con A causes agglutination of sperm of mouse, rat, guinea pig, rabbit and human and that fluorescein-conjugated ConA binds mainly to the acrosomal region of mouse sperm. Agglutination by Con A was also reported by Nicolson & Yanagimachi [ 161 who used sperm of rabbit and of hamster as materials. The polysaccharide-containing site on the mammalian sperm surface may play an essential role in the interaction of sperm and egg, as the present work has shown in sea urchin sperm, but no work has hitherto appeared on this matter. ConA does not reduce the fertilizability of egg of either species studied. It was previously suggested that the carbohydrate moiety of sperm-binding protein of sea urchin egg is not essential for sperm-binding [7], though it may help in the recognition of species. This is consistent with work which shows that the fertilizability of sea urchin egg is rather improved by sodium periodate

K.6 91. According to Wiese & Shoemaker [17], conjugation between opposite gametes of Chlamydomonas is entirely prevented when androgamete was preincubated with Con A. Gynogamete was insensitive to this reagent. On the other hand, trypsin was reported to affect the conjugal activity of gynogamete only [18]. A similar reciprocal relationship

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also exists between the opposite mating types (strains 5 and 21) of yeast, Hansenula wingei [19]. Agglutinability of strain 5 is resistant to trypsin and sensitive to periodate, whereas strain 21 is sensitive to the former and comparatively resistant to the latter. Sneath & Lederberg [20] arrived at an assumption that the carbohydrate specifically associates with the conjugal activity of male cells of Escherichia coli K 12. Periodate does not make female cells infertile. It goes without stating, however, that these works do not lead to the conclusion that polysaccharide residue is essential only for one sex gamete and protein component is essential only for the opposite. It was recently reported that both the protein and carbohydrate components contribute to the recognition and binding activity of 5agglutinin purified from Hansenula wingei type 5 [21], and that the carbohydrate component of the sperm-binding site on the zona pellucida of hamster egg may participate in the fertilization [22, 231. It seems of interest, nevertheless, that an apparent contrast exists beyond the taxonomic distance between the opposite gametes with respect to the sensitivity to some fertilization-affecting reagents. ConA interferes with the formation of the fertilization membrane and also blocks the first cleavage. Lallier [24] reported similar results in Paracentrotus lividus, though he did not distinguish fertilization sharply from the formation of the fertilization membrane and initiation of cleavage. Incomplete formation of the fertilization membrane is most probably caused by precipitation of the carbohydrate component derived from the cortical granules with ConA. The polysaccharide nature of the cortical granules was previously reported [25, 26, 271. It is of great interest that ConA blocks cleavage. Since this effect is cancelled by mannoside, some polysaccharide component seems to participate in advancing cleavage. According to Kinoshita Exptl

Cd

Res 90 (1975)

62 K. Aketa

PI> intracellular

release of heparin-like substance exerts a localized control of the sol-gel state of the cytoplasm and plays an important role in the cleavage of sea urchin egg. Recent work by Kojima [29] shows that the substance extracted by acidified sea water from unfertilized sea urchin egg promotes the cleavage and that this substance precipitates with ConA. This work was supported in part by a grant-in-aid of the Ministry of Education, Japan.

REFERENCES 1. Aketa, K, Exptl cell res 80 (1973) 439. 2. Aketa, K, Onitake, K & Tsuzuki, H, Exptl cell res 71 (1972) 27. 3. Wicklund, E, Ark zoo1 42A (1949) no. 11. 4. Hagstrom, B E, Ark zoo1 12 (1959) no. 10. 5. Aketa, K. Unpublished. 6. Onitake, K. Personal communication. 7. Tsuzuki, H & Aketa, K, Exptl cell res 55 (1969) 43. 8. Runnstrom, J & Kriszat, G, Exptl cell res 1 (1950) 355. 9. Hagstrom, B E & Hagstrom, B, Exptl cell res 6 (1954) 479. 10. Leblond, C P & Clermont, Y, Am j anat 90 (1952) 167.

Exptl

Cell Res 90 (1975)

11. Edelman, G M & Millette, C F, Proc natl acad sci US 68 (1971) 2436. 12. Nagai, Y, Oosawa, T & Hoshi, M, Zoo1 mag 81 (1972) 436. 13. Sharon, N & Lis, H, Science 177 (1972) 949. 14. Burger, M M & Noonan, K D, Nature 228 (1970) 512. 15. Tkacz, J S, Cybulska, E B & Lampen, J 0, J bact 105 (1971) 1. 16. Nicolson, G L & Yanagimachi, R, Science 177 (1972) 276. 17. Wiese, L & Shoemaker, D W, Biol bull 138 (1970) 88. 18. Wiese, L & Metz, C B, Biol bull 136 (1969) 483. 19. Brock, T D, J bacterial 78 (1959) 59. 20. Sneath, P H A & Lederberg, J, Proc natl acad sci US 47 (1961) 86. 21. Yen, P H & Ballou, C E, J biol them 248 (1973) 8316. 22. Oikawa, T, Yanagimachi, R & Nicolson, G L, Nature 241 (1973) 256. 23. Oikawa, T, ‘Nicolson, G L & Yanagimachi, R, Exptl cell res 74 (1974) 239. 24. Lallier, R, Exptl cell res 72 (1972) 157. 25. Monne. L & Harde. S. Ark zoo1 ser 2 (1951) \ I 487. ’ 26. Immers. J. Exntl cell res 19 (1960) 499. 27. Aketa, K,’ Embryologia 7 (1962) ‘223. 28. Kinoshita, S, Exptl cell res 56 (1969) 39. 29. Kojima, M K, Zoo1 mag 82 (1973) 241. I

I

Received June 7, 1974 Revised version received July 16, 1974

Physiological studies on the sperm surface component responsible for sperm-egg bonding in sea urchin fertilization. II. Effect of concanavalin A on the fertilizing capacity of sperm.

Printed in Sweden Copyright 0 1975 by Academic Press, Inc. A/I rights of rrproduction in any form reserwd Experimental PHYSIOLOGICAL COMPONENT Cell...
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