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rate peaks. It may be pointed out that the specific activities of the two enzymes recorded in Table 1 for various animals cannot be compared with each other since the supernatants of the homogenates contain only a portion of the total content of the respective hexosaminidases and since the instability of seryl-A-acetylgalactosaminidase3 may vary with the source. The relatively high specific activity of seryl-A-acetylgalactosaminidase of the oviduct of Rana

esculenta was observed only in frogs shortly before spawning.

This investigation was supported by the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t . The authors wish to thank Professor V. SCHWARTZ, Department of Zoo-logy, University of Tiibingen, for his valuable advice in the selection of the animals, and Dr. P E T E R D O H R N , Director of the Stazione Zoologica, Naples, for his generous cooperation in providing the maritime ani-mals.

Some Immunochemical Characteristics of Sea Urchin Gametes S T J E P A N K E Č K E Š 1 a n d M I R J A N A K R A J N O V I Ć

Institute „Ruder Bošković" and Institute of Marine Biology, Rovinj and Zagreb, Yugoslavia

(Z. Naturforschg. 22 b, 1032—1034 [1967] ; eingegangen am 27. Januar 1967)

By double diffusion in agar the antigenic composition of four sea urchin species was examined. At least one common antigenic determinant was found in both gametes of all the four species in-vestigated. Furthermore, in all eggs one and in all sperm another common "sex specific" antigenic determinant was established. In spite of the great number of antigenic determinants in each gamete extract, the existence of "species specific" determinants, common to sperm and eggs of one species, could not be determined with certainty.

Fertilization is at least partly due to the specific interaction between the chemical constituents of the sperm and egg surface. There are several methods for studying the chemical characteristics of the ga-metes, especially those which could play a certain role in fertilization. One of the best method is the immunochemical analysis introduced by T Y L E R 2 .

This method, which with regard to specificity is far beyond the classical chemical analyses, uses the very sensitive and highly specific antigen-antibody reac-tion to discover the chemical composition of bio-logical material.

Using the immunochemical analyses P E R L M A N N 3

found that in gamete extrats of Paracentrotus livi-dus, a common Mediterranean sea urchin, a great number of antigenically strongly different compo-nents are present. Furthermore, he showed that some of these components have a definite and specific ef-fect when combined with the corresponding antisera. K Ö H L E R and M E T Z 4 found a great number of dif-ferent antigens in sperm extracts of various sea urchins and some of them proved to be associated to the sperm surface 5.

1 Present address: Laboratory of Marine Radioactivity, International Atomic Energy Agency, Monaco.

2 A . TYLER, Physiol. Rev. 2 8 , 1 8 0 [ 1 9 4 8 ] . 3 P. PERLMANN, Experientia [Basel] 15, 41 [1959]. 4 K . KÖHLER and C . B. METZ , Biol. Bull. 1 1 8 , 9 6 [ I 9 6 0 ] .

It seemed worthwile to continue these investiga-tions and compare the results obtained with those of the investigations on homologous and hetero-logous fertilization 6 - 8 .

Material and Methods

Gametes of four different sea urchin species, com-mon along the west Istrian coast of the Adriatic sea (Paracentrotus lividus Lam., Arbacia lixula L., Sphaer-echinus granulans Lam. and Psammechinus microtuber-culatus Biv.) were used.

Freshly caught animals were opened by a circular cut with scissors and the ripe gonads were removed, se-parated according to sex and species, cut in smaller pieces and placed over dense gauze in a plastic funel. The extruded gametes were collected in bottles, cooled in ice and used freshly or freeze-dryed.

Antisera were obtained by hyperimmunization of rabbits with fresh or freeze-dryed gametes mixed with adjuvant 9. Ten days after the last injection of antigen the blood of rabbits was collected, sera were separated by centrifugation and stored at — 20 °C until use.

The following symbols were used for the antigens and the corresponding antisera:

5 G. P. FLAKE and C. B. METZ , Biol. Bull. 1 2 3 , 472 [1962]. 6 B. E. HAGSTRÖM and S. LGNNING, Sarsia 4, 5 [1961]. 7 S. KEČKEŠ, Ph. D. thesis, Univ. of Zagreb 1962. 8 B . E . HAGSTRÖM, Sarsia 1 7 , 3 3 [ 1 9 6 4 ] . 9 J . FREUND and K . MCDERMOTT, Proc. Soc. exp. Biol. Med.

49, 548 [1942].

Jp , Js, jps and JA — extracts of Paracentrotus, Sphaerechinus, Psammechinus and Arbacia eggs;

Sp , Ss , Sps and SA — extracts of Paracentrotus, Sphaerechinus, Psammechinus and Arbacia sperm;

Anti Jp and Anti Sp — antisera to Paracentrotus eggs and sperm;

Anti Js and Anti Ss — antisera to Sphaerechinus eggs and sperm.

Extracts of gametes were prepared by homogeniza-tion in a tissue grinder. Freeze-dryed gametes were homogenized as 1% suspensions. Fresh gametes were homogenized and diluted with sea water or water of the same osmotic value. Both homogenates were left standing 24 — 48 hours, filtered through rough filter paper, and the filtrates used as gamete extracts.

In absorption experiments, to each ml of antiserum 0.5 ml of undiluted fresh gametes was added. After 12 hours of standing at + 4 °C the mixture was centri-fuged and the clear supernatant used for experiments.

For diffusion in gel a slight modification of O U C H T E R -

L O N Y ' S diffusion method10 in Difco bacto agar was used. Agar plates of 6 mm thickness were prepared in Petri dishes. Holes ( 0 = 1 2 mm) in the agar were punched at distances of 15 mm. The wells were usually filled with antisera or gamete extracts only once to give a flat meniscus and the plates were then stored in humid atmosphere at room temperature or at + 4 ° C until precipitation lines were developed.

Results and Discussion

In the first series of experiments the reaction be-tween the extracts of Paracentrotus and Sphaerechi-nus gametes and the corresponding antisera were studied. Using various antisera and extract com-

Fig. 1. Scheme of precipitation lines between the gamete extracts and the corresponding antisera.

1 0 0 . OUCHTERLONY , Ark. Kemi, B 2 6 , 1 [ 1 9 4 9 ] .

binations in a great number of analyses the maxi-mally obtainable number of precipitation lines was established.

The results of these investigations are schemati-cally represented in Fig. 1 where the position, shape and relative thickness of the precipitation lines cor-respond to the actual situation. The lines are designated for easier orientation.

By this method it has been shown that eggs con-tain at least eight and sperm six different antigenic determinants (Table 1), because one should keep in mind that each precipitation line may represent more than one antigen-antibody combination.

obtained in com- antigenic determinants bination

Jp/Anti Jp AP BP Cp DP EP FP Gp Hp Js/Anti Js As Bs Cs Ds Es Fs Gs Hs Sp/Anti SP ap bp ep dp ep fp Ss/Anti Ss bs C s ds es fs

Table 1. Antigenic determinants of Paracentrotus and Sphaerechinus sperm and eggs. The symbols correspond to

those used in Fig. 1.

The relations between these antigenic components were studied in a further series of experiments in which the precipitation lines of various combina-tions of gamete extracts and antisera were obtained.

Analysing the precipitation lines it was possible to establish that Paracentrotus and Sphaerechinus eggs have two (or maybe even three) and their sperm also two antigenic determinants in common. Only one of these determinants was common to the sperm and eggs of both species investigated (Table 2 ) . Besides these identical antigenic determinants several similar, i. e. partially identical determinants were recorded. These investigations were extended

antigen antigenic determinants

Jp AP(?) Bp Cp Js Bs c s J P S + + J A + + S P M ? ) B P Cp Ss bs C s

Sps + + S A + +

Table 2. Common antigenic determinants of Paracentrotus, Sphaerechinus, Psammechinus and Arbacia gametes. The identical determinants are in the same vertical column. The identified determinants of Psammechinus and Arbacia game-

tes are marked only with + .

with the extracts of Arbacia and Psammechinus gametes. All comparisons were made only with anti Jp and Anti Sp sera. It was found that egg extracts of these species (Jps and JA) also have antigenic determinants corresponding to those denoted as Bp , Cp and B s , Cs in Paracentrotus and Sphaerechinus eggs. With sperm extracts (Sp3 and SA) only one precipitation line, corresponding to bp and bs in Paracentrotus and Sphaerechinus sperm, was ob-tained.

Thus it was established that all extracts had one common "group specific" antigenic determinant. Furthermore, all egg extracts as well as all sperm extracts had a common "sex specific" determinant. The absence of clear "species specific" antigenic determinants common to sperm and eggs of one species is surprising, but it can be partly attributed to generally weak reaction obtained with sperm ex-tracts.

The results of these analyses were confirmed in experiments in which antisera were absorbed with gametes in various combinations. Anti Jp, for

instance, absorbed with any sperm loses its capacity to precipitate Cp, but other precipitation lines re-mained, altough somewhat weakened.

The relative concentration of "group specific" antigenic determinants was measured using serial dilutions of gamete extracts and antisera to Para-centrotus gametes (Anti Jp and Anti Sp). On the basis of the highest dilutions which still gave the precipitation line, the relative concentration of the "group specific" antigenic determinants could be estimated as 100 : 80 : 25 : 1 between the gametes of Paracentrotus, Psammechinus, Sphaerechinus and Arbacia respectively.

A good correlation was found by comparing the relative concentration of "group specific" antigenic determinant with the cross fertilization rate in ex-periments where one of the gametes was from Para-centrotus 1. This coincidence should not, however, reflect a causal connection but shows that species with antigenically more different gametes are less compatible for successful cross-fertilization.