Z. Immun.-Forsch. vol. 154, pp. 155-161 (1978)

Institut fUr Biochemie und 1. Zoologisches Institut del' Urriversitat vVien, Vienna, Austria

Hemagglutinins in Female Fish Gonads: Comparative Investigations on Perch (Perea fluviatilis) Gonads of Different Stages of Development

H. SOHENKEL-BRUNNER and H. KOTHBAUER

Received August 29, 1977 . Accepted in Revised Form November 25, 1977

Abstract

Female gonads of the perch (Perea fluviatilis) in various stages of develop. ment are tested for hemagglutinin activity against human erythrocytes. Based on the different agglutination patterns obtained, and on inhibition tests with L-fucose and a mature gonad of a male perch, the appearance of two different agglutinins in the course of the development of the female gonad is assumed.

Introduction

In the eggs as well as in the female gonads of several fish species proteins have been detected, which are able to agglutinate erythro­cytes; some of these hem agglutinins are blood-group specific (review 3). In the gonads of the female perch (Perca fluviatilis) agglutinin ac­tivity has been found showing distinct anti-H specificity (8). Since the male gonads of these animals do not show any anti-H activity (12) but a distinct reaction with the agglutinin of the female gonad (5), it was postulated that the anti-H specific agglutinin of the female reproductive system, probably besides having other functions, could playa role in the fertilization process (5). Following this assumption it was supposed that the hemagglutinin activity might depend on the maturity of the female gonad. In this communication female gonads of Perca fllwiatilis of different stages of development were tested for hemagglutinin specificity in order to obtain a better insight into the formation and function of these compounds.

Materials and Methods

Female gonads - origin and preparation

Perch (Perea fluviatilis) of about equal size were supplied at the beginning of April. The stage of development (maturity) of their gonads was given as percent weight of the gonad with respect to the total weight of the animal. The gonads were frozen, lyophilized and pulverized.

156 . H. SCHENKEL-BRUNN~R and H. KOTHBAUER

The dry powder from the female gonads was extracted in phosphate-buffered saline (= PBS, 4) for 1 hour at room temperature (1 gjlO ml for hemagglutina­tion tests and 1 g/20 ml for hemagglutination inhibition tests). The super­natants obtained after centrifugation for 15 minutes at 3000 rpm were used for the experiments.

H ernaggl11,tination tests

The samples to be tested for hemagglutinin activity were serially diluted (1: 2") with PBS on Salk plates. The diluted samples (50 [t! each) were mixed with 50 ,ul of a 2 % suspension of human AI' Band 0 erythrocytes in saline and, after standing for 1 hour at room temperature, the degree of agglutination was read and graded from 4 (= maximum agglutination) to 1 (= minimum detect­able agglutination) and - (no agglutination).

H ernaggl1lt'ination inhibition tests

The serial dilutions of agglutinins (extracts of female gonads), as well as L-fucose or the suspension of a mature male gonad (maturity 9.7, 1 g dry powderjIO ml PBS) which were used as inhibitors, were prepared in test tubes. On Salk plates 25,u1 of the various agglutinin dilutions thus obtained were mixed with 25,u1 of the appropriate dilution of the inhibitor according to the pattern indicated in Table 2. In control experiments 25 ,ul of PBS were added instead of inhibitor. Following incubation at room temperature for 30 minutes, 25 ,ul aliquots of a 2 % suspension of human 0 erythrocytes in saline '.vere added. After further standing at room temperature for 1 hour the degree of agglutina­tion was assessed as mentioned above.

Results

When extracts from the female gonads of Perea fluviatilis were tested for hemagglutinin content, different agglutination patterns were observed, which depended on the stage of maturity (Table 1). The extract from an immature gonad (maturity 2.1) agglutinated human 0 erythrocytes best, but also, though with decreasing strength, B and Al cells. The extract was not able to agglutinate the erythro­cytes when used in initial high concentration, upon dilution during the titration procedure, however, a concentration range was reached where strong reaction occurred; upon further dilution the degree of agglutination again decreased. This agglutination pattern is known as prozone phenomenon (1). The same kind of agglutination pattern was found in the extract of a gonad of an even lower stage of maturity (maturity 0.9) where a weak reaction only with 0 erythrocytes was observed. Beginning with maturity 6.9 a different agglutination pat­tern was found. The agglutination was strongest at the highest con­centration and steadily decreased during the dilution steps. Also in this case the agglutinin reacted best with human 0 erythrocytes and less with B and Al cells, the respective titers rising steadily with in­creasing maturity of the gonad.

The agglutination of 0 erythrocytes as caused by extracts from an immature female gonad (maturity 2.1) and from a mature female

Hemagglutinins in female perch gonads . 157

gonad (maturity 28.1) was inhibited by L-fucose (Table 2, compare 8) and a suspension of a mature male gonad (Table 2, compare 5). Nevertheless, differences in the inhibition patterns could be observed: the agglutinin activity of the immature gonad was inhibited totally by 60 mM L-fucose, whereas the agglutinin activity of the mature gonad showed a distinct agglutination at this fucose concentration. It is remarkable that the prozone phenomenon was preserved with the extract of the immature gonad. Furthermore the appearance of a weak prozone phenomenon was noted with the extract of a mature gonad when inhibited by certain fucose concentrations. Also in the case where a suspension of a male gonad was used for inhibition tests, different inhibition patterns were found. The agglutinin reaction of the immature female gonad was distinctly inhibited, the prozone phenomenon again being preserved, whereas using the extract of the mature female gonad the weaker agglutination found with higher ag­glutinin dilutions was inhibited and the strong agglutination found with high concentrations of the extract remained unaffected.

"When the extracts were stored in the refrigerator overnight, or when erythrocytes from different donors were used, minor variations in the agglutination patterns were observed. Furthermore it was difficult to assess the exact endpoint of the titration as the degree of agglutination in the higher dilutions decreased very slowly. All the experiments, however, repeated several times, always gave the same overall reaction patterns.

Discussion

The agglutinin specificity of all extracts tested is obviously anti-H (compare 8), since their reactivity is strongest with human 0 erythro­cytes and weaker with B and Al cells, this decreasing agglutinability in the order 0 ~ B ~ Al parallels the decreasing blood-group H activity of these cells (11). Nevertheless, different stages of develop­ment of the female perch gonads show different reaction patterns. It is remarkable that the extract of the immature gonads (maturIty 0.9 and 2.1) shows a prozone phenomenon (1), i.e. the extract gives weak or no reaction when used in high concentrations. Beginning with maturity 6.9 no prozone phenomenon is detectable (Table 1).

The absence of the prozone phenomenon beginning with maturity 6.9 lead to the assumption of different agglutinins in immature and mature gonads. In order to test this possibility extracts of female gonads with maturity 2.1 (immature) and 28.1 (mature) were selected for inhibition tests (Table 2). The extract from the immature female gonad was totally inhibited by L-fucose and a suspension of a mature male gonad, whereas the extract of the mature female gonad reacted

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160 . H. SOHENKEL-BRUNNER and H. KOTHBAUER

in a distinctly different manner: using a suspension of a male gonad as inhibitor the titer could be reduced only to a certain extent; the inhibition experiments with L-fucose showed similar results.

These experiments can be possibly explained by the assumption that at least two different agglutinins are present: one agglutinin -<<agglutinin A,) - occming mainly in the immatme and in a smaller concentration in the matm·e gonad, and another agglutinin - <<ag­glutinin B» - only in the matme gonad.

The occmrence of the different agglutinins could possibly coincide with the development of the female gonad. The fish oocyte in the early phase has a small amount of cytoplasm. As oogenesis proceeds, the cytoplasm increases in volume. In a later phase of oogenesis, the formation of yolk begins with the appearance of yolk globules in the cytoplasm. These globules gradually increase in size and number (7). Following this, «agglutinin A,) in immatme gonads possibly coincides with the increase of cytoplasm and the appearance of «agglutinin B,) (starting with matmity 6.9?) with the increased formation of yolk as oogenesis proceeds. These agglutinins could, in the course of gonadal development, perhaps play a role in a kind of «transport mechanism,) (2) possibly binding certain substances (nutritients?) to the gonad similar to the proposal made for a possible function of the anti-A in the albumen gland of Helix pomatia (2). In mature female gonads «agglutinin A,), since reacting with matme male gonads, could playa role in the process of fertilization (5). These assumptions do not exclude, however, that these agglutinins have, to a certain extent, an immunobiological protective function (9, 10). But none of these discussed possibilities has yet been proved (6).

Zusammenfassung

Weibliche Barsch-Gonaden (Pel·ca (luV/:atil1:s) verschiedener Entwickhmgs­stadien \vurden vergleichend auf Hiimagglutinine gegen menschliche Erythro­cyten getestet. Auf Gnmd del' differierenden Agglutinationsmuster del' einzelnen Reifestadien sowie aus den Ergebnissen von Hemmtests mit L-Fucose und einer reifen miinnlichen Barsch-Gonade wurde angenommen, daD im Laufe del' Entwicklung del' weiblichen Gonade zwei verschiedene Agglutinine auftreten.

References

1. DUNSFORD, 1., und C. C. BOWLEY. 1969. ABC del' Blutgruppenkunde. J. F. Lehmarm, lVIiinchen.

2. FISOHER, K., A. POSOHMANN, K. REUTHER und O. PROKOP. 1972. Dber das gleichzeitige Vorkommen von «Antigen» und «Antik6rper» bei Schnecken: Autoantik6rper odeI' Transportmechanismus? Immun.-Information 2 (4): 20.

3. GOLD, E. R., and P. BALDING. 1975. Receptor-specific proteins. Plant and animal lectins. Excerpta Medica, Amsterdam.

Hemagglut,inins in female perch gonads . 161

4. KOTHBAUER, H., und H. SCHENKEL-BRUNNER. 1971. Haemagglutinine aus Schnecken: Zur Frage ihrer biologischen Funktion. Z. Naturforsch. 26b: 1082.

5. KOTHBAUER, H., lUld H. SCHENKEL·BRUNNER. 1974. Haemagglutinine aus weiblichen Fischgonaden: Zur Frage ihrer biologischen Funktion - Hemm­versuche mit mannlichen Gonaden. Z. Immun.-Forsch. 147: 87.

6. KOTHBAUER, H. 1975. Hiimagglutinine in Tieren. Biologische Funktionen und Verbreitung. Naturw. Rdseh. 28: 73.

7. NAKANO, E. 1969. Fishes; in: Fertilization (C. B. Metz and A. Monroy, eds.), Vol. II. Academic Press, New York - London. p. 296.

8. PROKOP, 0., S. SCHNITZLER und G. UHLENBRUCK. 1967. Uber einen krafti­gen H-Antikorper bei zwei Vertretern del' Percidae, aufgeflUlden im Rogen del' Tiere. Acta bioI. med. germ. 18: K 7.

9. PROKOP, 0., G. UHLENBRUCK, and W. KOHLER. 1968. A new source of anti­body-like substances having anti-blood-group specificity. A discussion on the specificity of Helix agglutinins. Vox Sang. (Basel) 14: 321.

10. PROKOP, 0., G. UHLENBRUCK und W. KOHLER. 1968. Protectine, eine neue Klasse antikorperahnlieher Verbindungen. Dtsch. Gesundh.vVes. 23: 318.

11. RACE, R. R., and R. SANGER. 1975. Blood groups in man. 6th ed., Blackwell, Oxford.

12. SCHENKEL-BRUNNER, H., H. SPLECHTNA lUld H. KOTHBAUER. 1972. -ober das Vorkommen von Hamagglutininen (Anti-H und Anti-B) beim FluB­barsch (Pe1'ca fluviatilis). Experientia (Basel) 28: 1479.

Univ.-Doz. Dr. H. SCHENKEL-BRUNNER, Institut fiir Biochemie del' Universi­tat vVien, WahringerstraLle 17, A-I090 Vienna, Austria.