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Soluble antigens in sea urchin gametes and developmental stages
394
SOLUBLE
ANTIGENS
IN SEA URCHIN
DEVELOPMENTAL
GAMETES AND
STAGES
P. PERLMANN The
Wenner-Gren
Institute
forExperimental
Biology,
University
ofStockholm,
Sweden
Received January 5, 1953
IN a previous
paper
(7)
results
have
been
reported
from a series
of se-
rological experiments on the eggs and developmental stages of the sea urchin Paracentrotus lividus. By means of the ordinary serological precipitation and absorption techniques, the appearance of at least one saline extractable molecular species of a new specificity, not occurring in unfertilized eggs and early developmental stages, could be demonstrated in 0.14 M saline extracts from 48 hours old plutei. In addition it appeared that egg extracts did not contain any precipitin producing antigens besides those in the extracts from the later stages up to 48 hours development. A further investigation of the serological properties of the sea urchin gametes and developmental stages has now been undertaken by means of Ouchterlony’s precipitation method in a salt-agar gel (4, 5, 6). By this method two or more antigen solutions can be tested at the same time. The identity or non-identity of the antigenic molecules in the different solutions can thus be tested qualitatively and semi-quantitatively. Rabbit antisera against sea urchin material and antigen solutions were prepared and standardized as previously reported. All antigen solutions were maintained by a phosphate buffer at pH 7.4. All solutions could be stored at -17” C in a refrigerated box for several months without losing activity. As a rule the serological reactions were carried out at 37” C in the same way as described by Ouchterlony (1. c.). Precipitation lines usually appeared on the agar plates after 2 days. After approximately 1 week all lines had appeared and grown strong enough for registration. The experiments were usually stopped after 2 weeks. During the second week no more changes occurred in the number of the precipitation lines. - Serological absorption experiments were carried out in the manner described by Bjorklund (1,2). After complete reaction the plates were photographed in a Busch-macrocamera and furthermore, drawings were made by means of a camera lucida.
When 0.14 hl saline extracts from jelly deprived and lyophilized eggs of the sea urchin Paracentrotus liuidus were allowed to react with their homologous antisera, a great number of precipitation lines was obtained. Further
395
Antigens in sea urchin gametes Fig. 1. Egg extract serum (right).
(left), reacting
with anti
-egg
Fig. 2. Egg extract, untreated (right) and after treatment with 40 per cent AmSO, (left), reacting with anti-egg serum (middle).
396
P. Perlmann
Fig. 4. Egg extract (right) and sperm extract (left), reacting with anti-egg serum (middle). Fig. 5. Sperm extract (left), egg extract (upper right) and jelly coat solution (lower right), reacting with anti-egg serum (middle).
Antigens in sea ud~in gametes
397
Fig. 6. Psammechinus extract (left) and Paracentrofus extract (right), reacting with swum anti-Psnmmechinus (middle).
extract Fig. 7. Arbaciu (upper) and Paracenfrotus extract (right), reacting with anti-drbacia serum (middle).
experiments showed that at least 10 of those lines really represented rnolccules with different chemical and serological properties. As can be seen from diagram 1 and from Fig. 1, most of the lines were arranged in at least four main fractions, indicated by the numbers I-I\‘. By ultracentrifugation at 63000 X g for 2 hours, some of the components in fraction I could be brought out of solution. Furthermore, in the top layer formed at this centrifugation (:I), chiefly components from fraction III and IIT could be found. - Dialysis o\-crnight against diluted HCl (pH 3.5) made a11 antigens insoluble, except most of those in fraction I\‘. - A further fractionation of the antig:ns could bc achieved by precipitation vith AmSO, in the cold. The experiments v-we performed as previously described by
P. Perlmann
Diagram 1: 1%:extract from unfertilize.l eggs. C: extract from 4X hours plutei. D: antiserum against 4X hours plutei. Diagram 2: IS: extract from unfertilized eggs. I$, IS,, IS,: extracts from unfertilized eggs after precipitation with 40 per cent, 55 per cent and 65 per cent AmSO,. A: antiserum against unfertilized eggs. Diagram 3: A: antiserum against unfertilized eggs. A,, A,, A,: antisera against unfertilized eggs, absorbed with IS,, I3,, and I&. 13: extract from unfertilized eggs.
Lindvall (3). As can he seen from diagram 2, precipitation with AmSO, at 40 per cent saturation brought out all antigens in fraction I (Fig. 2). In the saturation-range from 40-60 per cent all antigens in fraction III disappeared from the solution together lvith some of those in fraction II. Fraction-IIif the AmSO,-saturation \vas inantigens disappeared almost completely, ccrasetl ~111 to 65 per cent. 5lost of the antigens in fraction I\’ \\-ere still left in solution at this concentration hut clisaplwarcd completely if the AmSO, saturation \vas raised. So antigens \verc left in solution at AmSO, saturations higher than 75 per rent. \\‘hcn the antisera \vcre ahsorbed \vith antigen solutions, fractionated as tlcscri hcd ahove, and finally after the absorption \\-rre allo\vctl to react \vitli complctc antigens solutions, marked serological tliffcrcnccs hct\vccn the fractions I-I\’ ~~)ultl 1~1 ol~~rvecl (diagram 3). \vas IScsidw the cslwrinients rc~portctl above a scrics of cspcrimcnts undertaltcn \vith cstracts, made in 2 11 sodium c*hloritlc from the residue after the usual cstraction jvith 0.11 11 saline. ‘I’hc solutions jvcrc tlialyzctl and treated in tht usual manner. ‘l’hcse extracts containccl at lcast one soluble antigen, not cstractahlc \vitli sodium chloride in lo\vcr concentration. This fact could also he proved hy al~sorption c~xlwrimcnts. In another scrics of cslwrimcnts the changes in the serological pattern (luring clcvclopnicnt \\.erc stutliccl. ‘l’hc cslwrinicntr slio\\,ccl, that the “slxvtrum” of saline soli~l~lc antigens is more or less the sanic for tlif unfertilized eggs ant1 for the dcvclopnicntal stagw stutlicd. Llost of the antigens are in common anal of apl~roxiniatcly the sanic concentration in extracts from all stages. Some quantitative tlill’crcnccs swni, ho\\~cvrr, to exist. Alorc about these \\-ill lx rcportcd in a suhsccluvnt 1)uhlication. Ho\\c\cr, some changes
Antigens in sea urchin gametes
399
of a more qualitative nature could be observed: in 48 hours plutei, at least three soluble antigens were found, which were absent in the extracts from unfertilized eggs (diagram 1: a, b, c). Thus after absorption of an antiserum against 48 hours plutei with extracts from unfertilized eggs, still three lines were obtained in the reaction with extracts from 48 hours plutei (Fig. 3). These antigens seem to belong to fraction I and II. Apparently they are not present or at most they are present in very small quantities only, in the insoluble residues from the egg. Otherwise, antibodies should have been formed at immunisation. Their appearence in solution was first detected in the extracts from 30 hours old gastrulae stages. Besides the extracts mentioned above, 0.14 hl saline extracts from lyophilized sperm and from jelly coat, precipitated by AmSO,, were tested with antisera against eggs and developmental stages. As can be seen from Fig. 4, extracts from the sperm have one antigen fraction in common with the egg. The same is the case with the jelly solution (Fig. 5). This could also be proved by testing antisera against sperm and jelly coat with their homologous antigens and with egg extracts. In those cases, only l-2 lines, belonging to fraction II, were obtained with the egg solutions. In addition neither the sperm extract nor the jelly solution seemed to contain antigens, not occurring in the egg. In connection with a recently started work in collaboration with Drs. C. and D. Harding on the antigens of some sea urchin hybrids, eggs and developmental stages of the species Psnmmechinusmicrotuberculatus and Arbacia lixula have also been investigated. The experiments showed, that the antigenic set up of the two species Paracentrotus lividus and Psammechinus microtuberculatus is very similar. Both contain several antigens in common. Anyhow, in each species antigens exist which are lacking in the other species (Fig. 6). On the other hand, almost no similarity was found between the soluble antigens from those two species and from Arbacia lixula (Fig. 7). Only a very weak cross reaction could be obtained in the heterologous reactions between extracts and antisera from those species. The results of this investigation will be published later. REFERENCES
1. BJ~RLKUND, B., Proc. Sot. Expll. Viol. Med., 79, 319 (1952). ibid, 79, 324 (1952). 3. LINDVALL, S., and CARSJ~, A., Arkiv Kemi, 2, 293 (1950). 4. OUCIITERLONY, o., Arkio Kemi Mineral. Geol., 26 B, 1 (1949). 5. -Arkio Kemi, 1, 43 (1950). 2. -
6. -
ibid, 1, 55 (1950).
7. PERLMANN, P., and GUSTAFSON,T., Experientia, Y-533704
4, 481 (1948).
SOLUBLE
ANTIGENS
IN SEA URCHIN
DEVELOPMENTAL
GAMETES AND
STAGES
P. PERLMANN The
Wenner-Gren
Institute
forExperimental
Biology,
University
ofStockholm,
Sweden
Received January 5, 1953
IN a previous
paper
(7)
results
have
been
reported
from a series
of se-
rological experiments on the eggs and developmental stages of the sea urchin Paracentrotus lividus. By means of the ordinary serological precipitation and absorption techniques, the appearance of at least one saline extractable molecular species of a new specificity, not occurring in unfertilized eggs and early developmental stages, could be demonstrated in 0.14 M saline extracts from 48 hours old plutei. In addition it appeared that egg extracts did not contain any precipitin producing antigens besides those in the extracts from the later stages up to 48 hours development. A further investigation of the serological properties of the sea urchin gametes and developmental stages has now been undertaken by means of Ouchterlony’s precipitation method in a salt-agar gel (4, 5, 6). By this method two or more antigen solutions can be tested at the same time. The identity or non-identity of the antigenic molecules in the different solutions can thus be tested qualitatively and semi-quantitatively. Rabbit antisera against sea urchin material and antigen solutions were prepared and standardized as previously reported. All antigen solutions were maintained by a phosphate buffer at pH 7.4. All solutions could be stored at -17” C in a refrigerated box for several months without losing activity. As a rule the serological reactions were carried out at 37” C in the same way as described by Ouchterlony (1. c.). Precipitation lines usually appeared on the agar plates after 2 days. After approximately 1 week all lines had appeared and grown strong enough for registration. The experiments were usually stopped after 2 weeks. During the second week no more changes occurred in the number of the precipitation lines. - Serological absorption experiments were carried out in the manner described by Bjorklund (1,2). After complete reaction the plates were photographed in a Busch-macrocamera and furthermore, drawings were made by means of a camera lucida.
When 0.14 hl saline extracts from jelly deprived and lyophilized eggs of the sea urchin Paracentrotus liuidus were allowed to react with their homologous antisera, a great number of precipitation lines was obtained. Further
395
Antigens in sea urchin gametes Fig. 1. Egg extract serum (right).
(left), reacting
with anti
-egg
Fig. 2. Egg extract, untreated (right) and after treatment with 40 per cent AmSO, (left), reacting with anti-egg serum (middle).
396
P. Perlmann
Fig. 4. Egg extract (right) and sperm extract (left), reacting with anti-egg serum (middle). Fig. 5. Sperm extract (left), egg extract (upper right) and jelly coat solution (lower right), reacting with anti-egg serum (middle).
Antigens in sea ud~in gametes
397
Fig. 6. Psammechinus extract (left) and Paracentrofus extract (right), reacting with swum anti-Psnmmechinus (middle).
extract Fig. 7. Arbaciu (upper) and Paracenfrotus extract (right), reacting with anti-drbacia serum (middle).
experiments showed that at least 10 of those lines really represented rnolccules with different chemical and serological properties. As can be seen from diagram 1 and from Fig. 1, most of the lines were arranged in at least four main fractions, indicated by the numbers I-I\‘. By ultracentrifugation at 63000 X g for 2 hours, some of the components in fraction I could be brought out of solution. Furthermore, in the top layer formed at this centrifugation (:I), chiefly components from fraction III and IIT could be found. - Dialysis o\-crnight against diluted HCl (pH 3.5) made a11 antigens insoluble, except most of those in fraction I\‘. - A further fractionation of the antig:ns could bc achieved by precipitation vith AmSO, in the cold. The experiments v-we performed as previously described by
P. Perlmann
Diagram 1: 1%:extract from unfertilize.l eggs. C: extract from 4X hours plutei. D: antiserum against 4X hours plutei. Diagram 2: IS: extract from unfertilized eggs. I$, IS,, IS,: extracts from unfertilized eggs after precipitation with 40 per cent, 55 per cent and 65 per cent AmSO,. A: antiserum against unfertilized eggs. Diagram 3: A: antiserum against unfertilized eggs. A,, A,, A,: antisera against unfertilized eggs, absorbed with IS,, I3,, and I&. 13: extract from unfertilized eggs.
Lindvall (3). As can he seen from diagram 2, precipitation with AmSO, at 40 per cent saturation brought out all antigens in fraction I (Fig. 2). In the saturation-range from 40-60 per cent all antigens in fraction III disappeared from the solution together lvith some of those in fraction II. Fraction-IIif the AmSO,-saturation \vas inantigens disappeared almost completely, ccrasetl ~111 to 65 per cent. 5lost of the antigens in fraction I\’ \\-ere still left in solution at this concentration hut clisaplwarcd completely if the AmSO, saturation \vas raised. So antigens \verc left in solution at AmSO, saturations higher than 75 per rent. \\‘hcn the antisera \vcre ahsorbed \vith antigen solutions, fractionated as tlcscri hcd ahove, and finally after the absorption \\-rre allo\vctl to react \vitli complctc antigens solutions, marked serological tliffcrcnccs hct\vccn the fractions I-I\’ ~~)ultl 1~1 ol~~rvecl (diagram 3). \vas IScsidw the cslwrinients rc~portctl above a scrics of cspcrimcnts undertaltcn \vith cstracts, made in 2 11 sodium c*hloritlc from the residue after the usual cstraction jvith 0.11 11 saline. ‘I’hc solutions jvcrc tlialyzctl and treated in tht usual manner. ‘l’hcse extracts containccl at lcast one soluble antigen, not cstractahlc \vitli sodium chloride in lo\vcr concentration. This fact could also he proved hy al~sorption c~xlwrimcnts. In another scrics of cslwrimcnts the changes in the serological pattern (luring clcvclopnicnt \\.erc stutliccl. ‘l’hc cslwrinicntr slio\\,ccl, that the “slxvtrum” of saline soli~l~lc antigens is more or less the sanic for tlif unfertilized eggs ant1 for the dcvclopnicntal stagw stutlicd. Llost of the antigens are in common anal of apl~roxiniatcly the sanic concentration in extracts from all stages. Some quantitative tlill’crcnccs swni, ho\\~cvrr, to exist. Alorc about these \\-ill lx rcportcd in a suhsccluvnt 1)uhlication. Ho\\c\cr, some changes
Antigens in sea urchin gametes
399
of a more qualitative nature could be observed: in 48 hours plutei, at least three soluble antigens were found, which were absent in the extracts from unfertilized eggs (diagram 1: a, b, c). Thus after absorption of an antiserum against 48 hours plutei with extracts from unfertilized eggs, still three lines were obtained in the reaction with extracts from 48 hours plutei (Fig. 3). These antigens seem to belong to fraction I and II. Apparently they are not present or at most they are present in very small quantities only, in the insoluble residues from the egg. Otherwise, antibodies should have been formed at immunisation. Their appearence in solution was first detected in the extracts from 30 hours old gastrulae stages. Besides the extracts mentioned above, 0.14 hl saline extracts from lyophilized sperm and from jelly coat, precipitated by AmSO,, were tested with antisera against eggs and developmental stages. As can be seen from Fig. 4, extracts from the sperm have one antigen fraction in common with the egg. The same is the case with the jelly solution (Fig. 5). This could also be proved by testing antisera against sperm and jelly coat with their homologous antigens and with egg extracts. In those cases, only l-2 lines, belonging to fraction II, were obtained with the egg solutions. In addition neither the sperm extract nor the jelly solution seemed to contain antigens, not occurring in the egg. In connection with a recently started work in collaboration with Drs. C. and D. Harding on the antigens of some sea urchin hybrids, eggs and developmental stages of the species Psnmmechinusmicrotuberculatus and Arbacia lixula have also been investigated. The experiments showed, that the antigenic set up of the two species Paracentrotus lividus and Psammechinus microtuberculatus is very similar. Both contain several antigens in common. Anyhow, in each species antigens exist which are lacking in the other species (Fig. 6). On the other hand, almost no similarity was found between the soluble antigens from those two species and from Arbacia lixula (Fig. 7). Only a very weak cross reaction could be obtained in the heterologous reactions between extracts and antisera from those species. The results of this investigation will be published later. REFERENCES
1. BJ~RLKUND, B., Proc. Sot. Expll. Viol. Med., 79, 319 (1952). ibid, 79, 324 (1952). 3. LINDVALL, S., and CARSJ~, A., Arkiv Kemi, 2, 293 (1950). 4. OUCIITERLONY, o., Arkio Kemi Mineral. Geol., 26 B, 1 (1949). 5. -Arkio Kemi, 1, 43 (1950). 2. -
6. -
ibid, 1, 55 (1950).
7. PERLMANN, P., and GUSTAFSON,T., Experientia, Y-533704
4, 481 (1948).