FERTILIZATION IN BROWN ALGAE

Transcription

1 jf. Cell Sci. 6, (1983) 13 Printed in Great Britain Company of Biologists Limited 1983 FERTILIZATION IN BROWN ALGAE IV. APPEARANCE OF SPERM-SPECIFIC ANTIGENS ON FERTILIZED EGGS H. I. M. V. VITHANAGE*, ]. W. CATTf, J. A. CALLOW, MAUREEN E. CALLOW AND L. V. EVANSJ Department of Plant Sciences, The University, Leeds LS2 9JT, U.K. SUMMARY Flagella antigens from sperm of Fucus serratus have been used to raise antibodies in rabbits. The immunoglobulin G fraction inhibits fertilization with some degree of species specificity. The antigens detected on sperm are not present on unfertilized egg membranes, but appear after fertilization. The common antigens on the fertilized egg can be distinguished from the cell wall material that is also released on fertilization. INTRODUCTION There is increasing evidence that macromolecules on the surface of plant cells are involved in cellular recognition reactions (Albersheim & Anderson-Prouty, 1975; Callow, 1976; Heslop-Harrison, 1978; Clarke & Knox, 1979). Antibodies raised against various surface components are extremely useful tools in plant cell surface studies (Knox, 198). In animals, sperm-specific antisera have been used to follow the fate of sperm plasma membranes during fertilization (O'Rand, 1977), and in sea urchins, antisera to sperm-specific antigens have been used as probes for gamete receptors (Cordle & Metz, 1973; Maitra & Metz, 1974). Previous work in this laboratory, using lectins, polysaccharides and enzymes, has produced indirect evidence for the involvement of complementary surface receptors in the species-specific interaction between gametes of the brown alga Fucus serratus (Bolwell, Callow, Callow & Evans, 1979; Bolwell, Callow & Evans, 198). In the present study we have used antibodies raised against spermflagellato localize antigenic sites on sperm and to follow the initial binding of sperm to egg. This report describes a very early event in fertilization whereby common antigens are exposed on the egg surface following sperm entry. MATERIALS AND METHODS Mature plants of F. serratus L. were collected from the east coast of Yorkshire, England. Gametes were released as described by Callow, Coughlan & Evans (1978). Fertilization was assessed by Calcofluor staining (Bolwell, Callow, Callow & Evans, 1977) using incident fluorescence microscopy Present address: CSIRO Division of Horticultural Research, Merbein Laboratory, Private Mail Bag, P.O., Merbein, Victoria 355, Australia. f Present address: ARC Institute of Animal Physiology, Babraham, Cambridge, CB24AT, U.K. X Author to whom reprint requests should be sent.

2 14 H. I. M. V. Vithanage and others (Carl Zeiss IV FI Epifluorescence microscope with filter no ). For inhibition studies enough sperm were added to give 5-6 % fertilization in controls and a minimum of 5 eggs were counted. Species specificity was assessed by the same assay using gametes of Fucus vesiculosus and Ascophyllum nodosum. Crude flagella preparations were made by vortexing 5cm 3 of sperm (1 4-1* cells cm" 3 ) at 1s intervals for 3-6 s, pelleting the deflagellated cells by centrifugation (5 jf for lomin) and the flagella at 3 for 2min. Flagella membranes were solubilized in 2M-KC1 and the insoluble material was pelleted (3 g for 2min). The salt extract was dialysed, lyophilized, suspended in phosphate-buffered saline (PBS) and used as antigen source. Antisera were raised in rabbits and the immunoglobulin G (IgG) fraction was isolated. The IgG fraction from flagella antiserum was isolated by the caprylic acid method (Steinbuch & Audran, 1969) and lyophilized. This fraction was used to give increased sensitivity. The specificity of the serum was determined by the indirect haemagglutination assay (Bing, Wrygand & Stavistsky, 1967) using group O erythrocytes. The indirect method of fluorescent labelling was used for antigen localization. IgG was used because of non-specific effects caused by multivalent components in serum (Ackerman & Metz, 1972). Freshly released sperm (2cm 3 ) were centrifuged at 1g for 3 min and washed once in filtered sea water. A sample (5^1) of resuspended sperm (1 7 cm~ 3 ) in sea water was mixed with an equal volume of specific IgG or preimmune IgG (2mgcm~ 3 ) in PBS (ph 7-2) and the mixture was incubated for 15 min at room temperature, by which time the sperm were immobilized. This was then centrifuged (looog'for 3 min) washed twice and resuspended in 2 ;d of sea water. A total of 2fi\ of fluorescein isothiocyanate(fitc)-labelled goat anti-rabbit serum (Miles), diluted to 2 cm 3 with PBS (ph 7-2), was added and the mixture was incubated for 15 min before washing twice and examining in the fluorescence microscope with the Zeiss green filter combination Fab fragments of the IgG fraction were prepared by digestion with pepsin and separation on Sephadex G15 as described by Knox & Clarke (1978). Antigen localization on eggs was performed in disposable immunofluorescent slide trays (Sterilin). About 5 eggs were placed in each well; the quantities of reagents added and the incubation times were the same as for the sperm. After washing five times the eggs were removed for microscopy. RESULTS AND DISCUSSION Electron microscopy of negatively stained flagella (using 2 % ammonium molybdate) showed loss of membrane and other components after treatment with 2M-KC1. The indirect haemagglutination test showed that the serum reacted with sperm antigens isolated from flagella with a titre of 1/64. The low titre was probably caused by the Fig. 1. Immobilized sperm incubated with sperm-specific IgG followed by FITClabelled goat anti-rabbit IgG; sperm body (arrow),flagella(arrowheads). X1. Fig. 2. Line drawing of the sperm in Fig. 1 under bright-field conditions; sperm body (arrow), flagella (arrowheads). Fig. 3. Fertilized eggs (incubated as for Fig. 1), showing discrete patching over the surface. Even at this low magnification the large size of the egg (~15fim) prevents the whole surface from being in focus. X22. Fig. 4. Bright-field picture of fertilized (lower) and unfertilized (upper) eggs. The eggs have been treated with both FITC-labelled goat anti-rabbit IgG and Calcofluor (neither are revealed under bright-field conditions). X375. Fig. 5. Same eggs and field of view as in Fig. 4, but underfluorescentconditions, using a filter combination to reveal binding of FITC-labelled goat anti-rabbit IgG. Note the labelled sperm (arrowhead) entrapped in the developing wall. Fig. 6. Same eggs and field of view as in Figs 4 and 5, but underfluorescentconditions, using afiltercombination to reveal binding of Calcofluor White ST to the incipient zygote cell wall.

3 Fucoid gamete antigens 15 Figs 1-6

4 16 H. I. M. V. Vithanage and others relative insolubility of the antigen in PBS resulting in a low antigenic response, or by low effective concentration of antigen in the assay. Attempts to solubilize the antigen in detergents and 2M-KC1 seriously affected the assay. The sperm antigens were localized on both flagella and on the sperm body (Fig. 1). This may reflect the presence of sperm body fragments in the original flagella pellet or may be due to common antigenic determinants on the surface. Nofluorescencewas seen with unfertilized eggs, but if eggs were used within 3 min after fertilization, the sperm-specific IgG bound to many sites on the egg surface, but not to the incipient cell wall that was present (Figs 3, 5). When fertilized eggs had become surrounded by a complete wall (6 9 min after fertilization) no binding was detected, presumably because the zygote wall was impervious to the reagent. Controls with preimmune IgG showed no binding to the sperm or egg surface, even after fertilization. Addition of Calcofluor white to the same eggs used for antigen localization clearly showed early stages in the release of cell wall material, which eventually covered the surface (Fig. 6). These areas of Calcofluor white and IgG fluorescence (white and green, respectively) were distinctly separate when viewed in different focal planes under the two filter combinations used. The fluorescent patches seen on the fertilized egg surface could be due to dispersal of assimilated sperm antigens on the egg membrane after fusion, or to exposure of common antigens as a result of fertilization. Without quantitative measurement it is not possible to equate the observed fluorescence with the number of sperm involved, Table 1. The effect of IgG raised against flagella from F. serratus on fertilization in F. serratus, F. vesiculosus and A. nodosum IgG % Inhibition F. serratus yg Fab* F. vesiculosus A. nodosum " J k No inhibition In this instance Fab fragments were used.

5 Fucoid gamete antigens 17 but fertilization was always carried out under conditions in which only one or a small number of sperm would fuse with each egg (Callow, Evans, Bolwell & Callow, 1978). Unless the stoichiometry of antigen binding to dispersed as opposed to packed sperm antigens is very different, it would seem unlikely that the amount offluorescenceseen could have come from such a small number of sperm, so that it is perhaps more likely to be derived from unmasking of additional antigens on the egg surface after fertilization. However, until more data are available the question cannot be resolved. No binding of IgG raised toflagellaextracts of F. serratus could be detected for gametes of F. vesiculosus or A. nodosum. Fertilization in F. serratus was considerably reduced by the IgG, that in F. vesiculosus was slightly inhibited and no effect was seen with A. nodosum. No antibody binding was detected with F. vesiculosus gametes, so that the slight inhibition noted probably reflects the sensitivity of the fertilization assay over fluorescent antibody detection. The inhibition of fertilization by the IgG could be brought about by the masking of a sperm receptor by direct binding of IgG to that receptor, or by steric hindrance caused by IgG binding to surface moieties in close proximity to the sperm receptors. However, Fab fragments of the IgG inhibited fertilization to almost the same extent as the IgG (Table 1), indicating that steric hindrance near the receptor was minimal. Another possibility is that the IgG binding stops membrane fusion of the gametes after initial recognition has taken place. This possibility awaits further investigation, using more purified antigen preparations. Experiments are in progress to isolate more refined membrane components so that they may be used to investigate the appearance of these extra antigens, as well as broader aspects of gamete recognition at the species and generic levels. Financial support for this work was provided by SERC. We wish to thank Professor D. H. Lee for the use of animal-house facilities. REFERENCES ACKERMAN, N. R. & METZ, C. B. (1972). Effects of multiple antibody layers onarbacia eggs. Expl Cell. Res. 72, ALBERSHEIM, P. & ANDERSON-PROUTY, A. J. (1975). Carbohydrates, proteins, cell surfaces and the biochemistry of pathogenesis. A. Rev. PL Physiol. 26, BING, D. H., WRYGARD, J. G. M. & STAVISTSKY, A. B. (1967). Haemagglutination with an aldehyde-fixed erythrocytes for assay of antigens and antibodies. Proc. Soc. exp. Biol. Med. 124, BOLWELL, G. P., CALLOW, J. A., CALLOW, M. E. & EVANS, L. V. (1977). Cross fertilization in fucoid seaweeds. Nature, Land. 268, BOLWELL, G. P., CALLOW, J. A., CALLOW, M. E. & EVANS, L. V. (1979). Fertilization in brown algae. II. Evidence for lectin-sensitive complementary receptors involved in gamete recognition in Fucus serratus. J. Cell Set. 36, BOLWELL, G. P., CALLOW, J. A. & EVANS, L. V. (198). Fertilization in brown algae. III. Preliminary characterization of putative gamete receptors from eggs and sperm of Fucus serratus. J. Cell Set. 43, CALLOW, J. A. (1976). Recognition, resistance and the role of plant lectins in host-parasite interactions. Adv. bot. Res. 4, 2-5. CALLOW, M. E., COUGHLAN, S. J. & EVANS, L. V. (1978). The role of the Golgi bodies in polysaccharide sulphation in Fucus zygotes. J. Cell Set. 32,

6 18 H. I. M. V. Vithanage and others CALLOW, M. E., EVANS, L. V., BOLWELL, G. P. & CALLOW, J. A. (1978). Fertilization in brown algae. I. SEM and other observations on Fucus serratus.j. Cell Set. 32, CLARKE, A. E. & KNOX, R. B. (1979). Plants and immunity. Devi Comp. Immun. 3, CORDLE, C. T. & METZ, C. B. (1973). Isolation oiarbacia sperm antigens important in fertilization. Biol. Bull. mar. biol. Lab., Woods Hole 145, HESLOP-HARRISON, J. (1978). Cellular recognition systems in plants. In Studies in Biology, no. 1. London: E. Arnold. KNOX, R. B. (198). In The Antibody as a Tool (ed. A. Warr & J. J. Marchelonis). New York: John Wiley & Sons. KNOX, R. B. & CLARKE, A. E. (1978). Localization of proteins and glycoproteins by binding to labelled antibodies and lectins. In Electron Microscopy and Cytochemistry of Plant Cells (ed. J. L. Hall), pp Amsterdam: Elsevier/North Holland Biomedical Press. MAITRA, U. S. & METZ, C. B. (1979). Purification and characterization of an Arbacia sperm fertilization antigen. Biol. Bull. mar. biol. Lab., Woods Hole 147, O'RAND, M. (1977). The presence of sperm-specific surface iso-antigens on the egg following fertilization. J. exp. Zool. 22, STEINBUCH, M. & AUDRON, R. (1969). The isolation of IgG from mammalian sera with the aid of caprylic acid. Archs Biochem. Biophys. 34, (Received 23 July 1982)