Fertilization in brown algae. V. Further investigations of lectins as surface probes.

The binding of FITC- (fluorescein isothiocyanate), Au- and 125I-labelled lectins (conA, RCA120 (Ricinus communis agglutinin, MW 120 000) and FBP (fucose-binding protein from Lotus tetragonobolus)) to gametes of Fucus serratus and their physiological effects on fertilization have been studied. Results indicate that eggs strongly bind FITC- and Au-labelled conA and RCA120, whilst FITC-FBP binds strongly to sperm. All three iodinated lectins bound to eggs but this was apparently non-specific and similar in magnitude to the binding of iodinated bovine serum albumin. The results suggested the possibility of two distinct types of lectin receptor on egg surfaces: non-specific, highly abundant receptors and less abundant, specific receptors, possibly locally aggregated. All three lectins inhibit fertilization, FBP being the most effective.

Fertilization in brown algae. IV. Appearance of sperm-specific antigens on fertilized eggs.

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.

Immunocytochemical localization of water-soluble glycoproteins, including group 1 allergen, in pollen of ryegrass, Lolium perenne, using ferritin-labelled antibody.

The cellular sites of the glycoproteins Group 1 allergen (glycoprotein 1) and Antigen A (glycoprotein 2) in mature ryegrass pollen have been investigated by immunoelectron microscopy. Radioimmunoassays confirm previous findings of cross-reactivity between the purified glycoprotein antigens at the high immunoglobulin G (IgG) concentrations used for localization. Freeze-drying of anthers followed by anhydrous processing has been employed because of the water solubility and mobility of the glycoproteins. A double-embedding technique has been developed. This involves, first, embedding anthers in the water-soluble plastic resin JB-4, sectioning and incubating in ferritin-labelled antisera by the indirect method. The sections are then embedded in Spurr's resin for ultra-thin sectioning. Both glycoproteins are found in the following sites: (1) exine and intine wall layers; (2) pollen cytoplasm; (3) the orbicules and anther loculus; and (4) the anther cuticle. In the exine arcades and surface and in the anther loculus, the ferritin label is bound to pollenkitt. The finding that the glycoproteins are in similar sites is predictable in view of the cross-specificity of the antisera. The extent of antibody penetration of the plastic sections has been examined; labelling is confined to cut grains and absent from intact grains.

Immunofluorescent localization of two water-soluble glycoproteins including the major allergen from the pollen of ryegrass, Lolium perenne.

Two major glycoproteins have been localized in sectioned grains of ryegrass pollen by direct and indirect immunofluorescence methods using Fluorescein isothiocyanate (FITC)-labelled IgG fractions of antisera. These glycoproteins are the major allergen, Group 1 allergen, and a principal antigen, Antigen A. Four methods of fixation were employed: freeze-drying, methanol, 2.5% glutaraldehyde and 4% paraformaldehyde for 1 h at 4 degrees C. The post-embedding staining technique of immunocytochemistry was used: anthers were embedded directly, or after dehydration, in JB-4 plastic resin and antibody reacted with sectioned pollen. The effects of these fixatives on the antibody combining sites of the antigens were quantified by a solid phase radioimmunoassay using [125I]protein A to measure antibody binding. In mature pollen, these antigens were located in the cytoplasm and in the complex wall. In developing grains early in the maturation period, specific fluorescence was concentrated at the periphery of the cytoplasm.

The nature of callose produced during self-pollination inSecale cereale.

The response to incompatible (self) pollination in rye (Secale cereale L.) includes the rapid deposition in the germinating pollen grain and pollen tube of a substance that stains with aniline blue, resorcin blue and calcofluor, and in these respects resembles callose. This substance has been isolated and analysed by acid hydrolysis and methylation as well as specific enzyme hydrolysis. It contains a glucan component with 1,4-ß-glucosidic and 1,3-ß-glucosidic linkages within the same linear chains. The proportion of 1,4-to 1,3-glucosidic linkages in the preparation is 77∶9.

Pollen-wall proteins: quantitative cytochemistry of the origins of intine and exine enzymes in Brassica oleracea.

Simultaneous coupling methods for detection of acid phosphatase and non-specific esterase produce a coloured reaction product that is quantitatively related to enzyme content in freeze-sectioned Brassica pollen and tapetal cells. The intine-located acid phosphatase has 2 periods of synthesis: the first in late vacuolate period, associated with the completion of deposition of the intine polysaccharides; the second during pollen maturation, apparently reflecting cytoplasmic synthesis, Esterase activity accumulates in the tapetal cells until dissolution at early maturation period, when there is a dramatic rise in pollen-wall esterase activity, reflecting the transfer from tapetum to exine cavities. These quantitative studies confirm the gametophytic and sporophytic origins of the intine and exine proteins.