Antibodies to flowering-plant sperm

Abstract

Summary. The mechanisms by which sperm cells recognize and fusewith the egg and central cell during double fertilization in flowering plants are unknown. To identify membrane surface molecules that might function in fertilization, we immunized mice with isolated sperm of Brassica campestris and screened the polyclonal sera and monoclonal hybridoma supernatants by immunocytochemistry for binding to isolated sperm cells. We identified three cell lines producing hybridoma supematants which bind to sperm cell surfaces in B. campestris and further analyzed the properties of one of these, BRSP1. The molecular mass of the epitope to BRSP1 was 54 kDa, and was not glycosylated. Although the antibodies were immunoglobulin M, neither removal of carbohydrates nor competition with antibodies which recognize arabinogalactan decreased binding. BRSP1 recognized sperm of Plumbago zeylanica, Nicotiana tabacure, Arabidopsis thaliana, and Eruca vesicaria and generative cells of Lilium longiflorum and of Narcissus tazetta but did not recognize sperm of Helianthus annuus, Gerbera jamesonii, or Zea mays. Antibodies to plant sperm allow us to probe sperm membranes for functional components.

Experimental Analysis of the Fertilization Process

“…Presumably, sperm maturation involves multiple carbohydrate determinants on the cell surface that reduce the likelihood of random fusions but that do not impede sexual gamete fusion. Such surface changes are anticipated to include compounds such as arabinogalactan proteins (Southworth and Kwiatkowski, 1996) and other potentially immunogenic compounds (Southworth et al, 1999). In Plumbago, the polarity of the male germ unit is determined by the position of the vegetative nucleus and appears to be determined in the generative cell .…”

Fifty years of sperm cell isolations: from structural to omic studies

“…The first approach was performed with monoclonal antibodies in Brassica campestris ( Hough et al , 1986 ). Later on, this experiment led to immunohistochemical studies involving JIM8 and JIM13 antibodies, in which arabinogalactan proteins were identified as the first epitopes on the surface of sperm cells in Brassica campestris and L. longiflorum ( Southworth and Kwiatkowski, 1996 ; Southworth et al , 1999 ).…”

The Mechanisms of Pollination and Fertilization in Plants

“…If there is a specific surface protein or glycoprotein displayed on the surface of the cell, it is likely to be formed in such cells or under their control. Further evidence for an independent developmental program in sperm cells is the presence of specific arabinogalactan proteins on the sperm cell surface (Southworth & Kwiatkowski 1996) and heterogeneity of cell surface determinants in different flowering plant species (Southworth et al 1999). Nonrandom positioning of B chromosomes in the nuclei of maize sperm cells also points to independent developmental control (Rusche et al 2001).…”