Lectin staining and flow cytometry reveals female-induced sperm acrosome reaction and surface carbohydrate reorganization

Abstract

All cells are covered by glycans, an individually unique layer of oligo- and polysaccharides that are critical moderators of self-recognition and other cellular-level interactions (e.g. fertilization). The functional similarity between these processes suggests that gamete surface glycans may also have an important, but currently overlooked, role in sexual selection. Here we develop a user-friendly methodological approach designed to facilitate future tests of this possibility. Our proposed method is based on flow cytometric quantification of female-induced sperm acrosome reaction and sperm surface glycan modifications in the Mediterranean mussel Mytilus galloprovincialis. In this species, as with many other taxa, eggs release water-soluble factors that attract conspecific sperm (chemoattraction) and promote potentially measurable changes in sperm behavior and physiology. We demonstrate that flow cytometry is able to identify sperm from other seawater particles as well as accurately measure both acrosome reaction and structural modifications in sperm glycans. This methodological approach can increase our understanding of chemically-moderated gamete-level interactions and individual-specific gamete recognition in Mytilus sp. and other taxa with similar, easily identifiable acrosome structure. Our approach is also likely to be applicable to several other species, since carbohydrate-mediated cellular-level interactions between gametes are universal among externally and internally fertilizing species.

Figure 1

Flow cytometer gating strategy for sperm treated with seawater (control: (a,b)) and egg water (seawater + egg water, 1:1: (c,d)). Gating was based on FSC and SSC area of the sperm, which separated sperm from the other particles of the seawater (e) and egg water (f). The sub-population “Non-reacted” represents sperm with intact acrosomes, while the sub-population “AR” represents acrosome-reacted sperm. Numbers indicate proportions (%) of gated events of all events.

Figure 2. Measured flow cytometer variables for sperm acrosome reaction.

Dots represent mean (±s.e.) values of forward scatter (FSC) area (a), side scatter (SSC) area (b) and the proportion of sperm in the sub-population (see Fig. 1) “acrosome-reacted sperm” (c) in sea water (SW) and egg water (EW) treatments. N = 31 in all cases.

Figure 3

Fluorescent (a) and differential interference contrast (b) micrographs of sperm labeled with four lectins. Acrosome-intact (non-reacted) sperm are shown on the left (1), partially-reacted sperm in the middle (2) and fully acrosome-reacted sperm on the right (3).

Figure 4. Sperm lectin binding measured by flow cytometer.

Bars represent mean (±s.e.) fluorescence intensity of four lectins (PNA, WGA, DBA and LCH) in sperm samples treated with sea water (“SW”) or fresh egg water (“EW”). Asterisks indicate statistically significant differences between treatments (*P < 0.05; ***P < 0.001). Note differential scaling of the top and bottom segments of y-axis.

Figure 5

Fluorescent intensity of sperm labeled with PNA (a), WGA (b), DBA (c) and LCH (d). Blue colour = Sea water − treated sperm; Red colour = Egg water − treated sperm.

Figure 6. Specificity of lectin binding.

Bars represent mean (±s.e.) fluorescence of PNA (a), WGA (b), DBA (c) and LCH (d) in total (“All”) sperm and acrosome-reacted sperm population (“AR”) labeled with 10 μg/ml of lectins with or without pre-treatment of inhibiting sugars. Percentages indicate the relative proportion of fluorescence intensity of sugar-treated samples in relation sperm without pre-treatment.