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Fertilization in the sea : the chemical identity of an abalone sperm attractant

Jeffrey A. Riffell^*, Patrick J. Krug^* and Richard K. Zimmer

Department of Biology, University of California, Los Angeles, CA 90095-1606, USA
^* Order of authors was decided by tossing a coin

View larger version (30K): [in a new window]   Fig. 1. Orientation of abalone sperm to individual eggs. Sperm were videotaped for 30 s in the presence of a red abalone egg (A) or brine shrimp egg (B) (negative control). Polar plots show the vector mean angle of sperm swimming, relative to the egg surface (0°); the bar represents the vector mean length (r). On the left, representative swimming paths of individual sperm are plotted around an abalone or control egg. Circles correspond to video images captured at 0.033 s intervals, and arrowheads correspond to the directions of travel for individual cells.

View larger version (10K): [in a new window]   Fig. 2. Accumulation of the sperm attractant in sea water around live eggs. Freshly spawned eggs were suspended in filtered sea water (FSW) and incubated for 2 min or 5 min. The resulting egg-conditioned sea water (ESW) solutions were filtered to 0.22 µm and immediately bioassayed using fresh sperm. Swimming speed was quantified using computer-assisted video motion analysis. Values are means + S.E.M.; means labeled with different letters differed significantly (one-way ANOVA with post-hoc Scheffé test, P<0.005).

View larger version (17K): [in a new window]   Fig. 3. Partial purification of the sperm chemoattractant by RP-HPLC. The active fraction from egg-conditioned sea water (ESW) was eluted from a Sep-Pak cartridge in 25 % methanol, concentrated, and injected onto a C18 column. Compounds were eluted over a 30 min run using a gradient from 5 % to 50 % methanol in 0.1 % TFA. Fractions (0.5 ml) were pooled as indicated, lyophilized, and bioassayed for sperm motility and orientation. (A) Absorbance spectrum at 220 nm. (B) Sperm swimming response to isolated fractions. Fractions were diluted to the appropriate starting volume of sea water and tested in the sperm motility assay; swim speeds were measured using computer-assisted video motion analysis. Values are means + S.E.M. Fresh ESW and filtered sea water (FSW) were used as positive and negative controls, respectively. *Means significantly greater than negative controls (P<0.01). (C) Chemotactic response of abalone sperm to isolated fractions, represented as cell counts in a flat capillary bioassay. Values are means + S.E.M.

View larger version (17K): [in a new window]   Fig. 4. Final purification of the sperm chemoattractant by size-exclusion chromatography. (A) The bioactive fraction from RP-HPLC was chromatographed on a size-calibrated Bio-Sep S-2000 column, eluted at 1 ml min-1 in water and absorbance monitored at 220 nm. Fractions corresponding to detected peaks were pooled, lyophilized, resuspended in sea water, and bioassayed with freshly spawned sperm. Numbers indicate the elution times of size standards used to calibrate the column. (B) Sperm swimming response to isolated fractions. Values are means + S.E.M. Fresh egg-conditioned sea water (ESW) and filtered sea water (FSW) were used as positive and negative controls, respectively. *Means significantly greater than negative controls (P<0.0001). (C) Chemotactic response of abalone sperm to isolated size fractions. Values are means + S.E.M.

View larger version (11K): [in a new window]   Fig. 5. Analytical HPLC comparison of the natural attractant and a synthetic tryptophan standard. Samples were reacted with o-phthalaldehyde (OPA) prior to injection, producing derivatives that were identified by RP-HPLC with fluorescence detection at 400 nm (Em400). (A) A sample of the purified natural sperm attractant corresponding to 0.25 nmoll-1, showing the diagnostic retention time and purity of the sample. (B) Co-injection of the previous sample with 0.25 nmoll-1 of a synthetic standard of L-tryptophan, showing co-elution as a single peak.

View larger version (14K): [in a new window]   Fig. 6. Dose—response relationship of red abalone sperm to L-tryptophan. Values are means ± S.E.M. with the regression line calculated using the raw data. (A) Results of a linear regression of log10-transformed dose on sperm swim speed. (B) Results of a linear regression of log10-transformed dose on sperm density. The dose producing the half-maximal response (ED50) was calculated from the regressions to be 3x10-8 mol l-1 for both swim speed and cell density.

View larger version (16K): [in a new window]   Fig. 7. Structural specificity of the sperm response to L-tryptophan. Red abalone sperm were assayed for behavioral responses to natural egg-conditioned sea water (ESW), and to synthetic standards tested at 10-7 mol l-1 concentration. Both the natural L-enantiomer (L-trp) and the unnatural D-enantiomer (D-trp) of tryptophan were tested to determine the stereospecificity of sperm response. Serotonin, or 5-hydroxytryptamine, is a neuroactive metabolite of L-tryptophan known to activate sperm in other molluscan species. Filtered sea water (FSW) was used as a negative control. Values are means ± S.E.M.; means not linked by a horizontal bar differed significantly (one-way ANOVA with post-hoc Scheffé test, P<0.05). (A) Change in sperm swim speed in response to tested solutions. (B) Change in cell density due to sperm chemotaxis.

View larger version (16K): [in a new window]   Fig. 8. Effect of enzymatic treatment with tryptophanase on bioactivity of egg-conditioned sea water (ESW). Active ESW solutions were incubated with the enzyme tryptophanase, which cleaves the indole ring from L-tryptophan; solutions were boiled to quench the reaction after 10 min. In one positive control (boiled ESW), ESW solutions were boiled without enzyme, to determine effects of the deactivating step in enzyme treatments. In a second positive control (ESW+denatured enzyme), enzyme was added to ESW and immediately boiled, to ensure that the presence of denatured enzyme did not affect sperm behavior. Negative controls were run in filtered sea water (FSW). Values are means ± S.E.M.; means linked by a horizontal bar did not differ significantly (Scheffé test, P<0.05). (A) Change in sperm swim speed in response to tested solutions. (B) Change in cell density due to sperm chemotaxis.

View larger version (35K): [in a new window]   Fig. 9. Disruption of the tryptophan gradient around live eggs prevents navigation by sperm. Individual red abalone eggs were placed in 400 µl of filtered sea water (FSW) or the indicated test solution, after which sperm were added and video-recorded for 30s. Representative swimming paths are shown as in Fig. 1; the scale bar applies to all panels. Polar plots show the vector mean direction of sperm swimming, with angles measured relative to the egg surface (0°); the bar indicates the vector mean length (r). (A) Sperm in FSW near the surface of a live egg. (B) Sperm in the presence of the enzyme tryptophanase, which selectively digests L-tryptophan as it diffuses from the egg. Enzyme treatment had no effect on sperm viability. (C) Sperm in a uniform solution of 10-7 mol l-1 L-tryptophan. (D) Sperm in a uniform solution of 10-7 mol l-1 L-tyrosine. The tyrosine solution controlled for any effects of elevating the concentration of an aromatic amino acid in the sea water medium.