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Stereochemical specificity of lamoxirene, the sperm-releasing pheromone in kelp (laminariales, phaeophyceae).

INGO MAIER (1) (*)



Sexual reproduction in the large brown seaweeds of the Laminariales, commonly called kelp, involves signaling chemicals, or "pheromones," that induce sperm release from antheridia and subsequent chemotactic orientation of sperm towards the luring eggs. Lamoxirene (cis-2-cyclohepta-2',5'-dienyl-3-vinyloxirane) has been identified as the sperm-releasing pheromone in the largest and most advanced group of the Laminariales, comprising the Laminariaceae, Alariaceae, and Lessoniaceae. Recently, a stereoselective synthesis of lamoxirene has yielded pure substances for biological studies. Here, we used closed-loop stripping and chiral gas chromatography to establish which of the four possible stereoisomers of lamoxirene functions as the naturally occurring sperm-releasing pheromone in Undaria pinnatifida. In addition, the relationship between absolute configuration and sperm-releasing bioactivily in Laminaria, Alaria, Undaria, and Macrocystis was clarified in bioassays with lamoxirene stereoisomers. Our experiments e stablished (1'R,2S,3R)-lamoxirene as the most bioactive compound in all species tested and as the main component in egg secretions. Thus no species specificity in the stereochemistry of the sperm-releasing pheromone of the Laminariales has yet been found.

Sexuality in the Laminariales is strictly controlled by environmental cues and is coordinated by chemical interaction. Pheromones secreted by eggs produced on microscopic female gametophytes (Fig. 1) induce the release of sperm from antheridia on nearby male gametophytes and subsequently attract the sperm to the eggs (1-4). The species in the Laminariales belonging to the Laminariaceae, Alarmaceae, and Lessoniaceae are characterized by the exclusive possession of lamoxirene (cis-2-cyclobepta-2',5'-dienyl-3-vinyloxirane, Fig. 2) as the sperm-releasing pheromone (3, 5, 6). Lamoxirene may exist in four spatial variations (stereoisomers: enantiomers and diastereomers, Fig. 2), which may or may not occur in nature or function as pheromones, respectively. Supernatant taken from mature female cultures induces sperm release in many, if not all, interspecific combinations within this group (Maier, unpublished). These observations support the idea that pheromonal cross-reaction and even competition between kelp gametes may also occur in the field. In particular, this holds for sympatric species with overlapping reproductive periods, as exemplified by the four European Laminaria species listed in Table 1. However, experiments using culture supernatants or racemic synthetic pheromone do not rule out the possibility that kelps use specific mixtures of stereoisomers for differentiation of sympatric species (4, 7). Similar strategies, with different enantiomers or diastereomers used by different species, or specific mixtures being more active than single components, are well-known in insects (8). It was shown that two of the lamoxirenes, compound 1 and its diastereomer 3 (Fig. 2), are secreted in a ratio of 2.45:1 by eggs of Laminaria digitata, and preliminary bioassays revealed the highest biological activity in pheromone samples enriched in lamoxirene 1 (9).

Recently, an effective stereoselective synthesis of lamoxirene has become available (10), offering for the first time the chance of quantitative bioassays with pure stereoisomers of lamoxirene. Unfortunately, bioassay results on lamoxirene 1 and 3 were exchanged in the previous publication (10). In the present paper, we report the results of bioassays that correct this mistake and broaden the original data on Laminaria spp. to a number of other genera and families in the Laminariales. In addition to bioassays, the absolute configuration of naturally occurring lamoxirene 1 in Undaria pinnatifida is confirmed by chiral gas chromatography.

Clonal male gametophytes of several species of Laminariales (Table 1) were kept vegetatively in red fluorescent light (about 15 [micro]mol [m.sup.-2] [s.sup.-1], 16:8 h light/dark cycle) at 10[degrees]C with ASM-1 (11) as a culture medium. For the induction of gametogenesis, they were fragmented using a tissue homogenizer and transferred into white fluorescent light (30 [micro]mol [m.sup.-2] [s.sup.-1], 16 h light cycle) at 10[degrees]C. The gametophytes were used in sperm-release assays 12 days later, when numerous mature antheridia had been formed.

Lamoxirene stereoisomers 1-4 (Fig. 2) with high configurational purity (enantiomeric excess, e.e.) were available in dimethylsulfoxide (DMSO, puriss. p.a., FLUKA): lamoxirene 1: 97% e.e.; lamoxirene 2: 90% e.e.; lamoxirene 3: 95% e.e.; lamoxirene 4: 96% e.e. (10). The solutions were serially diluted (1 X [10.sup.-3] to 1 X [10.sup.-11] M, 5 X [10.sup.-4] M to 5 X [10.sup.-11] M, 2 X [10.sup.-4] M to 2 X [10.sup.-11] M) in DMSO. In addition, an equimolar mixture of lamoxirenes 1 and 3 was prepared and diluted accordingly.

For the stereochemical analysis of natural lamoxirene, female gametophytes of Undaria pinnatifida from Hokkaido, Japan, were cultivated as described above for the male gametophytes. Upon massive egg release, volatile egg secretions from the gametophyte suspension were adsorbed onto a bed of 1.5 mg activated carbon by closed loop stripping (2, 3), followed by elution with 20 [micro]l diethylether. Chiral capillary gas chromatography (FS-Lipodex E, Macherey-Nagel, Duren, Germany; 25 m X 0.32 mm, carrier [H.sub.2]) revealed a single lamoxirene peak (e.e. > 97%), identified as lamoxirene 1 by comparison with synthetic samples (12).

To compare the biological activity of the different stereoisomers in the induction of sperm release, a new semiquantitative assay was employed. Instead of preparing aqueous pheromone solutions by distribution from a waterimmiscible fluorocarbon phase as in the original assay (13), lamoxirene was directly diluted from stock solutions in DMSO into seawater. 0.5% of a lamoxirene solution was added to culture medium in 3-ml or 5-ml glass vials, carefully mixed, equilibrated to 12[degrees]C, and used immediately. All experiments were carried out in a climate-controlled culture room at 12[degrees]C. In the bioassay, 100 [micro]l of a suspension of male gametophytes were mixed with 100 [micro]l of an aqueous pheromone solution in a concavity slide. The final concentration of DMSO in all assays was 0.25%. DMSO alone did not induce sperm release, except in Alaria esculenta from Iceland (Table 1). This strain was thus excluded from all further experiments. The reaction to DMSO was not observed in A. esculenta from Newf oundland, which was used instead. Release of sperm was obseryed in a stereomicroscope at 40X magnification under dark-field illumination (Fig. 3). At saturating concentrations, release of an estimated several hundred sperm occurred within 7-15 s. A pheromone solution was considered inactive when no release occurred within 1 min. At the threshold, several spermatozoids were reliably released within 1 min, and stronger release occurred at the next higher concentration step. Assays were performed at least in triplicate and repeated in an independent experimental series. The results of bioassays on lamoxirene-induced sperm release in Laminaria digitata are shown in Table 2, and threshold concentrations for all species investigated are summarized in Table 3.

The response of the algae to pheromone was very reproducible (Table 2), and threshold concentrations were sharply defined. They were somewhat higher than those reported earlier (0.01 nM with racemic lamoxirene in L. digitata, L. hyperborea, and Macrocystis pyrifera (6); 0.002 nM in L. digitata with lamoxirene 1 (9)). The bioassay used here is simple and avoids inaccuracies introduced by solvent/water-distribution, but is probably more prone to pheromone loss by adsorption and volatilization than the original assay. The given thresholds should thus be regarded as conservative approximations.

In the species tested, lamoxirene 1 was generally the most active stereoisomer, followed by lamoxirene 3. L. saccharina was the only exception, with compounds 1 and 3 being equally effective. The relative biological activity of lamoxirene stereoisomers matches the composition of egg secretions, with lamoxirene 1 being the main or the only stereoisomer produced in L. digitata (9) and U. pinnatifida (this study), respectively. Lamoxirene 3 occurs as a by-product in L. digitata, while lamoxirene 2 and 4 have not yet been identified as natural products. Lamoxirene 4 is virtually inactive, which underlines the central importance of the spatial orientation of the epoxy group in relation to the terminal double bond in the side chain and the ring system. This has already been indicated in earlier studies on receptor specificity (13). In L. digitata, L. hyperborea, and L. ochroleuca, the diastereomer mixture of lamoxirenes 1 and 3 had no higher biological activity in sperm release than lamoxirene 1 alone (Table 4, A c ompared with B and C). On the contrary, weak competitive effects indicated by slightly increased thresholds were observed in L. digitata and L. hyperborea (Table 4, A compared with B).

In conclusion, the stereochemical specificity of lamoxirene action in pheromone-induced sperm release is conserved among all species tested. This holds not only for the sympatric European Laminaria and Alaria, but also for Undaria and Macrocystis from the North Pacific and thus probably for all species belonging to the Laminariaceae, Alariaceae, and Lessoniaceae. These families comprise the monophyletic "core group" of the most advanced Laminariales (14, 15). The origin of their pheromone system and its stereochemistry reaches back to at least the divergence of the group from a common ancestor, which dates to between 16 and 40 million years ago based on molecular clock estimations and various other considerations, including biogeography and paleooceanography (3, 14, 16-18).

Species specificity in gamete interaction in the Laminariales is thus not achieved by pheromone specificity in the induction of sperm release, but must be mediated by subsequent mechanisms. These may include differential sperm attraction to the egg, which is also under pheromonal control, and gamete surface recognition. In L. digitata, it was previously shown that different pheromone receptors are involved in sperm release and chemotaxis, and that desmarestene (6-(cis-1' ,3'-butadienyl)-cyclohepta-1,4-diene), not lamoxirene, is the most potent chemoattractant in this species (19). The possibility thus exists that a species-specific diversification of complex egg secretions and pheromone receptors is operative on the chemoattraction level.

(1.) Fachbereich Biologie, Universitat Konstanz, 78457 Konstanz, Germany;

(2.) Max-Planck-Institute for Chemical Ecology, Carl-Zeiss-Promenade 10, 07745 Jena, Germany

(*.) To whom correspondence should be addressed. E-mail:

Literature Cited

1. Maier, I. 1987. Environmental and pheromonal control of sexual reproduction in Laminaria. Pp. 66-74 in Algal Development-Molecular and Cellular Aspects, W. Wiessner, D. G. Robinson, and R. C. Starr, eds. Springer-Verlag, Berlin.

2. Maier, I., and D. G. Muller. 1986. Sexual pheromones in algae. Biol. Bull. 170: 145-175.

3. Maier, I., D. G. Muller, G. Gassmann, W. Boland, and L. Jaenicke. 1987. Sexual pheromones and related egg secretions in Laminariales (Phaeophyta). Z Naturforsch. Sect. C 42: 948-954.

4. Maier, I. 1995. Brown algal pheromones. Prog. Phycol. Res. 11:51-102.

5. Marner, F.-J., B. Muller, and L. Jaenicke. 1984. Lamoxirene. Structural proof of the spermatozoid releasing and attracting pheromone of Laminariales. Z. Naturforsch. Sect. C 39: 689-691.

6. Muller, D. G., I. Maier, and G. Gassmann, 1985. Survey on sexual pheromone specificity in Laminariales (Phaeophyceae). Phycologia 24: 475-477.

7. Boland, W., U. Flegel, G. Jordt, and D. G. Muller, 1987. Absolute configuration and enantiomer composition of hormosirene. Naturwissenschaften 74: 448-449.

8. Mori, K. 1997. Pheromones: synthesis and bioactivity. Chem. Commun. 1997: 1153-1158.

9. Maier, I., G. Pohnert, S. Pantke-Bocker, and W. Boland, 1996. Solid-phase microextraction and determination of the absolute configuration of the Laminaria digitata (Laminariales, Phaeophyceae) spermatozoid-releasing pheromone. Naturwissenschaften 83: 378-379.

10. Hertweck, C., and W. Boland, 2000. Tandem reduction-chloroallylboration of esters: asymmetric synthesis of lamoxirene, the spermatozoid releasing and attracting pheromone of the Laminariales (Phaeophyceae). J. Org. Chem. 65: 2458-2463.

11. Maier, I., and M. Calenberg. 1994. Effect of extracellular [Ca.sup.2+] and [Ca.sup.2+] -antagonists on the movement and chemoorientation of male gametes of Ectocarpus siliculosus (Phaeophyceae). Bot. Acta 107: 451-460.

12. Hertweek, C. 1999. Funktionalisierte Vinyloxirane durch Reduktive Allylierung von Estern: Stereoselektive Synthesen von Lamoxiren und Sphingoidbasen. Dissertation, University of Bonn, Germany.

13. Maier, L., D. G. Muller, C. Schmid, W. Roland, and L. Jaenicke. 1988. Pheromone receptor specificity and threshold concentrations for spermatozoid release in Laminaria digitata. Naturwissenschaften 75: 260-263.

14. Maier, I. 1984. Sexualitat bei Braunalgen aus der Ordnung Laminariales und die Phylogenie der Ordnung. Dissertation, University of Konstanz, Germany.

15. Boo, S. M., W. J. Lee, H. S. Yoon, A. Kato, and H. Kawai. 1999. Molecular phylogeny of Laminariales (Phaeophyceae) inferred from small subunit ribosomal DNA sequences. Phycol. Res. 47: 109-114.

16. Estes, J. A., and P. D. Steinberg. 1988. Predation, herbivory, and kelp evolution. Paleobiology 14: 19-36.

17. Luning, K., and I. tom Dieck. 1990. The distribution and evolution of the Laminariales: North Pacific-Atlantic relationships. Pp. 187-204 in Evolutionary Biogeography of the Marine Algae of the North Atlantic, D. J. Garbary, and G. R. South, eds. Springer-Verlag, Berlin,

18. Saunders, G. W., and L. D. Druehl. 1992. Nucleotide sequences of the small-subunit ribosomal RNA genes from selected Laminariales (Phaeophyta): implications for kelp evolution. J. Phycol. 28: 544-549.

19. Maier, I., D. G. Muller, and W. Boland. 1994. Spermatozoid chemotaxis in Laminaria digitata (Phaeophyceae). III. Pheromone receptor specificity and threshold concentrations. Z. Naturforsch. Sect. C 49: 601-606.

[Figure 1 omitted]

[Figure 2 omitted]

[Figure 3 omitted]
Table 1

Classification and geographic origin or species used in bioassays

Classification                       Origin

Family Laminariaceae
 Laminaria digitata (Huds.) Lamour.  Helgoland, German Bight,
                                      North Sea
 L. hyperborea (Gunn.) Fosl.         Helgoland
 L. saccharina (L.) Lamour.          Helgoland
 L. ochroleuca Pyl.                  Roscoff, Brittany, France
Family Alariaceae
 Alaria esculenta (L.) Grev.         Tjornes, Iceland
 A. esculenta (L.) Grey.             St. John's, Newfoundland,
 Undaria pinnatifida (Harv.)
 Suringar                            Hokkaido, Japan
Family Lessoniaceae
 Macrocystis pyrifera (L.) C. Ag.    Santa Barbara, California
Table 2

Results of bioassays on pheromone-induced sperm release in Laminaria

                           Pheromone concentration (nM)
Lamoxirene  0.025          0.05      0.1          0.25        0.5

    1       0, 0, 0      +, 0, +   +, +, +      ++, +, ++,  +++, +++
            0, 0         0, +, +   +, +, +      ++, ++      +++
    2         --           --      0, 0, 0      0, 0, 0     0, 0, 0

    3         --         0, 0, 0   0, 0, 0      +, +, 0     +, +, +
                                   0, 0, 0      0, +, +
    4         --           --        --            --         --

             Pheromone concentration (nM)
Lamoxirene     1        2.5        10

    1         --        --         --

    2       0, 0, 0   +, +, +    ++, ++
            0, +, 0   +, +, +    ++
    3       +, +, +   ++, ++     +++, +++
                      ++, +, ++  +++
    4       0, 0, 0   0, 0, 0    +, +, +
            0, 0, 0   0, +, 0    0, +, +

The thresholds are printed in bold.

--: not tested; 0, +, ++, +++: no max. 2, 3-20, 20-100, several hundred
sperm released, respectively.
Table 3

Threshold concentrations in seawater for the induction of spermatozoid
release by lamoxirene stereoisomers

                                Threshold concentrations (nM)
Species                  Lamoxirene 1  Lamoxirene 2      Lamoxirene 3

Laminaria digitata           0.05          2.5               0.25
L. hyperborea                0.01          0.1               0.02
L. saccharina                1             2.5               1
L. ochroleuca                0.02          1                 0.25
Alaria esculenta             0.1           1                 0.25
Undaria pinnatifida          0.1           1                 1
Macrocystis pyrifera         0.1           1                 1

Species                Lamoxirene 4

Laminaria digitata         10
L. hyperborea               2.5
L. saccharina             >20 (*)
L. ochroleuca              10
Alaria esculenta
Undaria pinnatifida       >20 (*)
Macrocystis pyrifera       20

(*)Highest test concentration available.
Table 4

Threshold concentrations of total lamoxirene (M) in sperm release

Experiment                  A                   B

Laminaria digitata  2.3 X [10.sup.-10]  1.1 X [10.sup.-10]
L. hyperborea       1.1 X [10.sup.-10]  5.7 X [10.sup.-11]
L. Ochroleuca       1.1 X [10.sup.-10]  1.1 X [10.sup.-10]

Experiment                  C

Laminaria digitata  1.2 X [10.sup.-10]
L. hyperborea       2.5 X [10.sup.-11]
L. Ochroleuca       1.2 X [10.sup.-10]

Experiment A: equimolar combination of lamoxirenes 1 and 3; B: same
lamoxirene 3 replaced by pure DMSO C: lamoxirene 1 only, but total
lamoxirene as in A. The total solvent concentration was identical in
all tests.
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Author:Maier, Ingo; Hertweck, Christian; Boland, Wilhelm
Publication:The Biological Bulletin
Article Type:Statistical Data Included
Geographic Code:1USA
Date:Oct 1, 2001
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