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Dual processing of sulfated steroids in the olfactory system of an anuran amphibian.

Sansone A, Hassenklöver T, Offner T, Fu X, Holy TE, Manzini I - Front Cell Neurosci (2015)

Bottom Line: Chemical communication is widespread in amphibians, but if compared to later diverging tetrapods the available functional data is limited.Furthermore, we found that larval and adult animals excrete multiple sulfated compounds with physical properties consistent with sulfated steroids.Breeding tadpole and frog water including these compounds activated a large subset of sensory neurons that also responded to synthetic steroids, showing that sulfated steroids are likely to convey intraspecific information.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurophysiology and Cellular Biophysics, University of Göttingen Göttingen, Germany ; Center for Nanoscale Microscopy and Molecular Physiology of the Brain Göttingen, Germany.

ABSTRACT
Chemical communication is widespread in amphibians, but if compared to later diverging tetrapods the available functional data is limited. The existing information on the vomeronasal system of anurans is particularly sparse. Amphibians represent a transitional stage in the evolution of the olfactory system. Most species have anatomically separated main and vomeronasal systems, but recent studies have shown that in anurans their molecular separation is still underway. Sulfated steroids function as migratory pheromones in lamprey and have recently been identified as natural vomeronasal stimuli in rodents. Here we identified sulfated steroids as the first known class of vomeronasal stimuli in the amphibian Xenopus laevis. We show that sulfated steroids are detected and concurrently processed by the two distinct olfactory subsystems of larval Xenopus laevis, the main olfactory system and the vomeronasal system. Our data revealed a similar but partially different processing of steroid-induced responses in the two systems. Differences of detection thresholds suggest that the two information channels are not just redundant, but rather signal different information. Furthermore, we found that larval and adult animals excrete multiple sulfated compounds with physical properties consistent with sulfated steroids. Breeding tadpole and frog water including these compounds activated a large subset of sensory neurons that also responded to synthetic steroids, showing that sulfated steroids are likely to convey intraspecific information. Our findings indicate that sulfated steroids are conserved vomeronasal stimuli functioning in phylogenetically distant classes of tetrapods living in aquatic and terrestrial habitats.

No MeSH data available.


Tadpole and frog breeding water contains sulfated compounds and activates neurons sensitive to synthetic steroids. (A) Following chromatographic extraction of steroids from breeding water, precursor ion scan mass spectrometry was used to analyze the abundance of peaks that fragment to produce negative ions with m/z of 80 (, left column) or 97 (, right column). (B) Representative calcium responses of five MOE neurons. The shown cells include the main subgroups of responding cells, i.e., cells responding to both synthetic steroids (200 μM) and breeding water (1:1 dilution), cells responding to amino acids (100 μM) and breeding water, cells responding to breeding water only, and cells responding to synthetic steroids, breeding water and amino acids. (C) Response matrix of neurons in the MOE responding to breeding water (1:1 dilution) and synthetic sulfated steroids (200 μM; 85 cells, 5 slices). The majority of the neurons responding to synthetic steroids also responded upon application of breeding water (16 out of 27 cells). Response intensity is coded by a color gradient. A mixture of amino acids (AA, 100 μM) was applied as a control for slice viability. TW, tadpole breeding water; FW, frog breeding water; TapW, tap water control; ctrl, bath solution control.
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Figure 6: Tadpole and frog breeding water contains sulfated compounds and activates neurons sensitive to synthetic steroids. (A) Following chromatographic extraction of steroids from breeding water, precursor ion scan mass spectrometry was used to analyze the abundance of peaks that fragment to produce negative ions with m/z of 80 (, left column) or 97 (, right column). (B) Representative calcium responses of five MOE neurons. The shown cells include the main subgroups of responding cells, i.e., cells responding to both synthetic steroids (200 μM) and breeding water (1:1 dilution), cells responding to amino acids (100 μM) and breeding water, cells responding to breeding water only, and cells responding to synthetic steroids, breeding water and amino acids. (C) Response matrix of neurons in the MOE responding to breeding water (1:1 dilution) and synthetic sulfated steroids (200 μM; 85 cells, 5 slices). The majority of the neurons responding to synthetic steroids also responded upon application of breeding water (16 out of 27 cells). Response intensity is coded by a color gradient. A mixture of amino acids (AA, 100 μM) was applied as a control for slice viability. TW, tadpole breeding water; FW, frog breeding water; TapW, tap water control; ctrl, bath solution control.

Mentions: Sulfated steroids were shown to be excreted in mouse urine and to activate receptor neurons of the VNO (Hsu et al., 2008; Nodari et al., 2008). To obtain some insight on whether Xenopus laevis also excretes sulfated steroids, we performed steroid extraction from tadpole and adult frog breeding water via solid-phase chromatography. A well-established protocol for steroid extraction from urine samples (Shackleton and Whitney, 1980; Nodari et al., 2008) was employed to recover and concentrate free steroids and steroid conjugates contained in the breeding water. These extracts were then analyzed by precursor ion mass spectrometry (see Materials and Methods) for ions that could dissociate to produce fragments with a mass-to-charge ratio (m/z) of 80 or 97, signatures of and ions produced by many sulfated steroids (Gaskell, 1988; Weidolf et al., 1988; Yan et al., 2014). This analysis revealed the presence of multiple sulfated compounds (Figure 6A), of which several have m/z (in the approximate range 350–450 Daltons) consistent with sulfated steroids.


Dual processing of sulfated steroids in the olfactory system of an anuran amphibian.

Sansone A, Hassenklöver T, Offner T, Fu X, Holy TE, Manzini I - Front Cell Neurosci (2015)

Tadpole and frog breeding water contains sulfated compounds and activates neurons sensitive to synthetic steroids. (A) Following chromatographic extraction of steroids from breeding water, precursor ion scan mass spectrometry was used to analyze the abundance of peaks that fragment to produce negative ions with m/z of 80 (, left column) or 97 (, right column). (B) Representative calcium responses of five MOE neurons. The shown cells include the main subgroups of responding cells, i.e., cells responding to both synthetic steroids (200 μM) and breeding water (1:1 dilution), cells responding to amino acids (100 μM) and breeding water, cells responding to breeding water only, and cells responding to synthetic steroids, breeding water and amino acids. (C) Response matrix of neurons in the MOE responding to breeding water (1:1 dilution) and synthetic sulfated steroids (200 μM; 85 cells, 5 slices). The majority of the neurons responding to synthetic steroids also responded upon application of breeding water (16 out of 27 cells). Response intensity is coded by a color gradient. A mixture of amino acids (AA, 100 μM) was applied as a control for slice viability. TW, tadpole breeding water; FW, frog breeding water; TapW, tap water control; ctrl, bath solution control.
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Related In: Results  -  Collection

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Figure 6: Tadpole and frog breeding water contains sulfated compounds and activates neurons sensitive to synthetic steroids. (A) Following chromatographic extraction of steroids from breeding water, precursor ion scan mass spectrometry was used to analyze the abundance of peaks that fragment to produce negative ions with m/z of 80 (, left column) or 97 (, right column). (B) Representative calcium responses of five MOE neurons. The shown cells include the main subgroups of responding cells, i.e., cells responding to both synthetic steroids (200 μM) and breeding water (1:1 dilution), cells responding to amino acids (100 μM) and breeding water, cells responding to breeding water only, and cells responding to synthetic steroids, breeding water and amino acids. (C) Response matrix of neurons in the MOE responding to breeding water (1:1 dilution) and synthetic sulfated steroids (200 μM; 85 cells, 5 slices). The majority of the neurons responding to synthetic steroids also responded upon application of breeding water (16 out of 27 cells). Response intensity is coded by a color gradient. A mixture of amino acids (AA, 100 μM) was applied as a control for slice viability. TW, tadpole breeding water; FW, frog breeding water; TapW, tap water control; ctrl, bath solution control.
Mentions: Sulfated steroids were shown to be excreted in mouse urine and to activate receptor neurons of the VNO (Hsu et al., 2008; Nodari et al., 2008). To obtain some insight on whether Xenopus laevis also excretes sulfated steroids, we performed steroid extraction from tadpole and adult frog breeding water via solid-phase chromatography. A well-established protocol for steroid extraction from urine samples (Shackleton and Whitney, 1980; Nodari et al., 2008) was employed to recover and concentrate free steroids and steroid conjugates contained in the breeding water. These extracts were then analyzed by precursor ion mass spectrometry (see Materials and Methods) for ions that could dissociate to produce fragments with a mass-to-charge ratio (m/z) of 80 or 97, signatures of and ions produced by many sulfated steroids (Gaskell, 1988; Weidolf et al., 1988; Yan et al., 2014). This analysis revealed the presence of multiple sulfated compounds (Figure 6A), of which several have m/z (in the approximate range 350–450 Daltons) consistent with sulfated steroids.

Bottom Line: Chemical communication is widespread in amphibians, but if compared to later diverging tetrapods the available functional data is limited.Furthermore, we found that larval and adult animals excrete multiple sulfated compounds with physical properties consistent with sulfated steroids.Breeding tadpole and frog water including these compounds activated a large subset of sensory neurons that also responded to synthetic steroids, showing that sulfated steroids are likely to convey intraspecific information.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurophysiology and Cellular Biophysics, University of Göttingen Göttingen, Germany ; Center for Nanoscale Microscopy and Molecular Physiology of the Brain Göttingen, Germany.

ABSTRACT
Chemical communication is widespread in amphibians, but if compared to later diverging tetrapods the available functional data is limited. The existing information on the vomeronasal system of anurans is particularly sparse. Amphibians represent a transitional stage in the evolution of the olfactory system. Most species have anatomically separated main and vomeronasal systems, but recent studies have shown that in anurans their molecular separation is still underway. Sulfated steroids function as migratory pheromones in lamprey and have recently been identified as natural vomeronasal stimuli in rodents. Here we identified sulfated steroids as the first known class of vomeronasal stimuli in the amphibian Xenopus laevis. We show that sulfated steroids are detected and concurrently processed by the two distinct olfactory subsystems of larval Xenopus laevis, the main olfactory system and the vomeronasal system. Our data revealed a similar but partially different processing of steroid-induced responses in the two systems. Differences of detection thresholds suggest that the two information channels are not just redundant, but rather signal different information. Furthermore, we found that larval and adult animals excrete multiple sulfated compounds with physical properties consistent with sulfated steroids. Breeding tadpole and frog water including these compounds activated a large subset of sensory neurons that also responded to synthetic steroids, showing that sulfated steroids are likely to convey intraspecific information. Our findings indicate that sulfated steroids are conserved vomeronasal stimuli functioning in phylogenetically distant classes of tetrapods living in aquatic and terrestrial habitats.

No MeSH data available.