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Olfactory receptor for prostaglandin F2α mediates male fish courtship behavior

Nature Neuroscience volume 19, pages 897–904 (2016)Cite this article

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Abstract

Pheromones play vital roles for survival and reproduction in various organisms. In many fishes, prostaglandin F2α acts not only as a female reproductive hormone, facilitating ovulation and spawning, but also as a sex pheromone inducing male reproductive behaviors. Here, we unravel the molecular and neural circuit mechanisms underlying the pheromonal action of prostaglandin F2α in zebrafish. Prostaglandin F2α specifically activates two olfactory receptors with different sensitivities and expression in distinct populations of ciliated olfactory sensory neurons. Pheromone information is then transmitted to two ventromedial glomeruli in the olfactory bulb and further to four regions in higher olfactory centers. Mutant male zebrafish deficient in the high-affinity receptor exhibit loss of attractive response to prostaglandin F2α and impairment of courtship behaviors toward female fish. These findings demonstrate the functional significance and activation of selective neural circuitry for the sex pheromone prostaglandin F2α and its cognate olfactory receptor in fish reproductive behavior.

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Figure 1: PGF2α attracts male zebrafish.
Figure 2: PGF2α activates ciliated OSNs expressing or114-1 or or114-2.
Figure 3: OR114-1 and OR114-2 are functional receptors for PGF2α with different sensitivities.
Figure 4: Olfactory neural pathways activated by PGF2α.
Figure 5: Generation and characterization of or114-1 mutant zebrafish.
Figure 6: Impaired courtship behavior in or114-1 mutant male zebrafish.

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  • 06 June 2016

    In the version of this article initially published online, the phrase "in various organisms" appeared twice in the first sentence of the abstract. The sentence should read, "Pheromones play vital roles for survival and reproduction in various organisms." The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank C. Yokoyama for critical reading of the manuscript; M. Shiozaki and T. Kajiyama for technical assistance; K. Touhara, S. Ihara (The University of Tokyo) and K. Kawakami (National Institute of Genetics, Japan) for reagents; Y. Niimura (The University of Tokyo) for mouse OR sequence data; members of the Research Resource Center of RIKEN BSI for fish maintenance and technical assistance; and members of the Yoshihara laboratory for fish care and discussion. Y. Yabuki is especially grateful to K. Watanabe and Y. Shimoda (Nagaoka University of Technology) for continuous support and encouragement. Y. Yabuki was supported by the RIKEN Junior Research Associate program. This work was supported by Grants-in-Aid for Scientific Research (KAKENHI 25430025 to N.M. and KAKENHI 21115504 to J.N.) and for Scientific Research on the Innovative Area “Memory Dynamism” (KAKENHI 25115005 to Y. Yoshihara) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Human Frontier Science Program (HFSP RGP0015/2010) to Y. Yoshihara, the Uehara Memorial Foundation to Y. Yoshihara, Japan Science and Technology Agency ERATO Program to Y. Yoshihara and M.M., and Regional Innovation Cluster Program (City Area Type, Central Saitama Area) to J.N.

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Authors and Affiliations

  1. RIKEN Brain Science Institute, Saitama, Japan

    Yoichi Yabuki, Tetsuya Koide, Nobuhiko Miyasaka, Noriko Wakisaka, Miwa Masuda & Yoshihiro Yoshihara

  2. Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan

    Yoichi Yabuki & Yoshihiro Yoshihara

  3. ERATO Touhara Chemosensory Signal Project, JST, The University of Tokyo, Tokyo, Japan

    Miwa Masuda & Yoshihiro Yoshihara

  4. Brain Science Institute, Saitama University, Saitama, Japan

    Masamichi Ohkura & Junichi Nakai

  5. Department of Pharmaceutical Biochemistry, Kumamoto University Graduate School of Pharmaceutical Sciences, Kumamoto, Japan

    Kyoshiro Tsuge, Soken Tsuchiya & Yukihiko Sugimoto

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  1. Yoichi Yabuki
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Contributions

Y. Yabuki, T.K., N.M., N.W., M.M., K.T., S.T., Y.S. and Y. Yoshihara performed the experiments. M.O. and J.N. provided unpublished reagents. Y. Yabuki, T.K., N.M. and Y. Yoshihara conceived this study and wrote the paper with help from all authors.

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Correspondence to Yoshihiro Yoshihara.

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Integrated supplementary information

Supplementary Figure 1 PGF2α activates OSNs that express or114-1 belonging to the OR group β.

(a) Double ISH of PGF2α-stimulated OE sections with OR mixture (magenta) and c-fos (green) probes. 600 μl of 10−4M PGF2α was added to 600 ml water in a tank containing a zebrafish (final concentration: 10−7M). PGF2α-induced c-fos signals co-localize with only the mixture of group β ORs (or112-1, or113-1, or113-2 and or114-1) (arrowheads), but not with other OR mixtures. Scale bar, 20 μm. Two fish examined for each OR showed similar results. (b) Quantification of the number of c-fos-positive (green), OR-positive (magenta), and c-fos/OR-double-positive (white) OSNs in PGF2α-stimulated OE sections (n = 3 fish). The double-positive OSNs are observed only for or114-1. Representative images of c-fos and individual OR expression are shown in Fig. 2b.

Supplementary Figure 2 Activation of or114-1- and or114-2-expressing OSNs by PGF2α as assessed by pERK immunohistochemistry and OR ISH.

(a) Double fluorescence labeling of PGF2α-stimulated OE sections with or114-1 and/or or114-2 cRNA probes (magenta in middle panels) and anti-pERK antibody (green in middle panels). 60 μl of 10−3M PGF2α was added to 600 ml water in a tank containing a zebrafish (final concentration: 10−7M). Closed and open arrowheads indicate OR/pERK-double-positive OSNs and pERK-single-positive OSNs, respectively. Scale bar, 50 μm. One fish was examined for observation. (b) Percentages of OR-positive OSNs among pERK-positive OSNs are shown (n = 1). or114-1 probe (red), or114-2 probe (blue), a mixture of or114-1 and or114-2 probes (purple) were used for double labeling with pERK antibody.

Supplementary Figure 3 PGF2α induces ERK phosphorylation in OB neurons.

(a) An OB section prepared from PGF2α-stimulated OMP:Gal4FF;UAS:GFP transgenic zebrafish was labeled with anti-pERK antibody (red), anti-GFP antibody (green), and DAPI (blue). 60 μl of 10−3M PGF2α was added to 600 ml water in a tank containing a zebrafish (final concentration: 10−7M). A pERK-positive glomerulus is observed in the ventromedial region of the right OB. Scale bar, 100 μm. (b) Magnification of two GFP-positive glomeruli in the ventromedial OB. One glomerulus (right) is pERK-positive, whereas the other (left) is negative. pERK signals are confined to somata and dendrites of post-synaptic OB neurons, but are not present in pre-synaptic olfactory axon terminals. Arrowheads indicate pERK-positive somata of OB neurons around the glomerulus. Scale bar, 20 μm. Four fish examined showed similar results.

Supplementary Figure 4 Comparison of higher olfactory centers activated by PGF2α (sex pheromone) and alanine (feeding cue).

(a) A schematic of the zebrafish brain. Vertical lines indicate the antero-posterior positions of the coronal sections in (b-f). (b-f) DAPI-stained coronal sections of a brain. Yellow boxes indicate the location of magnified views in (g-k). Scale bar, 500 μm. (g-k) pERK immunostaining of brain sections of zebrafish exposed to DMSO (left), PGF2α (middle), and alanine (right). 60 μl of 10−4M PGF2α or 600 μl of 10−2M alanine was added to 600 ml water in a tank containing a zebrafish [final concentration: 10−8M (PGF2α) or 10−5M (alanine)]. Blue- and yellow-encircled lines indicate individual brain nuclei (Vv, Vs, PPa, LH, Hd, Hc) with basal and up-regulated pERK signals, respectively. Scale bar, 200 μm. Four fish examined for each stimulus showed similar results.

Supplementary Figure 5 PGF2α stimulation fails to increase pERK signals in higher olfactory centers of OE-removed zebrafish.

(a) pERK immunostaining (red) of brain sections from intact (top) and OE-removed (bottom) zebrafish exposed to PGF2α. 60 μl of 10−4M PGF2α was added to 600 ml water in a tank containing a zebrafish (final concentration: 10−8M). Blue signals represent DAPI staining. Scale bar, 50 μm. Four fish examined for each stimulusexperimental group showed similar results. (b) Quantification of the number of pERK-positive neurons in the four brain regions. Values represent mean ± SEM (n = 3 fish). Unpaired t-test (Vv, p = 0.032; PPa, p = 0.022; LH, p = 0.22; Hc, p = 0.047). * p < 0.05.

Supplementary Figure 6 Impaired courtship behavior in OE-removed male zebrafish.

(a) Raster plots summarizing courtship behavior for 60 min of intact or OE-removed male fish paired with female fish. Yellow, red, and blue bars represent periods of chase, touch, and encircle behaviors of male fish, respectively. Gray bars indicate immobile periods of female fish. (b-i) Quantification of the courtship behavior in intact (green) and OE-removed (pink) fish (n = 6 pairs for each genotypeexperimental group). Means (solid line) and SEM (transparency) of cumulative chase duration (b), cumulative number of touches (c) and cumulative number of encircles (d) are shown. Box plots show median, quartiles (boxes) and range (whiskers) of total chase duration (e), total number of touches (f), total number of encircles (g), courtship duration per one bout (h), and total number of courtship bouts (i). Unpaired t-test (e, p = 0.0011; f, p = 0.048; g, p = 0.0049; h, p = 0.00083; i, p = 0.0087). * p < 0.05, ** p < 0.01.

Supplementary Figure 7 Comparison of PGF2α-induced ERK phosphorylation in higher olfactory centers between wild-type and or114-1 mutant zebrafish.

Quantification of the number of pERK-positive neurons in the four brain regions of wild-type (blue) and or114-1 mutant (red) zebrafish stimulated with PGF2α. 60 μl of 10−4M PGF2α was added to 600 ml water in a tank containing a zebrafish (final concentration: 10−8M). Values represent mean ± SEM (WT, n = 4; KO, n = 5 fish). Unpaired t-test (Vv, p = 0.021; PPa, p = 0.96; LH, p = 0.71; Hc, p = 0.42). * p < 0.05.

Supplementary Figure 8 Zebrafish group β ORs and putative pheromonal PGF2α receptor orthologs in other fish species.

By performing extensive database search with both zebrafish OR114-1 and OR114-2 amino acid sequences as queries, we found full-length orthologous ORs in common carp (Cyprinus carpio) and cavefish (Astyanax mexicanus), and a highly homologous EST clone in goldfish (Carassius auratus). (a) Neighbor-joining phylogenetic tree based on alignment of the amino acid sequences of zebrafish group β ORs and OR114 orthologs in other fishes. Bootstrap values shown at selected nodes were obtained from 1,000 replications. (b) Amino acid sequence alignment of zebrafish OR114-1, OR114-2 and their orthologs in common carp, cavefish and goldfish. The residues having 100% (black) or more than 50% (gray) similarities among the receptors are shaded.

Supplementary Figure 9 Zebrafish group β ORs show remarkable similarities with three class I ORs in mice.

Neighbor-joining phylogenetic tree based on alignment of all full-length ORs in zebrafish (136 ORs) and mouse (1130 ORs)23,54. Groups of zebrafish ORs are shown in different colors with Greek symbols. Two classes of mouse ORs are labeled. The tree shows high similarity between zebrafish group β ORs (OR112-1, OR113-1, OR113-2, OR114-1 and OR114-2) (red arrow) and particular class I ORs of MOR42 subfamily (Olfr543, Olfr544 and Olfr545) (black arrow).

54. Niimura, Y., Matsui, A. & Touhara, K. Extreme expansion of the olfactory receptor gene repertoire in African elephants and evolutionary dynamics of orthologous gene groups in 13 placental mammals. Genome Res. 24, 1485-1496 (2014).

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Supplementary Figures 1–9 (PDF 1637 kb)

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PGF2α attracts male zebrafish.

A representative example of zebrafish attractive behavior (8 adult males) to PGF2α. Upon PGF2α application to the right side of the tank (indicated by an arrow), fish immediately show robust attractive response and eventually make a tight shoal on the PGF2α-applied side. (MP4 493 kb)

PGF2α activates two ventromedial glomeruli.

Ca2+ imaging was performed from the ventral side of the OE-brain explant preparation prepared from OMP:Gal4FF;UAS:G-CaMP-HS transgenic fish. Two ventromedial glomeruli (vmG) show specific activation in response to 10−7M PGF2α. A graph shows time course of PGF2α-evoked calcium increase in the two glomeruli indicated with arrows. (MP4 472 kb)

Zebrafish courtship behavior.

A representative example of zebrafish courtship behavior. A wild-type male zebrafish shows chase, touch and encircle responses towards a female fish. (MP4 611 kb)

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Yabuki, Y., Koide, T., Miyasaka, N. et al. Olfactory receptor for prostaglandin F2α mediates male fish courtship behavior. Nat Neurosci 19, 897–904 (2016). https://doi.org/10.1038/nn.4314

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