Variation with reproductive status of PGE 2 receptor immunoreactivities in the Bostrichthys sinensis olfactory system

Journal of Fish Biology (2010) 77, 1542 1551 doi:10.1111/j.1095-8649.2010.02791.x, available online at wileyonlinelibrary.com Variation with reproductive status of PGE 2 receptor immunoreactivities in the Bostrichthys sinensis olfactory system X.-J. Lai and W.-S. Hong* State Key Laboratory of Marine Environmental Science and Department of Oceanography, Xiamen University, Xiamen, China (Received 18 July 2009, Accepted 15 August 2010) TheroleofPGE 2 as a putative sex pheromone in Chinese black sleeper Bostrichthys sinensis was investigated, using immunocytochemistry and how the immunoreactivities of the prostaglandin E 2 (PGE 2 ) receptor subtypes EP 1,EP 2,EP 3 and EP 4 varied with reproductive status in the olfactory system was determined. The results showed that PGE 2 receptors were present in the whole of the olfactory system of B. sinensis, and that the number of receptors was linked to the reproductive status of the fish. The densities of EP 1 immunoreactivity in the olfactory epithelium of mature fish were significantly (P <0 01) higher than those in immature fish of both sexes, and the densities of EP 2 and EP 3 immunoreactivities in mature fish were higher (but not significantly) than those in immature fish of both sexes. In the olfactory nerve, the density of EP 2 immunoreactivity in mature fish was higher (but not significantly) than that in immature fish in both sexes. In the olfactory bulb, the densities of EP 1 4 immunoreactivities in mature females were significantly (P <0 05 or <0 01) higher than those in immature females, and the density of EP 4 immunoreactivity in mature males was significantly (P <0 01) higher than that in immature males. As far as is known, the present study is the first report of the immunoreactivities of PGE 2 receptor subtypes in the olfactory system of a teleost, and offers new findings regarding the role of PGE 2 as sex pheromone and hormone in the reproductive behaviour and pheromonal communication of B. sinensis. Journal of Fish Biology 2010 The Fisheries Society of the British Isles Key words: immunocytochemistry; maturity; olfactory receptor; pheromone; teleost. INTRODUCTION Control of reproductive events is complex and involves both internal hormonal signals and external cues including pheromones. Gamete maturity and spawning, the controlled release of gametes by fishes, require synchronization of behaviour with gamete maturation both within and between the sexes (Kobayashi et al., 2002). Gonadal hormones, steroids, prostaglandins or their metabolites synchronize sexual behaviour with gamete maturation as endogenous signals or, excreted through the urine, signal the reproductive status of one fish to another as exogenous signals (pheromone) (Laberge & Hara, 2001; Kobayashi et al., 2002). At the time of ovulation, circulating and ovarian levels of F type prostaglandins (PGFs) increase in *Author to whom correspondence should be addressed. Tel.: +86 592 2186495; email: wshong@xmu.edu.cn 1542 Journal of Fish Biology 2010 The Fisheries Society of the British Isles

VA R I AT I O N O F P G E 2 RECEPTOR IMMUNOREACTIVITIES 1543 goldfish Carassius auratus (L.) probably reflecting their role in modulating follicular rupture. Circulating prostaglandin F2α (PGF2α) acts as a hormonal signal that triggers female spawning behaviour through direct actions on the brain (Sorensen et al., 1988). After ovulation, postovulatory PGF2α still acts hormonally to induce female receptivity and possibly pheromonally to trigger male behaviour (Stacey, 2003). Many fish species release gonadal hormones as sex pheromones (Resink et al., 1989; Kitamura et al., 1994; Sveinsson & Hara, 1995; Moore & Waring, 1996; Laberge & Hara, 2003; Bryan et al., 2008), as demonstrated in C. auratus (Dulka et al., 1987; Sorensen et al., 1988). These hormones are broadly termed hormonal pheromones (Yambe et al., 2006). Fishes demonstrate their reproductive readiness to find mating partners by employing these sex pheromones (Stacey, 2003; Yambe et al., 2006). Studies demonstrate that the fish olfactory system commonly uses hormonal pheromones (Laberge & Hara, 2001) and has been identified as an important seat for processing reproduction-related or pheromone-triggered signals (Biju et al., 2003). This view is primarily based on the evidence from electro-olfactogram (EOG) responses to the two C. auratus pheromones, 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P) and PGF. The same results are also obtained in >100 fish species (from the Cypriniformes, Siluriformes, Salmoniformes, Scorpaeniformes and Perciformes) (Sveinsson & Hara, 2000). It would be ideal to obtain information on the behavioural and physiological effects of a putative pheromone, since this exemplifies the role of the compound under natural conditions, and also the compounds likely to have pheromonal activity, which can be screened and identified rapidly using EOG techniques (Pottinger & Moore, 1997). Species-specific sex pheromones are found among C. auratus and other fishes (Sorensen et al., 1992) such as salmonids (Laberge & Hara, 2003). Even bile acid (Bryan et al., 2008) and some metabolites of amino acids (Yambe et al., 2006) are considered to be sex pheromones in the sea lamprey Petromyzon marinus L. and masu salmon Oncorhynchus masou (Brevoort). A different class of sex pheromones plays a distinct role in the spawning season. In female C. auratus, 17α,20β-P and its metabolite 17α,20β-P-S are considered to be primary components of the pre-ovulatory pheromone, while PGF2α and its metabolites are released as postovulatory pheromones that induce male spawning behaviour (Kobayashi et al., 2002). In teleosts, chemical information carried by pheromone molecules enters into the olfactory chamber with water and is transformed into an electrical signal. The process begins in the olfactory epithelium (OE), proceeds through the olfactory bulbs and reaches the telencephalon. The principal function of the biochemical pathways involved with olfactory transduction is to increase the sensitivity of the olfactory system by amplification of the signal received in pheromone receptors (de Souza & Antunes, 2007). Pheromone receptors bind pheromones and trigger signal transduction cascades and, therefore, characterization and computation of pheromone receptors in the olfactory system to a specific site offers an approach to understanding factors associated with pheromonal communication in fishes, which is complementary to electrophysiological and behavioural studies. The Chinese black sleeper Bostrichthys sinensis Lacépède belongs to the family Eleotridae, suborder Gobioidei. The fish is a seasonal breeder in the south-eastern regions of China and inhabits the intertidal zone. On the surface of mudflats, the fish build Y -shaped muddy burrows 40 65 cm deep. During the non-spawning season, females and males live in individual burrows, but during the spawning season, a pair

1544 X.-J. LAI AND W.-S. HONG of fish mate and spawn inside the same burrow (Zhong & Li, 2002). A behavioural study showed that in B. sinensis the synthetic hormone prostaglandin E 2 (PGE 2 ) attracted more males and females to the artificial nests than the control, and fish exposed to PGE 2 showed induced spawning (Hong et al., 2006). The PGE 2 levels in spawning waters were higher than those in the holding waters (Hong et al., 2006), suggesting that this compound is released from mature fish and the EOG values in response to PGE 2 are higher in mature fish than in immature ones (Ma et al., 2003). The results mentioned above suggested that PGE 2 might act as a putative sex pheromone in this species. The aim of this study was to investigate the presence of specific immunoreactivities of the PGE 2 receptor (PGE 2 R) subtypes and their variation with reproductive status in the olfactory system of B. sinensis. MATERIALS AND METHODS SAMPLING PROCEDURE Bostrichthys sinensis were collected from the Jiulong River, Fujian, China (24 45 N; 117 91 E), during both spawning and non-spawning seasons. Length (L) and body mass (M T ) were 142 167 mm and 41 6 73 2 g for females, and 152 186 mm and 45 5 76 7 g for males. Based on gonadal development, male and female fish were each divided into two groups: immature male and sexually mature male (milt could be stripped by gentle pressure on the abdomen), and immature female and sexually mature female (eggs could be stripped by gentle pressure on the abdomen). The fish were anaesthetized with 3-aminobenzoic acid ethyl ester (tricaine; MS-222, Sigma-Aldrich; www.sigmaaldrich.com) before being sacrificed. The gonado-somatic index (I G ) was calculated from I G = 100 M G MT 1,whereM G = gonad mass. The olfactory system was dissected out and post-fixed overnight in fresh fixative at 4 C. After dehydration, tissue samples were embedded in paraffin wax, cut serially at 5 μm and heated to 40 C for 8 h before use. IMMUNOCYTOCHEMISTRY Slides were deparaffinized, rehydrated through graded concentrations of alcohol to double distilled water and rinsed for 3 5 min in phosphate-buffered saline (PBS, ph = 7 4). They were then incubated in darkness for 10 min in H 2 O 2 (3% in double distilled water) to block endogenous peroxidase activity and then washed for 3 5 min with PBS. After this procedure, the slides were blocked with bovine serum albumin (BSA, catalogue number SA1022, Boster; www.boster.com.cn) in a humidified chamber for 20 min in order to block non-specific binding. The sections were then incubated overnight with the primary antibody in a humidified chamber at 4 C. The antibodies for PGE 2 R subtypes EP 1,EP 2 and EP 4 were rabbit polyclonal antibodies raised against synthetic peptides from the human EP 1,EP 2 and EP 4 receptors (catalogue numbers 101740, 101750 and 101770; Cayman Chemical; www.caymanchem.com). The antibody for the PGE 2 R subtype EP 3, however, was an affinity-purified rabbit antibody (catalogue number P9053-26M, United States Biological; www.usbio.com). As negative controls for PGE 2 R subtypes, the primary antibodies were replaced with PBS/BSA. The slides were washed in PBS/BSA for 3 5 min and then incubated with the secondary antibody (goat anti-rabbit, SABC-Kit, catalogue number AR1022, Boster) for 25 min at room temperature. The slides were then washed with PBS/BSA for 3 5 min before incubation with avidin biotin peroxidase complex (SABC-Kit, Boster) for 20 min at room temperature. After washing three times with PBS/BSA, freshly prepared diaminobenzidine hydrogen peroxide solution (DAB Kit, catalogue number AR1022, Boster) was added to the slides which were then rinsed with distilled water. The slides were counterstained with Erlich s haematoxylin, washed for 10 min in cold water, dried, then mounted with neutral balsam. Mouse intestine and kidney were used as the positive controls for EP 1 2 and EP 3 4, respectively.

VA R I AT I O N O F P G E 2 RECEPTOR IMMUNOREACTIVITIES 1545 MORPHOMETRIC ANALYSIS The population densities of the immunoreactive cells in the OE, olfactory nerve (ON) and olfactory bulb (OB) of the immature and mature groups were analysed. Immunoreactive cells in predetermined areas of the OE, ON and OB were counted according to the method described by Khan et al. (1998). A 5 mm 5 mm reticle, marked with 11 lines in each direction, making 121 intersections, was placed in a microscope ocular (Leica DM4500B; www.leica.com). Coronal sections through the olfactory system were examined. Immunoreactive cells under the intersections of the lines were counted; the fine focus was adjusted to examine different planes of focus so as to ensure that all the cells were counted. This semi-quantitative morphometric technique measured the number of 100 μm 3 of tissue volume selected from the front to the back of the sample. The repeatability of the technique was tested by re-measuring. The initial and repeat measurements were correlated and no significant difference was observed between the two sets of measurements. The data drawn from all the fish belonging to each reproductive phase were pooled and averaged. A one-way ANOVA was used to determine if significant differences existed among different groups (P <0 05 and <0 01). RESULTS THE ORGANIZATION OF THE OLFACTORY SYSTEM In B. sinensis, the olfactory system is composed of olfactory sac, ON and OB (Fig. 1). The olfactory sac (the rosette) is fusiform and present in the olfactory chamber. There are 10 16 primary olfactory lamellae radiating from the wall of the olfactory chamber, longitudinally arranged and parallel to each other (Fig. 2). Each olfactory lamella is composed of OE and central core. The axons of the primary olfactory receptor neurons in each rosette converge to form a single ON that exceeds 1 cm in length in a 17 cm fish. The single ON extends from the posterior ventral base of each rosette to the ipsilateral OB. The OBs, in close contact with the telencephalon, are slightly oval and sessile (Fig. 1). VA R I AT I O N O F P G E 2 R IMMUNOREACTIVITIES IN THE OLFACTORY SYSTEM WITH REPRODUCTIVE STATUS The mean ± s.d. values of I G were 0 044 ± 0 0143 and 0 164 ± 0 048 for immature males and mature males, and 7 654 ± 1 181 and 15 881 ± 6 406 for immature females and mature females. The I G of mature fish were significantly (P <0 05) higher than those of immature ones in both sexes. The EPs immunoreactivities in the olfactory system in mature B. sinensis are shown in Fig. 3. No olfactory neurons or fibres were immunostained in the negative controls for EP 1 4. The immunoreactivities for EP 1 4 were observed in the intestine or kidney tissues of mice. Variation of PGE 2 R immunoreactivities in the olfactory system with reproductive status was evident (Table I). In the OE, the density of EP 1 immunoreactivity in mature fish was significantly (P <0 01) higher than that in immature fish in both sexes. The densities of EP 2 and EP 3 immunoreactivities in mature fish were higher than those in immature fish in both sexes, but not significantly (P >0 05). The density of EP 1 immunoreactivity was significantly (P <0 05) higher than those of EP 2 and EP 3 in immature and mature fish in both sexes. No EP 4 immunoreactivity was observed. In the ON, immunoreactivity of EP 1 was observed in mature males as well as in immature and mature females, but not in immature males. The density of EP 2 immunoreactivity in

1546 X.-J. LAI AND W.-S. HONG OS ON OB T Fig. 1. Dorsal view of the olfactory system in Bostrichthys sinensis. OS, olfactory sac; ON, olfactory nerve; OB, olfactory bulb; T, telencephalon. Scale bar: 1 5 mm. mature fish was higher than that in immature fish in both sexes, but not significantly (P >0 05). No immunoreactivities of EP 3 and EP 4 were found in immature and mature fish of either sex. In the OB, immunoreactivities of EP 1 4 were encountered in all the fish tested except EP 2 immunoreactivity in immature males. EP 4 immunoreactivity was found only in the OB in the olfactory system of B. sinensis. The densities of EP 1 4 immunoreactivities in mature females were significantly (P <0 05 or 0 01) higher than those in immature females. The density of EP 4 immunoreactivity in mature males was significantly (P <0 01) higher than that in immature males. In the olfactory system, the density of EP 1 immunoreactivity in the OE was significantly (P <0 01) higher than that in the ON or OB in both sexes (Table I). DISCUSSION The results from this study revealed that the PGE 2 R subtypes EP 1,EP 2,EP 3 and EP 4 were present in the olfactory system of B. sinensis and that their immunoreactive densities varied with reproductive status. Overall, mature B. sinensis exhibited higher sensitivities to PGE 2 than immature B. sinensis. This was supported by the finding that in B. sinensis the EOG values of the OE in response to PGE 2 are higher in mature fish than in immature fish (Ma et al., 2003). The results from the present study, together with the evidence from previous investigations (Zhao et al., 2004; Hong et al., 2006), support the hypothesis that PGE 2 may act as a putative pheromone in the reproduction of B. sinensis. Variations with the reproductive status in response to putative sex pheromones are also found in some other teleosts (Irvine & Sorensen, 1993; Moore & Waring, 1995; Belanger et al., 2004). In the round goby Neogobius melanostomus (Pallas) (suborder Gobioidei), reproductive females respond with larger amplitude EOGs to water conditioned by reproductive

VA R I AT I O N O F P G E 2 RECEPTOR IMMUNOREACTIVITIES 1547 CC OL Fig. 2. Horizontal section through the olfactory sac in Bostrichthys sinensis. OL, olfactory lamella; CC, central core; ( ), olfactory epithelium. Scale bar: 200 μm. males compared with non-reproductive males (Zielinski et al., 2003; Belanger et al., 2004). The sensitivity thresholds of the male N. melanostomus in response to putative steroidal pheromone oestrone vary with their reproductive status (Belanger et al., 2007). The sensitivity of common carp Cyprinus carpio L. OE to putative pheromones is influenced by gonadal maturity (Irvine & Sorensen, 1993). A similar idea is suggested by Cardwell et al. (1995) for androgen treated South-east Asian cyprinids Puntius schwanenfeldi (Bleeker) and Puntius gonionotus (Bleeker). The crucian carp Carassius carassius (L.) and P. schwanenfeldi also vary with reproductive status in response to PGFs (Bjerselius & Olsén, 1993; Cardwell et al., 1995). Electrophysiological studies demonstrate that the sensitivities of male Atlantic salmon Salmo salar L. OE in response to PGF 1α and PGF 2α increase as the reproductive season advances (Moore & Waring, 1996). The amplitude of the olfactory sensitivity to PGFs in male brown trout Salmo trutta L. varies throughout the reproductive cycle (Moore et al., 2002). Pheromone receptors in the olfactory system bind pheromones and trigger signal transduction cascades, and the biochemical pathway followed increases the sensitivity of the olfactory system by the amplification of the signal received in the pheromone receptors (de Souza & Antunes, 2007). It is hypothesized that olfactory sensitivities can be modulated due to changes in the number of receptors and varying sensitivity of the receptors (Creese & Sibley, 1981; Habibi et al., 1989). The mechanisms controlling the changes in peripheral sensitivity to PGE 2 may involve the

1548 X.-J. LAI AND W.-S. HONG (a) (e) (i) (b) (f) (j) (c) (g) (k) (d) (h) (l) Fig. 3. Coronal sections through the olfactory system of Bostrichthys sinensis showing EPs immunoreactivities in olfactory receptor neurons ( )andfibres( ). (a), (b), (c), (d) olfactory epithelium; (e), (f), (g), (h) olfactory nerve; (i), (j), (k), (l) olfactory bulb; immunostaining for (a), (e), (i) EP 1 ; (b), (f), (j) EP 2 ; (c), (g), (k) EP 3 and (d), (h), (l) EP 4. (a) Many olfactory receptor neuron axon bundles immunostained with antibodies against EP 1 were observed at the base of the olfactory epithelium adjacent to the lamina propria. A few olfactory receptor neurons immunostained with antibodies against (b) EP 2 and (c) EP 3, but not (d) EP 4, were observed in the olfactory epithelium. A few fibres immunostained with antibodies against (e) EP 1 and (f) EP 2, but not (g) EP 3 and (h) EP 4 were observed in the olfactory nerve. In the olfactory bulb, immunoreactivities of (i) EP 1 and (l) EP 4 were intensive and dispersed. Immunoreactivity of (j) EP 2 was observed only in the fibres and immunoreactivity of (k) EP 3 was faint. Scale bar: (a) (d) 25 μm; (e) (l) 15 μm.

VA R I AT I O N O F P G E 2 RECEPTOR IMMUNOREACTIVITIES 1549 Table I. Reproductive status-related variation in the population of EPs positive olfactory receptor neurons in the olfactory system of Bostrichthys sinensis. Values are mean ± s.d. Sex and reproductive status Immature male Mature male Immature female Mature female Olfactory epithelium EP 1 106 6 ± 37 3 195 5 ± 45 0** 136 7 ± 30 2 211 5 ± 37 9** EP 2 7 5 ± 6 5 10 6 ± 9 1 8 7 ± 6 2 12 0 ± 10 6 EP 3 13 8 ± 11 1 20 8 ± 11 1 7 3 ± 6 5 11 1 ± 8 7 EP 4 0 0 0 0 Olfactory nerves EP 1 0 14 2 ± 6 2 14 2 ± 11 3 19 8 ± 14 5 EP 2 11 4 ± 11 2 12 4 ± 8 9 7 8 ± 6 4 11 2 ± 9 2 EP 3 0 0 0 0 EP 4 0 0 0 0 Olfactory bulb EP 1 10 5 ± 7 7 22 3 ± 16 8 10 4 ± 8 5 33 8 ± 23 7** EP 2 0 22 9 ± 20 4 7 6 ± 5 6 21 2 ± 15 6* EP 3 9 5 ± 8 3 8 2 ± 7 2 1 8 ± 1 7 15 7 ± 13 3** EP 4 15 7 ± 4 7 34 1 ± 21 5** 7 3 ± 7 1 21 2 ± 13 7** *Significant differences between immature and mature fish (ANOVA, n = 5, P<0 05). **Significant differences between immature and mature fish (ANOVA, n = 5, P<0 01). re-orientation of previously hidden or buried receptor sites, so that they become available to bind ligands, or conformational changes in receptor proteins in mature fishes (Moore & Waring, 1996). The finding in the present study that the densities of EP 1 3 immunoreactivities in the OE in mature fish were higher than those in immature fish in both sexes supports the hypothesis that olfactory sensitivities could be modulated due to changes in the number of receptors (Creese & Sibley, 1981; Habibi et al., 1989). The increased PGE 2 R neurons in mature B. sinensis may stimulate a sequence of neuronal events that lead reproductive individuals to display increased preference and locomotion in response to the putative pheromone PGE 2. These findings might explain the season-related variation of olfactory sensitivity in response to sex pheromones in the teleost. Teleosts use reproductive hormones both as endogenous signals to synchronize sexual behaviour with gamete maturation and as exogenous signals (pheromones) to synchronize spawning interactions between fishes (Kobayashi et al., 2002). At ovulation, oocytes stimulate the oviduct to increase synthesis of PGF 2 α,whichrises dramatically in the blood, and travels to the brain where it induces female receptivity (Stacey & Peter, 1979). PGE 2, as a pheromone, can be detected by B. sinensis OE (Ma et al., 2003). As prostaglandins are small molecules and lipid compounds, it is possible that some can enter the animal via the gills, as has been shown for steroids (Vermeirssen et al., 1996; Scott et al., 2005), and act as hormones internally. In summary, the present study indicated that the PGE 2 R subtypes were present in the olfactory system of B. sinensis and that the amounts of the receptors were linked to fish maturity. The presence of PGE 2 R in the OE suggested that PGE 2 might act as a pheromone. The presence of PGE 2 R in the ON and OB, however, suggested that it could equally act as a hormone. In the present study, the antibodies for PGE 2 R subtypes were rabbit polyclonal antibodies raised against synthetic peptides from the human PGE 2 R subtypes, and the immunostaining that represents PGE 2 receptors in B. sinensis remains to be confirmed.

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