Variation with Reproductive Status of PGE2 Receptor Immunoreactivities in the Bostrichthys Sinensis Olfactory System

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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 PGE2 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)

TheroleofPGE2 as a putative sex pheromone in Chinese black sleeper Bostrichthys sinensis was investigated, using immunocytochemistry and how the immunoreactivities of the prostaglandin E2 (PGE2) receptor subtypes EP1,EP2,EP3 and EP4 varied with reproductive status in the olfactory system was determined. The results showed that PGE2 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 EP1 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 EP2 and EP3 immunoreactivities in mature fish were higher (but not significantly) than those in immature fish of both sexes. In the olfactory nerve, the density of EP2 immunoreactivity in mature fish was higher (but not significantly) than that in immature fish in both sexes. In the olfactory bulb, the densities of EP1–4 immunoreactivities in mature females were significantly (P<0·05 or <0·01) higher than those in immature females, and the density of EP4 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 PGE2 receptor subtypes in the olfactory system of a teleost, and offers new findings regarding the role of PGE2 as sex pheromone and hormone in the reproductive behaviour and pheromonal communication of B. sinensis. © 2010 The Authors 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: [email protected] 1542 © 2010 The Authors Journal of Fish Biology © 2010 The Fisheries Society of the British Isles VA R I AT I O N O F P G E2 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 trig- gers 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 tech- niques (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 lam- prey 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 pro- cess 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 Lacep´ ede` 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

© 2010 The Authors Journal of Fish Biology © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 77, 1542–1551 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 E2 (PGE2) attracted more males and females to the artificial nests than the control, and fish exposed to PGE2 showed induced spawning (Hong et al., 2006). The PGE2 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 PGE2 are higher in mature fish than in immature ones (Ma et al., 2003). The results mentioned above suggested that PGE2 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 PGE2 receptor (PGE2R) 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 (MT) were 142–167 mm and 41·6–73·2 g for females, and 152–186 mm and 45·5–76·7g 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. = −1 = The gonado-somatic index (IG) was calculated from IG 100 MG MT ,whereMG 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 H2O2 (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 PGE2R subtypes EP1,EP2 and EP4 were rabbit polyclonal antibodies raised against synthetic peptides from the human EP1,EP2 and EP4 receptors (catalogue numbers 101740, 101750 and 101770; Cayman Chemical; www.caymanchem.com). The antibody for the PGE2R subtype EP3, however, was an affinity-purified rabbit antibody (catalogue number P9053-26M, United States Biological; www.usbio.com). As negative controls for PGE2R 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 EP1–2 and EP3–4, respectively.

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, mak- ing 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 μm3 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 ATI O N O F PG E2 R IMMUNOREACTIVITIES IN THE OLFACTORY SYSTEM WITH REPRODUCTIVE STATUS

The mean ± s.d. values of IG 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 IG 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 EP1–4. The immunoreactivities for EP1–4 were observed in the intestine or kidney tissues of mice. Variation of PGE2R immunoreactivities in the olfactory system with reproductive status was evident (Table I). In the OE, the density of EP1 immunoreactivity in mature fish was significantly (P<0·01) higher than that in immature fish in both sexes. The densities of EP2 and EP3 immunoreactivities in mature fish were higher than those in immature fish in both sexes, but not significantly (P>0·05). The density of EP1 immunoreactivity was significantly (P<0·05) higher than those of EP2 and EP3 in immature and mature fish in both sexes. No EP4 immunoreactivity was observed. In the ON, immunoreactivity of EP1 was observed in mature males as well as in immature and mature females, but not in immature males. The density of EP2 immunoreactivity in

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 EP3 and EP4 were found in immature and mature fish of either sex. In the OB, immunoreactivities of EP1–4 were encountered in all the fish tested except EP2 immunoreactivity in immature males. EP4 immunoreactivity was found only in the OB in the olfactory system of B. sinensis. The densities of EP1–4 immunoreactivities in mature females were significantly (P<0·05 or 0·01) higher than those in immature females. The density of EP4 immunoreactivity in mature males was significantly (P<0·01) higher than that in immature males. In the olfactory system, the density of EP1 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 PGE2R subtypes EP1,EP2,EP3 and EP4 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 PGE2 than immature B. sinensis. This was supported by the finding that in B. sinensis the EOG values of the OE in response to PGE2 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 PGE2 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

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 & Olsen,´ 1993; Cardwell et al., 1995). Electrophysiological studies demonstrate that the sensitivities of male Atlantic salmon Salmo salar L. OE in response to PGF1α and PGF2α 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 PGE2 may involve the

(a) (e) (i)

(b) (f) (j)

(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) EP1; (b), (f), (j) EP2; (c), (g), (k) EP3 and (d), (h), (l) EP4. (a) Many olfactory receptor neuron axon bundles immunostained with antibodies against EP1 were observed at the base of the olfactory epithelium adjacent to the lamina propria. A few olfactory receptor neurons immunostained with antibodies against (b) EP2 and (c) EP3, but not (d) EP4, were observed in the olfactory epithelium. A few fibres immunostained with antibodies against (e) EP1 and (f) EP2, but not (g) EP3 and (h) EP4 were observed in the olfactory nerve. In the olfactory bulb, immunoreactivities of (i) EP1 and (l) EP4 were intensive and dispersed. Immunoreactiv- ity of (j) EP2 was observed only in the fibres and immunoreactivity of (k) EP3 was faint. Scale bar: (a)–(d) 25 μm; (e)–(l) 15 μm.

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.

Immature Mature Immature Mature Sex and reproductive status male male female female

Olfactory epithelium EP1 106·6 ± 37·3 195·5 ± 45·0** 136·7 ± 30·2 211·5 ± 37·9** EP2 7·5 ± 6·510·6 ± 9·18·7 ± 6·212·0 ± 10·6 EP3 13·8 ± 11·120·8 ± 11·17·3 ± 6·511·1 ± 8·7 EP4 00 00 Olfactory nerves EP1 014·2 ± 6·214·2 ± 11·319·8 ± 14·5 EP2 11·4 ± 11·212·4 ± 8·97·8 ± 6·411·2 ± 9·2 EP3 00 00 EP4 00 00 Olfactory bulb EP1 10·5 ± 7·722·3 ± 16·810·4 ± 8·533·8 ± 23·7** EP2 022·9 ± 20·47·6 ± 5·621·2 ± 15·6* EP3 9·5 ± 8·38·2 ± 7·21·8 ± 1·715·7 ± 13·3** EP4 15·7 ± 4·734·1 ± 21·5** 7·3 ± 7·121·2 ± 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 EP1–3 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 PGE2R 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 PGE2. 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 PGF2α,whichrises dramatically in the blood, and travels to the brain where it induces female receptivity (Stacey & Peter, 1979). PGE2, 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 PGE2R 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 PGE2R in the OE suggested that PGE2 might act as a pheromone. The presence of PGE2R in the ON and OB, however, suggested that it could equally act as a hormone. In the present study, the antibodies for PGE2R subtypes were rabbit polyclonal antibodies raised against synthetic peptides from the human PGE2R subtypes, and the immunostaining that represents PGE2 receptors in B. sinensis remains to be confirmed.

This work was supported by the National Natural Science Foundation of China (No. 40776080) and the Natural Science Foundation of Fujian Province (No. 2007J0068). J. Hodgkiss is thanked for his help with English.

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