Fish Physiology and Biochemistry 26: 71–84, 2002. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 71 Hormonal and pheromonal control of spawning behavior in the goldfish Makito Kobayashi1,∗ , Peter W. Sorensen2,∗ & Norm E. Stacey3,∗ 1 Department of Biology, Division of Natural Sciences, International Christian University, Mitaka, Tokyo 1818585, Japan; 2 Department of Fisheries, Wildlife, and Conservation Biology, University of Minnesota, St. Paul, MN 55108 USA; 3 Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada Accepted: December 1, 2002 Abstract Species that employ sexual reproduction must synchronize gamete maturity with behavior within and between genders. Teleost fishes solve this challenge by using reproductive hormones both as endogenous signals to synchronize sexual behavior with gamete maturation, and as exogenous signals (pheromones) to synchronize spawning interactions between fish. This dual role of hormonal products is best understood in the goldfish, an external fertilizer with a promiscuous mating system. Female gonadal growth and vitellogenesis is stimulated by 17β-estradiol (E2) which also evokes release of a recrudescent pheromone. At the completion of vitellogenesis, ovarian E2 production drops and plasma testosterone increases, sensitizing the female gonadotropin II (luteinizing hormone; LH) system to environmental cues (temperature, spawning substrate, pheromones). These cues eventually trigger a LH surge that alters steroidogenesic pathways to favor the production of progestins including 17,20β-dihydroxy-4-pregnen3-one (17,20β-P). Plasma 17,20β-P stimulates oocyte maturation but is also released to the water along with sulfated 17,20β-P and androstenedione to serve as a preovulatory pheromone. This pheromone stimulates male behavior, LH release, and sperm production. At the time of ovulation, females become sexually active in response to prostaglandin F2α (PGF2α) synthesized in the oviduct. PGF2α and its metabolites are released as a postovulatory pheromone that induces male spawning behavior which further increases male LH and sperm production. Androgenic hormones are required for male behavior and LH release. Although goldfish are gonochorists, hormone treatments can induce heterotypical functions in adults. Similar findings in other fish demonstrate that a sexually bipotential brain is not restricted to hermaphroditic fishes. Introduction Spawning, the controlled release of gametes by fish, requires synchronization of behavior with gamete maturation both within and between the sexes. Not surprisingly, control of this key reproductive event is complex and involves both internal hormonal signals and external cues including pheromones (external signals that pass between member of the same species), photoperiod, temperature, and spawning substrate. Despite considerable interest in fish reproduction, the physiological control of spawning is well understood in only a few species, a frustrating situation given the enormous diversity of reproductive strategies pursued by the approximately 24,000 species of extant ∗ All authors share equal responsibility for this manuscript. fishes. Physiological control of spawning is probably best understood in the goldfish, Carassius auratus, an unremarkable member of the Family Cyprinidae (the largest family of fishes) which has been domesticated for at least a few thousand years. Because the goldfish is a suitable size for use in the laboratory where it readily matures and spawns, its reproductive endocrinology and behavior have been intensively studied (Van Der Kraak et al. 1998; Kobayashi et al. 1989a, 1997; Stacey and Sorensen 2002), often as a model for the edible carps (Sokolowska et al. 1984). Goldfish studies have confirmed basic aspects of hypothalamo-hypophyseal-gonadal function common to vertebrate reproductive systems, and illustrated the value of nonmammalian models by revealing novel features of reproductive control. Early discoveries that gonadotropin II (homologous to luteinizing hor- 72 mone [LH]; Yoshiura et al. 1997) release is inhibited by hypothalamic dopamine (Chang and Peter 1983), and that prostaglandin F2α (PGF2α) acts as a hormone to induce female sexual behavior (Stacey 1976), have fundamentally changed thinking about physiological events regulating ovulation and spawning. More recently, our understanding has been profoundly influenced by the discovery that hormonal products released by goldfish act as ‘hormonal pheromones’ that affect conspecific physiology and behavior (Dulka et al. 1987b; Sorensen et al. 1987, 1998; Stacey and Sorensen 2002). The dual and simultaneous actions of hormonal compounds as both internal and external signals obliges us to reconsider the classical notion that sex hormones synchronize reproductive processes only within individuals. Finally, because goldfish sex behaviors and LH release are sexually dimorphic and readily elicited in the laboratory, the goldfish serves as a valuable model for studying both the hormonal control of sexually dimorphic function, and the potential for heterotypical (opposite gender) function in a gonochoristic (separate gender) fish. Here, we review information on three aspects of spawning in goldfish: (1) hormonal control of behavior and gonadal maturation, (2) pheromonal control of behavior and sperm production, and (3) plasticity that exists within these reproductive systems. Space limitations preclude reference to any but the most recent and relevant information on other species. Hormonal control of spawning Gonadal maturation and endocrine events proceeding spawning Females. Because male goldfish remain in a state of spawning readiness throughout the spawning season, the timing of spawning is determined by the female’s ovulatory LH surge. The female LH surge follows an extended period of vitellogenesis during which females release a recrudescent pheromone that attracts males (Female ‘recrudescent’ pheromone, see below). The early and mid-vitellogenic period is characterized by elevated plasma 17β-estradiol (E2), whereas completion of vitellogenesis is marked by reduced E2 and increased levels of 19 carbon (‘C19’) sex steroids including testosterone (T) (Kagawa et al. 1984; Kobayashi et al. 1988). This postvitellogenic increase in T appears to prime the ovulatory LH surge system to respond to environmental cues that trigger LH release. For example, if sexually regressed, ovariectomized or vitellogenic females are implanted with T and moved into warm water, they exhibit female-typical LH surges, whereas females with blank implants, or sexually mature males with high plasma T, do not (Kobayashi et al. 1986a, 1989b; M. Kobayashi, unpublished). The positive influence of T on the LH surge in postvitellogenic female goldfish differs from the positive feedback effect of E2 on the LH surge in mammals (Kalra and Kalra 1997). Thus, whereas preovulatory E2 in mammals typically induces an LH surge after a predictable latency, preovulatory T in goldfish can remain high for months until they encounter exogenous ovulatory cues such as warm water, spawning substrate, or the odor of ovulating conspecifics (Stacey et al. 1979a, b; Sorensen and Stacey 1987). These environmental factors also appear to synchronize ovulation among females (Kobayashi et al. 1988). Although the ovulatory LH surge is facilitated by T and triggered by environmental and social cues, a circadian clock set by ambient photoperiod determines its precise timing. At typical spawning temperatures (18–20 ◦ C) on long (14–16 L) days, the surge commences late in photophase and terminates late in the following scotophase with the occurrence of ovulation (Stacey et al. 1979a, b; Kezuka et al. 1989). Goldfish therefore spawn synchronously in low light of early morning, likely a strategy to reduce predation. In addition to determining the timing of ovulation (and therefore spawning), onset of the LH surge determines the timing of release of the hormonally-derived preovulatory pheromone which is comprised of a changing mixture of steroids: androstenedione (AD), the maturation inducing steroid 17,20β-dihydroxy-4pregnen-3-one; (17,20β-P; Kobayashi et al. 1987; Stacey et al. 1989; Scott and Sorensen 1994), and 17,20β-dihydroxy-4-pregnen-3-one-20-sulfate in particular (17,20β-P-S; Sorensen et al. 1995a; see below). The goldfish LH surge appears remarkably similar to that of the common carp, Cyprinus carpio (Santos et al. 1986). Males. Prior to spawning, males increase LH if they make contact with the female preovulatory pheromone; thus, males and females that interact shortly before ovulation experience synchronous LH increase. The pheromone-induced LH increase in males stimulates testicular synthesis of 17,20βP (Kobayashi et al. 1986a, b; Dulka et al. 1987b), in turn increasing the number and motility of sperm 73 in the sperm ducts (DeFraipont and Sorensen 1993) and male fertility at spawning (Zheng et al. 1997). Male-typical LH release occurs within minutes of exposure to the preovulatory pheromone regardless of time of day (Dulka et al. 1987a, b), and appears to be regulated by plasma androgens; i.e., it occurs consistently in males but rarely in females unless they are implanted with 11-ketotestosterone (KT) (Kobayashi et al. 1997a). A similar male-typical, pheromonal 17,20β-P induced LH increase occurs in both common carp (Barry et al. 1990; Stacey et al. 1994) and crucian carp, Carassius carassius (Bjerselius et al. 1995). Endocrine control of spawning behavior Overview of goldfish spawning behavior. The goldfish mating system is typical of many other nonterritorial, non-parental cyprinids, such as the common carp, with which it will hybridize (Taylor and Mahon 1977). Females commence vitellogenesis during winter, exhibit group synchronous oocyte development, and ovulate one to several times over a protracted spring-summer spawning season (Kobayashi et al. 1986b, c). Starting several hours prior to ovulation (which occurs near dawn) small, disorganized groups of males actively ‘follow’ females, frequently initiating physical contact (‘nudging’) and inspecting their urogenital and gill regions where pheromones are released (Sorensen et al. 2000). Following behavior typically leads to active ‘chasing’, the intensity of which increases until the time of ovulation as males appear to compete to be closest to the female(s), at times ‘pushing’ each other away (DeFraipont and Sorensen 1993). Once ovulated, females become sexually receptive and initiate spawning acts by entering aquatic vegetation (Figure 1). Typically, one/few male(s) will enter vegetation with a receptive female and then swim rapidly with the female through a small arc, at which time the female will release (oviposit) eggs and the male(s) will release (ejaculate) sperm to complete a ‘spawning act’. Released eggs adhere to the vegetation. Female spawning behavior will continue until all ovulated eggs are released, and may involve a hundred or more oviposition acts over several hours with various males. In this manuscript, the female spawning act is referred to as ‘femaletypical behavior’ or ‘female sex behavior’, and chasing, nudging, and male spawning acts are referred to as ‘male-typical behaviors’ or ‘male sex behaviors’ (Figure 1). Female behavior. PGF2α is the key hormone regulating female sex behavior in goldfish (Stacey 1987; Sorensen et al. 1995b). At ovulation, oocytes stimulate the oviduct to increase synthesis of PGF2α, which rises dramatically in the blood, and travels to the brain where it induces female receptivity (Stacey and Peter 1979; Sorensen et al. 1988, 1995b, and unpublished) (Figure 2). PGF2α appears to play a critical role synchronizing female-typical behavior with ovulation. Thus, spawning behavior of ovulated females is terminated by manually stripping ovulated oocytes (Yamazaki 1965; Stacey and Liley 1974) or by injecting the prostaglandin synthesis inhibitor, indomethacin (Stacey 1976; Sorensen unpublished), and is rapidly induced by injecting nonovulated females (or males) with PGF2α (Stacey 1976, Stacey and Peter 1979) (Figure 2). In addition to functioning as a behavioral hormone in female goldfish, PGF2α and one of its major metabolites, 15-keto-prostaglandinF2α (15KPGF2α), are released to function as a potent pheromone that triggers male courtship and induces rapid endocrine and testicular responses (Sorensen et al. 1988, 1989, 1995b, unpublished; Zheng and Stacey 1997) (see Postovulatory Prostaglandin Pheromone below). It is not yet clear exactly how common this ‘hormonal pheromonal’ role for PGF2α might be amongst the fishes. However, PGF2α injection is known to induce spawning behaviors in nonovulated females of a wide variety of teleosts including other cypriniforms (lampan jawa, Puntius gonionotus; Liley and Tan 1985: loach, Misgurnus anguillicaudatus; Kitamura et al. 1994) and more highly derived species (two-spot cichlid, Cichlasoma bimaculatum; Cole and Stacey 1984: paradise fish, Macropodus opercularis; Villars and Burdick 1986: dwarf gourami, Colisa lalia; Yamamoto et al. 1997). Also, studies of the sensitivity of the olfactory epithelium to water-borne PGF2α and related compounds have found that many ostariophysan and salmonid fishes detect prostaglandins (Stacey and Sorensen 2002), suggesting widespread use of prostaglandins as both sex hormones and pheromones by freshwater fishes. Unlike the situation in tetrapods, where female sex behaviors typically are dependent on ovarian estrogen (Blaustein and Erskine 2002), female-typical behavior of goldfish appears to be independent of ovarian steroids. For example, PGF2α-induced behavior is unaffected by ovarian maturity, ovariectomy, or T or E2 replacement therapy and is exhibited by juvenile females tested prior to their first ovarian maturation 74 Figure 1. Diagrammatic representation (ethogram) of the spawning behavior of male and female goldfish. See text for details. (Kobayashi and Stacey 1993). However, in the internally fertilizing guppy (Poecilia reticulata), as in internally fertilizing tetrapods, E2 induces normal female-typical behaviors (Liley 1972). Gonadotropin-releasing hormone (GnRH) appears to play a role in spawning of female goldfish, because PGF2α-induced female spawning activity is increased by both the salmon (sGnRH) and chicken-II (cGnRHII) forms of the peptide, and decreased by a GnRH antagonist (Volkoff and Peter 1999). Unlike female goldfish, GnRH antagonist did not reduce spawning behavior of males. It is unclear which of the three neuronal GnRH populations in the goldfish brain (cranial nerve 0 or ‘terminal nerve’; TN; preoptic; mid-brain; Kim et al. 1995) might be involved with female behavior. However, because female goldfish ovulate and spawn after axonal transport of GnRH from TN to other brain areas is blocked by olfactory tract section (Stacey and Kyle 1983; Kobayashi et al. 1994; Kim et al. 2001), GnRH of TN origin may not be essential for display of female behavior. In the dwarf gourami, lesions of TN GnRH neurons affects male nest-building behavior, but does not impair spawning behavior per se (Yamamoto et al. 1997). In summary, the brain mechanism(s) underlying PGF2α-induced female-typical behavior of goldfish is functional in juveniles, appears independent of estrogen, and may be modulated by GnRH. Similar PGF-mediated mechanisms synchronizing female-typical behavior with ovulation are expected to be widespread among oviparous fish. Male behavior. As in tetrapods (Pfaff et al. 2002a), testicular androgens regulate male-typical behavior in fishes (Borg 1994). This role for androgens has been demonstrated by castration and androgen re- placement in some species (Liley and Stacey 1983; Borg 1994), but in goldfish and other species where testicular regeneration makes castration impractical, androgen effects have been examined in intact males and females (Borg 1994; Stacey and Kobayashi 1996). Non-aromatizable 11-oxygenated androgens, such as 11-ketoandrostendione and 11-ketotestosterone (KT), are more effective than testosterone in inducing maletypical behavior in intact female goldfish (Stacey and Kobayashi 1996) (Figure 2), castrated stickleback (Gasterosteus aculeatus; Borg 1994) and intact male bluegill (Lepomis macrochirus; Kindler et al. 1991). It perhaps is not surprising that male-specific 11-oxygenated androgens are more potent than T in induction of male-typical behavior, because T is produced by both testis and ovary (Borg 1994). However, treatment with exogenous T can nevertheless induce male-typical behaviors in female fish (Borg 1994; Kindler et al. 1991; Stacey and Kobayashi 1996), so it is not clear why ovulatory females with equivalent, endogenously generated concentrations of T do not exhibit male behaviors. Estrogenic endocrine disruptors in natural habitats may affect reproduction of male fishes (Kime 1998). Indeed, E2 administration or exposure suppresses male-typical sexual behaviors of goldfish (Bjerselius et al. 2001; Schoenfuss et al. 2002), guppy, Poecilia reticulata (Bayley et al. 1999), and medaka, Oryzias latipes (Oshima et al. 2002). E2 seems to directly inhibit testicular androgen synthesis (Trudeau et al. 1993) without affecting gene expression of pituitary gonadotropin subunits (M. Kobayashi unpublished). The common vertebrate paradigm of androgenregulated male-typical behaviors may not apply to all fish. For example, in the protogynous bluehead 75 Figure 2. Diagrammatic representation of the hormonal control and plasticity of sex behavior and luteinizing hormone (LH) secretion in goldfish. PGF (prostaglandin F2α); 11-KT (11-ketotestosterone); T (testosterone); 17,20β-P (water-borne 17,20β-dihydroxy-4-pregnen-3-one). (Thalassoma bifasciatum; Godwin et al. 1996) and cleaner wrasses (Labroides dimidiatus; Nakashima et al. 2000), behavioral sex change can precede gonadal sex change. Indeed, because ovariectomized bluehead wrasse will perform male spawning behavior when placed in the social conditions that normally induce female-to-male sex change (Godwin et al. 1996), gender-typical behaviors in some protogynous hermaphrodites may be influenced more by social factors than by endocrine milieu. As seems to be the case in at least some other teleosts (Liley and Kroon 1995), LH secretion in male goldfish is strongly stimulated by behavioral interaction with sexually active females (Sorensen et al. 1989; Zheng and Stacey 1997). Unlike the female preovulatory LH surge, this LH secretion in males can be induced at any time of the day (Sorensen et al. 1989; Zheng and Stacey 1997; Kobayashi et al. 1997), although the largest and most consistent responses occur in scotophase (Dulka et al. 1987a). It is not known what function this male-typical LH increase serves, or if it is androgen-dependent. In summary, studies in goldfish indicate that non-aromatizable androgens such as KT induce the potential for male-typical behaviors, which are triggered by hormonally derived pheromones. Pheromonal control of spawning Pheromones are chemical signals that transmit information between conspecifics and which do not require overt learning (Sorensen and Stacey 1999). Most fish including goldfish appear to rely heavily on pheromones, presumably because of the wealth of information that chemical cues can convey in poorly lit environments. Fish have evolved to use pheromones to mediate predator avoidance, aggregation, and reproduction. Reproductive pheromones are best understood in the goldfish which use at least three female cues and one produced by males, all four of which are comprised of mixtures of hormonal products (Figure 3). Pheromones of the goldfish’s most immediate relatives, the common and crucian carps, 76 Figure 3. Model of the goldfish hormonal pheromone system. In vitellogenic females, plasma E2 stimulates urinary release of a recrudescent pheromone that attracts males. In postvitellogenic females, exogenous cues induce an evening surge in LH that increases release of androstenedione (AD), suppressing potential male response to the small amounts of 17,20β-P released. The 17,20β-P: AD ratio soon increases, enabling the preovulatory pheromone to increase LH in males. Several hours later, 17,20β-P-S, a urinary metabolite of 17,20β-P dominates the prevoulatory pheromone mixture, enhancing its behavioral and endocrine effectiveness. Prior to ovulation, LH increase in pheromone-exposed males has increased both the quantity and quality of releasable sperm. At ovulation, eggs are released into the oviduct were they induce synthesis of prostaglandin F2α (PGF2α) which acts in the brain to stimulate female sex behavior and is cleared to the water with its principal metabolite (15K-PGF2α) to function as a postovulatory pheromone that stimulates male sex behavior, LH release and testicular responses. appear remarkably similar to those of the goldfish. Here, we discuss the cues released by females in the order of their release, then the male pheromone, neuroendocrine mediation of pheromone responses, and species-specificity. Female sex pheromones The female recrudescent pheromone. Goldfish, common, and crucian carp have no prominent sexual dimorphisms, typically live in turbid waters and spawn only a few times a year at somewhat unpredictable times and sites, factors that may have favored the evolution of several chemical cues which allow males to identify and remain close to reproductively mature females. Amongst pre-spawning, maturing goldfish, male attraction appears to be facilitated by a ‘recrudescent ’ pheromone released by vitellogenic fe- males (previously termed a ‘maturation’ pheromone; Stacey and Sorensen 2002). This cue was first described in the urine of females with high plasma E2 (Yamazaki and Watanabe 1979; Yamazaki 1990) (Figure 3). Recently, we (P.W. Sorensen, M. Kobayashi and R. Kihslinger) have confirmed this observation by showing that the odors of vitellogenic females and E2-treated ovariectomized females will induce large, equivalent increases in male physical contact/ inspection behavior. In contrast, the odors of mature males and ovariectomized, untreated females are without effect on male behavior (see Figure 4 for details). The chemical identity of the recrudescent pheromone is not known; however, as measured by extracellular electrophysiological (electroolfactogram or ‘EOG’) recording, the goldfish olfactory organ does not detect E2 or its common conjugates (Sorensen et al. 1987 and unpublished), suggesting it may be 77 Figure 4. Behavioral evidence for a recrudescent pheromone in goldfish (Sorensen et al., unpublished). Groups (N = 10) of five male goldfish were held in aerated 70 l aquaria overnight and exposed to one of five odor treatments (100 ml/min for 15 min): (1) Well water control; (2) Spermiated males; (3) Vitellogenic females; (4) Ovariectomized females that had been implanted (3 months post-surgery) for 30 days with silicone capsules containing 50 µl castor oil; (5) Ovariectomized females that had been implanted with capsules containing 200 ng of E2 dissolved in oil. Conspecific odors were holding water from pairs of fish held overnight in 2 l of well water. Male-male nudging (physical contact/inspection, a behavior typical of courtship; Poling et al. 2001) was observed for the 15 min immediately preceding (clear bars) and during (black bars) odor addition. ∗ ; P < 0.05: Wilcoxon matched pairs test. a novel steroidal metabolite. Further, because E2 induces morphological changes in the goldfish kidney (Yamazaki and Watanabe 1979) and the pheromone appears to be in the urine (Yamazaki and Watanabe 1979), it seems likely that some E2-induced specialization of the kidney is responsible for its production. The preovulatory steroid pheromone. When exposed to appropriate stimuli (spawning substrate, increased temperature, pheromones) (Stacey et al. 1979a, b, 1989; Kobayashi et al. 1987), postvitellogenic female goldfish exhibit a dramatic preovulatory LH surge that lasts about 15 h, terminating with ovulation and spawning. This surge induces rapid changes in steroid synthesis and a resumption of meiosis/oocyte maturation (Patiño et al. 2001). Plasma C19 steroids increase soon after the onset of the LH surge, decline as C21 steroids (including the oocyte maturation-inducing steroid, 17,20β-P) increase dramatically during the middle portion of the surge, and increase again when C21 steroids decline at its end when fish ovulate and spawn (Figure 3). Steroid release closely parallels Figure 5. Water-borne 17,20β-P increases the proportion of female goldfish that ovulate. Postvitellogenic female goldfish were removed from 12 ◦ C stock tanks, placed as groups of 4 into 70 l glass aquaria lacking spawning substrate in the early morning. Temperatures of incoming water were then increased to 18 ◦ C by late afternoon. At 18.00 h either 17,20β-P (50 µg; to create about 1 nM) (n = 12 groups of 4) or methanol carrier alone (100 µl) (n = 11 groups of 4) was then added to each aquarium and this treatment was repeated at 20.00 h. The next morning all fish were checked at 09.00 h to determine if they had ovulated (Sorensen and Stacey 1987; unpublished). ∗∗∗ , P < 0.001; Fisher exact test. steroid levels in the plasma, with the exception that conjugated metabolites are also released to the water (Scott and Sorensen 1994; Sorensen and Scott 1994). Of the several dozen reproductive steroids released by goldfish, only AD, 17,20β-P, and its metabolite 17,20β-P-S are detected with high specificity and sensitivity by the goldfish olfactory system (Sorensen et al. 1990, 1995a). Accordingly, we consider these steroids to be ‘primary’ components of the preovulatory pheromone and all have activity when tested alone and in combination with other steroids (see below). Interestingly, these pheromonal cues are released by different routes: the free steroids (AD and 17,20β-P) are released across the gills whereas glucuronidated and sulfated forms (17,20β-P-S) are released as controlled pulses in urine (Sorensen et al. 2000). In addition to these primary components, goldfish release at least a few other sex steroids that bind with moderate affinity to the same olfactory receptors as the primary components. We term these ’redundant’ components (Sorensen and Stacey 1999) because although these cues have some biologcial activity, it generally appears to mirror that of the primary components. As the mixture of steroids in the preovulatory pheromone changes during the female’s LH surge, the pheromone exerts different effects on the male. Initially, AD released early in the LH surge causes increases in aggressive interactions between males (Poling et al. 2001). However, as the surge progresses, 17,20β-P comes to be the dominant component within 78 about 6 h. Even brief exposure to 17,20β-P, or to steroid mixtures dominated by 17,20β-P (Stacey 1991), trigger immediate increases in male LH (Dulka et al. 1987b; Zheng and Stacey 1996, 1997; Sorensen, unpublished) as well as increased swimming and inspection behavior amongst males (DeFraipont and Sorensen 1993; Poling et al. 2001). These behavioral changes are persistent and accompanied by increased sperm production and motility by the time of ovulation/spawning, 4–8 h later (Dulka et al. 1987b; DeFraipont and Sorensen 1993). Dramatic increases in male fertility and spawning success are the final consequence (Zheng et al. 1997). The actions of water-borne 17,20β-P on LH release and sperm production have been tested in both the common carp (Stacey et al. 1994) and crucian carp (Bjerselius et al. 1995), and appear identical to those in goldfish. Possible behavioral effects and roles of steroid mixtures have not yet been examined in these species, however. Late in the female’s ovulatory LH surge, females start to release significant quantities of 17,20β-P-S in their urine. This steroid has somewhat different actions than 17,20β-P, stimulating intense bouts of chasing and following and small increases in LH (Sorensen et al. 1995a; Poling et al. 2001). Mixture composition appears important to the preovulatory signal with 17,20β-P and 17,20β-P-S synergizing each other’s behavioral activity (P. Sorensen, unpublished). Although it is now clear that the steroidal pheromone stimulates and synchronizes male reproductive behavior and physiology with that of the female, its precise composition and actions in the natural environment have yet to be fully resolved. In addition to its pheromonal effects on males, waterborne 17,20β-P appears to act as a ‘female’ pheromone to induce ovulation amongst other female goldfish, perhaps facilitatory synchrony amongst entire populations. This possibility is suggested both by the observation the female goldfish olfactory system detects sex steroids as well as males (Sorensen et al. 1987), and by a single experiment in which females experienced higher ovulation rates in the absence of spawning substrate when exposed to 17,20βP overnight (Sorensen and Stacey 1987; see Figure 5 for details). This preliminary finding is consistent with reports of ovulatory synchrony among groups of goldfish held in the laboratory (Kobayashi et al. 1988). Presumably the effects of the 17,20β-P pheromone on female LH levels are modulated by other environmental variables in a more complex manner than is the case with males but this must be studied. The postovulatory prostaglandin pheromone. At the time of ovulation, female LH titres decrease dramatically (Stacey et al. 1989), and steroid synthesis decreases dramatically (Scott and Sorensen 1994). Coincidentally, female goldfish become sexually active as a result of PGF2α produced in response to ovulated eggs in the oviducts (Stacey 1976; Stacey and Peter 1979; Sorensen and Goetz 1993; Sorensen et al. 1995b; Sorensen in preparation). Additionally, PGF2α and its metabolites are rapidly released as a postovulatory pheromone that attracts the male to the female while triggering male courtship, and activating a variety of unique (e.g., different from the processes stimulated by the 17,20β-P pheromone) physiological mechanisms that further increase milt stores (Sorensen et al. 1988, 1989; Zheng and Stacey 1996, 1997). So important is the F prostaglandin (PGF) pheromone for males that they typically fail to spawn if rendered anosmic (Stacey and Kyle 1983). The postovulatory PGF pheromone appears to be a mixture of PGF2α and 15K-PGF2α, with the latter being the primary component. These compounds are released in great quantity (>50 ng/h) by recentlyovulated goldfish and by nonovulated fish that have either been injected with PGF2α or had ovulated eggs placed into their oviducts (Sorensen et al. 1988, 1995b, unpublished). The PGF pheromone may be a specialized signal because PGFs are released exclusively in urine pulses which coincide with female spawning activity (Appelt and Sorensen 1999). EOG recording from the goldfish olfactory epithelium shows that PGF2α and 15K-PGF2α are detected with great sensitivity (nM and pM thresholds, respectively) and specificity by the male olfactory epithelium which possesses separate receptor mechanisms for these compounds (Sorensen et al. 1988). Interestingly, unlike the situation with steroids, the olfactory epithelium of male goldfish and carp is more sensitive to PGFs than is that of females (Irvine and Sorensen 1993; Sorensen and Goetz 1993), a sexual dimorphism mediated by androgen (Cardwell et al. 1995; Stacey et al. in press). EOG sensitivity and specificity in goldfish is consistent with results of behavioral assays (Sorensen et al. 1988) in which low concentrations (0.1 nM) of PGFs in aquarium water induce mature male goldfish to inspect and chase each other (Sorensen et al. 1988, 1989). No difference has yet been noted in the functions of PGF2α or 15K-PGF2α. Because PGF exposure does not induce LH release in isolated males (Sorensen et al. 1989), it is thought that the LH 79 The male sex pheromone Male goldfish direct most of their behavior towards females, but also exhibit agonistic behaviors towards males that likely are associated with sperm competition. Although mature males release little 17,20β-P or 17,20β-P-S, they release great quantities of androgenic steroids including AD (Sorensen and Scott 1994, unpublished), which increase agonistic behaviors when added to aquaria (Poling et al. 2001). AD released by males also appears to suppress milt levels in grouped males (Stacey 1991; Stacey et al. 2001; Fraser and Stacey 2002). Thus, water-borne AD appears to have several context-dependent functions; it serves (apparently by itself) as a male pheromone with both releaser and primer effects on other males, and as a component of a complex female preovulatory pheromone. Endocrine and neuroendocrine mediation of sex pheromone response Figure 6. Diagrammatic representation of sexual bipotentiality of the brain in fish (adapted from Kobayashi et al. 2000). In the rat, the undifferentiated brain sex is believed to normally be female but in the presence of androgen and estrogen during the perinatal period, the brain develops neural systems which regulate male functions and inhibit female functions (crossed out area). Teleost fish, on the other hand, appear to possess a sexually bipotential brain. When a protogynous hermaphroditic fish is in the female phase, the female portion of the brain is active and the male portion is quiescent (shaded area). At the time the individual starts to behave like a male, the male portion of the brain is activated and the female portion becomes quiescent (shaded area): External factors (age, social status, etc.) that regulate the sex change vary among species. Although gonochoristic and gynogenetic teleosts normally use only brain areas controlling homotypical behaviors during their lifetime, brain areas controlling heterotypical behaviors can be activated by hormonal treatments. increases measured in males actually result from PGFinduced socio-sexual interactions (Sorensen et al. 1989; Zheng and Stacey, 1996, 1997). Thus, unlike endocrine response to 17,20β-P, which can occur in isolated males (Fraser and Stacey 2002), the behavioral and endocrine responses exhibited to PGFs are highly dependent on the social context in which the cue is encountered. The physiological mechanisms that regulate pheromone responsiveness have been examined only in males, where they appear to be androgen-dependent secondary sex characters. For example, androgentreated female goldfish and crucian carp exhibit maletypical LH increase when exposed to pheromonal 17,20β-P (Kobayashi et al. 1997; Kobayashi and Nakanishi 1999). Masculinizing effects are seen both at the peripheral and central levels of the nervous system. Evidence for peripheral effects comes from EOG studies of several cyprinids including goldfish (Sorensen and Goetz 1993) showing that the olfactory epithelium of males is more sensitive to PGFs than is that of females, and that androgen treatment increases EOG responses to PGFs (Cardwell et al. 1995; Sorensen, unpublished; Stacey et al. in press). On the other hand, indirect evidence for central effects comes from the round goby (Neogobius melanostomus), where steroid pheromones induce sexually isomorphic EOG responses but dimorphic, androgendependent behavioral responses (Murphy et al. 2001; Murphy and Stacey 2002). Neuroendocrine mediation of pheromone action in goldfish has been examined for male-typical LH responses to pheromonal 17,20β-P and to spawning stimuli (stimuli received as a consequence of spawning). Pheromonal 17,20β-P appears to increase LH and releasable sperm through a pituitary-dependent mechanism involving tonic dopamine inhibition of the gonadotropes (Dulka et al. 1992; Zheng and Stacey 80 1997). In contrast, spawning stimuli appear to affect sperm through two mechanisms (Zheng and Stacey 1996, 1997): (1) an unidentified LH release mechanism that appears not to involve dopamine, and (2) a more rapid, pituitary-independent mechanism. Thus, injection of a GnRH antagonist does not affect the rapid increases in sperm supply to spawning stimuli, but does block LH responses to both pheromonal 17,20β-P and spawning stimuli (Zheng and Stacey 1997); however, it is not known whether tonic GnRH secretion plays an essentially permissive role, or whether 17,20β-P and spawning stimuli increase LH by increasing GnRH release. Species specificity The issue of species specificity is of particular relevance to hormonal pheromones, which are widely used by fishes yet appear to have limited chemical diversity (Stacey and Sorensen 2002). Although tests of species-specificity are few and the results varied, some behavioral studies using conspecific and heterospecific holding water (i.e., not synthetic hormonal products) of related species suggest sex pheromones can be species-specific (Sorensen and Stacey 1999). Although future studies of this important topic should target undomesticated species, the best available data are from goldfish, where the pheromonal components are commonly produced and detected by many fish (Sorensen and Stacey 1999; Stacey and Sorensen 2002). Therefore, pheromone specificity, if it exists in goldfish, is expected to reside in: (1) the use of/or discrimination of specific chemical mixtures, which may include non-hormonal components, and (2) the use of controlled pheromone release that determines the location of the signal’s active space (Sorensen et al. 2000). Future studies of specificity might be most profitable in species such as the gobies, which appear to employ more specialized signals, perhaps because of female sexual selection (Colombo et al. 1982; Murphy et al. 2001), a situation that may facilitate the evolution of species specificity (Sorensen and Stacey 1999). Sexual plasticity of spawning behavior Plasticity of behavior and LH secretion In mammals and birds, the potential for gender-typical gonadotropin secretion and behavior typically is determined by early organizational actions of steroids that induce sexual differentiation of the brain. Later, hormones may exert activational effects direcly on the adult brain (Pfaff et al. 2002b). Although gonochoristic (separate gender) teleost fish exhibit gender-typical reproductive functions and normally do not exhibit heterotypical patterns, heterotypical patterns of behavior and LH secretion can be induced in some species including the adult goldfish by hormone treatment. For example, PGF2α injection induces female spawning behavior within minutes in male goldfish and at a rate similar to that seen in females (Stacey and Kyle 1983; Figure 2) but does not inhibit performance of male-typical behaviors. Thus, PGF2α-injected males placed with both PGF2α-injected females and mature males alternate between engaging in male- and female-typical behaviors, the same pattern seen in simultaneous hermaphrodites (e.g., Serranus subligarius; Cheek et al. 2000). Similarly, androgen implants induce male-typical behaviors in mature females without reducing their capacity to exhibit PGF-induced female-typical behaviors (Stacey and Kobayashi 1996, Figure 2). Interestingly, such androgen treatments prevent neither ovarian development nor ovulatory LH surges (Stacey and Kobayashi 1996; Kobayashi et al. 1997). Finally, although female goldfish do not usually exhibit a male-typical LH surge in response to pheromonal 17,20β-P (at least outside the normal time for the preovulatory LH surge), they do if implanted with KT (Kobayashi et al. 1997, Figure 2). It is not yet known if androgen-treated females also exhibit the male-typical LH surge induced by spawning stimuli, or if hormone treatment can induce a female-typical ovulatory LH surge in males. Potential for heterotypical reproductive function also occurs in adult gynogenetic crucian carp, Carassius auratus langsdorfii, where KT treatment induces maletypical behaviors and 17,20β-P-induced LH secretion in females without inhibiting PGF2α-induced female behaviors (Kobayashi and Nakanishi 1999). Adult goldfish and gynogenetic crucian carp exhibit heterotypical reproductive functions in response to steroid and PGF2α treatments, and maintain homotypical function during these treatments. This sexual plasticity extends to the gonadal level: i.e., androgen treatment can induce development of testicular tissue within the ovary (Kobayashi et al. 1991). Such bipotentiality is comparable to that of hermaphroditic fishes (Strüssmann and Nakamura 2003). Gonochoristic teleosts other than goldfish and crucian carp also exhibit bipotentiality as adults. For instance, androgen treatment induces male-typical behavior in adult female stickleback (Gasterosteus ac- 81 uleatus; Wai and Hoar 1963), guppy (Landsman et al. 1987), and gobies (Murphy and Stacey 2002), and exposure to paper mill effluents containing androgenic chemicals causes female mosquitofish, Gambusia affinis holbrooki, to display male sex behavior (Howell et al. 1980). Adult teleosts thus appear to retain relatively more bisexual potential than mammals (Yamanouchi 1997; Pfaff et al., 2002b), where normal processes of male development typically involve loss of female-typical potential. in fish, but rather as the opportunistic expression of latent behavioral, endocrine and gonadal bisexuality in ecological situations where hermaphroditism is more adaptive than gonochorism. From this perspective, the interesting questions pertain not to adult teleost bisexuality but to the evolutionary and physiological mechanisms by which this bisexuality has been lost in other vertebrate groups. Acknowledgements Sexual bipotentiality in the fish brain Although the neuroanatomical basis for the sexual bipotentiality of teleost behavior is unclear, studies of hermaphroditic fish suggest that brain GnRH and arginine vasotocin (AVT) are involved. After sex change, the number and size of GnRH-immunoreactive neurons change in bluehead wrasse (Grober et al. 1991), ballan wrasse, Labrus berggylta (Elofsson et al. 1999), and dusky anemone fish, Amphiprion melanopus (Elofsson et al. 1997). Similar changes are seen in the number and size of AVT neurons in bluehead wrasse (Godwin et al. 2000) and marine goby, Trimma okinawae (Grober and Sunobe 1996) at the time of sex change. It is unknown, however, whether these peptidergic cell changes are the cause or result of behavioral sex change. When female goldfish were implanted with KT and induced to perform male sex behavior, no change was observed in GnRH- and AVT-immunoreactive cells (Parhar et al. 2001). The mechanisms mediating display of heterotypical behaviors are not known, but short response latencies suggest activation of preexisting mechanisms rather than development of new ones (Figure 6). In protogynous hermaphrodites, for example, display of heterotypical behavior occurs rapidly in response to social cues (1–2 h in cleaner wrasse, Nakashima et al. 2000; 1–2 days in bluehead wrasse, Godwin et al. 1996), and in the gonochoristic goldfish, KT can induce male-typical behavior in females within 3 days (Kobayashi, unpublished data) whereas PGF2α induces female behavior in males within minutes (Stacey and Kyle 1983). 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