Male primer endocrine responses to preovulatory female cyprinids under natural conditions in Sweden.

This study investigated two related aspects of male-female reproductive interactions in the family Cyprinidae: (1) whether ovulating female rudd Scardinius erythrophthalmus (subfamily Leuciscinae) induce endocrine and gonadal priming responses in conspecific males, a phenomenon which has been described only in species from the subfamily Cyprininae such as goldfish, Carassius auratus, crucian carp Carassius carassius and common carp, Cyprinus carpio and (2) whether the stimuli mediating these responses are species-specific. Field studies of three sympatric European cyprinids, two leuciscins (S. erythrophthalmus and white bream Blicca bjoerkna) and one cyprinin (C. carassius), were conducted on fishes captured in Sweden in the spawning season and held in net pens under natural conditions. As previously reported in C. carassius, male S. erythrophthalmus increased milt (sperm and seminal fluid) volume and plasma concentrations of the sperm maturation hormone 4-pregnen-17,20ß-diol-3-one (17,20ß-P) when they were held with female S. erythrophthalmus induced to ovulate by injection of Ovaprim (GnRH analogue plus dopamine antagonist). Male S. erythrophthalmus had larger milt volumes than male C. carassius prior to and following exposure to ovulatory conspecifics, but exhibited a smaller proportional milt increase in response to stimulation, suggesting species differences in sperm allocation at spawning. The presence of female S. erythrophthalmus and B. bjoerkna did not affect milt volumes of C. carassius under two experimental conditions: (1) ovulating S. erythrophthalmus and B. bjoerkna did not increase the milt volumes of C. carassius and (2) S. erythrophthalmus and B. bjoerkna did not interfere with the milt volume increase induced in male C. carassius by ovulating conspecifics. These results suggest that, as in C. auratus, C. carassius and C. carpio (subfamily Cyprininae), female S. erythrophthalmus (subfamily Leuciscinae) release a preovulatory pheromone that exerts priming effects on male hormones and sperm allocation. The findings also indicate that C. carassius discriminate between the reproductive odours of conspecifics and heterospecifics.

Endocrine and milt responses of male crucian carp (Carassius carassius L.) to periovulatory females under field conditions.

Laboratory studies in domesticated goldfish (Carassius auratus) show that, during the preovulatory luteinizing hormone (LH) surge, females release a complex steroidal pheromone that induces in males a rapid increase of plasma LH, which in turn increases strippable milt (sperm and seminal fluid) prior to ovulation and spawning. The objective of this study was to determine if the same phenomenon occurs in a wild congener, the crucian carp (Carassius carassius), under field conditions where fish are held in natural waters under ambient temperature and photoperiod. During the spawning season in June 2003, crucian carp were trapped in a small pond near Uppsala, Sweden, and held separately by sex in floating net pens. Addition of untreated females to male pens did not change male LH concentrations or milt volume during the 17 h sampling period. In contrast, addition of females injected with Ovaprim (to induce an LH surge and ovulation) increased male LH concentrations at all sample times (5, 9, 13, and 17h) following female addition and increased milt volumes at all but the first (5h) sample time. Similar increases in male LH and milt that also occurred when untreated females ovulated spontaneously after addition to male pens suggest it is female ovulatory condition, rather than injection of ovaprim per se, that induced male LH and milt responses. Males also increased LH and milt 9h after addition of females injected with the goldfish pheromonal steroid 4-pregnen-17,20beta-diol-3-one (17,20betaP), suggesting that similar responses to ovaprim-injected females were due, at least in part, to release of preovulatory pheromonal steroid(s). The clear and consistent effects of ovulatory females on male LH and milt, and the fact that crucian carp adapted well to confinement, ovulated spontaneously, and exhibited apparently normal spawning behavior, all suggest that this species can serve as a useful cyprinid model to study reproductive processes in natural conditions.

Putative steroidal pheromones in the round goby, Neogobius melanostomus: olfactory and behavioral responses.

To identify potential hormonal pheromones of the round goby (Neogobius melanostomus), a species recently introduced to the Great Lakes, we used electro-olfactogram (EOG) recording to examine olfactory responsiveness to more than 100 steroids and prostaglandins. Neogobius detected free and conjugated 18-, 19- and 21-carbon steroids, but did not detect prostaglandins. EOG cross-adaptation, used to determine if Neogobius can discriminate the detected compounds at the sensory level, suggested that the detected steroids act on four classes of olfactory receptor mechanisms named (according to the most potent ligand for each): estrone, 17 beta-estradiol-3 beta-glucuronide, etiocholanolone, and dehydroepiandrosterone-3-sulfate. Although none of the detected steroids induced reproductive behaviors, exposure to steroids from three of the four receptor classes (estrone, 17 beta-estradiol-3 beta-glucuronide, or etiocholanolone) increased ventilation rate in males, whereas only etiocholanolone increased ventilation rate in females. Using the ventilation increase as a behavioral bioassay of steroid detection, behavioral cross-adaptation studies in males demonstrated that steroids discriminated at the sensory level are also discriminated behaviorally. These findings suggest the round goby may use steroids as putative pheromones.

Discrimination of pheromonal cues in fish: emerging parallels with insects.

Many fish species employ hormonal products as sex pheromones, and these cues are often mixtures that are released with a temporal pattern. This behavior is strikingly similar to that of insects, as moths use precise blends of odorants as sex pheromones and are skillful at tracking them in spite of changes in odor intensity associated with aerial dispersal. New studies in both groups of animals suggest many parallels in the functional anatomy of olfactory pathways and the organization of information-coding circuits.

A steroidal pheromone and spawning stimuli act via different neuroendocrine mechanisms to increase gonadotropin and milt volume in male goldfish Carassius auratus.

In goldfish (Carassius auratus), pheromonal 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) and spawning stimuli (interaction with a sexually active female releasing prostaglandin pheromone) both increase gonadotropin-II (GtH-II) and milt volume. In the goldfish pituitary, GtH-II release is stimulated by gonadotropin-releasing hormone (GnRH) and inhibited by dopamine (DA). In this study, we investigated the possibility that 17,20beta-P and spawning stimuli act via separate neuroendocrine mechanisms by determining whether their effects on GtH-II could be selectively disrupted by injection of DA type-2 receptor (D-2) agonists (bromocryptine and LY171555) or a goldfish GnRH antagonist, [Ac-Delta3-Pro1, 4FD-Phe2, d-Trp3,6]-mGnRH (analog E). D-2 agonists blocked 17,20beta-P-induced increases in GtH-II and milt volume but did not affect spawning-induced responses. GnRH antagonist blocked 17,20beta-P-induced increases in GtH-II and milt volume, and spawning-induced GtH-II increase, but did not affect spawning-induced increase in milt volume. These results suggest that (1) pheromonal 17,20beta-P and spawning stimuli increase GtH-II increase via distinct neuroendocrine mechanisms, (2) the effect of pheromonal 17,20beta-P on increasing milt volume is GtH-II-dependent, and (3) the effect of spawning stimuli on increasing milt volume is GtH-II-independent.

Sulfated 17,20 beta-dihydroxy-4-pregnen-3-one functions as a potent and specific olfactory stimulant with pheromonal actions in the goldfish.

This study demonstrates that in addition to using the maturational steroid hormone 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) as a potent sex pheromone, the goldfish uses its sulfated metabolite 17,20 beta-dihydroxy-4-pregnen-3-one 20-sulfate (17,20 beta-P-20S). As measured by electro-olfactogram recording (EOG), the goldfish olfactory epithelium is extremely sensitive to both free and sulfated 17,20 beta-P (detection thresholds of approximately 10(-12) and 10(-11) M) but not to glucuronidated or phosphated 17,20 beta-P. Furthermore, both structure-activity and cross-adaptation studies using EOG indicated that 17,20 beta-P-20S and 17,20 beta-P are detected by different olfactory receptor sites, suggesting that these cues function as a distinguishable mixture. Finally, although the pheromonal activity of 17,20 beta-P-20S appears to be slightly less than that of 17,20 beta-P, it too stimulates gonadotropin release and sperm production in male goldfish. The precise function of 17,20 beta-P-20S remains somewhat enigmatic, however, because it does not appear to enhance the actions of 17,20 beta-P and is released by a different mechanism. In any case, our findings clearly establish that a fish olfactory system can detect particular conjugated steroidal compounds in a highly specific manner and that naturally released conjugated steroids can function as components of a pheromonal mixture.

A reduction in pituitary dopamine turnover is associated with sex pheromone-induced gonadotropin secretion in male goldfish.

In goldfish, the gonadal steroid, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P), functions as a potent preovulatory female sex pheromone which stimulates rapid elevations in serum gonadotropin (GtH) levels and subsequent increases in milt production in males. GtH secretion in goldfish is known to be regulated by the stimulatory actions of gonadotropin-releasing hormone (GnRH) and the inhibitory actions of dopamine (DA). This study specifically examined whether the 17,20 beta-P-induced elevation in male GtH is caused by pheromone-mediated changes in DA inhibition at the level of the pituitary. First, we have demonstrated that dihydroxyphenylacetic acid (DOPAC) is the primary metabolite of DA catabolism in the brain and pituitary gland of goldfish. Second, we measured changes in circulating levels of GtH and changes in pituitary content of DA and its metabolite, DOPAC, as well as possible alterations in DA turnover rate (DOPAC/DA ratio) following short-term exposure of male goldfish to water-borne 17,20 beta-P. Water-borne 17,20 beta-P consistently increased serum GtH levels in males within 20 min of exposure and maintained elevated levels for up to 120 min. Although changes in pituitary DA content were not observed during periods of high GtH release, coincident reductions in pituitary levels of DOPAC were measured within 45 min of exposure to the pheromone. More importantly, there was a significant decrease in the rate of DA turnover in the pituitary, as assessed by comparing the ratio of DOPAC to DA present, at 20, 45, and 120 min of exposure. Since the reduction of DA turnover in the pituitary is inversely correlated with periods of increased GtH release, the present results suggest that water-borne 17,20 beta-P causes an abatement of DA release to the pituitary. Based on the latency of the GtH response to water-borne 17,20 beta-P, a rapid reduction of DA turnover in the pituitary appears to be at least part of the neuroendocrine trigger for 17,20 beta-P-induced GtH release in male goldfish.

Sex pheromones selectively stimulate the medial olfactory tracts of male goldfish.

The olfactory tracts of teleost fish are comprised of medial and lateral sub-tracts which previous studies suggest convey responses to pheromones and food odors respectively. This study tested this possibility by recording electrical responses from the medial and lateral tracts of male goldfish exposed to sex pheromones and food odors. Only the medial olfactory tract responded to pheromones and both tracts responded to an L-amino acid and crude food odor. These findings verify earlier studies of peripheral olfactory sensitivity to pheromones and confirm that pheromonal information is carried within the medial tracts. They also suggest that the neural processes responsible for food recognition are more complex than previously supposed.

The olfactory system, not the terminal nerve, functions as the primary chemosensory pathway mediating responses to sex pheromones in male goldfish.

Studies of the neural mechanisms underlying responsiveness to sex pheromones in male goldfish suggest that, contrary to a currently popular hypothesis, the olfactory system (cranial nerve 1), and not the terminal nerve (cranial nerve 0), mediates chemosensory responses to pheromones. When the olfactory epithelium of male goldfish was exposed to two identified sex pheromones, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one and a mixture of prostaglandin F2 alpha and its metabolite 15-keto-prostaglandin F2 alpha, the spontaneous activity of olfactory neurons located in the medical portion of the olfactory bulb changed, while activity of terminal nerve cell bodies did not. A variety of other synthetic and natural odors also failed to alter the activity of terminal nerve cell bodies as did visual, magnetic, thermal, and auditory cues. Terminal nerve activity was, however, inhibited by tactile stimulation, suggesting that this system may have a modulatory role associated with the physical interactions that characterize goldfish spawning behavior.

Differing behavioral and endocrinological effects of two female sex pheromones on male goldfish.

Ovulatory female goldfish sequentially release at least two sex pheromones: 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta P) and a mixture of F prostaglandins (PGFs). This study sought to determine whether these pheromones have different endocrinological and behavioral actions and whether the PGF pheromone, which is released by spawning females, is responsible for increasing the gonadotropin (GtH) and milt (sperm and seminal fluid) levels of spawning males. Grouped and isolated males were exposed to combinations of these pheromones, food odor, and spawning and nonspawning females. 17,20 beta P stimulated GtH increases in both grouped and isolated males but had only minor effects on behavior; because its principal function appears to be physiological it may be considered a "primer" pheromone. In contrast, exposue to the PGFs elicited large increases in sexual behavior but increased GtH only when fish were exposed as groups; this pheromone's principal action appears to be behavioral and it should be considered a "releaser" pheromone. Although males had increased GtH and milt levels after 1 hr of spawning, males allowed to interact with nonspawning females also had elevated GtH; thus, behavioral interactions appear capable of elevating GtH in the absence of either pheromone. The existence of an independent behavioral mechanism which stimulates GtH was supported by the fact that males exposed to 17,20 beta P while spawning had GtH levels much greater than males exposed to only one of these stimuli.

Direct evidence that 17 alpha,20 beta-dihydroxy-4-pregnen-3-one functions as a goldfish primer pheromone: preovulatory release is closely associated with male endocrine responses.

This study directly tested the hypothesis that 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) is a goldfish preovulatory pheromone (pheromone released at peak levels during oocyte final maturation) which increases blood gonadotropin (GtH) and milt volume in males. During spontaneous ovulation, GtH and 17,20 beta-P in female blood and 17,20 beta-P released to the water increased dramatically 7-10 hr prior to ovulation, peaked 1-4 hr prior to ovulation, and then rapidly declined. Males held with these females, or exposed to their odors, had increased GtH levels and milt volumes at approximately the time when increased 17,20 beta-P release by ovulatory females commenced. Although these findings strongly support the hypothesis that 17,20 beta-P is a preovulatory female sex pheromone in goldfish which stimulates male GtH levels and milt production prior to spawning, the milt increases occurred earlier than predicted, suggesting either that preovulatory 17,20 beta-P release begins earlier than the data indicate or that other steroids known to have pheromonal activity are released before 17,20 beta-P.

Periovulatory female goldfish release three potential pheromones: 17 alpha,20 beta-dihydroxyprogesterone, 17 alpha,20 beta-dihydroxyprogesterone glucuronide, and 17 alpha-hydroxyprogesterone.

Our previous studies have shown that 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) produced by preovulatory female goldfish functions both as a hormone promoting oocyte final maturation and as a primer sex pheromone stimulating rapid reproductive endocrine responses in the male. In the present study, the amounts of free and glucuronated 17,20 beta-P as well as free 17 alpha-hydroxyprogesterone (17P) released to the holding water by female goldfish throughout the periovulatory period were determined. Compared to nonovulating female goldfish, ovulating goldfish released very high levels of each of these steroids. This study confirmed that 17,20 beta-P is released to the water by ovulating fish in sufficient amounts to have pheromonal activity and indicated that 17P may also function as a pheromone. Although considerable quantities of 17,20 beta-P glucuronide were also released, its physiological actions are unknown.

F prostaglandins function as potent olfactory stimulants that comprise the postovulatory female sex pheromone in goldfish.

This study establishes that ovulated female goldfish release F type prostaglandins (PGFs) to the water where they stimulate male spawning behavior and comprise the goldfish postovulatory pheromone. We first demonstrated that ovulated and prostaglandin-injected female goldfish release immunoreactive PGFs to the water. Next, using electro-olfactogram recording (EOG), we determined that waterborne prostaglandins function as potent olfactory stimulants for mature male goldfish. Prostaglandin F2 alpha (PGF2 alpha) and its metabolite 15-keto-prostaglandin F2 alpha (15K-PGF2 alpha) were the most potent prostaglandins; the former had a detection threshold of 10(-10) M and the latter a detection threshold of 10(-12) M. Studies of prostaglandin-injected fish indicated that PGF metabolites are an important component of the pheromone. Cross-adaptation experiments using the EOG demonstrated that goldfish have separate olfactory receptor sites for PGF2 alpha and 15K-PGF2 alpha that are independent from those that detect other olfactory stimulants. Finally, we established that male goldfish exposed to low concentrations of waterborne PGFs exhibit reproductive behaviors similar to those elicited by exposure to the odor of ovulated fish. Together with our recent discovery that a steroidal maturational hormone functions as a preovulatory "priming" pheromone for goldfish, these findings suggest that hormones and their metabolites may commonly serve as reproductive pheromones in fish.

Elevations in gonadotrophin concentrations and milt volumes as a result of spawning behavior in the goldfish.

In many male mammals and birds, exposure to sexual stimuli results in acute elevations of circulating luteinizing hormone (LH) and testosterone (T); a similar phenomemon has now been observed in the male goldfish (Carassius auratus). Mature males placed with either a receptive female or stimulus pairs of spawning goldfish had gonadotrophin (GtH) concentrations and expressible milt (sperm) volumes that were significantly greater than those of males kept in all-male groups. This stimulatory effect lasted from 20 min to at least 2 hr for GtH (20 degrees) and from less than or equal to 1 hr to greater than or equal to 24 hr for milt (14 degrees). When males were separated from the spawning pair by either a solid or perforated clear partition, no elevations of GtH or milt levels occurred. In contrast, these values increased in males placed in contact with a spawning pair, even when that pair contained no female, but a male induced to perform female sexual behavior by treatment with prostaglandin. These results suggest that, in goldfish, access to a spawning situation is necessary for rapid elevations in GtH and milt. Furthermore, it appears that the males must be sexually active in order for these physiological changes to occur, as males that failed to engage in courtship behavior with a spawning pair had GtH and milt values not different from isolated fish. This suggests that male sexual behavior and elevations in milt and GtH are concurrent events that share a common activation pathway in the brain. The increase in milt may be due to both neurally and hormonally mediated events that ensure milt availability for imminent spawning activity.

Endocrine changes during natural spawning in the white sucker, Catostomus commersoni. I. Gonadotropin, growth hormone, and thyroid hormones.

White suckers (Catostomus commersoni; Cypriniformes, Teleosteii) spawning in a small stream in central Alberta were captured during different stages of their spawning migrations in 1981 and 1982, blood was sampled, and the fish were examined to determine their reproductive condition. Blood samples were analyzed for gonadotropin (GtH), growth hormone (GH), triiodothyronine (T3), and thyroxine (T4) by radioimmunoassay. GtH levels in both sexes were lowest prior to the onset of spawning, increased significantly in spawning males, females in which germinal vesicle migration had begun, and ovulated females and then dropped significantly in spent fish of both sexes. GH was lowest in prespawning females, increased significantly at ovulation, and remained high in spent females. In contrast, GH levels in males were relatively constant throughout spawning. In both sexes, highest T4 levels were found in prespawning fish, and T4 decreased significantly in spent fish. Although a similar decline was seen in T3 in 1981, in 1982 there were no T3 changes associated with changes in reproductive condition. No significant diurnal variations were detected in the levels of GtH or T3; T4 levels appeared to vary on a diurnal basis in prespawning males only. Spawning activity in both sexes therefore appears to be associated with increases in GtH occurring at ovulation in females and at the initiation of spawning in males. GH levels may also be related to reproductive condition in females, but not in males. The relationship of thyroid hormone levels to reproductive condition is less clear, however, and these levels may reflect both endocrine and environmental influences on thyroid function.

Endocrine changes during natural spawning in the white sucker, Catostomus commersoni. II. Steroid hormones.

Blood samples were taken from white suckers (Catostomus commersoni) during their annual spring spawning migration and analyzed by radioimmunoassay for gonadotropin (GtH), estradiol-17 beta (E2), testosterone (T), 11-ketotestosterone (11-KT), 17 alpha-hydroxyprogesterone (17-P), 17 alpha-hydroxy-20 beta-dihydroprogesterone (17, 20-P), androstenedione (A), and cortisol. GtH, 17-P, and 17,20-P levels were low in prespawning fish of both sexes, rose to their highest levels in ovulated females and spawning males, and then fell to low levels in spent fish. In females, E2, T, and A levels were high in prespawning fish and declined significantly at ovulation, dropping to lowest levels in spent fish. In males, 11-KT, T, and A levels were highest in prespawning fish, and lowest in spent fish. Cortisol levels were highest in spermiating males and ovulated females. Plasma profiles of the sex steroids in the white sucker are very similar to those observed in the rainbow trout (Salmo gairdneri).

Prostaglandin induction of spawning behavior in Cichlasoma bimaculatum (Pisces cichlidae).

Prostaglandin (PG) stimulates female spawning behavior in goldfish and in some other teleosts in which female reproductive behaviors consist of postovulatory oviposition acts. This study examined the effects of PG on female sexual behavior in a teleost fish, Cichlasoma bimaculatum, in which female reproductive behaviors involve both preovulatory courtship and substrate cleaning behaviors, and post-ovulatory oviposition behavior. In females of established pairs, PGF2 alpha injection (5 micrograms, im) at any stage of the spawning cycle, or in the parental phase, rapidly induced substrate cleaning which soon merged into oviposition behavior (without egg release). These results support a role for PG in oviposition behavior of Cichlasoma. However, indomethacin (1 mg, ip), a PG synthesis inhibitor, did not block oviposition in ovulated females which had begun to spawn. Indomethacin may not have lowered PG levels sufficiently. Alternatively, as shown by J.J. Polder (1971, Neth. J. Zool. 21, 265), oviposition behavior may be induced or maintained by other factors associated with the spawning situation.

Clonidine inhibits female spawning behavior in ovulated and prostaglandin-treated goldfish.

Previous studies have indicated that female spawning behavior in goldfish is stimulated by prostaglandin (PG) synthesized in the reproductive tract shortly after ovulation and acting within the brain. In this study, clonidine inhibited PG-induced spawning behavior of nonovulated female goldfish in a dose-dependent manner and also inhibited spawning of ovulated goldfish. In male goldfish, clonidine also inhibited PG-induced female spawning behavior but did not affect male spawning behavior. The results provide further support for a role of endogenous PG in female spawning behavior of goldfish and are consistent with the findings [15] that clonidine antagonizes the action of PG on sexual behavior in the female guinea pig.

Effects of olfactory tract lesions on sexual and feeding behavior in the goldfish.

The paired olfactory tracts (OTs) of goldfish each have a lateral (LOT) and medial (MOT) olfactory tract, the latter with medial (mMOT) and lateral (lMOT) subdivisions. Bilateral OT section (OTX) reduced, while unilateral OT section (UNI) did not impair, male sexual behavior and feeding responses to a complex food odor; in contrast, female sexual behavior, induced in both sexes by prostaglandin treatment, was little affected by OTX. Experiments in which control fish were given UNI and treatment groups received UNI plus selective section of the remaining OT assessed the role of the OT subdivisions in the two olfactory-influenced behaviors (male sexual behavior and feeding). LOT section did not affect courtship, while MOT section reduced courtship to the low levels seen in OTX males; within the MOT, the mMOT may be more important than the lMOT for courtship expression. In contrast to male sexual behavior, feeding responses were less affected by MOT than LOT section. With respect to male sexual behavior, these findings demonstrate differential functions for the anatomically distinct subdivisions of the goldfish olfactory tracts, possibly related to their distinct terminal fields within the telencephalon.