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.

Stimulatory effects of selected PAHs on testosterone production in goldfish and rainbow trout and possible mechanisms of action.

This study investigates the effects of selected polycyclic aromatic hydrocarbons (PAHs) on testicular function in fish. In vitro incubations of goldfish testis tissue exposed to three selected PAHs [naphthalene, beta-naphthoflavone (betaNF), or retene] resulted in biphasic potentiation of gonadotropin (GtH)-stimulated testosterone (T) production, with no effect on basal levels. In vivo exposure of goldfish to betaNF also resulted in biphasic increases in plasma T levels. Experiments with rainbow trout revealed increased levels of in vitro T production when exposed to naphthalene, albeit only at higher concentrations than the responses observed in goldfish. Studies of possible sites of action at the level of the goldfish testis showed that betaNF exerted its most potent action when T production was initiated by a ligand such as GtH or prostaglandin E(2). Within the steroidogenic pathway, betaNF potentiation of GtH-stimulated T production may be partially mediated by increases in cAMP production, but appear to be independent of cytosolic calcium content. These studies demonstrate that PAHs may function as disruptors of reproductive endocrine function in teleosts through the unexpected effect of enhancing testicular steroidogenesis.

An interlaboratory study on the use of steroid hormones in examining endocrine disruption.

In recent years, there has been an increased use of the measurement of sex steroid hormone levels in the blood of animals exposed to chemicals as an indicator of reproductive impairment or an alteration in endocrine function. Although levels of hormones are often compared among animals and laboratories, there has been no study to examine the between-laboratory variability in actual steroid measurements. Therefore, we initiated a study with white sucker collected from a site receiving pulp mill effluent, previously documented as having reduced steroid levels, to address this issue. Samples of plasma and media from in vitro gonadal incubations were delivered to eight outside laboratories with the ability to measure steroid hormones. These laboratories ranged from well-established fish endocrine laboratories to wildlife toxicology laboratories, which have recently implemented the methods to measure steroid hormones. In this study, we have considered both the absolute measure of steroid content between laboratories as well as the ability to discriminate between reference and exposed populations as important criteria when evaluating the utility of these measures. Of the eight outside laboratories conducting the analyses, six detected identical site differences in circulating levels of testosterone and 17beta-estradiol to those documented by our Burlington laboratory (ON, Canada). However, the absolute value of the steroid hormones measured in the plasma varied significantly (plasma testosterone 0.6-23.1 ng/ml, 17beta-estradiol 77.6-1782.7 pg/ml) with coefficients of variation of 70.4% and 60.3% respectively. Similar results were demonstrated for the measurement of steroid hormones in media following in vitro gonadal incubation. Although there was a fair amount of variability in the absolute measure of steroid hormone levels, we would predict a far greater coherence of interlaboratory results through the sharing of reagents and the use of a common methodology between laboratories. These results are very promising, providing evidence for the inclusion of steroid hormones in monitoring endocrine disruption in wildlife species.

Apoptosis and ovarian function: novel perspectives from the teleosts.

Apoptosis is a fundamental mechanism in follicular atresia and postovulatory regression in mammals, but its role in teleost ovarian function is currently unknown. This study tested the hypotheses that apoptosis mediates follicular atresia in teleosts and is inducible in vitro by incubation in serum-free conditions. Vitellogenic follicles from rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus) were incubated overnight in serum-free medium and examined for apoptosis by 3'-end-labeling and/or TUNEL analysis. Primary, postovulatory, and oocytectomized vitellogenic trout follicles and atretic goldfish follicles were evaluated in similar fashion. Overall, goldfish follicles had lower levels of DNA fragmentation than trout follicles. The DNA fragmentation in atretic goldfish follicles was similar to that measured in healthy vitellogenic and prematurational follicles; DNA fragmentation did not change after incubation. In the trout, postovulatory and oocytectomized vitellogenic follicles showed significantly greater in vitro susceptibility to apoptosis than intact vitellogenic follicles, whereas primary follicles were least susceptible. The TUNEL analyses revealed that in trout vitellogenic follicles, more thecal/epithelial cells than granulosa cells showed fragmented DNA in vivo, but incubation (24 h) did not result in increased apoptosis in cells of either type. These results indicate that apoptosis is involved in normal ovarian growth and postovulatory regression in teleosts, but that it does not appear to be an early event in teleost follicular atresia.

An overview of recent studies on the potential of pulp-mill effluents to alter reproductive parameters in fish.

In the early 1990s, many Canadian pulp and paper mills implemented process changes to comply with new regulations that came into effect in 1993. These regulations placed stricter guidelines on a number of parameters in effluent discharges, including limits on acute toxicity, on the discharges of suspended solids, and on biochemical oxygen demand. To meet these new regulations, many of the older Canadian pulp and paper mills had to install secondary treatment systems. The investment by the Canadian pulp and paper industry was in excess of $5 billion, and the implementation of the new regulations and the process changes took several years. The new regulations were an extension of regulations designed in the early 1970s and were not designed specifically to address the reproductive responses recently reported in fish collected downstream of mills in Scandinavia and North America. This report describes a series of projects conducted between 1991 and 1996 to evaluate the effectiveness of the new regulations to address the issue of reproductive responses in fish associated with exposure to pulp-mill effluents. These studies have shown that the existing short-term bioassays do not adequately predict the potential of effluents to affect reproduction in wild fish. Laboratory testing using fathead minnows exposed over a full life cycle confirmed depression in sex steroid production, delay in sexual maturity, reduced egg production, and changes in secondary sex characteristics documented at some sites. Our studies demonstrated that both steroid hormone changes and induction of liver detoxification enzymes take place quickly. While short-term exposures can predict the potential of some effluents to impact steroid hormone production, there is no readily available assay that can be widely applied. In the absence of a usable and transferable laboratory bioassay, field collections were conducted at a number of sites. Generalizations are not possible at this time, but impacts have been seen at a variety of sites, and partial recovery has been documented at five sites in North America following various process and waste treatment changes. Data gaps and critical research areas are identified.

Health of tree swallows (Tachycineta bicolor) nesting in pesticide-sprayed apple orchards in Ontario, Canada. II. Sex and thyroid hormone concentrations and testes development.

To investigate the effects of pesticides on wild birds, sex (17beta-estradiol; testosterone) and thyroid (triiodothyronine (T3) hormone concentrations, body mass, and testes mass were measured and the development of testes was evaluated in wild tree swallows (Tachycineta bicolor) nesting in four sprayed apple orchards and three nonsprayed sites in southern Ontario, Canada, in 1995-1996. In orchards, birds were exposed to asmany as 11 individual spray events and five sprays of mixtures of chemicals. Residues of organochlorine pesticides, PCBs, lead, and arsenic concentrations were low and not variable among sites except p,p'-DDE concentrations, which ranged from 0.36 to 2.23 microg/g wet weight in eggs. These persistent compounds were not correlated with any endocrine response measured in tree swallows. In 16-d-old male tree swallow chicks, body mass and concentrations of 17beta-estradiol (estradiol), testosterone, and T3 in plasma showed no significant differences between sprayed and nonsprayed groups and among sites within those groups. However, T3 concentrations were slightly elevated in the sprayed group compared to the nonsprayed group, and there was a significant and positive correlation between T3 and the number of mixtures of sprays applied during egg incubation through chick rearing. In 16-d-old female chicks, there were no significant differences among spray treatments or sites and no correlations with spray exposure for testosterone, estradiol, or T3 in plasma. Body mass was correlated positively with T3 and negatively with estradiol but showed no differences among spray exposure groups or sites. Histology of testes of 16-d-old male chicks indicated there were no significant differences among sprayed and nonsprayed birds in testes mass, area, or diameter, or the presence of Leydig cells in the interstitium, the distribution of the Sertoli cells, or the occurrence of heterophils in the testicular interstitium. For the percentage of spermatogonia present on the basement membrane, there were significant differences among sites, but these differences were not specifically associated with spray exposure. However, there was a marginally significant trend between increasing occurrence of a disrupted Sertoli cell population on the seminiferous tubular basement membranes as the number of mixtures of pesticides sprayed during chick rearing increased. In adult male and female parent tree swallows, there were no differences in hormone concentrations between birds from sprayed and nonsprayed sites. Nor were there any significant correlations between the concentration of any hormone and collection date, body mass, or any type of spray exposure for adults. The correlations between increasing pesticide exposure and abnormal thyroid hormone and testes development in male chicks indicate that further reductions of pesticide use in orchards may benefit the health of birds that nest there. However, it is unclear which of these pesticides or spray mixtures are responsible for these effects, and this needs to be examined in future studies.

Dopamine causes ultrastructural changes in prolactin cells of tilapia (Oreochromis niloticus).

This study was undertaken to examine ultrastructural changes induced by dopamine in fish prolactin cells. Tilapia adenohypophyses were incubated with dopamine and evaluated by electron microscopy. The quantities of rough endoplasmic reticulum (RER) in prolactin cells increased and the number of secretory granules were decreased by dopamine (10(-6) mol/l) treatment. Another set of adenohypophysial tissues was placed back into control medium for 10 min following a 3 h incubation period with dopamine (10(-6) mol/l) (RE10 min group). This group had significantly less RER than the 3 h dopamine-treated tissue, and the shape of many granules in the RE10 min group changed from spherical to rod-like. In addition, some of the granule content appeared to diffuse out of granules since some were not fully surrounded by membrane. It was therefore hypothesized that the rod-shaped granules might be the result of prolactin secretion by diffusion.

Capture and confinement stress in white sucker exposed to bleached kraft pulp mill effluent.

This study evaluates the effects of handling and confinement stress and a 3-day recovery period on a number of biochemical parameters used to monitor exposure of fish to bleached kraft mill effluent (BKME). Plasma was collected at four times of the day from male and female white sucker subjected to four levels of handling stress during their spawning migration at a BKME and a reference site. Indicators of a general response to stress (plasma cortisol, glucose, lactate, and total protein) and of reproductive fitness (plasma testosterone, 11-ketotestosterone, and 17 beta-estradiol) were measured. With the exception of 17 beta-estradiol in females, all of the parameters measured varied with time of day and stress level. The general stress indicators ranged from being highly to marginally responsive to handling and confinement stress and were inconsistent in terms of indicating a site difference. The reproductive steroids were moderately responsive to stress and gave the most consistent site difference with testosterone and 11-ketotestosterone levels reduced in BKME-exposed fish under low levels of stress. There was limited evidence for recovery from capture, handling, and transport in the spring for plasma cortisol, lactate, and 17 beta-estradiol at the BKME site, plasma glucose at the reference site, and plasma testosterone at both sites. Fish were also more responsive to an additional acute stress on Day 1 and Day 3 of recovery compared to that on Day 0. This study emphasizes the need for standardized methods in field collections and sampling with the least amount of stress possible, and suggests that holding white sucker for 1 or 3 days does not allow them to recover from the stress of capture.

Effect of dominance status on sex hormone levels in laboratory and wild-spawning male trout.

We investigated the relationship between male social status and hormone levels in salmonids spawning under laboratory and field conditions. In small groups of rainbow trout (Onchorhynchus mykiss) spawning in the laboratory, dominant males had higher plasma levels of testosterone (T) and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) compared with subordinates. Steroid levels increased in subordinate males that became dominant after dominant males were experimentally removed; higher steroid levels in dominant males appears to be a result rather than a cause of their social status. In free-ranging brown trout (Salmo trutta) sampled in the field, we found higher levels of 11-ketotestosterone (11KT) but not T in dominant males. No significant differences in levels of either androgen were found between dominant and subordinate male brook trout (Salvelinus fontinalis) sampled at the same field location. Furthermore, in marked contrast with the laboratory fish, there were no significant differences in plasma 17,20 beta-P between dominant and subordinate males in either species of fish in the wild. The different findings in the laboratory and field may indicate species differences in behavioral endocrinology among brook, brown, and rainbow trout. Alternatively, the greater differential in hormonal profile of dominant and subordinate males in the laboratory may reflect the relative uniformity of the laboratory environment; this simple environment may allow competitively superior males to more completely dominate less competitive tank-mates and to exclude them from female sexual cues. In any case, these results suggest that the relationship between steroid hormones and spawning behavior in male salmonids is likely more complex than suggested by experiments conducted solely on laboratory-held rainbow trout.

The phytoestrogen beta-sitosterol alters the reproductive endocrine status of goldfish.

There is a growing awareness that chemicals in the environment may function as hormone mimics and affect endocrine function in wildlife. In this study, the effects of beta-sitosterol, a phytoestrogen present in high concentration in bleached kraft pulp mill effluent (BKME), on reproductive fitness of goldfish were investigated. Plasma reproductive hormone levels were measured in male and female goldfish on Day 4 following two intraperitoneal injections of beta-sitosterol or an oxidized sitosterol preparation. In some experiments, plasma hormone levels were also measured after fish were injected with Ovaprim, which contains a superactive analog of salmon GnRH and the dopamine receptor antagonist domperidone and leads to increased secretion of gonadotropin (GtH)-II (LH-type GtH). Plasma testosterone (T) and 11-ketotestosterone levels in males and T and 17 beta-estradiol levels in females were significantly decreased in beta-sitosterol-treated fish on Day 4 and 24 hr after an injection of Ovaprim. Plasma GtH-II levels were elevated in male fish treated with beta-sitosterol on Day 4 and further increased in response to Ovaprim, suggesting that reduced plasma steroid levels were not due to effects on pituitary function. In other studies, testes pieces from beta-sitosterol-treated goldfish produced reduced levels of T and pregnenolone in vitro both basally and in response to the GtH-II agonist human chorionic gonadotropin (hCG) when compared to the testes from control fish. Basal and hCG-stimulated pregnenolone and hCG-stimulated T were reduced in follicles from beta-sitosterol-treated fish; however, basal T production was not different from controls. These results suggest that beta-sitosterol reduces the gonadal steroid biosynthetic capacity through effects on cholesterol availability or the activity of the side chain cleavage enzyme P450SCC. These findings raise the possibility that beta-sitosterol could be a contributing factor to the reproductive dysfunction observed in fish exposed to BKME.

Impact of low-level sampling stress on interpretation of physiological responses of white sucker exposed to effluent from a bleached kraft pulp mill.

In this study the authors evaluated the effects of handling and confinement stress on a number of biochemical parameters in prespawning male white sucker collected from a bleached kraft mill effluent (BKME) exposed and a reference site. There were no effects of site or stress level on plasma cholesterol or glutamic oxalacetic transaminase activity; all other parameters were affected by either sampling stress or overnight confinement. Unstressed BKME fish reflected higher levels of plasma lactate, and lower levels of plasma testosterone, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, and cortisol. Acute handling stress or overnight confinement created a site difference in plasma glucose, protein, and 11-ketotestosterone, reversed the site effect for plasma lactate, and eliminated site differences for 17 alpha,20 beta-dihydroxy-4-pregnen-3-one. Since it is very difficult to standardize capture and handling stress in field studies impacted by industrial effluents, biochemical differences must be interpreted carefully. This study has also demonstrated for the first time that BKME exposure resulted in reduced circulating levels of the stress steroid cortisol, similar to the decreases in reproductive sex steroids previously reported.

Endocrine changes associated with spawning behavior in male kokanee salmon (Oncorhynchus nerka) and the effects of anosmia.

This study examines the relationship between social status, behavioral activity, and hormone levels through the spawning cycle in male kokanee salmon (Oncorhynchus nerka). Shortly after arrival at the spawning grounds, male kokanee undergo dramatic changes in morphological and physiological condition: there is an increase in body depth, a bright silvery appearance is replaced by an intense red coloration, and spermiation commences. Males become associated with nesting females which they defend against other males. The androgens, testosterone and 11-ketotestosterone, decline slowly over the spawning period from high levels observed in the silvery males; gonadotropin (GtH II) and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one increase as males approach final maturation. Androgen and GtH levels in males placed in pens with females over the spawning bed were higher than those in males penned without females. Among males competing for females, amounts of milt and plasma concentrations of GtH were higher in those males that were successful in gaining access to nesting females. A marked reduction in milt and plasma hormones following cauterization of the olfactory nares indicates that olfactory stimuli play a role in mediating endocrine and spermiation responses to the nesting females. Anosmic males were less vigorous and persistent in their courtship, suggesting that, either a female pheromone and/or high levels of hormone are necessary to maintain full reproductive behavior in male kokanee. The role of socially induced hormone changes in modulating reproductive behavior is discussed.

Exposure to bleached kraft pulp mill effluent disrupts the pituitary-gonadal axis of white sucker at multiple sites.

Our recent studies have demonstrated reproductive problems in white sucker (Catostomus commersoni) exposed to bleached kraft pulp mill effluent (BKME) at Jackfish Bay on Lake Superior. These fish exhibit delayed sexual maturity, reduced gonadal size, reduced secondary sexual characteristics, and circulating steroid levels depressed relative to those of reference populations. The present studies were designed to evaluate sites in the pituitary-gonadal axis of prespawning white sucker affected by BKME exposure. At the time of entry to the spawning stream, plasma levels of immunoreactive gonadotropin (GtH)-II (LH-type GtH) in male and female white sucker were 30- and 50-fold lower, respectively, than the levels in fish from a reference site. A single intraperitoneal injection of D-Arg6, Pro9N-Et sGnRH (sGnRH-A, 0.1 mg/kg) increased plasma GtH levels in male and female fish at both sites, although the magnitude of the response was greatly reduced in BKME-exposed fish. Fish at the BKME site did not ovulate in response to sGnRH-A, while 10 of 10 fish from the reference site ovulated within 6 hr. Plasma 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) levels were depressed in BKME-exposed fish and unlike fish at the reference site, failed to increase in response to sGnRH-A. Testosterone levels in both sexes and 11-ketostestosterone levels in males were elevated in fish from the reference site but were not further increased by GnRH treatment. In contrast, BKME-exposed fish exhibit a transitory increase in testosterone levels in response to the GnRH analog. In vitro incubations of ovarian follicles obtained from fish at the BKME site revealed depressed basal secretion of testosterone and 17,20 beta-P and reduced responsiveness to the GtH analog human chorionic gonadotropin and to forskolin, a direct activator of adenylate cyclase. By comparison, ovarian follicles from fish collected at BKME and reference sites produced similar levels of prostaglandin E basally and in response to a phorbol ester and calcium ionophore A23187, suggesting that BKME effects on ovarian function are selective and do not reflect a general impairment of ovarian function. BKME-exposed fish had plasma levels of testosterone glucuronide proportionately lower than those of reference fish, suggesting that there are site differences in the peripheral metabolism of steroids. These studies demonstrate that BKME exposure affects reproduction by acting at multiple sites in the pituitary-gonadal axis.

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.