Jennifer L. Specker

The hypothalamic-pituitary-thyroid (HPT) axis in fish and its role in fish development and reproduction.

Bony fishes represent the largest vertebrate class and are a very diverse animal group. This chapter provides a thorough review of the available scientific literature on the thyroid system in these important vertebrate animals. The molecular components of the hypothalamic-pituitary-thyroid (HPT) axis in this group correspond closely to those of mammals. The thyroid tissue in the fishes is organized as diffuse follicles, with a few exceptions, rather than as an encapsulated gland as is found in most other vertebrate species. The features of this diffuse tissue in fishes are reviewed with an emphasis on feedback relationships within the HPT axis, the molecular biology of the thyroid system in fishes, and comparisons versus the thyroid systems of other vertebrate taxa. A review of the role of thyroid hormone in fish development and reproduction is included. Available information about the HPT axis in fishes is quite detailed for some species and rather limited or absent in others. This review focuses on species that have been intensively studied for their value as laboratory models in assays to investigate disruption in normal function of the thyroid system. In addition, in vitro and in vivo assay methods for screening chemicals for their potential to interfere with the thyroid system are reviewed. It is concluded that there are currently no in vitro or in vivo assays in fish species that are sufficiently developed to warrant recommendation for use to efficiently screen chemicals for thyroid disruption. Methods are available that can be used to measure thyroid hormones, although our ability to interpret the causes and implications of potential alterations in T4 or T3 levels in fishes is nonetheless limited without further research.

Vitellogenin in tilapia (Oreochromis mossambicus): Induction of two forms by estradiol, quantification in plasma and characterization in oocyte extract

Two forms of vitellogenin were isolated by DEAE agarose ion-exchange chromatography from plasma of the tilapia, Oreochromis mossambicus. The monomers have apparent molecular masses of 200 and 130 kDa, as indicated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and a total amount of phosphorus of 1.7 and 0.1%, respectively. Antibodies specific to the two forms, designated tVTG-200 and tVTG-130, were generated in rabbits and used to develop enzyme-linked immunosorbent assays (ELISAs) and in Western blot analyses of plasma and oocyte extract. SDS-PAGE of the oocyte extract showed a major protein band at 106.6, minor bands at 26.6, 24.2, and 23.7 kDa, and very faint bands at 83.4 and 17.5 kDa. Western blots of the oocyte extract revealed that the antiserum to tVTG-200 recognized strongly the protein bands at 24.2 and 23.7 kDa, and less strongly the bands at 25.1 and 22.6 kDa, whereas the antiserum to tVTG-130 recognized mainly the protein band at 106.6 kDa. The presence of both VTGs in untreated male tilapia was detected with the ELISAs using relatively high plasma volumes. Their presence in males was confirmed by VTG-like immunoreactive materials eluting from the ion-exchange column at the same positions as tVTG-200 and tVTG-130. The concentrations of the VTGs in males were several orders of magnitude lower than in vitellogenic females. Treatment of male tilapia with estradiol-17β (E2) induced both VTGs within 24h. After 7 days, tVTG-130 reached a maximum concentration in plasma, whereas tVTG-200 continued to increase. Our findings demonstrate that the two vitellogenins are biochemically distinct, possibly differentially regulated, and made by both sexes.

Induction by β-estradiol of vitellogenin in striped bass (Morone saxatilis): Characterization and quantification in plasma and mucus

Striped bass (Morone saxatilis) were implanted with beta-estradiol to induce the production of vitellogenin, the egg yolk precursor produced by the liver. Electrophoretic analysis revealed that beta-estradiol caused marked production of a plasma protein of apparent molecular mass 170 kDa. Size exclusion chromatography suggested that the estradiol-induced protein circulated as a dimer. This protein was purified from the plasma of estradiol-treated fish by DEAE-agarose column chromatography and used to induce antibodies in rabbits and goats. Western blots revealed that the antiserum bound to the putative vitellogenin in plasma from estradiol-treated fish and adult females, but not with any proteins in male plasma. Western blot of ovarian extract revealed several smaller immunoreactive protein bands and supported the identity of the purified protein as vitellogenin. A competitive ELISA was developed with sensitivity in a range from 8 to 1000 ng/ml. Plasma concentrations of adult females during their spawning migration ranged from 100 to 600 micrograms/ml. Western blot of mucus extract revealed the presence of a 170-kDa protein in vitellogenic female fish along with several minor bands ranging from 50 to 110 kDa. Positive immunoreactivity was present in the surface mucus of all females and in none of the males collected during a spawning migration in the Hudson River.

Effects of photoperiod on growth and smolting in juvenile Atlantic salmon (Salmo salar)

Abstract Juvenile Atlantic salmon were reared under simulated natural photoperiod (LDN) from first feeding in May until late August. Three 16-h daylength regimes were started in August or September and continued until November or December (LD16:8 A-N; LD16:8 S-N; LD16:8 S-D). A control group remained on LDN. The fish reared under LD16:8 regimes became significantly larger soon after the start of photoperiod manipulation; these differences increased during autumn and persisted until completion of smolting in May. The LD16:8 A-N regime had a significantly higher proportion of fish in the upper-modal group than all other treatment groups. Based on length > 13.5 cm, all LD16:8 regimes produced significantly more smolt-sized fish than LDN. Thyroid hormone (T3, T4) levels rose in October-November in all treatment groups and declined in December-January. T3 and T4 levels rose slightly in February. T4, but not T3, continued to rise in March. Pituitary somatotrop and thyrotrop activity (mitotic figures and granulation) was observed in all groups in September-October, with most activity in LD16:8 groups. There was within-group variation in somatotrop activity, possibly in relation to faster and slower growing fish. All groups had transitory, high levels of salinity tolerance in early December. Although gill Na+K+-ATPase activity and salinity tolerance rose earlier and to a greater extent in LDN than in LD16:8 treatments, all groups appeared to reach smolt status in May as judged by high survival and growth rates during 6 months in seawater. The LD16:8 S-D group appeared to complete smolting later than the LD16:8 A-N and S-N groups and LDN. Thus, the application of LD16:8 daily light cycles to salmon during their first autumn increases the proportion of fish in the uppermodal group (presumptive smolts) and has only minor effects on the timing of completion of smolting the following spring.

Changes in gonadal hormones during oocyte development in the striped bass,Morone saxatilis

Wild striped bass,Morone saxatilis, were collected from coastal waters and spawning areas to describe the endocrine correlates of oocyte development in non-captive, migratory fish. The fish were classified according to their most advanced oocytes. Serum levels of estradiol (E2), testosterone (T) and 17α-20β-dihydroxyprogesterone (DHP) were measured by radioimmunoassay (RIA). Females in the primary growth phase and early secondary growth phase (pre-vitellogenic) had low levels of plasma steroids, ovarian lipid content and gonadosomatic indices (GSIs). Significant increases in E2, T, ovarian lipid content and GSIs occurred during the vitellogenic phase. Maximum levels of all reproductive parameters were found in prespawning fish sampled in the Hudson River. Mean levels of E2, T, ovarian lipids and GSIs for these fish were 2.0±0.5 ng/ml, 3.0±0.3 ng/ml, 24±1 mg/g, and 5.6±0.3% (mean±SEM), respectively. In fish induced to spawn with human chorionic gonadotropin (HCG), DHP levels (1.9±0.4 ng/ml) were significantly elevated. Similar levels were found in two fish captured during the spawning season, suggesting that DHP may serve as the maturation-inducing steroid in this species.

Metamorphosis in the summer flounder, Paralichthys dentatus: thyroidal status influences salinity tolerance.

Metamorphosis in the summer flounder (Paralichthys dentatus) is controlled by thyroid hormones (TH) and takes place as the larvae move from a salinity of about 35 parts per thousand (ppt) in the ocean to salinity ranging from 0-35 ppt in estuaries. Historically, the role of TH in juvenile and adult teleost osmoregulation has been ambiguous, and it is not known if TH influences larval teleost osmoregulatory development. This study addresses the influence of thyroxine (T4) on the development of tolerance to low (5 ppt) and high salinity (45 and 50 ppt) as determined by salinity tolerance tests. In untreated larvae, tolerance to both low and high salinity was high during early premetamorphosis (early pre-M) and decreased or was very low from late prometamorphosis (late pro-M) through mid-metamorphic climax (mid-MC). Salinity tolerance increased 2-3-fold during late MC when whole-animal T4 levels are highest, and reached maximum tolerance at the juvenile stage. The early induction of metamorphosis by exposing larvae in pre-M to exogenous T4 reduced tolerance to low salinity during early and mid-MC, though tolerance of fish that had developed into juveniles was not impaired. In contrast, T4 increased high salinity tolerance during early and mid-MC, and the juvenile stage. This T4-induced heterochrony in salinity tolerance with regards to developmental stage suggests that the effects of T4 on salinity tolerance may be uncoupled from accelerated metamorphosis. Treatment of larvae with thiourea (TU, an inhibitor of T4 synthesis) inhibited metamorphosis and reduced tolerance to high salinity, but did not affect tolerance to low salinity. Reduced tolerance to high salinity by TU was only partially counteracted by T4 treatment, suggesting that TU also affects hypoosmoregulatory activity by an extrathyroidal mechanism. Our findings suggest that in the summer flounder T4 plays a more important role in the development of hypoosmoregulatory ability than hyperosmoregulatory ability. J. Exp. Zool. 284:414-424, 1999.

Methodology for implanting cortisol in Atlantic salmon and effects of chronically elevated cortisol on osmoregulatory physiology

Abstract A method for achieving high physiological concentrations of cortisol in the plasma of Atlantic salmon (Salmo salar) is outlined. Cortisol was emulsified in vegetable shortening:vegetable oil (1 : 1). This mixture is fluid at 24°C and can be injected into the peritoneum where it solidifies into a slow-releasing cortisol implant. Three doses of cortisol implants were used: 25, 50 and 100 μg/g body weight. Salmon in the post-smolt stage were serially sampled in short-term (1 week) and long-term (1 month) experiments. There was significant variability in the vehicle-implanted control fish over time in both experiments. Except for day of the implant, there were no differences between the implanted controls and the non-implanted controls, indicating that the implant itself was probably not stressful. The medium dose (50 μg/g body weight) was found to elevate plasma cortisol within 1 day and to maintain plasma cortisol above vehicle-implanted salmon for as long as 1 month. The low dose was ineffective after 1 week and the high dose resulted in plasma cortisol concentrations above physiological levels. Exogenous cortisol increased the fluid uptake rate of the non-everted posterior intestinal sac after 1 month and increased the gill Na + K + - ATPase activity after 1 week. These implants might be useful in allowing culturists to move salmon to sea cages at times outside the typical smolt window.

In vitro effects of homologous prolactins on testosterone production by testes of tilapia (Oreochromis mossambicus)

The tilapia, Oreochromis mossambicus, produces two prolactins designated tPRL177 and tPRL188 to indicate the number of amino acid residues in each. The direct in vitro actions of tPRL177 and tPRL188 on basal and ovine luteinizing hormone (LH)-induced testosterone production in minced testes of courting and noncourting (bachelor) tilapia were examined. Courting males were housed with females and were used as soon as they built a spawning pit; noncourting males were housed with other males and were used when they appeared female-like in coloration. The in vitro culture system consisted of two phases. In phase 1 (0-10 hr), testicular tissue was incubated without (control) or with the tPRLs--both alone and together and in doses ranging from 0.5 to 32 microM. In phase 2 (10-16 hr), the culture medium was replaced with medium containing 6.25 microM (0.25 micrograms/ml) ovine LH. Testosterone production during both phases was determined by radioimmunoassay of the medium. The tPRLs stimulated testosterone production during phase 1 in courting males (by 25%) but not in noncourting males. Exposure to either or both tPRLs in phase 1 enhanced the response of testicular tissue from courting males to ovine LH (by 10%) and inhibited the response of minced testes taken from noncourting males (by 12%). This is the first evidence to demonstrate direct gonadal activity of homologous PRL in a teleost.

Asynchrony of changes in tissue and plasma thyroid hormones during the parr-smolt transformation of coho salmon

The relationship between plasma thyroid hormone concentrations and the thyroid hormone concentrations in selected tissues was examined throughout the spring during the typical course of parr-smolt transformation in coho salmon (Oncorhynchus kisutch) in fresh water and also in coho salmon moved prematurely to seawater. The thyroid hormones thyroxine (T4) and triiodothyronine (T3) were extracted from brain, liver, and muscle tissue. The T4 and T3 concentrations in the extracts and plasma were measured by radioimmunoassay. The peak in plasma T4 occurred in late April; however, the concentration of T4 in the brain and liver increased before levels of T4 in plasma increased. During the rise in plasma T4, the T4 content in muscle decreased. Plasma T3 concentrations were unchanged in March and April, but decreased in May. Transfer to seawater eliminated the late April peak in plasma T4 levels, indicating suppressed thyroid activity; however, the tissues of salmon in seawater contained more T3 than tissues of salmon in fresh water at this time. These findings indicate complex peripheral regulation of thyroidal status in this teleost and represent the first bridge between compartmental models of thyroid hormone kinetics and actual measurement of tissue pools of thyroid hormones in an ectothermic vertebrate. In summary, tissue concentrations of thyroid hormones did not echo plasma concentrations, indicating that thyroidal status cannot be inferred from plasma data alone.

Metamorphosis in summer flounder: manipulation of thyroid status to synchronize settling behavior, growth, and development

In the aquaculture of summer flounder (Paralichthys dentatus), the inherent variation in growth and settling behavior during metamorphosis may lead to cannibalism and necessitate increased labor due to grading. Our goal was to use thyroid hormone manipulation to synchronize settling behavior and produce a uniformly sized cohort of juvenile summer flounder. Premetamorphic flounder were treated with exogenous thiourea (TU, 30 ppt, an inhibitor of thyroxine (T4) synthesis). Following removal of TU, metamorphosing flounder were exposed to either exogenous T4 (T4-Na salt, 100 ppb, dissolved in DMSO) or DMSO only. A control (no TU, then DMSO only, when appropriate) was used for comparison. Settling behavior in the control was prolonged, with the Peak Settlement Interval (defined as the interval beginning on the day the first 20% settled until the day 80% had settled) requiring 9 days, and a settling rate of 4.1±0.2 fish/day. Settling behavior was synchronized by thyroid hormone treatment. The Peak Settlement Interval was reduced (5 days) and settling rate increased (8.5±0.6 fish/day) by TU treatment. Treatment with TU followed by T4 further reduced the Peak Settlement Interval (3 days) and increased settling rate (11.2±0.6 fish/day). Development rate (DR) and growth rate (GR) were reduced by TU treatment, but following TU with T4 treatment restored DR and GR to control levels. However, TU treatment eliminated the larger, potentially cannibalistic juveniles found in control tanks. Both treatments reduced variance in GR, whereas only TU-T4 reduced variance in DR. Percent survival was unaffected by treatment. In aquaculture, the effective synchronization of settling behavior by thyroid hormone manipulations should reduce the labor costs associated with grading. We recommend a treatment regime that could aid in the production of more uniform cohorts of juvenile summer flounder.