Prolactin--thyroid interaction in Fundulus heteroclitus

Injections of adult Fundulus heteroclitus with 0.2 IU of oTSH over 4 days caused an increase in serum T4, with no effect on serum T3. Administration of 12 IU of oPRL in conjunction with the same dosage of TSH prevented the TSH-induced rise in T4, without affecting serum T3 levels. The failure of TSH to cause T4 levels to rise in the presence of PRL may result from either inhibition of release or acceleration of metabolic clearance of T4. We therefore conducted three experiments to examine potential effects of PRL on the kinetics of peripheral clearance of thyroid hormones. 125I T4 was cleared from serum in a biphasic pattern that was unaltered by PRL. Clearance of labeled T3 followed a similar pattern that was also not influenced by PRL treatment. Generation of labeled T3 by deiodination of a dose of 125I T4 was quantified over a 24-h period. Again, PRL-treated fish showed no significant differences. Since PRL is without effects on thyroid hormone clearance patterns or deiodination rate, we conclude that PRL prevents TSH-induced increases in serum T4 in this species by directly affecting thyroid function.     

Growth hormone is thyrotropic in Fundulus heteroclitus.

Ovine growth hormone elevates serum T₄ in both intact and hypophysectomized Fundulus heteroclitus. This activity appears to be an intrinsic property of oGH and is not due to contamination by either oTSH or other glycoprotein hormones. Moreover, while hypophysectomy prevents the oTSH-induced increase in serum T₄ found in intact fish, simultaneous treatment with oGH fully restores the normal response. Unlike oTSH, oGH lacks the ability to elevate thyroidal radioiodine uptake in hypophysectomized Fundulus. Thus, oGH appears to act at a level beyond the iodine trap stimulating thyroid function.     

Functional specificity of gonadotropin and thyrotropin in Fundulus heteroclitus

Male rough-skinned newts (Taricha granulosa) were used to investigate the hormonal responses associated with stress-induced inhibition of reproduction. When male newts were confined for 1 hr, using a procedure that previously had elicited physiological stress responses, androgen concentrations decreased in the plasma and immunoreactive (ir) LH-RH concentrations increased in the infundibulum and rostral hypothalamus. Likewise, when male newts were injected with 25 micrograms of corticosterone, androgen concentrations decreased and hypothalamic irLH-RH concentrations increased. These data, which are from experiments in February, support the hypothesis that in this amphibian, exposure to acute stress or to exogenous corticosterone can suppress plasma androgen titers by inhibiting the release of LH-RH from the hypothalamus. The effects of the confinement procedure and the injection of corticosterone on the concentrations of irLH-RH and androgens were different for newts in September than for newts in February.     

Clonal hybrids of the common laboratory fish Fundulus heteroclitus.

All-female hybrids of the killifishes Fundulus heteroclitus and Fundulus diaphanus, known from two sites in Nova Scotia, Canada, are shown to reproduce clonally. Isozyme analysis of crosses between female hybrids and male F. heteroclitus reveals that their progeny are genetically identical and show no evidence of recombination or paternal inheritance. Flow cytometric measurement of DNA content shows the hybrids to be diploid, with DNA values intermediate to those of the parental species. Because they are related to F. heteroclitus, a fish used widely as a model organism in experimental biology, the clonal hybrids are potentially valuable for experimental studies requiring subjects with a constant genetic background. In addition, the discovery of unisexuality and cloning in a fish whose reproductive physiology and development are so well characterized provides a unique opportunity to examine the underlying causes of clonal reproduction in vertebrates.     

Pigment particle translocation in detergent-permeabilized melanophores of Fundulus heteroclitus.

Melanophore preparations of Fundulus heteroclitus that have been treated with the detergent Brij 58 can aggregate their pigment in response to epinephrine. On the basis of several criteria, it appears that cell lysis occurs under the detergent conditions used. Electron microscopic examination of detergent-treated cells shows progressive disruption of the melanophore plasma membrane during the time in which pigment aggregation occurs. Brij-treated cells are accessible to ferritin, a large electron-dense probe that is effectively excluded from non-detergent-treated controls. In cells incubated with detergent, fixed, and treated first with rat monoclonal antibodies against tubulin and then with fluorescein-isothiocyanate-labeled goat anti-rat IgG, a characteristic radical pattern of microtubule staining can be visualized by fluorescence microscopy. Control preparations treated similarly, but without detergent, do not stain. Vanadate, an inhibitor of ciliary and flagellar dynein ATPase, blocks melanosome aggregation in response to epinephrine in detergent-treated preparations but has no effect on intact melanophores. erythro-9-[3-(2-hydroxynonyl)]Adenine, another inhibitor of dynein ATPase, also inhibits pigment aggregation in Fundulus melanophores. The possibility that a dynein-like molecule plays a role in pigment aggregation is discussed.     

The liver of Fundulus heteroclitus expresses deiodinase type 1 mRNA

The presence of a type 1 deiodinase (D1) in the liver of teleosts has been a controversial issue. Recently we characterized the deiodinase activity in rainbow trout and killifish liver and found that the liver of both species co-expresses the two enzymes (D1 and D2) that catalyze the outer ring-deiodinating pathway. We here report the cloning and characterization of an mRNA from the liver of the killifish Fundulus heteroclitus that encodes a D1 (FhD1). The cDNA amplified by RT-PCR from F. heteroclitus liver is 1314 nt long and encodes a protein of 248 aa. It contains a TGA codon in its open reading frame and a selenocysteine insertion sequence in its 3(') untranslated region, consistent with the structure of a selenoenzyme mRNA. The deduced peptide sequence is 73% identical to that encoded by the tilapia D1 cDNA cloned from kidney and 46% identical to the D1s reported in other vertebrates. Northern blot analysis shows that FhD1 mRNA is expressed in F. heteroclitus liver, consistent with prior biochemical evidence for hepatic D1 activity. Furthermore, heterologous expression of the FhD1 cDNA resulted in a protein with properties similar to the D1-like activity in F. heteroclitus liver. The cloned enzyme, like the native species, is relatively insensitive to inhibition by PTU, but mutation of Ser-159 in FhD1 to the Pro residue found in D2 and D3 isoforms increased the sensitivity to PTU. Our results show that, under basal conditions, killifish liver indeed expresses a D1 enzyme that is homologous to mammalian D1s, establishing this as a useful model in which to study the regulation of D1 and D2 concurrently.     

Photoperiod-dependent negative feedback effects of thyroid hormones in Fundulus heteroclitus

In Fundulus heteroclitus, an annual cycle in the response of the thyroid to ovine thyroid-stimulating hormone (oTSH) is characterized by maximal thyroxin (T4) secretion in mid-winter and minimal T4 secretion in summer. Four daily injections of oTSH, given in winter caused serum T4 to plateau at elevated levels for several days, while in summer fish similar treatment resulted in far more fluctuating titers of serum T4; maximum levels were similar in both groups. The difference in sustenance rather than magnitude of Peak T4 led to an examination of the negative feedback effects of thyroid hormones as they might relate to these seasonal changes. Radioiodine uptake by thyroid follicles served as a simple, but effective bioassay for endogenous TSH. Fish collected in summer were more sensitive to negative feedback of T3 than those collected in winter; feedback effects of T4 in the two groups were not significantly different. The effects of specific photoperiods on negative feedback sensitivity to T3 and T4 were also tested. Exposure of winter fish for one month to long days (LD 14:10) enhanced the degree of reduction of iodine uptake caused by T4 in the aquarium water (10 micrograms/100 ml). Negative feedback in short-day (LD 8:16) winter fish was not demonstrated. It is concluded that long days increase and short days diminish the negative feedback sensitivity of the hypothalamus-pituitary axis to thyroid hormones in F. heteroclitus. Such photoperiodically induced changes may act to aid in the year-round maintenance of T4 levels necessary for seasonal adaptation and survival.     

Cloning and characterization of estrogen receptor alpha in mummichog,Fundulus heteroclitus

Developmental exposure to 17 beta-estradiol (E(2)) induced the death of embryos and fry, malformations, sex reversal, and incomplete ossification of vertebrae and cranial bones in the cyprinodont fish, the mummichog (Fundulus heteroclitus). To clarify the mechanism by which exogenous estrogens caused these developmental effects, we determined the sequence of an estrogen receptor (ER) coding region, encoded by 620 amino acid residues. This region shared 80% identity to that of ER alpha of medaka (Oryzias latipes). Northern blot analysis showed that two ER alpha mRNAs with 5.5 and 4 kb were expressed in the liver. These mRNAs were strongly induced by E(2) stimulation. The 4 kb mRNA was expressed 8 h after treatment, whereas the 5.5 kb mRNA was not induced until 12 h after E(2) stimulation. Vitellogenin (VTG) was expressed 8 h after E(2) stimulation in the male liver. Receptor binding assays using the protein of F. heteroclitus ER alpha (fh ER alpha) ligand binding domain showed that alkylphenols bind to fh ER alpha with a higher affinity (50 times or more) as compared with the human ER alpha. The present results demonstrate that the fh ER alpha has a sequence very similar to that of medaka, and the mRNA for this receptor was induced by E(2)-stimulation, followed subsequently by VTG expression. Furthermore, alkylphenols bind to fh ER alpha more efficiently than to human ER alpha.     

Oogenesis in Fundulus heteroclitus II. The transition from vitellogenesis into maturation

Female Fundulus heteroclitus collected in the vicinity of Woods Hole have asynchronous ovaries which continuously ovulate eggs during the breeding season (early May until early July). At this time, vitellogenic oocytes become competent to respond to exogenous deoxycorticosterone (DOC) by undergoing maturation when follicles reach a diameter of 1.3 mm. Follicular oocytes with a diameter of 1.35 ± 0.05 mm can also give a 100% response to exogenous gonadotropins under appropriate conditions in vitro, indicating that endogenous gonadotropin(s) most likely acts directly on the ovary in situ by promoting the synthesis of maturation-inducing steroid. A response equivalent to one female pituitary is provided by 0.49 μg ovine luteinizing hormone, 6.7 μg human chorionic gonadotropin (hCG), and 31 μg ovine follicle-stimulating hormone. The response to gonadotropins is slower than that achieved by exogenous DOC alone and is potentiated (23–24 times) by the addition of physiological levels (100 ng/ml) of cortisol to the incubation medium. The available cytological evidence suggests that the granulosa cells rather than “special cells” in the theca are the progenitors of maturation-producing steroid. Once follicular oocytes reach a diameter greater than 1.4 mm diameter in situ, they become irreversibly committed to complete the process of maturation and ovulation. Cessation of feeding eliminates the competence of 1.3-mm-diameter follicles to respond to exogenous DOC and also the recruitment of 1.4-mm-diameter follicles into maturational stages, but hCG injection or refeeding restores both processes. Two hormonal mechanisms which can regulate recruitment of vitellogenic follicles into maturation have thus been identified (gonadotropin and/or cortisol production), but the sensitivity of these mechanisms to the nutritional state of the animal has not yet been elucidated.     

Serum hormone levels associated with spawning activity in the mummichog, Fundulus heteroclitus

Daily collections of the mummichog, Fundulus heteroclitus, from field populations during the first 45 days of the breeding season revealed a semilunar cycle in the sperm index. Peaks in the sperm index were preceded by 6 days with peaks in the serum testosterone concentration. Bihourly sampling of field populations during a 72-hr period at the new moon showed both diel cycling and an upward trend in serum progesterone levels in female mummichogs. Male mummichogs had 12-hr cycles in serum 17 beta-hydroxy-4-androstene-3,11-dione levels, the peaks of which preceded high tide by 4 hr. The physiological significance of these cycles are discussed.     

Pituitary regulation of serum calcium levels in the killifish, Fundulus heteroclitus L

Hypophysectomy produced hypocalcemia in male killifish, Fundulus heteroclitus, adapted to calcium-deficient sea water. Such changes in serum calcium levels were corrected by intraperitoneal injections of a homogenate of whole pituitary glands or by transplantation of the gland to the musculature of the caudal peduncle. Injections of homogenates of the anterior or posterior parts of the pituitary gland were also effective while the homogenate of the middle part of the gland produced a smaller change. Injections with prolactin at the doses of 5, 2.5, and 0.5 μg per gram body weight per injection significantly increased the serum calcium levels of the hypocalcemic hypophysectomized fish. Cortisol (2.5 μg per gram body weight per injection) and ACTH (0.05 IU per gram body weight) were also hypercalcemic, but lower doses of ACTH (0.02 and 0.005 IU per gram body weight) and two doses of MSH (2.5 and 5 μg per gram body weight) were without effect. It is suggested that either ACTH, prolactin or both may have hypercalcemic effects on fish in calcium-deficient environments.     

Serum androgens during the annual reproductive cycle of the male mummichog, Fundulus heteroclitus

A technique for the simultaneous detection of serum concentrations of testosterone (T), 17 beta-hydroxy-4-androstene-3,11-dione (11-KT), and 11 beta, 17 beta-dihydroxy-4-androsten-3-one (11 beta-HT) was established using a combination of high performance liquid chromatography separation and radioimmunoassay. In the mummichog, Fundulus heteroclitus, serum concentrations of T, 11-KT, and 11 beta-HT varied from 11.3, 10.1, and 27.9 ng/ml, respectively, during the breeding season to 0.2, 0.3, and 0.9 ng/ml, respectively, during the nonbreeding season. The changes in serum androgen levels, determined weekly, did not coincide with changes in the gonosomatic index or measurement of sperm/gram testes. Increases in serum androgen levels did coincide with the rise in sperm index, but the decreases in serum concentrations of androgens preceded the fall in sperm index by more than a month. A model is proposed to explain the reproductive cycle of male mummichogs.     

Effects of sex steroids on plasma calcium levels in male killifish,Fundulus heteroclitus

Injection of 0.5 or 2 μg of estradiol benzoate/g of body wt produced hypercalcemia in male killifish while injection of the same doses of testosterone proprionate had no effect on plasma calcium levels. Injection of these sex steroids had no consistent effect on hepatosomatic index, gonadosomatic index, percentage hematocrit, or water, sodium, or potassium content of muscle.     

Cloning of the gene and complete cDNA encoding a type 2 deiodinase from Fundulus heteroclitus

Recently, we reported the cloning of a cDNA fragment from Fundulus heteroclitus liver encoding the open reading frame of type 2 deiodinase (FhD2). We here report the cloning of 14 kb of genomic sequence from F. heteroclitus that includes the previously reported coding region of the F. heteroclitus Dio2 gene (FhDio2), the 5(') and 3(') untranslated regions, and flanking regions and introns. This FhDio2 gene comprises two exons divided by a 4.8-kb intron. The position of the intron is similar to that of introns in other Dio2 genes. The analysis of approximately 1.3 kb of genomic sequence upstream of the mRNA start site revealed that, in contrast to mammalian Dio2 genes, there were no apparent TATA or CRE sequences. Nevertheless, a putative Sp1 site was found, similar to that in other F. heteroclitus TATA-less promoters. We have also cloned the complete FhD2 cDNA, which spans 4652 bp and contains a sequence adjacent to its poly(A) tail that is highly similar to the selenocysteine insertion sequence (SECIS) found in human D2 cDNA. The expression of a construct containing the FhD2 ORF plus the native SECIS resulted in a protein with deiodinase activity similar to that of the native FhD2. Analysis of the regulation of this gene, combined with ongoing studies of the F. heteroclitus D1 gene, will allow us to elucidate the functions of the colocalized deiodinases in teleost liver.     

Concordant mitochondrial and nuclear DNA phylogenies for populations of the teleost fish Fundulus heteroclitus.

Molecular phylogenies using mitochondrial DNA and nuclear alleles of the lactate dehydrogenase B locus were found to be concordant for populations of Fundulus heteroclitus ranging from Canada to Florida. Both mitochondrial DNA and lactate dehydrogenase alleles show a clear separation between the northern individuals (from Nova Scotia and Maine) and the southern ones (from Georgia and Florida), with a mixed population found in the geographic intermediate (New Jersey). An historical isolation, possibly as ancient as 0.5-1 million years old, may have played a role in shaping the situation observed today.     

Genetic variation and relative catalytic efficiencies: lactate dehydrogenase B allozymes of Fundulus heteroclitus.

In order to evaluate whether functional differences exist between allelic variants of a B type lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) in the teleost fish Fundulus heteroclitus (Linnaeus), the kinetic properties of pyruvate reduction were examined. While the pH dependence and the temperature dependence for maximal catalysis were indistinguishable among the allozymes, reaction velocities at low pyruvate concentrations were significantly different. At pH values below 8.00, the LDH-BbBb allozyme showed a greater reaction rate at lower temperatures (e.g., 10 degrees C) than LDH-BaBa. The phenomenon was reversed at higher temperatures (e.g., greater than 25 degrees C) for pH values between 6.50 and 7.00. The rates for the heterozygous phenotype, LDH-BaBb, were not the arithmetic average of the two homotetrameric allozymes. When reaction rates were compared at constant relative alkalinity, that is, a constant [OH-]/[H+] ratio, the findings were similar. The differences in the temperature dependence and the pH dependence for pyruvate reduction found between the LDH-B allozymes may reflect a selective adaptation and help explain the geographical variation in the Ldh-B gene frequencies of F. heteroclitus.     

Molecular basis of evolutionary adaptation at the lactate dehydrogenase-B locus in the fish Fundulus heteroclitus.

At the extremes of its natural distribution, populations of the common killifish Fundulus heteroclitus experience a difference of more than 15 degrees C in mean annual temperature. These populations are virtually fixed for two different codominant alleles at the heart-type lactate dehydrogenase locus (Ldh-B) which code for allozymes with different and adaptive kinetic responses to temperature. Two populations near the extremes of the species range (i.e., Maine and Georgia) were further studied for thermal adaptation at this locus. In the absence of any kinetic differences one would predict that to maintain a constant reaction velocity, 2 to 3 times as much enzyme would be required for each 10 degrees C decrease in environmental temperature. Consistent with this adaptive strategy and in addition to the adaptive kinetic characteristics, the LDH-B4 enzyme (EC 1.1.1.27) concentration and its mRNA concentration were approximately twice as great in the northern population as in the southern population. Acclimation experiments allow us to conclude that these differences are due to a combination of fixed genetic traits (evolutionary adaptation) and plastic responses to temperature (physiological acclimation). Furthermore, our calculations show that the LDH-B4 reaction velocities are essentially equivalent for these two populations, even though they live in significantly different thermal environments.