Widespread alterations to hypothalamic-pituitary-gonadal (HPG) axis signaling underlie high temperature reproductive inhibition in the eurythermal sheepshead minnow (Cyprinodon variegatus).

Fish experiencing abnormally high or prolonged elevations in temperature can exhibit impaired reproduction, even for species adapted to warm water environments. Such high temperature inhibition of reproduction has been linked to diminished gonadal steroidogenesis, but the mechanisms whereby hypothalamic-pituitary-gonadal (HPG) axis signaling is impacted by high temperature are not fully understood. Here, we characterized differences in HPG status in adult sheepshead minnow (Cyprinodon variegatus), a eurythermal salt marsh and estuarine species of eastern North America, exposed for 14 d to temperatures of 27 °C or 37 °C. Males and females at 37 °C had lower gonadosomatic index (GSI) values compared to fish at 27 °C, and females at 37 °C had fewer spawning capable eggs and lower circulating 17ß-estradiol (E2). Gene transcripts encoding gonadotropin-inhibitory hormone (gnih) and gonadotropin-releasing hormone-3 (gnrh3) were higher in relative abundance in the hypothalamus of both sexes at 37 °C. While pituitary mRNAs for the ß-subunits of follicle-stimulating hormone (fshß) and luteinizing hormone (lhß) were lowered only in males at 37 °C, Fsh and Lh receptor mRNA levels in the gonads were at lower relative levels in both the ovary and testis of fish at 37 °C. Females at 37 °C also showed reduced ovarian mRNA levels for steroid acute regulatory protein (star), P450 side-chain cleavage enzyme (cyp11a1), 3ß-hydroxysteroid dehydrogenase (3ßhsd), 17ß-hydroxysteroid dehydrogenase (hsd17ß3), and ovarian aromatase (cyp19a1a). Females at the higher 37 °C temperature also had a lower liver expression of mRNAs encoding estrogen receptor α (esr1) and several vitellogenin and choriogenin genes, but elevated mRNA levels for hepatic sex hormone-binding globulin (shbg). Our results substantiate prior findings that exposure of fish to high temperature can inhibit gonadal steroidogenesis and oogenesis, and point to declines in reproductive performance emerging from alterations at several levels of HPG axis signaling including increased hypothalamic Gnih expression, depressed gonadal steroidogenesis, and reduced egg yolk and egg envelope protein production in the liver.

Reproductive effects of estrogenic and antiestrogenic chemicals on sheepshead minnows (Cyprinodon variegatus).

Environmental estrogens can activate genes of the reproductive system, such as vitellogenin (VTG), a precursor to egg yolk protein, by activating the estrogen receptor (ER), whereas antiestrogens can inhibit ER activation. Adult lab-reared male sheepshead minnows (Cyprinodon variegatus) were exposed to estrogenic 4-tert-octylphenol (OP) and females to antiestrogenic cadmium (Cd), and the effects on four potential indicators of impaired reproductive function were examined: VTG in F0 male blood as sign of feminization, F0 generation fecundity/fertility, embryonic development/egg hatching/survival rate of F1 generation fry, and F0 gonadal histology. Mean VTG in the control, 11.5, 33.6, and 61.1 microg/L OP male fish were 0, 10.7, 38.7, and 65.6 mg/ml postexposure and 0, 2.5, 19.4, and 30.0 mg/ml postreproduction. A significant inverse relationship between increasing VTG in male blood and reproductive success of mating groups involving these males was shown, with higher OP decreasing percent viable eggs (fertility) by approximately 60%. Histology showed increased testis anomalies and decreased spermatozoa with increasing OP exposure. No effects on F1 embryonic development, egg hatching, or fry survival rate were observed. A significant decline in percent viable egg production involving Cd-exposed females occurred only when mated with OP-exposed males, with no eggs produced by fish exposed to the highest OP and Cd concentration. A three-week field exposure near a wastewater treatment plant outfall showed no elevated VTG in male plasma, but significantly higher total egg production per female per collection day (approximately 45%) was observed at the site furthest from the outfall.

DNA adducts in hematopoietic tissues and blood of the mummichog (Fundulus heteroclitus) from a creosote-contaminated site in the Elizabeth River, Virginia.

Hydrophobic DNA adducts were examined in liver, anterior kidney, spleen, and blood of tumor-prone mummichog (Fundulus heterclitus) from the creosote-contaminated Atlantic Wood (AW) site (Elizabeth River, Virginia). DNA adducts eluted in a diagonal radioactive zone, characteristic of polycyclic aromatic hydrocarbon exposure, in all examined tissues of AW fish. Mummichog demonstrated significantly higher levels of DNA adducts in spleen (394 +/- 109 nmol adducts/mol nucleotides) than in liver (201 +/- 77 nmol adducts/mol nucleotides) or anterior kidney (211 +/- 68 nmol adducts/mol nucleotides; P = 0.036). The levels of DNA adducts in the pooled blood (pool of four) were 142 nmol adducts/mol nucleotides. DNA adducts were not detected in the liver, anterior kidney, spleen and blood of fish collected from the reference site (< 2 nmol adducts/mol nucleotides). The high levels of DNA adducts detected in tissues of AW mummichog may be linked to the increased cancer incidence and immunosuppression in this population.

Serological alterations in carcinogen-exposed teleosts: procedures for preparation and analysis of samples from small fish.

To study the effects of environmental carcinogens on the immune system of Cyprinodon variegatus, we had to miniaturize or modify standard immunological procedures due to the small size of the fish. Modifications in standard bleeding procedures allowed collection of sufficient serum for most serological procedures. Serum electrophoresis showed considerable variation between exposed and unexposed fish as did qualitative immunoelectrophoresis techniques. We successfully adapted a bacteriophage neutralization procedure for use with the C. variegatus system to measure antiviral antibodies. The presence of antibody-forming cells in spleen suspensions from fish immunized with human type O erythrocytes was demonstrated by a modified immune rosette procedure. A capillary tube procedure was developed for separation of leukocytes from erythrocytes in blood from C. variegatus.

Bioassay for salmon prolactin using hypophysectomized Fundulus heteroclitus.

A bioassay for salmon prolactin (PRL) is described. This assay which is based on the sodium-retaining action of PRL in the hypophysectomized killifish, Fundulus heteroclitus, has proved to be rapid, sensitive (250 pg PRL per gram of fish), and specific. The procedure has been used to characterize the biological activity of a highly purified PRL from the pituitaries of the chinook salmon, Oncorhynchus tschawytscha, and a similar PRL isolated (by acid buffer polyacrylamide disc gel electrophoresis) from pituitaries of coho salmon (O. kisutch) (MW ca. 22,000; isoelectric point greater than 9).

Hemoglobins of the killifish Fundulus heteroclitus. Separation, characterization, and a model for the subunit compositions.

Polyacrylamide and starch gel electrophoresis of the hemoglobin of the killifish Fundulus heteroclitus reveal the presence of four clearly distinguishable components. These isohemoglobins, each tetramers consisting of alpha and beta chains, can be preparatively separated by ion exchange chromatography on DEAE-cellulose and are homogeneous according to isoelectric focusing in polyacrylamide gels. Oxygen equilibria of the isolated hemoglobin components (Hb I, Hb II, Hb III, and Hb IV) show only minor differences in the magnitude of the Bohr effect and in the effect of ATP on the binding of oxygen. Four different globin chains, alphaa, alphab, betaa, and betab, can be separated by ion exchange on CM-cellulose. Hb I is a homotetramer of alphab and betab chains, Hb IV consists of alphaa and betaa subunits, and components II and III are heterotetramers consisting of all four chains. The alpha and beta chains differ significantly in amino acid composition. A model suggesting the existence of 10 different isohemoglobins, 6 of which have stable intersubunit contacts, has been proposed to account for the qualitative and quantitative aspects of the electrophoretic behavior of the components. Separations of the isohemoglobins on DEAE-cellulose under slightly modified conditions provide additional support for the model.