Bruce A. Adams

Contaminant effects on the teleost fish thyroid.

Numerous environmentally relevant chemicals, including polychlorinated hydrocarbons, polycyclic aromatic hydrocarbons, organochlorine pesticides, chlorinated paraffins, organophosphorous pesticides, carbamate pesticides, cyanide compounds, methyl bromide, phenols, ammonia, metals, acid loads, sex steroids, and pharmaceuticals, exert acute or chronic effects on the thyroid cascade in the approximately 40 teleost fish species tested to date. Thyroid endpoints, therefore, serve as biomarkers of exposure to environmental pollutants. However, the mechanisms underlying thyroid changes and their physiological consequences are poorly understood because the thyroid cascade may respond indirectly and it has considerable capacity to compensate for abuses that otherwise would disrupt thyroid hormone homeostasis. Indeed, a xenobiotic-induced change in fish thyroid function has yet to be conclusively causally linked to decreased fitness or survival. Other complications in interpretation arise from the diversity of test conditions employed and the often indiscriminate use of numerous thyroid endpoints. Future work should be directed toward standardizing test conditions and thyroid endpoints and investigating causal links between thyroid changes and fish growth, reproduction, and development. Development may be particularly susceptible to thyroid disruption, and thyroid endpoints appropriate for early life stages need to be applied.

Three Forms of Gonadotropin-Releasing Hormone, Including a Novel Form, in a Basal Salmonid, Coregonus clupeaformis

Abstract Multiple forms of GnRH within individual brains may have different functions. However, some vertebrates such as salmonids continue to reproduce even though they have lost or do not express 1 of the 3 forms of GnRH found in most other teleosts. We examined a basal salmonid, lake whitefish, to determine the mechanism by which a reduction in the number of GnRH forms occurs. We identified for the first time 3 distinct GnRHs in a salmonid. One form is novel and is designated whitefish GnRH. The primary structure is pGlu-His-Trp-Ser-Tyr-Gly-Met-Asn-Pro-Gly-NH2. HPLC and RIA were used for purification followed by Edman degradation for sequence determination. Mass spectroscopy was used to confirm the sequence and amidation of the peptide. The other 2 forms, salmon GnRH and chicken GnRH-II, are identical to the 2 forms found in salmon, which evolved later than whitefish. Synthetic whitefish GnRH is biologically active, as it increased mRNA expression of growth hormone and the α-subunit for LH and thyroid-stimulating hormone in dispersed fish pituitary cells. Our data support the hypothesis that the ancestral salmonid had a third GnRH form when the genome doubled (tetraploidization), but the third form was lost later in some salmonids due to chromosomal rearrangements. We suggest that the salmon GnRH form compensated for the loss of the third form.

Knocked down and out: PACAP in development, reproduction and feeding

One approach to understanding the role of PACAP in vivo is to knockdown the translation of PACAP mRNA to protein or to knock out the PACAP gene by targeted disruption. In this paper, we review the effect of PACAP knockdown with morpholinos on early brain development in zebrafish. Also reviewed is the role of PACAP at several stages of reproduction as assessed in mice with a disrupted PACAP gene. New data are presented to analyze PACAPs action in energy homeostasis (body mass, food intake, endocrine parameters) using female PACAP-null mice. The evidence suggests PACAP is important for brain development in zebrafish and is required for normal reproduction, but not for body mass or food intake in mice maintained near thermoneutrality.

Cloning and Localization of Three Forms of Gonadotropin-Releasing Hormone, Including the Novel Whitefish Form, in a Salmonid, Coregonus clupeaformis

Abstract Cells containing different GnRH peptides currently are thought to have distinct locations and functions in the brain. Lake whitefish is the first salmonid species to have three forms of GnRH peptide in contrast to later-evolving salmonids (salmon and trout) in which only two forms have been identified. Our objective was to isolate the cDNAs that code for these transcripts and to localize the transcripts for the three forms of GnRH in adult lake whitefish brain. Also, we provide phylogenetic analysis of these three whitefish genes based on their preprohormone sequence. From whitefish we isolated cDNAs encoding chicken (c)GnRH-II, salmon (s)GnRH, and the novel whitefish (wf)GnRH. The three cDNAs each encode only one GnRH and are placed in separate groups with phylogenetic analysis. A combination of in situ hybridization and immunocytochemistry with two antisera revealed neurons that expressed protein and/or mRNA for cGnRH-II in the midbrain and hindbrain; sGnRH in the olfactory nerve and bulb, ventral telencephalon, and preoptic area; and wfGnRH in the same latter two brain regions and the hypothalamus. Thus, in the anterior brain, cells containing sGnRH and wfGnRH were in the same brain areas but not at identical locations in the ventral telencephalon and preoptic area. Based on our results, we speculate that both sGnRH and wfGnRH have gonadotropin-releasing roles in the lake whitefish brain.

Role of Two Genes Encoding PACAP in Early Brain Development in Zebrafish

Abstract:  To study the role of pituitary adenylate cyclase‐activating polypeptide (PACAP) in early brain development, we examined PACAP and its receptors for first expression and then separately knocked down the two forms of PACAP in zebrafish where development is rapid and observable. We injected morpholinos (antisense oligonucleotides) into fertilized eggs to block PACAP. Morphological changes in the brain were observed in embryos at 27 h post fertilization (hpf). Using in situ hybridization of early brain marker genes, we found that the most striking effects were an increase in pax2.1 expression in eye stalks associated with absence of either form of PACAP or an increase in eng2 and fgf8 in the midbrain–hindbrain boundary after loss of PACAP2. These marker genes are among the earliest factors in the formation of the midbrain–hindbrain boundary, an early organizing center. We suggest that PACAP is a target gene with feedback inhibition on pax2.1, eng2, or fgf8 in specific brain areas. In the hindbrain, the absence of either form of PACAP had little effect, as shown by expression of ephA4 and meis1.1. During midbrain development, our evidence suggests that PACAP1 can activate mbx. In both the diencephalon and/or forebrain, lack of PACAP1 or PACAP2 led to an increase in fgf8, again suggesting a suppressive effect of PACAP during development on these important genes that help to define cells in the forebrain. The early expression of transcripts for PACAP and its receptors by 0.5–6 hpf make both PACAP1 and PACAP2 candidates for factors that influence brain development.

Transcription and Translation of the Salmon Gonadotropin-Releasing Hormone Genes in Brain and Gonads of Sexually Maturing Rainbow Trout (Oncorhynchus mykiss)

Abstract Rainbow trout sexually mature at the end of Year 3. The form of GnRH that controls gonadotropin release in trout is salmon GnRH (sGnRH). In the tetraploid rainbow trout, two genes encode an identical sGnRH peptide. The sGnRH gene-1 produces one mRNA, whereas sGnRH gene-2 can produce more than one. This study asks whether the transcripts and their protein products are expressed in the brain and gonads and whether the pattern correlates with sexual maturity over the final year leading to first spawning. Brain sGnRH mRNA and protein were continuously present throughout the third year. We show for the first time that the long sGnRH-2 mRNA transcript is expressed in neural tissue and not exclusively in gonadal tissue. Expression of the long sGnRH-2 mRNA in the brain coincides with high levels of sGnRH peptide in the brain during a time of increased gonadal growth. Thus, the long sGnRH-2 mRNA in the brain may act to regulate sGnRH production in a stage-specific rather than a tissue-specific manner. In gonads, local sGnRH is thought to play an autocrine/paracrine role in regulating gonadal maturation and spawning. In the maturing gonads, sGnRH gene-1 and -2 are expressed intermittently. Strikingly, sGnRH peptide was not detected in the gonads at any time during Year 3. These results suggest that either the sGnRH transcripts in the gonads are not translated into protein or, if translated, the protein is rapidly released, resulting in gonadal content below 1 fM per fish.

Egg Production, Fertility, and Hatch Success of American Plaice Held in Captivity

Abstract The reproductive performance of individual female American plaice Hippoglossoides platessoides was monitored during a spawning season under laboratory conditions. The majority (74%) of females produced eggs at some point in a 2-month period (May–July) during which they were checked every 2 d for ovulated eggs. Most females produced several batches of eggs during an average production period of 26 d. Approximately 12% of the total number of eggs produced by each female were obtained in the first egg batch. There were periodic peaks of egg production for the group in increasing intervals at about 8, 20, and 34 d after the first eggs were collected. The mean relative fecundity was 1.5 × 105 eggs/ kg body weight, but there was no relationship between actual fecundity and either length or weight. The mean percentages of viable and fertile eggs were 44.7% and 13.7%, respectively. A significant decline in the percentage of fertile eggs was observed with time, such that percent of fertile eggs by the six...

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP)/Glucagon Superfamily

The pituitary adenylate cyclase-activating polypeptide (PACAP)/ glucagon superfamily includes nine hormones in humans that are related by structure, distribution (especially the brain and gut), function (often by activation of cAMP), and receptors (a subset of seven-transmembrane receptors). The nine hormones include glucagon, glucagon-like peptide-1 (GLP-1), GLP-2, glucose-dependent insulinotropic polypeptide (GIP), GH-releasing hormone (GRF), peptide histidine-methionine (PHM), PACAP, secretin, and vasoactive intestinal polypeptide (VIP). The origin of the ancestral superfamily members is at least as old as the invertebrates; the most ancient and tightly conserved members are PACAP and glucagon. Evidence to date suggests the superfamily began with a gene or exon duplication and then continued to diverge with some gene duplications in vertebrates. The function of PACAP is considered in detail because it is newly (1989) discovered; it is tightly conserved (96% over 700 million years); and it is probably the ancestral molecule. The diverse functions of PACAP include regulation of proliferation, differentiation, and apoptosis in some cell populations. In addition, PACAP regulates metabolism and the cardiovascular, endocrine, and immune systems, although the physiological event(s) that coordinates PACAP responses remains to be identified.