Penny Swanson

Biochemistry and physiology of fish gonadotropins

Pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), play central roles in regulating gametogenesis and the production of gonadal hormones required for the development of sexual behavior and secondary sex characters in all vertebrates. Studies in fish conducted nearly 50 years ago demonstrated that removal of the pituitary caused cessation of gametogenesis in both sexes (Pickford and Atz 1957); however, the exact chemical nature two fish gonadotropins homologous to mammalian LH and FSH was not determined until the mid to late 1980’s. Since that time, a great deal of information has emerged on the structure of FSH and LH from numerous species within Teleostei, one Chondrostean and one Chondricthyan (Querat et al. 2000, 2001; Yaron et al. 2003). In this paper, the present status of our understanding of structure and function of LH and FSH in fish is briefly reviewed. For a comprehensive review of fish gonadotropin regulation and gene structures the reader is referred to Yaron et al. 2003.

The duality of teleost gonadotropins.

The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.

Involvement of gonadotropin in the uptake of vitellogenin into vitellogenic oocytes of the rainbow trout, Oncorhynchus mykiss

The effects of two fully characterized, structurally distinct gonadotropins, GtH I and GtH II, on the uptake of vitellogenin (VTG) into oocytes of the rainbow trout, Oncorhynchus mykiss, were investigated both in vivo and in vitro. GtH I, injected into maturing vitellogenic females at a dose of 10 micrograms.kg body wt-1, increased the rate of [3H]VTG uptake into oocytes by more than two-fold, effectively doubling their rate of growth. Ovaries from females similarly treated with GtH II sequestered VTG at rates similar to controls. In vitro, GtH I stimulated VTG uptake in a dose-dependent manner. At a GtH I concentration of 100 ng.ml-1 and above, the rate of VTG uptake was significantly greater than that of the controls and at 1000 ng.ml-1 the rate of uptake was more than doubled. GtH II did not significantly increase VTG sequestration into isolated oocytes at concentrations up to, and including, 1000 ng.ml-1. These data provide the first evidence that GtH I has a primary function in stimulating VTG uptake and strongly support the contention that at least two functionally distinct GtHs occur in fish.

Gonadotropins I and II in juvenile coho salmon

The present study was designed to obtain basic endocrine information on GTH I and GTH II in previtellogenic and prespermatogenic coho salmon (immature). Levels of GTH II in pituitary extracts were 6.5 ± 2.0 and 6.7 ± 2.0 pg/μg pituitary protein in male and female fish, respectively. In contrast, the pituitary content of GTH I was approximately 100-fold higher than GTH II (1.302 ± .22 and 1.173 ± .21 ng/μg pituitary protein in male and female fish, respectively). Plasma levels of GTH II in immature salmon were not detectable by RIA whereas plasma GTH I levels were approximately 0.62 ± 0.12 and 0.78 ± 0.13 ng/ml in male and female fish, respectively. Highly purified coho salmon GTH I and GTH II stimulated testicular testosterone production and ovarian estradiol productionin vitro in a similar manner, though GTH II appeared more potent than GTH I. Therefore, it appears that although the salmon pituitary contains predominantly GTH I prior to puberty, the gonad can respond to both GTH I and GTH II.

Dietary exposure to 2,2´,4,4´-Tetrabromodiphenyl Ether (PBDE-47) alters thyroid status and thyroid hormone–regulated gene transcription in the pituitary and brain

Background Polybrominated diphenyl ether (PBDE) flame retardants have been implicated as disruptors of the hypothalamic-pituitary-thyroid axis. Animals exposed to PBDEs may show reduced plasma thyroid hormone (TH), but it is not known whether PBDEs impact TH-regulated pathways in target tissues. Objective We examined the effects of dietary exposure to 2,2′,4,4′-tetrabromodiphenyl ether (PBDE-47)—commonly the highest concentrated PBDE in human tissues—on plasma TH levels and on gene transcripts for glycoprotein hormone α-subunit (GPHα) and thyrotropin β-subunit (TSHβ) in the pituitary gland, the autoinduced TH receptors α and β in the brain and liver, and the TH-responsive transcription factor basic transcription element-binding protein (BTEB) in the brain. Methods Breeding pairs of adult fathead minnows (Pimephales promelas) were given dietary PBDE-47 at two doses (2.4 μg/pair/day or 12.3 μg/pair/day) for 21 days. Results Minnows exposed to PBDE-47 had depressed plasma thyroxine (T4), but not 3,5,3′-triiodothyronine (T3). This decline in T4 was accompanied by elevated mRNA levels for TStHβ (low dose only) in the pituitary. PBDE-47 intake elevated transcript for TH receptor αin the brain of females and decreased mRNA for TH receptor β in the brain of both sexes, without altering these transcripts in the liver. In males, PBDE-47 exposure also reduced brain transcripts for BTEB. Conclusions Our results indicate that dietary exposure to PBDE-47 alters TH signaling at multiple levels of the hypothalamic-pituitary-thyroid axis and provide evidence that TH-responsive pathways in the brain may be particularly sensitive to disruption by PBDE flame retardants.

Previtellogenic Oocyte Growth in Salmon: Relationships among Body Growth, Plasma Insulin-Like Growth Factor-1, Estradiol-17beta, Follicle-Stimulating Hormone and Expression of Ovarian Genes for Insulin-Like Growth Factors, Steroidogenic-Acute Regulatory Protein and Receptors for Gonadotropins, Growth Hormone, and Somatolactin

Abstract Body growth during critical periods is known to be an important factor in determining the age of maturity and fecundity in fish. However, the endocrine mechanisms controlling oogenesis in fish and the effects of growth on this process are poorly understood. In this study interactions between the growth and reproductive systems were examined by monitoring changes in various components of the FSH-ovary axis, plasma insulin-like growth factor 1 (Igf1), and ovarian gene expression in relation to body and previtellogenic oocyte growth in coho salmon. Samples were collected from females during two hypothesized critical periods when growth influences maturation in this species. Body growth during the fall-spring months was strongly related to the degree of oocyte development, with larger fish possessing more advanced oocytes than smaller, slower growing fish. The accumulation of cortical alveoli in the oocytes was associated with increases in plasma and pituitary FSH, plasma estradiol-17beta, and ovarian steroidogenic acute regulatory protein (star) gene expression, whereas ovarian transcripts for growth hormone receptor and somatolactin receptor decreased. As oocytes accumulated lipid droplets, a general increase occurred in plasma Igf1 and components of the FSH-ovary axis, including plasma FSH, estradiol-17beta, and ovarian mRNAs for gonadotropin receptors, star, igf1, and igf2. A consistent positive relationship between plasma Igf1, estradiol-17beta, and pituitary FSH during growth in the spring suggests that these factors are important links in the mechanism by which body growth influences the rate of oocyte development.

Salmonid pituitary gonadotrophs. I, Distinct cellular distributions of two gonadotropins, GTH I and GTH II

Using antisera specific for the beta subunits of two distinct coho salmon gonadotropins, GTH I and GTH II, an immunocytochemical study of rainbow trout and Atlantic salmon pituitaries was done. Cells which immunostained with anti-GTH I beta were distributed in the periphery of the glandular cords of the proximal pars distalis (PPD), in close association with somatotrophs. On the other hand, cells immunostained with anti-GTH II beta were located in the central parts of the glandular cords of the PPD. Neither the GTH I-producing nor the GTH II-producing cells stained with antisera against chum salmon growth hormone or the beta subunit of human thyroid-stimulating hormone. Moreover, GTH I and GTH II were localized in distinctly different cells. In no case was colocalization of these GTHs in the same cell observed. Finally, it was concluded that classification of GTH cells as globular and vesicular forms does not reflect the type of hormone produced by the cell, but may reflect differences in the physiological conditions of the cells.

Exon Skipping in the Gene Encoding Pituitary Adenylate Cyclase-Activating Polypeptide in Salmon Alters the Expression of Two Hormones that Stimulate Growth Hormone Release1

In mammals, GRF and pituitary adenylate cyclase-activating polypeptide (PACAP) are encoded in separate genes. We report here that in the salmon a 4.5-kilobase gene contains five exons that encode the biologically active part of the GRF-like peptide (amino acids 1–32) on exon 4 and PACAP on exon 5. Analysis of two fish messenger RNAs reveals that a long precursor containing GRF and PACAP and a short precursor containing only PACAP are both expressed in the brain of at least five species of salmon, whereas mice express only the long precursor encoded by the PACAP gene. Synthetic salmon PACAP-38 and salmon GRF-like peptide-45 both stimulated GH release from cultured salmon pituitary cells; PACAP stimulated a concentration-dependent release of GH at both 4 and 24 h of incubation, whereas GRF-like peptide did not. Alternative splicing, resulting in the short precursor in which GRF-32 is excised, may provide a means for differential control of GH secretion with higher production of the more potent PACAP. A dupl...

The effect of whole body lipid on early sexual maturation of 1+ age male chinook salmon (Oncorhynchus tshawytscha)

Abstract Early sexual maturation of male chinook salmon (maturation 1 to 4 years prior to females in the same age class) results in reduced effectiveness of stock enhancement programs and a financial loss to the salmon farming industry. Previous studies in Atlantic salmon have shown that the age of maturity in males is affected by growth and/or body energy stores, but the relative roles of these two factors are not well understood. Therefore, an experiment was designed to determine when spermatogenesis was initiated, to characterize the endocrine changes during the onset of puberty in male salmon, and to determine if the level of whole-body lipid affects the incidence of early male maturation in a wild stock (Yakima River) of 1+ spring chinook salmon. Fry were fed a commercial diet from February until August and were then divided into groups of 320 fish (mean weight, 5.6 g) and fed one of five experimental diets (two replicate groups/diet) containing 4%, 9%, 14%, 18% or 22% lipid and 82%, 77%, 73%, 69%, or 65% protein for 13 months. Fish were reared on natural photoperiod and ambient temperature (6°C to 16°C), and pair-fed to a level based on the tank with the lowest feed consumption. Fish were weighed monthly and sampled to determine body composition, pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, plasma insulin-like growth factor I (IGF-I) levels, and stage of gonadal development. Throughout the experimental period the mean fish weight was similar among treatment groups. However, from December through the end of the experiment in the following September, maturing males were significantly larger than nonmaturing fish. Initial lipid levels in 0-age experimental fish were near 6%, which is similar to wild fish of the same stock and age captured in the Yakima River during August. Fish fed diets containing more than 4% lipid increased in whole-body lipid content during the first 2 months of feeding and then maintained at relatively constant levels during the course of the experiment. Whole-body lipid levels for the dietary treatment groups averaged 5.6%, 7.1%, 8.2%, 9.4%, and 9.6% from October through the following September. Based on histological examination of the testes of experimental fish, type B spermatogonia and primary spermatocytes were first observed in some of the yearling males during November. These were designated maturing males. Pituitary FSH levels were significantly higher in maturing than nonmaturing males at this time and for the remainder of the study. Pituitary FSH levels increased as spermatogenesis proceeded in maturing fish, whereas pituitary LH levels increased in maturing 1+ males only during July and August, when testes were in late stages of spermatogenesis and in September during spermiation. Plasma IGF-I levels were significantly higher in maturing males than nonmaturing fish from December through the end of experiment. Since maturing males were significantly larger than nonmaturing fish of both sexes from December through September, the difference in IGF-I levels could be due to differences in growth or due to maturation. The percentage of maturing males was significantly influenced by whole-body lipid, increasing from 34% in fish fed the 4% lipid diet to 45% in fish fed the 22% lipid diet. These data suggest that whole-body lipid levels influenced the incidence of maturation of male spring chinook salmon. In addition, both endocrine and histological indicators suggest that maturation was initiated in males approximately a full year prior to the time the fish will spawn.

Salmonid pituitary gonadotrophs. II. Ontogeny of GTH I and GTH II cells in the rainbow trout (Salmo gairdneri irideus).

Immunocytochemistry of rainbow trout pituitary gonadotrophs (GTH I- and GTH II-producing cells) during gametogenesis was investigated. GTH I and GTH II were found in distinctly different cells in all stages of reproductive development that were examined. Only GTH I cells were present in trout prior to puberty. GTH II appeared in addition to GTH I coincident with the onset of vitellogenesis and spermatogenesis. Both GTH I and GTH II cells were found in trout at the time of final reproductive maturation, although the number of GTH II cells was greater than that of GTH I cells. These data indicate that GTH I and GTH II are localized in separate cells in the trout pituitary throughout gametogenesis, and that synthesis of GTH I and GTH II varies during reproductive development.