Jon T. Dickey

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.

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.

Endocrine Changes During Onset of Puberty in Male Spring Chinook Salmon, Oncorhynchus tshawytscha

Abstract In male salmonids, the age of maturation varies from 1 to 6 years and is influenced by growth during critical periods of the life cycle. The endocrine mechanisms controlling spermatogenesis and how growth affects this process are poorly understood. Recent research has indicated that gonadotropins, 11-ketotestosterone, and insulin-like growth factor I play roles in spermatogenesis in fish. To expand our understanding of the roles of these endocrine factors in onset of puberty, male spring chinook salmon (Oncorhynchus tshawytscha) were sampled at monthly intervals 14 mo prior to spermiation. This sampling regime encompassed two hypothesized critical periods when growth influences the initiation and completion of puberty for this species. Approximately 80% of the males matured during the experimental period, at age 2 in September 1999. An initial decline in the ratio of primary A to transitional spermatogonia was observed from July to December 1998, and during this period plasma levels of 11-ketotestosterone and pituitary levels of FSH increased. From January 1999 onward, males with low plasma 11-ketotestosterone levels (<1 ng/ml) had low pituitary and plasma FSH levels and no advanced development of germ cells. Conversely, from January through September 1999, males with high plasma 11-ketotestosterone levels (>1 ng/ml) had testes with progressively more advanced germ cell stages along with elevated pituitary and plasma FSH. Plasma levels of insulin-like growth factor I increased during maturation. These data provide the first physiological evidence for activation of the pituitary-testis axis during the fall critical period when maturation is initiated for the following year.

Follicle-stimulating hormone regulation of ovarian transcripts for steroidogenesis-related proteins and cell survival, growth and differentiation factors in vitro during early secondary oocyte growth in coho salmon

Little is known about follicle-stimulating hormone (FSH) function during oocyte growth in fishes. The goal of this study was to gain a fundamental understanding of FSH action on ovarian follicles during early secondary oocyte growth by examining changes in ovarian gene expression and steroidogenesis in response to FSH. Coho salmon (Oncorhynchus kisutch) mid to late cortical alveolus stage follicles were incubated with or without salmon FSH in time-course and concentration-response experiments. Steroid levels were determined in the culture medium by immunoassay and levels of target ovarian mRNAs were determined by quantitative RT-PCR. Medium estradiol-17β (E2) levels increased in response to FSH and plateaued by 36h, while testosterone levels increased similarly but were lower and more variable than E2. Gonadotropin receptor transcripts were differentially regulated, with fshr and lhcgr being down- and up- regulated, respectively. Transcripts encoding proteins involved in steroidogenesis, such as star and hsd3b were significantly upregulated by FSH, whereas aromatase (cyp19a1a) mRNA was unaffected by FSH and declined over time in culture. A recently identified teleost gene, bmp16, was suppressed by FSH and an anti-apoptotic factor, clusterin 1 (clu1), was upregulated by FSH. Lesser FSH effects were observed on igf2, cyp11a1 and cyp17a1, which were stimulated, and igf1ra, inhbb, amh and apoe, which were suppressed. As evident by the significant increases in steroid production and transcripts for specific steroidogenesis-related proteins, FSH influences steroidogenesis during early secondary growth and may be a critical signal for puberty onset. Effects of FSH on ovarian anti-apoptotic and growth factor genes suggest roles for FSH in cell survival, growth and differentiation in teleosts.

Metabolic hormones regulate basal and growth hormone-dependent igf2 mRNA level in primary cultured coho salmon hepatocytes: effects of insulin, glucagon, dexamethasone, and triiodothyronine

Igf1 and Igf2 stimulate growth and development of vertebrates. Circulating Igfs are produced by the liver. In mammals, Igf1 mediates the postnatal growth-promoting effects of growth hormone (Gh), whereas Igf2 stimulates fetal and placental growth. Hepatic Igf2 production is not regulated by Gh in mammals. Little is known about the regulation of hepatic Igf2 production in nonmammalian vertebrates. We examined the regulation of igf2 mRNA level by metabolic hormones in primary cultured coho salmon hepatocytes. Gh, insulin, the glucocorticoid agonist dexamethasone (Dex), and glucagon increased igf2 mRNA levels, whereas triiodothyronine (T(3)) decreased igf2 mRNA levels. Gh stimulated igf2 mRNA at physiological concentrations (0.25x10(-9) M and above). Insulin strongly enhanced Gh stimulation of igf2 at low physiological concentrations (10(-11) M and above), and increased basal igf2 (10(-8) M and above). Dex stimulated basal igf2 at concentrations comparable to those of stressed circulating cortisol (10(-8) M and above). Glucagon stimulated basal and Gh-stimulated igf2 at supraphysiological concentrations (10(-7) M and above), whereas T(3) suppressed basal and Gh-stimulated igf2 at the single concentration tested (10(-7) M). These results show that igf2 mRNA level is highly regulated in salmon hepatocytes, suggesting that liver-derived Igf2 plays a significant role in salmon growth physiology. The synergistic regulation of igf2 by insulin and Gh in salmon hepatocytes is similar to the regulation of hepatic Igf1 production in mammals.

Regulation of pituitary GnRH receptor and gonadotropin subunits by IGF1 and GnRH in prepubertal male coho salmon

Insulin-like growth factor 1 (IGF1) is a key somatotropic hormone that may convey growth status to the reproductive endocrine system. This study examined effects of IGF1 alone or in combination with gonadotropin-releasing hormone (GnRH) on pituitary transcripts for GnRH receptor (GnRHR) variants, follicle-stimulating hormone (FSH), luteinizing hormone (LH), growth hormone (GH), and IGF, as well as secretion of FSH in vitro. Three experiments were conducted with dispersed pituitary cells of prepubertal male coho salmon (Oncorhynchus kisutch) to determine the time course of the response to IGF1, IGF1 concentration response, and GnRH concentration response. IGF1 consistently elevated pituitary transcripts for gnrhr1 and the four gonadotropin subunits (fshb, lhb, cga1, and cga2) by day 10 of culture, while suppressing gh and igf2. Short-term treatment with GnRH (24h) induced minor increases in transcripts for fshb, cga1, and cga2, but suppressed lhb and strongly inhibited gnrhr1 expression. IGF1 significantly increased GnRH-stimulated FSH protein release by the pituitary cells, although not as robustly as previously observed in more reproductively advanced salmon. Our results demonstrate that IGF1 increases steady-state mRNA levels of gnrhr1 and four gonadotropin subunits, and may act alone or with GnRH to increase pituitary FSH release in male coho salmon, over 1year before puberty. These findings suggest that IGF1 may prime pituitary gonadotrope cells of prepubertal salmon to respond to GnRH by stimulating synthesis of GnRHR and FSH during puberty onset.

Reproductive Investment and Growth History in Female Coho Salmon

Abstract The body size, ovary mass, egg mass, and fecundity of coho salmon Oncorhynchus kisutch from three different rearing environments (wild, ranched, and captive) were compared to determine which aspects of growth history affect changes in these reproductive characters. In addition, the growth of individually tagged females reared in captivity throughout the seawater growth phase was measured and related to final reproductive investment. A strong positive relationship existed between ovary mass and body length at spawning; this relationship was maintained over a wide range of final body sizes, and the regression slope was consistent among the rearing environments. However, the relative investment in ovary mass and fecundity (regression intercepts) differed between rearing environments. Fish reared in captivity throughout their life cycle had higher values than did fish reared in the other two environments. A relatively weak positive relationship between egg mass and final body length was common to fem...

Development of a multiplex gene expression assay for components of the endocrine growth axis in coho salmon.

This study explores the efficacy of the Quantigene plex (QGP) technology for measuring a panel of endocrine growth-related transcripts in coho salmon, Oncorhynchus kisutch. The QGP technology permits the simultaneous quantification of multiple targeted mRNAs within a single tissue homogenate using sequence-specific probes and requires no reverse transcription (RT) or amplification as is required for RT-quantitative PCR (RT-qPCR). Using liver homogenates from coho salmon under fed and fasted conditions, we compared the detectable fold differences of steady-state mRNA levels between the QGP and probe-based RT-qPCR assays for insulin-like growth factors (igf1 and igf2), insulin-like growth factor binding proteins (igfbp1b, igfbp2a, and igfbp2b), somatolactin receptor (slr), and growth hormone receptors (ghr1 and ghr2). Significant, positive correlations for all genes between the two assays were found. In addition, the relatively low variance of results from the QGP assay suggests that this is a suitable method for a comprehensive analysis of endocrine growth-related transcripts and could potentially be used to develop assays for other gene networks in teleosts.

Use of Morphological and Physiological Indices to Characterize Life History Diversity in Juvenile Hatchery Winter-Run Steelhead

AbstractSteelhead Oncorhynchus mykiss exhibit significant life history plasticity, including variation in freshwater residence time, age of smoltification, and maturation that are influenced by exceeding specific thresholds in size or body energy stores. Fish that do not reach minimum thresholds for smoltification or exceed thresholds for maturation may “residualize” in freshwater. Understanding life history diversity is important in steelhead hatchery programs. The objective of this investigation was to characterize life history diversity in Hood River, Oregon, hatchery winter-run steelhead prior to release. We used both morphological and physiological indices to assess degree of smoltification in both sexes and maturation in males. Smoltification was categorized by measuring size, visual smolt index, and gill Na+/K+-ATPase activity. All females were determined to be immature. Male reproductive development required histological staging of testes. Other indices of maturation—including gonadosomatic index,...