Andrew L. Pierce

Gender-specific expression of multiple estrogen receptors, growth hormone receptors, insulin-like growth factors and vitellogenins, and effects of 17β-estradiol in the male tilapia (Oreochromis mossambicus)

Gender-specific expression of estrogen receptors (ER alpha and ER beta), growth hormone receptors (GHR1 and GHR2), insulin-like growth factors (IGF-I and IGF-II) and three vitellogenins (Vgs A-C) was examined in the liver, gonad, pituitary, and brain of sexually mature male, female, and 17 beta-estradiol (E2)-treated male tilapia (Oreochromis mossambicus). Reflecting greater growth rate in male tilapia, hepatic expression of GHR1, GHR2, IGF-I and IGF-II as well as plasma IGF-I levels were higher in males than in females, whereas the expression of Vgs A-C and ER alpha was higher in females. On the other hand, expression of all genes measured was higher in the ovary than in testis. Forty eight hours after E2 injection (5 microg/g) into male fish, hepatic expression of most transcripts measured were altered to levels that were similar to those seen in females. The changes included decreased expression of GHR1, GHR2, IGF-I, and IGF-II, and increased expression of ER alpha and Vgs A-C. E2 treatment also increased Vg and decreased IGF-I in the plasma. Brain expression of ER alpha, ER beta, GHR1, and IGF-I was higher in females than in males, whereas pituitary expression of GHR2 and IGF-I was lower in females; only brain expression of GHR1 was increased by E2 treatment. These findings suggest that E2 stimulates Vg production primarily through activation of ER alpha and down-regulation of the GH/IGF-I axis, thus shifting energy from somatic growth towards vitellogenesis at the level of the liver.

Induction of Three Vitellogenins by 17beta-Estradiol with Concurrent Inhibition of the Growth Hormone-Insulin-Like Growth Factor 1 Axis in a Euryhaline Teleost, the Tilapia (Oreochromis mossambicus)

Abstract The objective of the present study was to utilize the male Mozambique tilapia (Oreochromis mossambicus) as a model for examining the molecular mechanisms that mediate the physiological transition between somatic and gonadal growth in female teleost fish, and in vertebrates in general. Partial cDNAs that encode multiple forms of vitellogenin (Vtg), which is the major precursor of yolk proteins, were cloned from estrogen-treated males and utilized to develop real-time quantitative RT-PCR assays, which were supplemented by an assay for Vtg immunoreactivity in the plasma. Alignment analyses of the amino acid sequences deduced from the vtg cDNAs revealed three distinct tilapia Vtgs, which were categorized as Aa-, Ab-, and C-type Vtgs. A single injection of male tilapias with 17beta-estradiol (E2) at 5 μg/g body weight significantly increased the plasma E2 and hepatic levels of all three vtg transcripts within 1 day. Plasma E2 levels declined after 3 days, whereas the plasma Vtg immunoreactivity and hepatic levels of the three vtg transcripts continued to increase. Hepatic expression of the estrogen receptor (esr) 1 gene, but not the esr2 gene, also increased markedly 1 day after E2 injection and remained elevated for 5 days. While plasma growth hormone (Gh) levels were unaffected, hepatic expression of transcripts that encoded the Gh receptor and insulin-like growth factor 1 (Igf1) was suppressed by E2, as were the plasma Igf1 levels. These results clearly suggest a distinct negative interplay between the growth and reproductive axes at the molecular level of key hepatic regulatory pathways involved in the control of energy utilization by gonadal and somatic growth processes.

Tissue-specific regulation of the growth hormone/insulin-like growth factor axis during fasting and re-feeding: Importance of muscle expression of IGF-I and IGF-II mRNA in the tilapia.

The effects of prolonged nutrient restriction (fasting) and subsequent restoration (re-feeding) on the growth hormone (GH)/insulin-like growth factor (IGF) axis were investigated in the tilapia (Oreochromis mossambicus). Mean weight and specific growth rate declined within 1 week in fasted fish, and remained lower than controls throughout 4 weeks of fasting. Plasma levels of IGF-I were lower than fed controls during 4 weeks of fasting, suggesting a significant catabolic state. Following re-feeding, fasted fish gained weight continuously, but did not attain the weight of fed controls at 8 weeks after re-feeding. Specific growth rate increased above the continuously-fed controls during the first 6 weeks of re-feeding, clearly indicating a compensatory response. Plasma IGF-I levels increased after 1 week of re-feeding and levels were not otherwise different from fed controls. Plasma GH levels were unaffected by either fasting or re-feeding. No consistent effect of fasting or re-feeding was observed on liver expression of GH receptor (GH-R), somatolactin (SL) receptor (SL-R), IGF-I or IGF-II. In contrast, muscle expression of GH-R increased markedly during 4 weeks of fasting, and then declined below control levels upon re-feeding for weeks 1 and 2. Similarly, muscle expression of SL-R increased after 4 weeks of fasting, and reduced below control levels after 1 and 2 weeks of re-feeding. On the other hand, muscle expression of IGF-I was strongly reduced throughout the fasting period, and levels recovered 2 weeks after re-feeding. Muscle expression of IGF-II was not affected by fasting, but was reduced after 1 and 2 weeks of re-feeding. These results indicate that GH/IGF axis, particularly muscle expression of GH-R, SL-R and IGF-I and -II, is sensitive to nutritional status in the tilapia.

Differential regulation of Igf1 and Igf2 mRNA levels in tilapia hepatocytes: effects of insulin and cortisol on GH sensitivity

Igf1 and Igf2 stimulate growth and development of vertebrates. In mammals, liver-derived endocrine Igf1 mediates the growth promoting effects of GH during postnatal life, whereas Igf2 stimulates placental and fetal growth and is not regulated by GH. Insulin enhances Igf1 production by the mammalian liver directly, and by increasing hepatocyte sensitivity to GH. We examined the regulation of igf1 and igf2 mRNA levels by GH, insulin, and cortisol, and the effects of insulin and cortisol on GH sensitivity in primary cultured hepatocytes of tilapia, a cichlid teleost. GH increased mRNA levels of both igf1 and igf2 in a concentration-related and biphasic manner over the physiological range, with a greater effect on igf2 mRNA level. Insulin increased basal igf2 mRNA level, and strongly increased GH-stimulated igf2 mRNA level, but slightly reduced basal igf1 mRNA level and did not affect GH-stimulated igf1 mRNA level. Cortisol inhibited GH stimulation of igf1, but increased GH stimulation of igf2 mRNA level. The synergistic effect of insulin and GH on igf2 mRNA level was confirmed in vivo. These results indicate that insulin and cortisol differentially modulate the response of igf1 and igf2 mRNA to GH in tilapia hepatocytes, and suggest that the regulation of liver Igf2 production differs between fish and mammals. Regulation of liver Igf2 production in fish appears to be similar to regulation of liver Igf1 production in mammals.

Gene expression of growth hormone family and glucocorticoid receptors, osmosensors, and ion transporters in the gill during seawater acclimation of Mozambique tilapia, Oreochromis mossambicus

This study characterized endocrine and ionoregulatory responses accompanying seawater (SW) acclimation in Mozambique tilapia (Oreochromis mossambicus). Changes in plasma hormones and gene expression of hormone receptors, putative osmosensors, and ion transporters in the gill were measured. Transfer of freshwater (FW)-acclimated tilapia to SW resulted in a marked elevation in plasma osmolality and a significant rise in plasma growth hormone (GH) levels at 12 hr and 14 days after transfer. Significant reductions in plasma prolactin (PRL(177) and PRL(188)) levels also occurred in SW-transferred fish; no effect of transfer upon plasma cortisol or insulin-like growth factor I was observed. Gene expression of GH receptor increased strongly 6 hr after transfer, whereas PRL receptor was lower than controls at 12 hr. By contrast, mRNA levels of somatolactin and glucocorticoid receptors were unaffected by SW transfer. Osmotic stress transcription factor 1 mRNA levels rose significantly between 3 and 12 hr, whereas the calcium-sensing receptor was unaffected. Aquaporin-3 gene expression was strongly down-regulated during SW acclimation from 12 hr until the conclusion of the experiment. Na(+)/K(+)/2Cl(-) cotransporter gene expression increased significantly 3 hr after transfer, whereas expression of Na(+)/Cl(-) cotransporter, specific to FW-type chloride cells, declined by 6 hr into SW acclimation. The response of Na(+)/H(+) exchanger was less pronounced, but showed a similar pattern to that of the Na(+)/Cl(-) cotransporter. These results suggest that acquisition of hyposmoregulatory mechanisms in Mozambique tilapia entails the coordinated interaction of systemic hormones with local factors in the gill, including hormone receptors, ion transporters, and osmosensors.

cDNA cloning and isolation of somatolactin in Mozambique tilapia and effects of seawater acclimation, confinement stress, and fasting on its pituitary expression.

Somatolactin (SL) is a member of the growth hormone (GH)/prolactin (PRL) family of pituitary hormones, and is found in a variety of teleost species. Somatolactin is thought to be involved in a wide range of physiological actions, including reproduction, stress response, the regulation of Ca(2+) and acid-base balance, growth, metabolism, and immune response. We report here on the cDNA structure of SL from the pituitary of Mozambique tilapia, Oreochromis mossambicus, and its gene expression in response to seawater acclimation, stress, and fasting. Tilapia SL cDNA (1573bp long) encoded a prehormone of 230 amino acids. Sequence analysis of purified SL revealed that the prehormone is composed of a signal peptide of 23 amino acids and a mature protein of 207 amino acids, which has a possible N-glycosylation site at position 121 and seven Cys residues. Tilapia SL shows over 80% amino acid identity with SLalpha of advanced teleosts such as medaka and flounder, and around 50% identity with SLbeta of carp and goldfish. Acclimation to seawater had no effect on pituitary expression of SL or on hepatic expression of the putative tilapia SL receptor (GHR1). By contrast, seawater acclimation resulted in significant increases in pituitary GH expression and in hepatic expression of tilapia GH receptor (GHR2). Confinement stress had no effect on pituitary expression of either SL or GH, or on hepatic expression of GHR1, whereas a significant increase was seen in GHR2 expression in the liver. Fasting for 4 weeks resulted in significant reductions in SL transcripts both in fresh water and seawater. It is highly likely that SL is involved in metabolic processes in tilapia along with the GH/IGF-I axis.

Survival and Traits of Reconditioned Kelt Steelhead Oncorhynchus mykiss in the Yakima River, Washington

Abstract We evaluated the traits and survival to release of reconditioned kelt steelhead Oncorhynchus mykiss in the Yakima River (Washington State, USA). From 2001 to 2011, we captured a total of 9,738 downstream-migrating kelts at an irrigation diversion facility, an average about 27% of each annual wild steelhead return. Captured kelts were reared for 4.5–10 months in an artificial environment, treated for diseases and parasites, and fed both krill and pellets. Surviving reconditioned fish were released into the Yakima River during the peak of the upstream migration of prespawn steelhead. Reconditioned steelhead kelts were predominantly (>92%) female. Annual survival to release ranged from 20% to 62% and averaged 38% over the course of the study, the surviving reconditioned kelts showing increases in FL, weight, and Fultons K condition factor compared with their preconditioning status. Kelts in good condition and those with bright coloration at the time of collection were more likely to survive than th...

Regulation of growth hormone (GH) receptor (GHR1 and GHR2) mRNA level by GH and metabolic hormones in primary cultured tilapia hepatocytes

Growth hormone (GH) regulates essential physiological functions in teleost fishes, including growth, metabolism, and osmoregulation. Recent studies have identified two clades of putative receptors for GH (GHR1 clade and GHR2 clade) in fishes, both of which are highly expressed in the liver. Moreover, the liver is an important target for the anabolic effects of GH via endocrine IGFs, and liver sensitivity to GH is modulated by metabolic hormones. We investigated the effects of GH, insulin, glucagon, cortisol and triiodothyronine on GHR1 and GHR2 mRNA levels in primary cultured tilapia hepatocytes. Physiological concentrations of GH strongly stimulated GHR2 mRNA level (0.5-50×10(-9) M), but did not affect GHR1 mRNA level. Insulin suppressed stimulation of GHR2 mRNA level by GH (10(-8)-10(-6) M). Insulin increased basal GHR1 mRNA level (10(-8)-10(-6) M). Cortisol increased basal GHR2 mRNA level (10(-7)-10(-6) M), but did not consistently affect GH-stimulated GHR2 mRNA level. Cortisol increased basal GHR1 mRNA level (10(-9)-10(-6) M). Glucagon suppressed GH-stimulated GHR2 mRNA level and increased basal GHR1 mRNA level at a supraphysiological concentration (10(-6) M). A single injection of GH (5 μg/g) increased liver GHR2 mRNA level, and insulin injection (5 μg/g) decreased both basal and GH-stimulated GHR2 mRNA levels after 6 h. In contrast, insulin and GH injection had little effect on liver GHR1 mRNA level. This study shows that GHR1 and GHR2 gene expression are differentially regulated by physiological levels of GH and insulin in tilapia primary hepatocytes.