E. Gordon Grau

Metabolic Responses Associated with Confinement Stress in Tilapia: The Role of Cortisol

We examined the effect of 2- or 24-hr confinement stress and cortisol treatment on plasma cortisol, glucose, lactate and free amino acids concentration and hepatic glycogen content and activities of certain enzymes involved in the intermediary metabolism in tilapia (Oreochromis mossambicus). Confinement of tilapia for 2 or 24 hr resulted in significantly higher plasma cortisol, glucose, lactate (2 hr), total and some of the free amino acids concentration (especially at 24 hr) and hepatic pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK; 24 hr) and lactate dehydrogenase activities (24 hr). Hepatic glycogen content was lower at 2 and 24 hr in the confined fish compared with the unstressed fish. Tilapia given cortisol implants (50 mg·kg−1 body wt) had significantly higher plasma cortisol, glucose and some of the free amino acids concentration and hepatic PEPCK and aspartate aminotransferase activities, whereas PK activity ratio was significantly lower compared with the sham group. The results suggest that glucose production 2 hr after confinement may be due to glycogenolysis, whereas the maintenance of higher glucose at 24 hr after confinement is essentially due to gluconeogenesis. Furthermore, the changes in plasma metabolites and hepatic enzyme activities with cortisol implantation suggest that cortisol plays a role in the metabolic adjustment to 24-hr confinement stress in tilapia.

Histological and ultrastructural evidence for the role of gonadal steroid hormones in sex change in the protogynous wrasse Thalassoma duperrey

SynopsisThe process of sex change in the protogynous wrasse, Thalassoma duperrey, was investigated through histological and ultrastructural observations on the gonads of females changing sex to male. Changes in plasma steroid levels concomitant with structural changes were measured by radioimmunoassay. The process of sex change from ovary to testis was divided into six stages on the basis of changes in the structure of the germinal and somatic elements. Ovaries of females were filled with vitellogenic oocytes during the breeding season, but contained no spermatogenic tissue (Stage 1). At the commencement of sex change (Stage 2), vitellogenic oocytes began to degenerate, and were ingested by macrophagous cells. This stage was accompanied by a rapid drop in plasma levels of estradiol-17β. Thereafter, previtellogenic oocytes (Stage 3) also began to degenerate, and aggregations of stromal tissue, and loose connective tissue were observed in the central region of the lamellae. Steroid producing cells (Leydig cells), developed at the border of this loose connective tissue. Presumed spermatogonia proliferated on the periphery of the lamellae, and Leydig cells increased in size and number (Stage 4). Spermatogonia formed cysts, and underwent spermatogenesis (Stage 5). Finally, sex change to male was considered complete, with the beginning of active spermatogenesis and spermiation (Stage 6). Plasma levels of testosterone remained low throughout the sex change, but a second androgen, 11-ketotestosterone increased gradually in parallel to the increased numbers of Leydig cells and spermatogonia. Preliminary in vitro incubation of gonads with salmon gonadotropin, revealed that sex-changed males had higher levels of 11-ketotestosterone production than did females, while females had higher levels of estradiol-17β production than did males. Production of both these steroids increased in a dose-related fashion with increasing doses of gonadotropin.

Identification of tilapia ghrelin and its effects on growth hormone and prolactin release in the tilapia, Oreochromis mossambicus.

We have identified ghrelin and cDNA encoding precursor protein from the stomach of a euryhaline tilapia, Oreochromis mossambicus. The sequence of 20-amino acid tilapia ghrelin is GSSFLSPSQKPQNKVKSSRI. The third serine residue was modified by n-decanoic acid. The carboxyl-terminal end of the peptide possessed an amide structure. RT-PCR analysis revealed high levels of gene expression in the stomach and low levels in the brain, kidney and gill. Tilapia ghrelin stimulated growth hormone (GH) and prolactin (PRL) release from the organ-cultured tilapia pituitary at a dose of 10 nM. Thus, a novel regulatory mechanism of GH secretion by gastric ghrelin seems to be conserved in the tilapia. Stimulation of PRL release by homologous ghrelin has been reported in human, bullfrog and eel, and suggests the presence of growth hormone secretagogue receptor not only on somatotrophs but also on PRL cells of the tilapia pituitary.

Physiological and respiratory responses of the Mozambique tilapia (Oreochromis mossambicus) to salinity acclimation

Abstract We have examined several physiological variables related to salinity acclimation in the euryhaline tilapia, Oreochromis mossambicus. Tilapia reared in fresh water (FW) were transferred to FW, isosmotic salinity (ISO, 12‰) and 75% seawater (SW, 25‰). Oxygen consumption, plasma levels of cortisol, growth hormone (GH), prolactins (tPRL177 and tPRL188), glucose, ions (Na+, K+, Cl−), and gill Na+,K+-ATPase activities were measured for up to 4 days in each salinity treatment. Plasma Na+ and Cl− concentrations were elevated 1 day after transfer to SW, but returned to FW values on day 4. Plasma cortisol and glucose levels were higher in FW and ISO than in SW 1 day after transfer. Plasma GH levels of tilapia in SW increased above FW and ISO values after 4 days, whereas plasma PRL levels decreased in ISO and SW compared to FW at 4 days. These results are consistent with the possible osmoregulation roles of GH and PRL in SW and FW, respectively. Gill Na+,K+-ATPase activity of tilapia in SW increased more than 2-fold over the FW value after 4 days, but activity of this enzyme did not change in ISO. Oxygen consumption rates of tilapia in SW were significantly elevated 4 days after transfer compared to FW and ISO. The results of this study indicate that the physiological changes associated with SW acclimation in tilapia represents a significant short-term energetic cost, and may account for as much as 20% of total body metabolism after 4 days in SW.

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.

Estradiol-17β and thyrotropin-releasing hormone stimulate prolactin release from the pituitary gland of a teleost fish in vitro

The effects of estradiol-17 beta (E2) and thyrotropin-releasing hormone (TRH) on prolactin (PRL) release were investigated using the organ-cultured rostral pars distalis (RPD) of the tilapia, Oreochromis mossambicus. Spontaneous PRL release into hyperosmotic medium increased in a dose-related manner following E2 pretreatment in vitro. In addition, TRH stimulated a dose-related increase in PRL release from E2-preincubated RPDs, but had no effect on tissues not previously exposed to E2. The maximal PRL response, nearly three times control levels, occurred at 50 nM TRH. Higher doses of TRH were less effective in stimulating PRL release. These findings indicate that TRH may be an important hypothalamic prolactin-releasing factor in the tilapia. Furthermore, the marked potentiation of the action of TRH on PRL release following exposure to E2 suggest that there may be a shift in the control of PRL secretion with changes in the reproductive state of the tilapia.

Histology, ultrastructure, and In vitro steroidogenesis of the testes of two male phenotypes of the protogynous fish, Thalassoma duperrey (labridae)

Species with multiple male reproductive phenotypes may serve as model systems to study the relationship between form and function in reproduction. Large and small males of the protogynous wrasse, Thalassoma duperrey differ in reproductive behavior, gonad morphology, and gonadal steroid production. Initial-phase (IP) males are small males that spawn in groups. They have large testes with high sperm production. Terminal-phase (TP) males are large, defend temporary spawning territories, and spawn individually with females. TP males are derived from either IP males or from sex-changed females. Regardless of origin, TP males have much smaller testes than do IP males, but steroid-producing Leydig cells in the gonads of TP males appear more numerous and better developed. Testes of TP males produce more testosterone (T) and especially 11-ketotestosterone (11-KT) in vitro than do testes of IP males, and the production is more responsive to salmon gonadotropin. 11-KT was the major metabolite produced by incubating the gonads of TP males with 14C-labeled steroid precursors. In vitro 11-KT production was correlated with plasma levels of 11-KT in TP males and these levels were significantly higher than those of IP males. The in vitro conversion of 17 alpha-hydroxyprogesterone to 17 alpha, 20 beta-progestogen (17 alpha, 20 beta-P) for both types of males was similar, and was highest in winter when spawning occurred every day. Basal production of 17 alpha, 20 beta-P was similar in IP and TP male testes, and was enhanced by gonadotropin. The enzyme 20 beta-hydroxysteroid dehydrogenase, responsible for the conversion of 17 alpha-hydroxyprogesterone to 17 alpha, 20 beta-P resided in the sperm. These results indicate a function of 17 alpha, 20 beta-P in male reproductive function, probably spermiation, and a relationship of Leydig cell development and high levels of 11-KT production to the terminal male phenotype, perhaps reproductive or aggressive behavior, rather than to male gametogenesis per se.

Rat Ghrelin Stimulates Growth Hormone and Prolactin Release in the Tilapia, Oreochromis mossambicus

Abstract Recently, ghrelin (Ghr), a new peptide which specifically stimulates growth hormone (GH) release from the pituitary, was identified in the rat and human stomach. Ghrelin has been shown to stimulate GH release by acting through a growth hormone secretagogue (GHS) receptor in the rat. The present study describes the in vitro effect of rat Ghr on the release of GH and two forms of prolactin (PRL177 and PRL188) in the tilapia, Oreochromis mossambicus. Rat Ghr stimulated the release of GH in a dose-related manner after 8 and 24 hr of incubation. Rat Ghr also significantly stimulated the release of PRL177 and PRL188 in a dose-related manner after 24 hr. Rat Ghr had no effect on the pituitary content of GH or PRL188, but significantly increased PRL177 content. These results show for the first time that rat Ghr significantly stimulates GH and PRL release in teleosts, and suggest that Ghr and a GHS receptor are present in fish.

Effect of 17α-methyltestosterone on the growth of the euryhaline tilapia, Oreochromis mossambicus, in fresh water and in sea water

Abstract These studies were aimed at determining whether: (1) rearing salinity influenced the growth of the tilapia, Oreochromis mossambicus , and (2) long-term 17α-methyltestosterone (MT) treatment of tilapia enhanced growth beyond that produced by masculinization alone. These studies showed that tilapia raised in sea water (34–36 ppt) grew to twice the size attained by freshwater fish under the conditions of our experiment ( P P P These findings suggest that continuous treatment with MT offers considerable advantage over sex reversal alone in the commercial culture of the tilapia. Overall, the greatest growth was observed in seawater-reared tilapia which were continuously treated with MT and, these fish were 5–7-fold larger than freshwater controls. The practical applicability of the findings from our tank investigations was evaluated in full-scale pond studies in which tilapia were cultivated in freshwater polyculture with common carp ( Cyprinus carpio ), grass carp ( Ctenopharyngdon idellus ), mud carp ( Cirrhina molitorella ), silver carp ( Hypophthalmichthys molitrix ), and bighead carp ( Aristichthys nobilis ). The administration of 17α-methyltestosterone consistently and significantly augmented the growth of tilapia (22%) and the carp species as well.

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.