Warmer waters masculinize wild populations of a fish with temperature-dependent sex determination.

Southern flounder (Paralichthys lethostigma) exhibit environmental sex determination (ESD), where environmental factors can influence phenotypic sex during early juvenile development but only in the presumed XX female genotype. Warm and cold temperatures masculinize fish with mid-range conditions producing at most 50% females. Due to sexually dimorphic growth, southern flounder fisheries are dependent upon larger females. Wild populations could be at risk of masculinization from ESD due to globally increasing water temperatures. We evaluated the effects of habitat and temperature on wild populations of juvenile southern flounder in North Carolina, USA. While northern habitats averaged temperatures near 23 °C and produced the greatest proportion of females, more southerly habitats exhibited warmer temperatures (>27 °C) and consistently produced male-biased sex ratios (up to 94% male). Rearing flounder in the laboratory under temperature regimes mimicking those of natural habitats recapitulated sex ratio differences observed across the wild populations, providing strong evidence that temperature is a key factor influencing sex ratios in nursery habitats. These studies provide evidence of habitat conditions interacting with ESD to affect a key demographic parameter in an economically important fishery. The temperature ranges that yield male-biased sex ratios are within the scope of predicted increases in ocean temperature under climate change.

Endocrine regulation of prolactin cell function and modulation of osmoreception in the Mozambique tilapia.

Prolactin (PRL) cells of the Mozambique tilapia, Oreochromis mossambicus, are osmoreceptors by virtue of their intrinsic osmosensitivity coupled with their ability to directly regulate hydromineral homeostasis through the actions of PRL. Layered upon this fundamental osmotic reflex is an array of endocrine control of PRL synthesis and secretion. Consistent with its role in fresh water (FW) osmoregulation, PRL release in tilapia increases as extracellular osmolality decreases. The hyposmotically-induced release of PRL can be enhanced or attenuated by a variety of hormones. Prolactin release has been shown to be stimulated by gonadotropin-releasing hormone (GnRH), 17-ß-estradiol (E2), testosterone (T), thyrotropin-releasing hormone (TRH), atrial natriuretic peptide (ANP), brain-natriuretic peptide (BNP), C-type natriuretic peptide (CNP), ventricular natriuretic peptide (VNP), PRL-releasing peptide (PrRP), angiotensin II (ANG II), leptin, insulin-like growth factors (IGFs), ghrelin, and inhibited by somatostatin (SS), urotensin-II (U-II), dopamine, cortisol, ouabain and vasoactive intestinal peptide (VIP). This review is aimed at providing an overview of the hypothalamic and extra-hypothalamic hormones that regulate PRL release in euryhaline Mozambique tilapia, particularly in the context on how they may modulate osmoreception, and mediate the multifunctional actions of PRL. Also considered are the signal transduction pathways through which these secretagogues regulate PRL cell function.

Involvement of phospholipase C and intracellular calcium signaling in the gonadotropin-releasing hormone regulation of prolactin release from lactotrophs of tilapia (Oreochromis mossambicus).

Gonadotropin-releasing hormone (GnRH) is a potent stimulator of prolactin (PRL) secretion in various vertebrates including the tilapia, Oreochromis mossambicus. The mechanism by which GnRH regulates lactotroph cell function is poorly understood. Using the advantageous characteristics of the teleost pituitary gland from which a nearly pure population of PRL cells can be isolated, we examined whether GnRH might stimulate PRL release through an increase in phospholipase C (PLC), inositol triphosphate (IP3), and intracellular calcium (Ca(i)2+) signaling. Using Ca(i)2+ imaging and the calcium-sensitive dye fura-2, we found that chicken GnRH-II (cGnRH-II) induced a rapid dose-dependent increase in Ca(i)2+ in dispersed tilapia lactotrophs. The Ca(i)2+ signal was abolished by U-73122, an inhibitor of PLC-dependent phosphoinositide hydrolysis. Correspondingly, cGnRH-II-induced tPRL188 secretion was inhibited by U-73122, suggesting that activation of PLC mediates cGnRH-II's stimulatory effect on PRL secretion. Pretreatment with 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of Ca2+ release from intracellular stores, impeded the effect of cGnRH-II on Ca(i)2+. To further address the possible involvement of intracellular Ca2+ stores, IP3 concentrations in the tilapia rostral pars distalis (RPD containing 95-99% PRL cells) was determined by a radioreceptor assay. We found that GnRH-II induces a rapid (<5min) and sustained increase in IP3 concentration in the RPD. Secretion of tPRL(188) in response to cGnRH-II was suppressed by Ca2+ antagonists (TMB-8 and nifedipine). These data, along with our previous findings that show PRL release increases with a rise in Ca(i)2+, suggest that GnRH may elicit its PRL releasing effect by increasing Ca(i)2+. Furthermore, the rise in Ca(i)2+ may be derived from PLC/IP3-induced mobilization of Ca2+ from intracellular stores along with influx through L-type voltage-gated Ca2+ channels.

Insulin-like growth factor-I augments prolactin and inhibits growth hormone release through distinct as well as overlapping cellular signaling pathways.

We recently discovered a new role for insulin-like growth factor-I (IGF-I) as a specific and direct stimulator of prolactin (PRL) release in addition to its recognized function as an inhibitor of growth hormone (GH) release and synthesis. Little is known of the mechanisms that transduce the actions of IGF-I on PRL and GH release in vertebrates. The present study was undertaken to determine the cellular pathways that mediate the disparate actions of IGF-I on PRL and GH release in hybrid striped bass (Morone saxatilis X M. chrysops). When regulating cellular function, IGF-I may activate two primary pathways, phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase (MAPK). The specific MAPK inhibitor, PD98059, blocked IGF-I-evoked PRL release as well as GH release inhibition over an 18-20-h incubation. LY294002, a specific PI 3-K inhibitor, overcame IGF-I's inhibition of GH release but was ineffective in blocking PRL release stimulated by IGF-I. These studies suggest IGF-I disparately alters PRL and GH by activating distinct as well as overlapping signaling pathways central for mediating actions of growth factors on secretory activity as well as cell proliferation. These results further support a role for IGF-I as a physiological regulator of PRL and GH.

Cortisol suppresses prolactin release through a non-genomic mechanism involving interactions with the plasma membrane.

In the classical theory of steroid hormone action, steroids diffuse through the membrane and alter transcription of specific genes resulting in synthesis of proteins important for modulating cell function. Most often, steroids work solely through the genome to exert their physiological actions in a process that normally takes hours or days to occur. In tilapia (Oreochromis mossambicus), cortisol inhibits prolactin (PRL) release within 10-20 min in vitro. This action is accompanied by similarly rapid reductions in cellular Ca(2+) and cAMP levels, second messengers known to transduce the membrane effects of peptide hormones. We further examined whether cortisol might inhibit PRL release through a non-genomic, membrane-associated mechanism using the protein synthesis inhibitor, cycloheximide, and a membrane impermeant form of cortisol, cortisol-21 hemisuccinate BSA (HEF/BSA). Cycloheximide (2 and 10 microg/ml) was ineffective in overcoming PRL release induced by hyposmotic medium or that inhibited by cortisol over 4 h static incubations. These dosages reduced protein synthesis as measured by amino acid incorporation in pituitaries by 75 and 99%, respectively. During 4-h incubation, HEF/BSA and HEF significantly reduced PRL release in a dose-dependent fashion. These studies suggest that cortisol inhibits PRL release through a plasma membrane-associated, protein-synthesis independent (non-genomic) pathway.

Nongenomic membrane actions of glucocorticoids in vertebrates.

For decades, it was widely assumed that glucocorticoids (GCs) work solely through changes in gene expression to exert their physiological actions, a process that normally takes several hours to occur. However, recent evidence indicates that GCs might also act at the membrane through specific receptors to exert multiple rapid effects on various tissues and cells. GCs modulate hormone secretion, neuronal excitability, behavior, cell morphology, carbohydrate metabolism and other processes within seconds or minutes. These early actions occur independent of the genome and are transduced by the same biochemical effector pathways responsible for mediating rapid responses to neurotransmitters. The biological significance of most rapid GC effects are not well understood, but many might be related to the important functions that this hormone plays in modulating stress responses.

Insulin-like growth factor I disparately regulates prolactin and growth hormone synthesis and secretion: studies using the teleost pituitary model.

Although insulin-like growth factor I (IGF-I)'s inhibition of GH release is well documented, little is known of its control of GH synthesis at the posttranscriptional level. The manner by which IGF-I alters PRL synthesis and secretion is also unclear. This study was undertaken to examine the role IGF-I plays in regulating in vitro PRL and GH synthesis and release using the teleost pituitary model system. This model allows for isolation of nearly homogenous populations of distinct pituitary cell types that can be cultured in a completely defined, hormone-free medium. Tissues containing PRL cells and those consisting of GH cells were dissected from pituitaries of hybrid striped bass and exposed to varying concentrations of IGF-I, IGF-II, and insulin for 18-20 h. Exposure to graded doses of IGF-I markedly stimulated fractional, total, and newly synthesized PRL release in a dose-dependent fashion (ED50 for fractional release, 35 ng/ml or 4.6 nM; P < 0.0001). IGF-II and insulin also increased PRL release, but only at 10-fold higher concentrations than the lowest effective IGF-I dose. The total PRL content in the incubations and PRL synthesis, as measured by [35S]methionine incorporation, were not altered by IGF-I. By contrast, IGF-I potently reduced GH release (ED50, 29 ng/ml or 3.8 nM; P < 0.0001) and synthesis. Both 100 and 1000 ng/ml IGF-I decreased newly synthesized GH and total GH content (P < 0.001). Insulin and IGF-II mimicked IGF's action in attenuating GH release, but only at 10- to 11-fold higher concentrations. Taken together, these findings clearly indicate that IGF-I disparately regulates PRL and GH synthesis and secretion. We show that the effects of IGF-I on pituitary hormone release occur in a variety of species, suggesting that its actions are well conserved. The inhibition of GH release and synthesis by IGF-I probably reflects a negative feedback loop for maintaining tight control over GH cell function. These findings further indicate that IGF-I is a potent and specific secretagogue of PRL release in vertebrates.

Induction of growth hormone (GH) mRNA by pulsatile GH-releasing hormone in rats is pattern specific.

Growth hormone-releasing hormone (GHRH) is a main inducer of growth hormone (GH) pulses in most species studied to date. There is no information regarding the pattern of GHRH secretion as a regulator of GH gene expression. We investigated the roles of the parameters of exogenous GHRH administration (frequency, amplitude, and total amount) upon induction of pituitary GH mRNA, GH content, and somatic growth in the female rat. Continuous GHRH infusions were ineffective in altering GH mRNA levels, GH stores, or weight gain. Changing GHRH pulse amplitude between 4, 8, and 16 microg/kg at a constant frequency (Q3.0 h) was only moderately effective in augmenting GH mRNA levels, whereas the 8 microg/kg and 16 microg/kg dosages stimulated weight gain by as much as 60%. When given at a 1.5-h frequency, GHRH doubled the amount of GH mRNA, elevated pituitary GH stores, and stimulated body weight gain. In the rat model, pulsatile but not continuous GHRH administration is effective in inducing pituitary GH mRNA and GH content as well as somatic growth. These studies suggest that the greater growth rate, pituitary mRNA levels, and GH stores seen in male compared with female rats are likely mediated, in part, by the endogenous episodic GHRH secretory pattern present in males.

Regulation of somatic growth and the somatotropic axis by gonadal steroids: primary effect on insulin-like growth factor I gene expression and secretion.

The site-specific regulation of somatic growth by sex steroids is poorly understood. The aim of the present study was to assess the influence of 17 beta-estradiol (E2) and 5 alpha-dihydrotestosterone (DHT) on somatic growth and pituitary GH and hepatic insulin-like growth factor I (IGF-I) secretion and synthesis in the adult female rat. Animals (200-250 g) underwent sham surgery or bilateral ovariectomy. Some ovariectomized (OVX) rats were given sc implants that provided almost physiological female E2 (OVX/E2) and male DHT (OVX/DHT) levels. Animals were killed 3, 7, 14, and 26 days later. Body weight gain was calculated, and pituitary GH content, pituitary GH messenger RNA (mRNA) levels, plasma GH, and circulating IGF-I concentrations were measured. Levels of hepatic IGF-I mRNA were measured at 26 days. Ovariectomy increased body weight gain (P < 0.001) in parallel with a significant elevation in plasma IGF-I (P < 0.001). Replacement of E2 markedly suppressed somatic growth (P < 0.001), plasma IGF-I concentrations (P < 0.001), and liver IGF-I gene expression (P < 0.002). However, circulating GH concentrations were high in OVX/E2 animals (P < 0.001), whereas pituitary GH stores were significantly attenuated (P < 0.05). In contrast, DHT exposure increased body weight gain (P < 0.001), circulating IGF-I concentrations (P < 0.05), and steady state hepatic IGF-I mRNA levels (P < 0.05). Pituitary GH stores were markedly elevated (P < 0.001) in DHT-treated animals, but circulating GH levels remained very low. Pituitary GH mRNA rose transiently at 7 days in OVX and OVX/E2 rats, but no consistent changes between the groups were observed thereafter. We conclude that 1) gonadal steroids have disparate effects on somatic growth in female rats, with E2 suppressing and DHT stimulating body weight gain; 2) these effects are likely to be primarily mediated at the level of IGF-I synthesis and secretion; and 3) changes in pituitary GH content and secretion probably reflect normal adjustment to changes in the intensity of IGF-I negative feedback.

Upregulation of GH receptor and GH binding protein during pregnancy in the GH deficient rat.

During pregnancy there are dramatic changes in the endocrine and metabolic status of the mother: growth hormone (GH) is an important regulator of growth and development. A proportion of GH is bound by specific GH binding proteins (GHBP) that closely resemble the GH receptor (GHR). In the rodent both GHBP and the GHR are considered to be GH dependent, and consequently during pregnancy the increase in serum GH is associated with an increase in GHBP. To examine whether an increase in maternal GH is obligatory for elevation of maternal GHBP or GHR during pregnancy, we used a unique GH-deficient (GHD) strain of rats, to avoid the methodological complications of hypophysectomy and assessed serum GH, GHBP and hepatic GHR binding during the course of pregnancy. In GH normal rats, serum GH concentrations increased twofold and GHBP levels increased threefold; there was no change in hepatic GHR binding. In GHD rats, serum GH concentrations were low and did not increase during pregnancy. Nonetheless, levels of both serum GHBP and hepatic GHR binding increased to that measured in normal rats. Thus, an increase in maternal GH concentration is not required for the gestational upregulation of maternal GHBP or hepatic GHR binding, suggesting that other hormones may be essential in modulating the GH axis during pregnancy.

Effects of environmental salinity on pituitary growth hormone content and cell activity in the euryhaline tilapia, Oreochromis mossambicus.

Studies were undertaken to determine whether several indicators of growth hormone (GH) cell activity, namely GH content, fine structure, and volume of the GH region, differ in the pituitaries of freshwater (FW) and seawater (SW) tilapia, Oreochromis mossambicus. Tilapia raised from the stage of yolk-sac absorption for 7 months in SW contain significantly more GH in their pituitaries than in those of fish reared in FW. Pituitary growth hormone content in tilapia raised in FW for 7 months and transferred to SW for 49 days is greater than that in sibling tilapia retained in FW. Conversely, GH content is significantly lower in the pituitaries of SW-reared tilapia transferred to FW for 49 days than that in the pituitaries from fish retained in SW. Likewise, the volume of the GH region and activity of the GH cells are enhanced in pituitaries from SW-reared tilapia over that seen in pituitaries from FW fish. Taken together, all data indicate heightened GH cell activity in SW-raised tilapia and suggest that GH may play a causal role in the greater growth rates observed in SW tilapia compared to FW fish and/or that GH may be involved in SW osmoregulation. The latter suggestion is supported, in part, by our observation that in vivo oGH treatment (2 micrograms/g body wt) stimulated gill Na+,K(+)-ATPase activity.

Effects of osmotic pressure and somatostatin on the cAMP messenger system of the osmosensitive prolactin cell of a teleost fish, the tilapia (Oreochromis mossambicus).

Altered osmotic pressure and somatostatin (SRIF) rapidly alter prolactin (PRL) release from the pituitary gland of the euryhaline teleost, the tilapia. The present studies were undertaken to determine whether altered osmotic pressure and SRIF influence cAMP metabolism in a manner that is correlated with the pattern of PRL release observed previously. Although PRL release is stimulated within 10-20 min when medium osmotic pressure is reduced, cAMP metabolism was not altered. However, following 1 hr of incubation in the presence of IBMX, cAMP accumulation was higher in PRL tissue exposed to medium of reduced osmotic pressure. This suggests that cAMP does not initiate an increase in PRL release in response to reduced osmotic pressure. By contrast, SRIF reduced the forskolin-stimulated increase in cAMP levels in a manner consistent with its rapid effects on PRL release. Moreover, the ability of SRIF to suppress the forskolin-stimulated increase in cAMP levels suggests that SRIF may act to render adenylate cyclase less responsive to direct stimulation by forskolin.

Cortisol rapidly reduces prolactin release and cAMP and 45Ca2+ accumulation in the cichlid fish pituitary in vitro.

During in vitro incubation, prolactin release is inhibited in a dose-related manner by cortisol. This action is mimicked by the synthetic glucocorticoid agonist dexamethasone but not by other steroids tested. Perifusion studies indicate that the inhibition of [3H]prolactin release by cortisol occurs within 20 min. Cortisol (50 nM) also inhibits cAMP accumulation and reduces 45Ca2+ accumulation in the tilapia rostral pars distalis within 15 min. Cortisol's action on prolactin release is blocked in the presence of either the Ca2+ ionophore A23187 or a combination of dibutyryl cAMP and 3-isobutyl-1-methylxanthine, which increase intracellular Ca2+ and cAMP, respectively. Taken together, these findings suggest that cortisol may play a physiologically relevant role in the rapid modulation of prolactin secretion in vivo. Our studies also suggest that the inhibition of prolactin release by cortisol is a specific glucocorticoid action that may be mediated, in part, through cortisol's ability to inhibit intracellular cAMP and Ca2+ metabolism.