Immunomodulatory effects of prolactin and growth hormone in the tilapia,Oreochromis mossambicus

To clarify the roles of prolactin (PRL) and GH in the control of the immune system, the effects of environmental salinity, hypophysectomy, and PRL and GH administration on several immune functions were examined in tilapia (Oreochromis mossambicus). Transfer from fresh water (FW) to seawater (SW) did not alter plasma levels of immunoglobulin M (IgM) and lysozyme. The superoxide anion (O(2)(-)) production in head kidney leucocytes accompanied by phagocytosis was elevated in SW-acclimated fish over the levels observed in FW fish. Hypophysectomy of the fish in FW resulted in a reduction in O(2)(-) production in leucocytes isolated from the head kidney, whereas there was no significant change in plasma levels of IgM or lysozyme. Treatment with tilapia GH and PRLs (PRL(177) and PRL(188)) enhanced O(2)(-) production in vitro in head kidney leucocytes in a dose-related manner. Extrapituitary expression of two PRLs, GH and IGF-I mRNA was detected in lymphoid tissues and cells such as head kidney, spleen, intestine and leucocytes from peripheral blood and head kidney. PRL-receptor mRNA was detected in head kidney leucocytes, and the level of expression was higher in SW-acclimated fish than that in FW fish. Treatment with PRL(177) caused higher production of O(2)(-) in the head kidney leucocytes isolated from SW tilapia than that from FW fish. In view of the fact that PRL acts antagonistically to osmoregulation in SW, its immunomodulatory actions in this euryhaline fish would appear to be independent of its osmoregulatory action.     

In vitro release of growth hormone from the pituitary gland of tilapia, Oreochromis mossambicus

The release of growth hormone from the proximal pars distalis of the tilapia, Oreochromis mossambicus, was significantly stimulated by cortisol (1 microgram/ml) in an in vitro system. Growth hormone released into the medium and remaining in the tissue was measured by densitometry after gel electrophoresis. Neither triiodothyronine (6.7 ng/ml) nor equimolar concentrations of thyroxin altered the release of growth hormone. In combination with cortisol, triiodothyronine did not alter the effect of cortisol alone.     

Effects of angiotensin II and natriuretic peptides of the eel on prolactin and growth hormone release in the tilapia,Oreochromis mossambicus

The effects of angiotensin II (ANG II) and natriuretic peptides (NPs) of the eel (ANP, atrial natriuretic peptide; CNP, C-type natriuretic peptide; and VNP, ventricular natriuretic peptide) on prolactin (PRL(188) and PRL(177)) and growth hormone (GH) release from the organ-cultured tilapia pituitary were examined. Eel ANG II at concentrations greater than 1 nM stimulated the release of PRL(188) and PRL(177) in a dose-related manner during the first hour of incubation. Significant stimulation by 100 nM ANG II on PRL(177) release was observed until 4h of incubation, and on PRL(188) release until 12 h. No effect of ANG II was seen on GH release. None of the NPs altered the release of PRLs at any time point. On the other hand, eel VNP at concentrations greater than 1 nM stimulated GH release in a dose-related manner after 4 h, and significant stimulation was observed until 48 h. Eel CNP was less effective than eel VNP; significant stimulation of GH release was observed at 1 and 10 nM during 24-48 h of incubation. No significant effect of eel ANP on GH release was seen at any concentration. ANG II had no effect on GH release at any time point. There was no change in mRNA levels of PRLs or GH in the pituitaries incubated with ANG II for 8 h or those incubated with the NPs for 48 h. These results indicate rapid and short-lasting stimulation by ANG II on PRL release and slow and long-lasting stimulation by VNP and CNP on GH release from the tilapia pituitary.     

Effects of fasting on growth hormone, growth hormone receptor, and insulin-like growth factor-I axis in seawater-acclimated tilapia, Oreochromis mossambicus

Effects of fasting on the growth hormone (GH)--growth hormone receptor (GHR)-insulin-like growth factor-I (IGF-I) axis were characterized in seawater-acclimated tilapia (Oreochromis mossambicus). Fasting for 4 weeks resulted in significant reductions in body weight and specific growth rate. Plasma GH and pituitary GH mRNA levels were significantly elevated in fasted fish, whereas significant reductions were observed in plasma IGF-I and hepatic IGF-I mRNA levels. There was a significant negative correlation between plasma levels of GH and IGF-I in the fasted fish. No effect of fasting was observed on hepatic GHR mRNA levels. Plasma glucose levels were reduced significantly in fasted fish. The fact that fasting elicited increases in GH and decreases in IGF-I production without affecting GHR expression indicates a possible development of GH resistance.     

Effects of insulin-like growth factors (IGF-I and -II) on growth hormone and prolactin release and gene expression in euryhaline tilapia,Oreochromis mossambicus

We investigated in vitro effects of insulin-like growth factors (IGF-I and -II) on growth hormone (GH) and prolactin (PRL) release and gene expression in euryhaline tilapia, Oreochromis mossambicus. Pituitaries were removed from freshwater-acclimated adult males and incubated for 2-24h in the presence of human IGF-I or -II at doses ranging from 1-1000 ng/ml (0.13-130 nM). IGF-I at concentrations higher than 10 ng/ml and IGF-II higher than 100 ng/ml significantly inhibited GH release after 8, 16, and 24h. No effect of IGFs was seen during the first 4h of incubation. IGFs at the same concentrations also significantly attenuated GH gene expression after 24h, although no effect was seen at 2h. By contrast, PRL(188) release was stimulated significantly and in a dose-related manner by IGF-I at concentrations higher than 10 ng/ml and by IGF-II at concentrations higher than 100 ng/ml within 2h. No stimulation was observed after 4h. Similarly, both IGFs at concentrations higher than 10 ng/ml increased PRL(177) release within 2h. However, no significant effect of IGF-I or -II was observed on mRNA levels of both PRLs after 2 and 24h at all concentrations examined. These results clearly indicate differential regulation of GH and PRL release and synthesis by IGFs in the tilapia pituitary, i.e., rapid-acting, stimulatory effects of IGFs on PRL release and slow-acting, inhibitory effects on GH release and synthesis.     

Effects of somatostatin and urotensin II on tilapia pituitary prolactin release and interactions between somatostatin, osmotic pressure Ca++, and adenosine 3',5'-monophosphate in prolactin release in vitro

Both somatostatin (SRIF) and urotensin II, a dodecapeptide from the teleost caudal neurosecretory system, inhibit PRL release from the organ-cultured rostral pars distalis of the tilapia, Sarotherodon mossambicus, in a dose-related manner. The inhibitory action of SRIF on PRL release was completely prevented by the presence of the calcium ionophore A23187. PRL release was also blocked when Ca++ was excluded from the incubation medium, even in the presence of the ionophore. Both dibutyryl cAMP (dbcAMP) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, alone or in combination, stimulated PRL release during incubation in high osmotic pressure medium. The effect of dbcAMP appeared to be dose related. Together, dbcAMP and 3-isobutyl-1-methylxanthine were also effective in preventing the inhibition of PRL release by SRIF. These results are consistent with the notion that Ca++, and possibly cAMP, may be important mediators of PRL secretion, and it is likely that SRIF may inhibit PRL release by blocking a Ca++- or cAMP-mediated mechanism.     

Effects of fasting, medium glucose, and amino acid concentrations on prolactin and growth hormone release, in vitro, from the pituitary of the tilapia Oreochromis mossambicus.

Previous investigations have shown that the release of PRL and GH from the tilapia pituitary is directly sensitive to osmotic pressure and a variety of endocrine and neuroendocrine factors. The present studies were aimed at determining whether the spontaneous release of PRL and GH, in vitro, is: (1) sensitive to the nutritional status of the fish, and (2) responsive to variations in the D-glucose and total amino acid content of the incubation medium. In the first series of experiments, male fish (50 to 60 g) were divided into two groups. One group was fed twice daily for 2 weeks while the second received no food. A nearly homogeneous mass of PRL-secreting cells was dissected from the rostral pars distalis (RPD) and incubated for 18 to 20 hr in either hyposmotic (300 mOsmolal) or hyperosmotic (355 mOsmolal) medium. Similarly, a mass of GH-secreting cells was dissected from the proximal pars distalis (PPD) and incubated for 18 to 20 hr in isosmotic (320 mOsmolal) medium. Fasting was found to alter the total amount of PRL and GH in the culture well (tissue + medium) at the end of the incubations, decreasing PRL and increasing GH. Fasting was also found to both reduce spontaneous PRL release in vitro and suppress its stimulation by reduced osmotic pressure (P less than 0.01). By contrast, fasting resulted in a substantial increase in spontaneous GH release from the PPD in vitro (P less than 0.01). In the second series of experiments, GH release was found to increase as the D-glucose concentration of the medium decreased (P less than 0.01), while prolactin release was unresponsive.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro effects of homologous prolactins on testosterone production by testes of tilapia (Oreochromis mossambicus).

The tilapia, Oreochromis mossambicus, produces two prolactins designated tPRL177 and tPRL188 to indicate the number of amino acid residues in each. The direct in vitro actions of tPRL177 and tPRL188 on basal and ovine luteinizing hormone (LH)-induced testosterone production in minced testes of courting and noncourting (bachelor) tilapia were examined. Courting males were housed with females and were used as soon as they built a spawning pit; noncourting males were housed with other males and were used when they appeared female-like in coloration. The in vitro culture system consisted of two phases. In phase 1 (0-10 hr), testicular tissue was incubated without (control) or with the tPRLs--both alone and together and in doses ranging from 0.5 to 32 microM. In phase 2 (10-16 hr), the culture medium was replaced with medium containing 6.25 microM (0.25 micrograms/ml) ovine LH. Testosterone production during both phases was determined by radioimmunoassay of the medium. The tPRLs stimulated testosterone production during phase 1 in courting males (by 25%) but not in noncourting males. Exposure to either or both tPRLs in phase 1 enhanced the response of testicular tissue from courting males to ovine LH (by 10%) and inhibited the response of minced testes taken from noncourting males (by 12%). This is the first evidence to demonstrate direct gonadal activity of homologous PRL in a teleost.     

Studies on the regulation of growth hormone release from the proximal pars distalis of male tilapia, Oreochromis mossambicus, in vitro

The in vitro effects of several factors, including cortisol, somatostatin (SRIF), and medium osmotic pressure, on growth hormone (GH) release from the tilapia pituitary were examined in relation to fish size. Spontaneous GH release from the proximal pars distalis (PPD) of approximately 60-g fish was significantly less than that from tissue of fish weighing either approximately 120 or approximately 280 g when incubated in 340 m phi smolal medium. While GH content of the PPD cultures (tissue + medium measured by densitometry) increased consistently with fish size, GH concentration (per microgram of tissue protein) was variable, being highest in 120-g fish and lowest in 280-g fish. Moreover, GH concentration was not related to GH release. Fish size also appeared to be important in the responsiveness of GH cells to stimulation by cortisol (Nishioka et al., 1985) and by increased osmotic pressure. In cultures of PPD from approximately 60-g fish, in which spontaneous release was relatively low, cortisol and increased medium osmotic pressure significantly enhanced release. Cortisol and hyperosmotic medium were without significant effect, however, on GH release from PPD of approximately 120-g fish, which showed high spontaneous release. In contrast, SRIF, a potent inhibitor of GH secretion, was effective in lowering GH release regardless of fish size. Nevertheless, SRIF was apparently more effective in inhibiting GH release from tissue of 60-g fish than from tissue of 120-g fish. Our data suggest that GH secretion may be augmented when smaller tilapia (approximately 60 g) are transferred to seawater, a situation in which blood cortisol and osmotic pressure would presumably be elevated.     

Novel effect of vasoactive intestinal polypeptide and peptide histidine isoleucine: inhibition of in vitro secretion of prolactin in the tilapia, Oreochromis mossambicus

The effects of vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) on the in vitro secretion of two prolactins (PRL) from the rostral pars distalis (RPD) and of growth hormone (GH) from the proximal pars distalis (PPD) of the pituitary of the tilapia (Oreochromis mossambicus) were studied. RPDs were incubated for 20 hr in hypoosmotic (280-300 mOsm) or hyperosmotic (340-350 mOsm) Krebs-Ringer bicarbonate medium with added peptide concentrations of 0 (control), 0.3, 3.0, 30, and 300 nM; similarly, PPDs were incubated with the same peptide concentrations in isoosmotic (325 mOsm) medium supplemented with cortisol. PRL and GH in the tissue and secreted into the medium were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by soft laser densitometry of the protein band(s). Neither VIP nor PHI has a detectable effect on the secretion of GH. Secretion of the two PRLs is significantly inhibited by VIP and PHI in both hyperosmotic and hypoosmotic medium. In hyperosmotic medium, 300 nM VIP inhibits secretion of both PRLs by 47%, whereas in hypoosmotic medium, 300 nM VIP inhibits their secretion by 27%. PHI inhibits their secretion by ca. 65% in hyperosmotic medium and by 40% in hypoosmotic medium. There is preliminary immunocytochemical evidence for some VIP-like immunoreactivity (IR), but no conclusive indication of PHI-like IR, in the hypothalamo-hypophysial area. The inhibitory actions of VIP and PHI on PRL secretion in tilapia are in contrast to the known stimulatory actions of VIP and PHI on PRL secretion in tetrapods.     

Control of prolactin secretion in the teleost Oreochromis mossambicus: effects of water acidification

Prolactin secretion is stimulated markedly in fish exposed to water of pH 4. This phenomenon was used to study the control of prolactin secretion. Activation occurs irrespective of changes in plasma osmolarity or plasma sodium and calcium concentrations. After acute acidification of the water, which leads to a substantial fall in plasma osmolarity and plasma electrolyte levels, the activation of the prolactin cells is less marked than after gradual acidification of the water, when plasma osmolarity, plasma sodium, and plasma total and ionic calcium levels are not noticeably affected. When fish bearing an implanted rostral pars distalis of the pituitary gland are exposed to water of pH 4, both the in situ prolactin cells and the prolactin cells of the implant become activated only when the drop in water pH is acute and followed by a reduction in plasma osmolarity and electrolyte levels. When the rate of reduction of the pH is slow and not changing plasma osmolarity or sodium and calcium levels, the in situ prolactin cells are stimulated, but not those of the implants. We conclude that the activation of the prolactin cells in situ in fish in acid water is not mediated by reductions in plasma osmolarity, plasma sodium, total calcium, or ionic calcium, but by hypothalamic mechanisms. The drop in plasma osmolarity and electrolytes probably reflects excessive osmoregulatory stress and this may hamper instead of stimulate the response of the prolactin cells to osmoregulatory disturbance. The physiological significance of the in vitro activation of prolactin cells by reduced ambient osmolarity is unclear.     

Effects of salinity, hypophysectomy, and prolactin on whole-animal transepithelial potential in the tilapia Oreochromis mossambicus

We have examined whether two recently isolated forms of tilapia (Oreochromis mossambicus) prolactin exert similar effects on osmoregulatory physiology. The effects of salinity, hypophysectomy, and replacement therapy with tilapia prolactins on whole-animal transepithelial potential (TEP), gill Na+, K+-ATPase activity, and plasma ions were determined. When intact fish adapted to 25% seawater (SW) were transferred to different salinities, TEP reached a steady state after 10 hr; TEP increased with increasing salinity from fresh water (FW) to 75% SW but was stable from 75 to 125% SW. Plasma osmolality, [Na+], and [Cl-] of these fish 24 hr after salinity change showed that fish in 100 and 125% SW had greater osmotic perturbation than those transferred to lower salinities. Following a 5-day recovery period in 25% SW, hypophysectomized fish transferred to FW for 10 hr had significantly lower TEP and plasma ion levels than either sham-operated fish or intact fish under the same conditions. Injection of hypophysectomized fish with "small" prolactin (tPRL177), "large" prolactin (tPRL188), or a combination of both (0.5 micrograms/g body weight) 22 hr and again 20 min prior to transfer from 25% SW to FW, restored TEP and plasma ion levels to those of sham-operated fish. Neither prolactin affected the TEP or plasma ions of sham-operated (intact) fish. Hypophysectomized fish had lower gill Na+,K+-ATPase activity than sham-operated fish in FW, but prolactin injections as described above did not affect gill Na+,K+-ATPase activity in either hypophysectomized or sham-operated fish. Our results indicate that the two forms of prolactin are indistinguishable with regard to several aspects of tilapia osmoregulation.     

The effects of growth hormone, prolactin, corticotropin, and thyrotropin on the production and secretion of somatostatin by hypothalamic cells in vitro

The influence of GH and several other pituitary hormones on the secretion and intracellular content of immunoreactive somatostatin (IRS) in cultured hypothalamic cells was examined. Hypothalamic cells prepared from 17-day-old rat embryos and maintained as reaggregate monolayers for 12 days in vitro were used. Short term release of IRS from these cultures was found to be stable and relatively constant. IRS release was enhanced by 60 mM K+ in a calcium-dependent fashion and by dopamine at concentrations as low as 1 nM. GH (1 microM) increased medium IRS content above baseline 12, 24, and 48 h after the incubation was begun, but had no effect after only 4 h. Hypothalamic cell content of IRS was increased by a 24-h incubation in 0.3 and 1.0 microM GH, but not by 0.1 microM GH. TSH (1 microM) induced an increase in IRS release comparable to 1 microM GH. However, intracellular IRS content was decreased after exposure to TSH. Cosyntropin, an ACTH analog, inhibited both IRS release and cell content, whereas PRL had no effect on either medium or cellular IRS content. Our results demonstrate that developing somatostatinergic neurons in the hypothalamus have the capacity to respond to pituitary hormones, indicating that short-loop feedback pathways may exist in perinatal hypothalamic cells.     

Reciprocal interactions of somatostatin with thyrotropin-releasing hormone and vasoactive intestinal peptide on prolactin and growth hormone secretion in vitro

Reciprocal interactions of somatostatin (SRIF) and vasoactive intestinal peptide (VIP) or TRH on in vitro PRL and GH release from male rats hemipituitaries were investigated. SRIF did not modify basal PRL release, but TRH- or VIP-induced release was inhibited by SRIF in a dose-dependent manner [effective concentration-fifty (EC50) = 1.7 +/- 0.9 nM for SRIF inhibition of TRH stimulation and EC50 = 0.8 +/- 0.5 nM for SRIF inhibition of VIP stimulation]. VIP and TRH did not affect GH release by themselves, but reduced the inhibition of GH secretion elicited by SRIF (EC50 = 7.6 +/- 3.4 nM for TRH blockade of SRIF inhibition and EC50 = 4.6 +/- 3.1 nM for VIP blockade of SRIF inhibition). Secretin, a partial structural analog of VIP, also blocked SRIF-induced inhibition of GH and stimulated PRL release. Secretin stimulation of PRL release was also prevented by SRIF. [D-Trp8,D-Cys14]SRIF, a potent analog of SRIF, antagonized VIP stimulation of PRL secretion with the same apparent affinity as the native peptide. The maximal stimulation, but not the apparent affinity of VIP action on prolactin release was reduced by SRIF, suggesting that the interaction is of a noncompetitive nature. This conclusion as further substantiated by the observation that neither TRH nor VIP were able to displace specific 125I-labeled [Tyr1] SRIF high affinity binding to pituitary membranes. The three peptides tested thus appear to exhibit reciprocal interactions mediated by independent receptor sites on GH as well as on PRL-producing cells.     

Effects of estrogens in vitro and in vivo on cartilage growth in the tilapia (Oreochromis mossambicus)

To study the effects of estrogens on cartilage growth in the tilapia Oreochromis mossambicus, an epiceratobranchial cartilage radioisotope incorporation assay was employed to measure proteoglycan synthesis and prechondrocyte proliferation by incorporation of radiolabeled sulfate and thymidine, respectively. Cartilage explants were cultured with estrogens with or without recombinant bovine insulin-like growth factor-I (IGF-I). In vitro experiments using the natural teleost estrogen, 17beta-estradiol (E2), showed a trend toward inhibition of sulfate incorporation and an inhibition of thymidine incorporation at higher doses (10 micrograms/ml), but not at physiological levels. E2 also showed a trend toward inhibition of sulfate and thymidine incorporation in the presence of IGF-I. Similar results were found with other estrogenic compounds in vitro: ethinylestradiol, diethylstilbestrol (DES), genistein, and nonylphenol. Ethinylestradiol inhibited sulfate and thymidine incorporation at 1000 ng/ml in the presence of IGF-I. DES inhibited thymidine incorporation at 1000 ng/ml in untreated or IGF-I-exposed cartilage. Genistein inhibited sulfate incorporation at 100 micrograms/ml in IGF-I-exposed cartilage and inhibited thymidine uptake at 1, 10, and 100 micrograms/ml in untreated and IGF-I-exposed cartilage. Nonylphenol inhibited sulfate uptake at 100 microM in untreated and IGF-I-exposed cartilage. Nonylphenol alone at 10 and 100 microM inhibited thymidine uptake. In IGF-I-exposed cartilage nonylphenol inhibited thymidine uptake at 100 microM. Fish receiving estrogen injections (E2 or DES) in vivo at a concentration of 2 micrograms/g body weight showed increased sulfate incorporation by cartilage in vitro. Stimulation in vivo by estrogens, in contrast to the inhibition by high doses in vitro, may be a result of the influence of estrogen on pituitary growth hormone release.     

Effects of dopamine and somatostatin on phorbol ester-stimulated prolactin and growth hormone secretion

Incubation of cultured ovine pituitary cells with the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) (0.1-100 nM), caused a dose-related stimulation of both growth hormone (ED50 approximately 4 nM) and prolactin (ED50 approximately 14 nM) secretion. Stimulation by TPA (100 nM) produced a substantial 10-fold increase in growth hormone with a smaller, 2-fold rise in prolactin secretion over 30 min; significant effects on the release of both hormones occurred within 2 min. Treatment with TPA also produced a small, time- and concentration-dependent rise in cellular cyclic AMP content which reached, at maximum, a level 20-30% over basal values. Non-tumor-promoting phorbol esters did not stimulate the secretion of either growth hormone or prolactin. In the presence of TPA (10 nM), dopamine (1-1000 nM) suppressed prolactin secretion to a level close to that observed for maximal inhibition of unstimulated cells. At high concentrations (0.1-1.0 microM) dopamine also partially attenuated (by 43%) the TPA-induced stimulation of growth hormone secretion. Somatostatin (0.01-1.0 microM) completely inhibited the substantial (approximately 9-fold) TPA-induced stimulation of growth hormone secretion (inhibitory ED50 approximately 47 nM), and also suppressed TPA-stimulated prolactin secretion to the control level. Our results suggest that activation of protein kinase-C may be involved in the stimulatory regulation of both growth hormone and prolactin secretion in sheep pituitary cells. Failure of TPA to attenuate the inhibitory activity of dopamine and somatostatin suggests that inhibitory regulation occurs at, or beyond, the point in the secretory process regulated by protein kinase-C.     

Growth hormone binding sites in tilapia (Oreochromis mossambicus) liver.

125I-labeled bovine and tilapia growth hormones were used to assess the presence of growth hormone receptors in membranes prepared from tissues of the tilapia Oreochromis mossambicus. The highest level of specific binding was detected in liver membranes from animals of both sexes and the binding was protein-dependent. Tilapia growth hormone, bovine growth hormone, and ovine prolactin, but not tilapia prolactin, potently inhibited the hepatic binding of 125I-labeled bovine growth hormone. Scatchard analysis of the 125I-labeled bovine growth hormone binding data revealed a Bmax (maximum binding) value of 180 fmol/mg protein and a Kd (dissociation constant) value of 13 nM. Tilapia growth hormone potently inhibited hepatic binding of 125I-labeled tilapia growth hormone. Scatchard analysis revealed a single class of binding sites with Bmax and Kd values of 390 fmol/mg protein and 2.5 nM, respectively. Bovine growth hormone and ovine prolactin were less potent while tilapia prolactin was inactive in inhibiting hepatic 125I-labeled tilapia growth hormone binding.