Tilapia Pituitary Research Articles

Characterization of tilapia FSHβ gene and analysis of its 5′ flanking region

The objective of the current study was to unveil molecular mechanisms underlying transcriptional regulation of the FSHβ gene expression in the pituitary of tilapia (Oreochromis mossambicus). The full-length sequence of tilapia FSHβ (tFSHβ) gene was determined. Its transcriptional unit (2.7 kb) exhibits the conserved genomic organization, i.e. three exons and two introns. Primer extension and RT-PCR analysis revealed heterogeneity of the tFSHβ transcripts, due to alternate mRNA splicing and multiple initiation sites for transcription. Examination of the 5′ flanking region (5′FR) of the tFSHβ gene identified potential CAAT and TATA promoter proximal elements as well as several sequences of cis-acting motifs known to dictate inducible and tissue-specific transcriptional regulation in other gonadotropin genes. Chimeric constructs containing 1.7 kb of the tFSHβ 5′FR fused to a luciferase (LUC) reporter gene were transiently transfected into primary culture of tilapia pituitary cells. The tFSHβ-LUC construct was efficiently expressed under basal conditions and was rapidly induced by GnRH stimulation. Our data indicate that the 5′FR contains a functional promoter, which is responsive to GnRH treatment. In addition, 5′ deletion analysis showed that the 1.7 kb, DNA sequence of the FSHβ 5′FR encompasses both positive and negative regulatory elements.

Oreochromis mossambicus (tilapia) corticotropin-releasing hormone: cDNA sequence and bioactivity.

Although hypothalamic corticotropin-releasing hormone (CRH) is involved in the stress response in all vertebrate groups, only a limited number of studies on this neuroendocrine peptide deals with non-mammalian neuroendocrine systems. We determined the cDNA sequence of the CRH precursor of the teleost Oreochromis mossambicus (tilapia) and studied the biological potency of the CRH peptide in a homologous teleost bioassay. Polymerase chain reaction (PCR) with degenerate and specific primers yielded fragments of tilapia CRH cDNA. Full-length CRH cDNA (988 nucleotides) was obtained by screening a tilapia hypothalamus cDNA library with the tilapia CRH PCR products. The precursor sequence (167 amino acids) contains a signal peptide, the CRH peptide and a motif conserved among all vertebrate CRH precursors. Tilapia CRH (41 aa) displays between 63% and 80% amino acid sequence identity to CRH from other vertebrates, whereas the degree of identity to members of the urotensin I/urocortin lineage is considerably lower. In a phylogenetic tree, based on alignment of all full CRH peptide precursors presently known, the three teleost CRH precursors (tilapia; sockeye salmon, Oncorhynchus nerka; white sucker, Catostomus commersoni) form a monophyletic group distinct from amphibian and mammalian precursors. Despite the differences between the primary structures of tilapia and rat CRH, maximally effective concentrations of tilapia and rat CRH were equally potent in stimulating adrenocorticotropic hormone (ACTH) and alpha-MSH release by tilapia pituitaries in vitro. The tilapia and salmon CRH sequences show that more variation exists between orthologous vertebrate CRH structures, and teleost CRHs in particular than previously recognized. Whether the structural differences reflect different mechanisms of action of this peptide in the stress response remains to be investigated.

Calcium Ionophores Lead to Apoptotic-like Changes in Tilapia Pituitary Cells

Influx or mobilization of Ca2+plays an important part in the signal transduction mechanisms regulating release of gonadotropin (GtH) and growth hormone (GH) in teleost fish. In mammals it may also mediate a stimulatory effect on the transcription of the genes encoding these hormones (i.e., LHβ, FSHβ, and GH). In the present study, exposure of tilapia pituitary cells in primary culture to two ionophores, A23187 and ionomycin, increased GtH and GH secretion over 5–24 h but led to a significant drop in mRNA levels of GtH IIβ and GH. The mRNA levels of β actin were also reduced by this treatment, suggesting a general, nonspecific effect in these cells. The morphology of the ionophore-exposed cells also differed markedly; they lacked cytoplasmic extensions, appeared smaller, and were less aggregated than control cells. Staining the nuclei of these cells with 4,6-diamidino-2-phenyl-dihydrochloride revealed that they had undergone condensation and fragmentation, typical of programmed cell death. Extraction of DNA from the ionophore-exposed cells and its separation on ethidium bromide-stained gels revealed that, unlike in control cells, the DNA had been broken into fragments in multiples of approximately 180–200 bp, providing further evidence of apoptotic-like effects of the ionophores on the cells. It is speculated that Ca2+, which mediates stimulation of GtH and GH release by the hypothalamic regulatory hormones, may, under certain conditions, have apoptotic-like effects, which specifically regulate the sizes of gonadotroph and somatotroph cell populations. In addition, the fact that pituitary cells exposed to ionophores may become apoptotic should be borne in mind when experiments on signal transduction are carried out with these substances.

Possible Interactions between Gonadotrophs and Somatotrophs in the Pituitary of Tilapia: Apparent Roles for Insulin-Like Growth Factor I and Estradiol

Possible Interactions between Gonadotrophs and Somatotrophs in the Pituitary of Tilapia: Apparent Roles for Insulin-Like Growth Factor I and Estradiol

Possible interactions between gonadotrophs and somatotrophs in the pituitary of tilapia: apparent roles for insulin-like growth factor I and estradiol.

The unique organization of the teleost pituitary, in which cells are grouped according to their characteristic hormone, makes this a suitable model for studying pituitary paracrine interactions. In a number of fish, including tilapia, there are variations in the circulating levels of the gonadotropins and GH, which are elevated during the reproductive season, suggesting interactions between the reproductive and growth axes. The aim of this study was to investigate paracrine interactions between the gonadotrophs and somatotrophs in the tilapia pituitary. Initially, dispersed pituitary cells were separated on a density gradient in which the gonadotrophs were found in the least dense fractions, and the somatotrophs were concentrated in the densest fraction. After 4 days in culture, cells in the least dense fractions showed characteristic cytoplasmic extensions not seen in the somatotrophs, which appeared small and failed to form aggregates; somatotrophs were found, however, attached to other non-GH cells. Staining of the nuclei with 4,6-diaminidino-2-phenyl-dihydrochloride revealed that the isolated somatotrophs had undergone nuclear condensation and fragmentation typical of apoptosis. Addition of either estradiol or human recombinant insulin-like growth factor I (IGF-I; 10 nM) to the somatotroph cultures increased the number of cell aggregates and reduced the number of condensed or fragmented nuclei. Immunocytochemical studies on pituitary sections revealed IGF-I immunoreactivity in regions of the proximal pars distalis that stain with gonadotropin IIbeta antisera and also in regions of the rostral pars distalis characteristic of corticotrophs; immunoreactive IGF-I was never seen in the region of the somatotrophs. Incubation of cells from the different fractions with testosterone (10 nM; 24 h) revealed that cells of the least dense fractions, which were rich in gonadotrophs, possessed aromatizing ability, which was absent in the somatotroph-enriched fraction. These results suggest that estradiol and IGF-I, both generated from cells other than the somatotrophs, may exert antiapoptotic effects and thus possibly control the size of this population of cells.

Induced Breeding of Two Clariid Catfishes Clurius gariepinus and Heterobranchus bidorsalis Using Tilapia Pituitary Extracts

AbstractInduced breeding trials of two clariid catfishes, Clarias gariepinus and Heterobranchus bidorsalis, were conducted using five doses (2, 4, 6, 8 or 10 mg/kg) of acetone‐dried tilapia pituitary (ADTP) extracts. Oocyte maturation and ovulation were induced in female catfishes by single intramuscular injection of 6–10 mgkg ADTP; optimum results were obtained with 8 mgkg in both catfishes. At ambient temperature (27 ± 1 C). ovulation occurred within 14–18 h post‐injection resulting in 16–20% increase in egg diameter. Fertilization and hatching percentages increased (P < 0.05) with increases in hormone dosage. Survival of fry fed a mixed zooplankton diet was high (ranging from 79% to 85%) after 30 d of rearing.

Melanin-concentrating hormone gene-related peptide stimulates ACTH, but not alpha-MSH, release from the tilapia pituitary.

Tilapia (Oreochromis mossambicus; teleostei) melanin-concentrating hormone gene-related peptide (tMgrp) was tested for tropic actions on adenocorticotropin hormone (ACTH) and alpha-melanocyte stimulating hormone (alpha-MSH) producing cells in the tilapia pituitary gland in vitro. Up to 100 microM synthetic tilapia Mgrp (tMgrp) had no effect on alpha-MSH release from tilapia neuro-intermediate lobes in a superfusion set up. However, at concentrations above 1 microM, tMgrp concentration dependently stimulated ACTH release from tilapia anterior lobes. This is the first evidence that Mgrp modulates ACTH release from teleost corticotropes, and this might implicate the peptide in the regulation of the pituitary-interrenal axis of fish.

Possible sites of dopaminergic inhibition of gonadotropin release from the pituitary of a teleost fish, tilapia

The present study is an attempt to find sites of dopaminergic inhibition along the transduction cascades culminating in gonadotropin (GtH) release in a teleost fish, tilapia. Experiments were carried out on perifused pituitary fragments and in primary culture of trypsinized pituitary cells. Salmon GnRH, chicken GnRH I and II stimulated GtH release in culture with estimated ED 50 values of 15.56 pM, 2.55 nM and 8.65 pM, respectively. Apomorphine (APO; 1 μM) totally abolished this stimulation. Dopamine (DA; 1 μM) reduced both basal and GnRHa-stimulated GtH release from perifused pituitary fragments but did not alter the formation of cAMP. In a similar perifusion experiment DA abolished GtH release in response to forskolin (10 μM) with no reduction in cAMP formation. This indicates that one site of the dopaminergic inhibition is distal to cAMP formation, an indication not compatible with the classic characteristic of DA D 2 type mode of action. The inhibition of GtH release in culture, caused by 1 μM APO, the specific DA D 2 agonists LY 171555 (LY) or bromocryptine (BRCR) could not be reversed by activating protein kinase C (PKC) by DiC8 or the phorbol ester TPA. This would indicate a site for DA action distal to PKC. However, the stimulatory effect of arachidonic acid (AA; 50 μM) in perifusion was not reduced by DA (1 μM) or by APO, LY or BRCR in culture, which suggests a site for DA action proximal to AA formation. APO, LY and BRCR reduced GtH release in response to the Ca 2+ ionophore A23187, however, their inhibitory effect was reversed by 10 μM ionomycin. The stimulatory effect of ionophore A23187 differed from that of ionomycin in that A23187 was able to stimulate GtH release only in the presence of Ca 2+ in the medium, whereas ionomycin stimulated the release also in the absence of the ion, and even in the presence of EGTA. It is assumed that ionomycin at 10 μM promotes the mobilization of Ca 2+ from intracellular stores. As neither ionomycin nor AA caused any leakage of lactic dehydrogenase from cultured pituitary cells, the GtH released in response to this agonist is specific and cannot be attributed to damage of the cells' membrane. It is proposed that, in addition to sites distal to cAMP formation and proximal to AA formation, DA may inhibit GtH release from the pituitary of tilapia at sites distal to Ca 2+ influx and at a step proximal to Ca 2+ mobilization from intracellular sources.

Biphasic effect of MCH on α-MSH release from the tilapia (Oreochromis mossambicus) pituitary

The effect of melanin-concentrating hormone (MCH) on the release of alpha-melanocyte stimulating hormone (alpha-MSH) from the tilapia pituitary gland was studied in vitro. In a superfusion set up, 10 nM to 1 microM synthetic salmon MCH caused a concentration-dependent inhibition of alpha-MSH release from tilapia neurointermediate lobes (NILs). Immunoneutralization of MCH in tilapia NILs further indicated that endogenous MCH has an inhibitory effect on the melanotropes. The release of monoacetylated alpha-MSH release was more strongly inhibited by MCH than that of des-, and diacetylated alpha-MSH, indicating that MCH modulates the secretory signal of the melanotropes in a quantitative and qualitative manner. A high concentration of MCH (10 microM) substantially increased the release of alpha-MSH. Further evidence in support of a stimulatory action of high concentrations of MCH was provided by the observation that the MCH analogue MCH(2-17) at 10 and 35 microM enhanced alpha-MSH release as well. Therefore, we conclude that the response of pituitary melanotropes to MCH is biphasic, as was reported previously for the effects of MCH on other targets in fish and mammals. Under physiological conditions the inhibitory action of MCH on fish melanotropes most likely dominates.

Identification, cellular localization and in vitro release of a novel teleost melanin-concentrating hormone gene-related peptide.

The melanin-concentrating hormone (MCH) precursor encodes MCH and a second peptide named neuropeptide EI (NEI) in mammals, neuropeptide EV (NEV) in salmonids and MCH gene-related peptide (Mgrp) in other fish. The primary structure of the putative Mgrp of the cichlid fish tilapia (Oreochromis mossambicus) appears to be very different from mammalian NEI and salmonid NEV. To investigate the processing and release of tilapia Mgrp (tMgrp), in the present study an antiserum was raised against synthetic tMgrp. By immunocytochemistry, tMgrp immunoreactivity was colocated with MCH immunoreactivity in the tilapia hypothalamus and pituitary. In addition, a tMgrp enzyme-linked immunosorbent assay in combination with reversed phase HPLC was used to demonstrate the presence of processed tMgrp in tilapia hypothalamus and pituitary. The release of tMgrp from neuro-intermediate lobes (NILs) of tilapia pituitaries was demonstrated after in vivo incubation of chopped NILs. Depolarizing concentrations of potassium significantly stimulated tMgrp release. Six weeks of adaptation of tilapia to white or black backgrounds had no effect on in vitro tMgrp release or on the tMgrp content of NIL and hypothalamus. Tilapia Mgrp, unlike MCH, had no effect on tilapia scale melanophores, nor did it modulate the melanin-concentrating effect of MCH. We conclude that tMgrp is processed from the MCH preprohormone, that it is released in vitro, and that the peptide has no direct role in the melanin concentration of fish scale melanophores. Therefore a neuroendocrine or neuromodulatory function is proposed for tMgrp.

Central dopaminergic neurons in tilapia: effects of gonadectomy and hypothalamic lesion

The effects of gonadectomy, testosterone and estrogen on the dopamine (DA) neurons were examined by measuring the concentrations of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) in the brain and pituitary of male tilapia. The tuberal area and the pituitary had significantly high levels of DA and low levels of DOPAC, indicating the existence of a rich dopaminergic innervation in these areas. Gonadectomy and sex steroid replacement had no effect on DA and DOPAC levels. Preoptic lesions (14 days survival period) significantly increased DA levels of the pituitary, indicating a possible existence of a preoptico-hypophysial neural system that inhibits pituitary DA synthesis in tilapia. The lack of effect by preoptic (4 days survival period) and posterior hypothalamic lesions on the DA content of the pituitary indicates the absence of dopaminergic innervation of the pituitary by the preoptic and the posterior hypothalamus. Instead, the overall results do suggest the anterior periventricular area as a possible source of pituitary dopaminergic innervation.

Immunoelectron Microscopical Study of Prolactin in Pituitary of Tilapia (Orochromis mossambicus).

Protein A-gold method was used for cytochemical localization of prolactin (PRL) in the secretory cells in the tilapia (Oreochromis mossambicus) pituitary. Antiserum to chum salmon prolactin (sPRL) was used for this study. Effects of fixation on the antigencity of PRL were examined, and the antigenicities of PRL in pituitaries of seawater (SW) -and freshwater (FW) -adapted tilapia were compared.The antiserum to sPRL reacts selectively only with the secretory granules in the specific cells, i, e., the presumptive PRL cells, in the rostral pars distalis, but never reacts with the other cellular compartment of the PRL cells nor with other cell types in the tilapia pituitary. The findings have been confirmed by the control experiments in which preincubating the antiserum with purified sPRL abolished all the immunoreactivity. PRL cells which have been fixed with 4% paraformaldehyde for 2 to 12 hr, do not show any significant difference in antigenicity preservation, but show a better ultrastructure in longer fixation periods. Addition of 2.5% glutaraldehyde to paraformaldehyde could greatly enhance the ultrastructural preservation but concomitantly might cause a loss of 30% of the antigenicity. Post fixation with 1% osmium tetroxide improves the membranous ultrastructure of organelles, however reduced the PRL antigenicity by 50%. The PRL antigenicity (number of protein A-gold binding per PRL granule) in FWadapted tilapia is 1.5 times that in SW-adapted one. Thus, protein A-gold immunocytochemical method is suggested to be suitable for evaluation of the activity of PRL cells in tilapia reared in different environments.

Localization of mRNAs for a Pair of Prolactins and Growth Hormone in the Tilapia Pituitary Using in Situ Hybridization with Oligonucleotide Probes

Oligonucleotide probes were synthesized for the mRNAs of a pair of tilapia prolactins (tPRL 177 and tPRL 188) and growth hormone (tGH) based on cDNAs for the hormones of Oreochromis niloticus and amino acid sequences for the hormones of O. mossambicus. The three 45mer probes were labeled with 35S for hybridization studies on pituitary sections of O. mossambicus adapted to fresh water (FW) or seawater (SW). Expression of tPRL mRNA in the rostral pars distalis was clearly evident with either PRL probe in adjacent sections in PRL cells of the rostral pars distalis; mRNAs of both PRLs were colocalized in the same cells. In addition, the tGH probe demonstrated expression of tGH mRNA specifically in GH cells in the proximal pars distalis. The hybridization signals for both PRLs were significantly greater in the rostral pars distalis of FW fish than in that of SW fish, as judged by computer-aided analysis. In addition, grain concentration for both PRLs was significantly greater over centrally located PRL cells of FW fish. In addition, although overall grain concentrations were lower in SW fish, there were significantly more grains over the centrally located PRL cells with the tPRL 177 probe, whereas there was no difference with the tPRL 188 probe. There was no detectable difference in the occurrence of tGH mRNA between FW and SW fish.

Differential processing of the two prolactins of the tilapia (Oreochromis mossambicus) in relation to environmental salinity

AbstractProlactin (PRL) is thought to play a significant role in the freshwater (FW) adaptation of euryhaline teleost fishes, including the tilapia, Orechromis mossambicus. Two PRLs (tPRL188 and tPRL177) have been characterized in the anteriormost portion of the tilapia pituitary, the rostral pars distalis (RPD; Specker et al., '85a,b; Yamaguchi et al., '88). Our studies were undertaken to determine whether the in vivo RPD content of tPRL188 and tPRL177 varied with environmental salinity. To this end, tilapia were reared from the period of yolk‐sac absortion for 7 months or acclimated for 21 and 35 days in FW or seawater (SW). In a parallel study, we also examined whether the release of the 2 tilapia PRLs from the RPD of FW or SW tilapia might be differentially sensitive to changes in medium osmotic pressure during 18–20 hr incubations. In agreement with earlier work, we found that the smaller tPRL177 molecule was present in greater quantities in the RPD of FW tilapia compared to the content in RPD of SW fish. For the first time, we show here that the content of tPRL188, like that of tPRL177, is also significantly greater in the RPD of FW fish compared to the quantity in RPD of SW tilapia. Nevertheless, the relative content or ratio of the 2 PRLs in the pituitary was dependent on rearing salinity. Thus, the relative content or ratio of the larger tilapia PRL to smaller PRL (tPRL188/tPRL177) in the RPD of FW tilapia was significantly higher (1.5:1) than that seen in SW fish (0.75:1). This ratio was not altered after 18–20 hr in vitro incubations in hyposmotic or hyperosomtic medium, suggesting that the shift in ratios did not result from differential release by osmotic pressure. During 18–20 hr incubation in either hyposmotic or hyperosmotic medium, the RPD of FW tilapia released more of both forms of PRL than did the RPD of SW fish. Consistent with previous studies, reductions in medium osmotic pressure within the physiological range of the tilapia stimulated the release of botg tPRL188 and tPRL177 from RPD of FW fish over levels observed during exposure to hyperosmotic medium. We report here, for the first time, that the reduction of medium osmotic pressure also stimulates the release of both tPRL188 and tPRL177 from RPD of SW fish. Throughout the experiments we did not observe differences between the release of tPRL188 and tPRL177 under our incubation conditions. The transfer of FW‐reared fish to SW and vice versa for 49 days altered the ratio of the 2 PRLs (tPRL188/tPRL177) to that observed in the RPD of SW‐reared and FW‐reared fish, respectively. Overall, our studies indicate that the processing of the 2 PRLs may be differentially sensitive to environmental salinity. © 1992 Wiley‐Liss, Inc.

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 < 0.01). By contrast, fasting resulted in a substantial increase in spontaneous GH release from the PPD in vitro ( P < 0.01). In the second series of experiments, GH release was found to increase as the d-glucose concentration of the medium decreased ( P < 0.01), while prolactin release was unresponsive. The spontaneous release of both PRL and GH in vitro was inversely related to the quantity of essential amino acids (Eagle's minimum essential medium; MEM) added to the incubation medium ( P < 0.01). These findings suggest that the activity and regulation of PRL and GH cells in the tilapia pituitary may be sensitive to the nutritional status of the fish. It would also appear that GH cells, but not PRL cells, may respond to changes in circulating d-glucose levels and that both may respond to variations in circulating amino acids. Additional studies will be required to determine whether the latter response is directed by specific amino acids.

Effects of GnRH-associated peptide and its component peptides on prolactin secretion from the tilapia pituitary in vitro

The rostral pars distalis (RPD), containing mainly prolactin (PRL)-secreting cells, of the pituitary from immature and mature tilapia was incubated for 16 hr at 27° in hypoosmotic medium (300 mOsm/kg) in the presence (10 −8 and 10 −11 M) or absence of the human GnRH-associated peptide (GAP) molecule, a potent PRL-inhibiting factor in mammals (Nikolics et al., Nature ( London) 316, 511, 1985), and of a series of its component peptides. The release of the two forms of PRL in tilapia into the medium was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by densitometry. The variability inherent in this method was normalized by calculating PRL release as the percentage of the total hormone present in both tissue and medium. Newly synthesized PRL was detected by incorporation of [ 35S]methionine, introduced into the culture medium, by the PRL molecules. In immature tilapia, GAP inhibited the release of total PRL while stimulating the release of newly synthesized large PRL. Among the GAP fragments tested, 28–36 was the fragment that most significantly affected PRL secretion. Both concentrations of fragment 28–36 stimulated the release of newly synthesized PRL from immature rostral pars distalis (RPDs). This stimulation appears to be dependent on the osmotic pressure of the medium since this fragment did not affect PRL secretion in hyperosmotic medium (340 mOsm/kg). Fragment 38–49 inhibited total PRL release from mature RPDs. Fragment 51–66 stimulated the release of total PRL from mature RPDs. Examination of tissue and medium values in densitometric units after incubation with fragments 28–36 and 51–66 indicated that while the tissue content of PRL was decreased, the medium content of PRL was not affected. This suggests that fragments 28–36 and 51–66, in opposition to the situation found when the data are expressed as percentage release of PRL, may not stimulate PRL release but may instead decrease the tissue content of PRL. These results suggest that the entire human GAP molecule, as well as some of its fragments, may have direct effects on the PRL cells in the tilapia pituitary.

Gonadotropin secretion from perifused tilapia pituitary in relation to gonadotropin-releasing hormone, extracellular calcium, and activation of protein kinase C

Gonadotropin (taGTH) secretion from perifused fragments of tilapia pituitaries was stimulated in a dose-dependent manner by an analog of gonadotropin-releasing hormone ([ d-Ala 6] des Gly 10 ethylamide LHRH; GnRHa) in a dose range of 1.28 to 128 p M. The baseline secretion rate and taGTH secretion in response to GnRHa were both reduced when the perifusion medium lacked Ca 2+. Calcium ionophore (A23187; 0.1 m M mimicked the effect of GnRHa but only in the presence of Ca 2+. The addition of cobalt chloride to the medium at 0.6 m M initially caused an increase in taGTH secretion which was followed by its decrease. At a CoCl 2 concentration of 1.3 m M, the baseline secretion rate remained low and the effect of GnRHa on taGTH secretion was attenuated. Withdrawal of CoCl 2 from the medium was followed by an elevated basal secretion rate. Five-minute pulses of the protein kinase C activator, 1 oleyl-2-acetyl-rac-glycerol (OAG; 0.25 to 10.4 m M) stimulated taGTH secretion in the presence of Ca 2+. With the reservation that the experiments were performed on fragments containing more than one pituitary cell type, the results indicate that the stimulation of GTH secretion in this fish is dependent, as in mammals, on extracellular Ca 2+ and probably involves the activation of protein kinase C. However, the fact that taGTH may be stimulated to some extent in the absence of extracellular calcium or in the presence of 1.3 m M Co 2+ may point to the possibility that Ca 2+ is mobilized from intracellular stores as a result of GnRH stimulation or to the involvement of an additional mechanism of GnRH action in fish independent of calcium.

Aromatase is concentrated in the proximal pars distalis of tilapia pituitary

Aromatase has been identified in the teleostean, avian, and mammalian pituitaries, although its cellular location(s) is not yet certain. To address this question, experiments were performed in tilapia ( Oreochromis mossambicus), a species which has been well characterized with respect to the intraglandular distribution of the different pituitary cell types. To estimate aromatase, glands were microdissected into rostral pars distalis (RPD), proximal pars distalis (PPD), and neurointermediate lobe (NIL) and organs were cultured in the presence of [ 3H]androstenedione for 16–24 hr. [ 3H]Estrogen products were isolated and quantified after ether extraction, hydrolysis with glucuronidase-sulfatase, thin-layer chromatography, and phenolic partition. Authentic estrone or estradiol-17β were produced by all pituitary regions and also by the urophyseal region of the spinal cord. Aromatase was two to five times higher in PPD than in RPD or NIL and similar to activity in adjacent hypothalamus-preoptic area (HPOA). Much lower estrogen yields were obtained in cultures of cerebellum, urophysis, and other cord regions. Since the PPD contains most of the somatotropes, these data are consistent with earlier studies implicating GH 3/GH 4 cell strains as an enriched enzyme source, although its presence in other cell types cannot be ruled out. The unusually high and localized aromatase in tilapia pituitary renders this species a useful model for studying the targets and functional importance of estrogen as a parahormone in the pituitary.

Development of radioimmunoassay for eel growth hormone.

A highly specific and homologous radioimmunoassay (RIA) for the measurement of growth hormone (GH) in the plasma and the pituitary of the eel was developed using a rabbit antiserum to eel GH. Two forms of eel GH: eGHI and II, were purified from the media from organ-cultures of pituitaries of the Japanese eel Anguilla japonica. An antiserum against eGH I, which does not distinguish between the two forms, was used to measure total GH levels. Pituitary extracts from the Japanese eel and the European eel A. anguilla exhibited displacement curves parallel to the eGHI standard, whereas extracts from chum salmon, rainbow trout, Japanese charr, carp, goldfish and tilapia pituitaries showed no cross-reactivity. GH or prolactin preparations from sheep, whale, chum salmon and tilapia also did not cross-react with the antibody. RIA sensitivity was 0.2ng/ml of plasma when 50μl of plasma were employed. Most of the plasma GH levels were low or undetectable in starved eels either in fresh water or in seawater, or in fed eels in fresh water. On the other hand, plasma samples from seawater-adapted male eels receiving injections of human chorionic gonadotropin showed high GH levels. Apparent plasma half-life of eGH I injected intravascularly through a cannula into the pneumogastric artery was about 15 min.

Somatostatin and altered medium osmotic pressure elicit rapid changes in prolactin release from the rostral pars distalis of the tilapia, Oreochromis mossambicus, in vitro

Prolactin (PRL) cells in the rostral pars distalis of the tilapia Oreochromis mossambicus respond to somatostatin (SRIF) and reduced medium osmotic pressure within 10–20 min of exposure during perifusion incubation. Pieces of rostral pars distalis tissue were removed from freshwater-adapted tilapia and were preincubated in [ 3H]leucine in static culture (355 mØsmolal) for 48 hr. Following preincubation, they were placed in the perifusion apparatus and baseline release was established for 3 hr in hyperosmotic medium (355 mØsmolal). Exposure to hyposmotic medium (280 mØsmolal) resulted in a rapid and steep rise in the release of [ 3H]PRL, which remained elevated for more than 2 hr. When SRIF was added simultaneously with hyposmotic medium, the rise in PRL release normally initiated by reduced osmotic pressure was prevented. Somatostatin also quickly reduced release that had been previously elevated by exposure to hyposmotic medium. The time course of these changes suggests that SRIF and altered osmotic pressure act on PRL secretion in at least partial independence of effects which they may have on PRL synthesis in the tilapia pituitary.