Genomic Structure and Functional Analysis of Promoter Region of Somatolactin Gene of Sea Bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>)

Somatolactin (SL) is a pituitary hormone belonging to the growth hormone–prolactin family and is produced in the intermediate lobe of teleosts. The SL gene was isolated from a sea bream genomic library and found to be composed of 5 exons distributed within a 9-kb length of DNA. Sequence analysis of the proximal promoter region showed the presence of a classical TATA box located 59 bp upstream from the initial start ATG codon, 5 consensus sequences corresponding to the Pit-1 binding element, and a putative CREB site. In CHO cells cotransfected with the DNA from 2 plasmids, one encoding sea bream Pit-1 under Rous sarcoma virus long terminal repeat regulation and one encoding the SL promoter driving the expression of luciferase, Pit-1 was found to enhance the expression of luciferase. Only one Pit-1 binding site was necessary for enhancement. Analysis by immunoblots of in vitro culture of pituitaries of Sparus aurata showed that several agents, including estradiol, verapamil, and phorbol myristate acetate, had different inhibitory effects on SL and growth hormone released to the culture medium.     

Grass carp somatolactin: I. Evidence for PACAP induction of somatolactin-alpha and -beta gene expression via activation of pituitary PAC-I receptors

Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. At present, SL can be identified only in fish but not in tetrapods and its regulation at the pituitary level has not been fully characterized. Using grass carp as a model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL secretion and synthesis at the pituitary cell level. As a first step, the structural identity of grass carp SL, SLalpha and SLbeta, was established by 5'/3'-rapid amplification of cDNA ends. These two SL isoforms are single-copy genes and are expressed in two separate populations of pituitary cells located in the pars intermedia. In the carp pituitary, PACAP nerve fibers were detected in the nerve tracts of the neurohypophysis and extended into the vicinity of pituitary cells forming the pars intermedia. In primary cultures of grass carp pituitary cells, PACAP was effective in stimulating SL release, cellular SL content, and total SL production. The increase in SL production also occurred with parallel rises in SLalpha and SLbeta mRNA levels. With the use of a combination of molecular and pharmacological approaches, PACAP-induced SL release and SL gene expression were shown to be mediated by pituitary PAC-I receptors. These findings, as a whole, suggest that PACAP may serve as a hypophysiotropic factor in fish stimulating SL secretion and synthesis at the pituitary level. Apparently, PACAP-induced SL production is mediated by upregulation of SLalpha and SLbeta gene expression through activation of PAC-I receptors.     

Insulin-like growth factor as a novel stimulator for somatolactin secretion and synthesis in carp pituitary cells via activation of MAPK cascades

Somatolactin (SL), a member of the growth hormone/prolactin family, is a pituitary hormone unique to fish models. Although SL is known to have diverse functions in fish, the mechanisms regulating its secretion and synthesis have not been fully characterized. Using grass carp pituitary cells as a model, here we examined the role of insulin-like growth factor (IGF) in SL regulation at the pituitary level. As a first step, the antisera for the two SL isoforms expressed in the carp pituitary, SLα and SLβ, were produced, and their specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the carp pituitary. Western blot using these antisera revealed that grass carp SLα and SLβ could be N-linked glycosylated and their basal secretion and cell content in carp pituitary cells could be elevated by IGF-I and -II treatment. These stimulatory effects occurred with parallel rises in SLα and SLβ mRNA levels, and these SL gene expression responses were not mimicked by insulin but blocked by IGF-I receptor inactivation. In carp pituitary cells, IGF-I and -II could induce rapid phosphorylation of IGF-I receptor, MEK1/2, ERK1/2, MKK3/6, and p38 MAPK; and SLα and SLβ secretion, protein production, and mRNA expression caused by IGF-I and -II stimulation were negated by inactivating MEK1/2 and p38 MAPK. Parallel inhibition of PI3K and Akt, however, were not effective in these regards. These results, taken together, provide evidence that IGF can upregulate SL secretion and synthesis at the pituitary level via stimulation of MAPK- but not PI3K/Akt-dependent pathways.     

Cellular localization of somatolactin in the pars intermedia of some teleost fishes

Summary We report here on the cellular localization in the fish pituitary of somatolactin (SL), a putative new pituitary hormone related to growth hormone and prolactin, which has been recently identified in the piscine pituitary gland. Immunocytochemical staining, using anti-cod SL serum, revealed that in the cod pituitary gland, SL is produced by cells in the intermediate lobe, bordering the neural tissue. These cells, staining weakly with periodic-acid-Schiff (PAS), are distinct from the melanocyte stimulating hormone (MSH) cells which, as in all teleosts, are PAS-negative. SL-immunoreactivity was observed in the same location in all other teleost species examined: flounder, rainbow trout, killifish, molly, catfish and eel. In most fish the SL-immunoreactive cells are either strongly or weakly PAS-positive but in rainbow trout are chromophobic, indicating that the SL protein can probably exist in glycosylated and non-glycosylated forms. Thus, in demonstrating the cellular localization of SL, this study provides the first identification of the enigmatic, second cell-type of the fish pars intermedia.     

Electron-microscopic study on the anterior pituitary gland of spontaneous dwarf rats

Summary The spontaneous dwarf rat is a novel experimental model animal on the study of pituitary dwarfism. The fine structure of the anterior pituitary cells was studied in the immature and mature dwarf rats. Pituitary glands were removed from 5-, 10-, 20-day-old immature dwarfs, adult (45 days-16 weeks) dwarfs and normal 3-month-old rats and processed for electron-microscopic observation. In the control animals, growth hormone cells were readily identified by their ultrastructural characteristics, such as the presence of numerous electron-dense secretory granules, 300–350 nm in diameter, well developed rough endoplasmic reticulum and a prominent Golgi complex. In contrast, growth hormone cells were not found in the anterior pituitary gland of the spontaneous dwarf rat at any age examined. Other pituitary cell types, i.e., luteinizing hormone/ follicle stimulating hormone, thyroid stimulating hormone, adrenocorticotropic hormone and prolactin cells, appeared similar in their fine structure to those found in the control rats. In the pituitary gland of dwarf rats, a number of polygonal cells were observed either with no or relatively few secretory granules. The rough endoplasmic reticulum was arranged in parallel cisternae and the Golgi complex was generally prominent in these cells. In addition, many were found to have abundant lysosomes. A few minute secretory granules were occasionally observed; however, the immunogold technique failed to localize growth hormone or prolactin in the granules. The nature of these cells remained obscure in this study. Since their incidence and fine structural features, other than the secretory granules, were quite similar to those of the growth hormone cells in normal rats, we postulate that these cells are dysfunctional growth hormone cells. These results suggest that the cause of the growth impairment in the spontaneous dwarf rat is due to a defect in the functional growth hormone cells in the pituitary gland, and since other pituitary cell types appeared normal, the disorder seems to be analogous to the isolated growth hormone deficiency in the human.     

Bacterial Production and Purification of the Fish Pituitary Hormone Somatolactin

Somatolactin, a pituitary hormone belonging to the growth hormone/prolactin family, is produced in the intermediate lobe of teleost pituitary. To date, the functions of this new hormone and the target tissues are unknown. ASolea senegalensissomatolactin (ssSL) cDNA has previously been cloned and isolated. Here we have inserted this cDNA into a pET-3a plasmid in order to produce recombinant ssSL inE. coliBL21 (DE3) cells. The protein induced was isolated from inclusion bodies by a solubilization–renaturation procedure originally developed to generate native disulfide bonds, to get putative active proteins. The recombinant somatolactin was further purified to homogeneity by gel filtration on FPLC. The estimated molecular weight of 26 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis agrees well with the molecular mass calculated from the translated cDNA sequence and with native somatolactin (SL). The recombinant protein showed electrophoretic mobility identical to that of one of the native forms of SL secretedin vitroby cultured pituitaries from sole. Another native SL expressed inS. senegalensisrepresented a glycosylated modified hormone as shown byN-glycosidase treatment. Further, recombinant SL was recognized by an anti-native SL antibody and used to generate polyclonal sera reactive with the native pituitary hormone. To date, this represents the first recombinant SL protein isolated in sufficient quantities for biophysical and biochemical investigation and for studies on its physiological actions.     

Morphometric evaluation of subcellular changes induced by in vivo TRH treatment in the pituitary gland of Rana perezi: Effects on prolactin and thyrotropic cells

Summary The effects of synthetic thyrotropin-releasing hormone on pituitary prolactin and thyrotropic cells were investigated in adult male Rana perezi (formerly Rana ridibunda) frogs. Animals were given daily injections of synthetic thyrotropin-releasing hormone into the dorsal lymph sac. Prolactin and thyrotropic cells were identified by the colloidal-gold method, using anti-human prolactin and anti-human--thyrotropin hormone as primary antisera. The stereological parameters of the rough endoplasmic reticulum, Golgi complex, and secretory granules of prolactin and thyrotropic cells were evaluated by ultrastructural morphometry (point-counting method). Thyrotropin-releasing hormone caused cytological changes in both cell-types which were consistent with increased synthesis and release of both prolactin and thryrotropin. These changes were still significant after 48 h treatment in the case of thyrotropic cells, while in prolactin cells the thyrotropin-releasing hormone increased the number of secretory granules. After 6 days, the cells resembled essentially those used as controls. These results indicate that thyrotropin-releasing hormone stimulates the synthesis and release of prolactin and thyrotropin, and that the response of each cell type to this hypothalamic stimulus follows a different time-course.This work has been supported by grants no. 2184-83 and PB 86-0095 from the Comisión Interministerial para la Ciencia y Tecnología, Spain     

Grass carp somatolactin: II. Pharmacological study on postreceptor signaling mechanisms for PACAP-induced somatolactin-alpha and -beta gene expression

Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. However, the signal transduction mechanisms responsible for SL expression are still largely unknown. Using grass carp as an animal model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL gene expression at the pituitary level. In primary cultures of grass carp pituitary cells, SLalpha and SLbeta mRNA levels could be elevated by PACAP via activation of PAC-I receptors. With the use of a pharmacological approach, the AC/cAMP/PKA and PLC/inositol 1,4,5-trisphosphate (IP(3))/PKC pathways and subsequent activation of the Ca(2+)/calmodulin (CaM)/CaMK-II cascades were shown to be involved in PACAP-induced SLalpha mRNA expression. Apparently, the downstream Ca(2+)/CaM-dependent cascades were triggered by extracellular Ca(2+) ([Ca(2+)](e)) entry via L-type voltage-sensitive Ca(2+) channels (VSCC) and Ca(2+) release from IP(3)-sensitive intracellular Ca(2+) stores. In addition, the VSCC component could be activated by cAMP/PKA- and PLC/PKC-dependent mechanisms. Similar postreceptor signaling cascades were also observed for PACAP-induced SLbeta mRNA expression, except that [Ca(2+)](e) entry through VSCC, PKC coupling to PLC, and subsequent activation of CaMK-II were not involved. These findings, taken together, provide evidence for the first time that PACAP can induce SLalpha and SLbeta gene expression in fish model via PAC-I receptors through differential coupling to overlapping and yet distinct signaling pathways.     

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on prolactin and somatolactin release from the goldfish pituitary in vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays a role in mediating growth hormone and gonadotropin release in the teleost pituitary. In the present study, we examined the immunohistochemical relationship between PACAP nerve fibers and prolactin (PRL)- and somatolactin (SL)-producing cells in the goldfish pituitary. Nerve fibers with PACAP-like immunoreactivity (PACAP-LI) were identified in the neurohypophysis in close proximity to cells containing PRL-LI or SL-LI. Several cells with PRL-LI or SL-LI showed PACAP receptor (PAC(1)R)-LI. The cell immunoblot assay method was used to examine the effect of PACAP on PRL and SL release from dispersed goldfish pituitary cells. Treatment with PACAP increased the immunoblot area for PRL- and SL-LI from individual pituitary cells in a dose-dependent manner. The effect of PACAP on the expression of mRNAs for PRL and SL in cultured pituitary cells was also tested. Semiquantitative analysis revealed that the expression of SL mRNA, but not PRL mRNA, was increased significantly by the treatment with PACAP. The effect of PACAP on intracellular calcium mobilization in isolated pituitary cells was also investigated using confocal laser-scanning microscopy. The amplitude of Ca(2+) mobilization in individual cells showing PRL- or SL-LI was increased significantly following exposure of cells to PACAP. These results indicate that PACAP can potentially function as a hypophysiotropic factor mediating PRL and SL release in the goldfish pituitary.     

Effect of salmon melanin-concentrating hormone and mammalian gonadotrophin-releasing hormone on somatolactin release in pituitary culture of Cichlasoma dimerus

We detected a close morphological association between melanin-concentrating hormone (MCH)-immunoreactive (ir) fibers and somatolactin (SL)-ir cells in the pars intermedia of the cichlid fish Cichlasoma dimerus by double-label immunofluorescence. Male pituitaries obtained from adult C. dimerus were incubated with 0.1-10 muM salmon MCH, and the amount of SL released into the culture medium was semi-quantified by Western blot. This assay showed an increase of SL release in a dose-dependent manner (linear regression: P < 0.05). A close association of GnRH-ir fibers with SL-ir cells was also detected at the pars intermedia level. Male pituitaries were also incubated with 0.1-10 muM of mammalian GnRH, and SL release was semi-quantified by Western blot, showing an increase of released SL levels in a dose-dependent manner (linear regression: P < 0.05). In contrast, SL release was unaffected from female pituitaries incubated with salmon MCH; however, an increasing tendency was observed when mammalian GnRH was used. Hypothalamic close association of MCH-ir perikarya and GnRH-ir fibers was found by double-label immunofluorescence indicating a possible relationship between them. These results suggest that SL, like other pituitary hormones, is under hypothalamic control and is involved in diverse physiological processes including background adaptation and reproduction. This study has also shown that the in vitro culture of a single C. dimerus pituitary is a feasible method for studying the control of SL release and other pituitary hormones.     

A systematic immunohistochemical survey of the distribution patterns of GH, prolactin, somatolactin, β–TSH, β–FSH, β–LH, ACTH, and α–MSH in the adenohypophysis of Oreochromis niloticus, the Nile tilapia

Fish pituitary plays a central role in the control of growth, development, reproduction and adaptation to the environment. Several types of hormone-secreting adenohypophyseal cells have been characterised and localised in diverse teleost species. The results suggest a similar distribution pattern among the species investigated. However, most studies deal with a single hormone or hormone family. Thus, we studied adjacent sections of the pituitary of Oreochromis niloticus, the tilapia, by conventional staining and immunohistochemistry with specific antisera directed against growth hormone (GH), prolactin (PRL), somatolactin (SL), thyrotropin (beta-TSH), follicle-stimulating hormone (beta-FSH), luteinising hormone (beta-LH), adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (alpha-MSH). The pituitary was characterised by a close interdigitating neighbourhood of neurohypophysis (PN) and adenohypophysis. PRL-immunoreactive and ACTH-immunoreactive cells were detected in the rostral pars distalis. GH-immunoreactive cells were present in the proximal pars distalis (PPD). A small region of the PPD contained beta-TSH-immunoreactive cells, and beta-LH-immunoreactive cells covered approximately the remaining parts. Centrally, beta-FSH-immunoreactive cells were detected in the vicinity of the GH-containing cells. Some of these cells also displayed beta-LH immunoreactivity. The pars intermedia was characterised by branches of the PN surrounded by SL-containing and alpha-MSH-immunoreactive cells. The ACTH and alpha-MSH antisera were observed to cross-react with the respective antigens. This cross-reactivity was abolished by pre-absorption. We present a complete map of the distinct localisation sites for the classical pituitary hormones, thereby providing a solid basis for future research on teleost pituitary.     

Immunocytochemical localisation of prolactin and growth hormone in the ovine pituitary

Summary Using the peroxidase-antiperoxidase immunocytochemical staining technique, prolactin and growth hormone cells have been identified and described in the ovine pituitary. The image analysing computer, Quantimet 720, was used to assess accurately the size range of the secretory granules in these cell types. The area size distributions of the prolactin and growth hormone granules are similar. An increased proportion of larger granules was observed in the prolactin cells post-partum. Serial sections stained alternately for prolactin or growth hormone confirmed that the cells contain either prolactin or growth hormone but not both.     

The kinetics of YOYO-1 intercalation into single molecules of double-stranded DNA

Recent studies have suggested that treatment of glucocorticoid to immature growth hormone (GH)-producing cell line, MtT/S cells, dramatically induced the accumulation of GH-containing secretory granules in the cytosol and differentiated into mature GH-producing cells. However, the molecular mechanism of glucocorticoid-induced GH-containing secretory granule biogenesis in the MtT/S cells remains unknown. In the present study, we found that GH mRNA expression was facilitated by application of glucocorticoid. We artificially increased GH synthesis by transfection of green fluorescent protein-tagged GH (GH-GFP) gene. We found that the artificial elevation of GH expression in the cells did not accumulate the secretory granules in the cytosol, whereas glucocorticoid-induced the biogenesis of granules in GH-GFP-expressing MtT/S cells. We next performed DNA microarray and real-time RT-PCR analysis and found that glucocorticoid significantly altered the expression of membrane trafficking-related protein, syntaxin11 (Syx11). Immunocytochemical analysis further demonstrated that Syx11 positive structures were well colocalized with GH-containing granules in both MtT/S cells and rat anterior pituitary gland. Our findings indicate that glucocorticoid regulate the expression of Syx11 and facilitate the biogenesis and the trafficking of GH-containing granules in the MtT/S cells.     

Electron microscopic autoradiographic localization of prolactin mRNA in rat pituitary

Recent immunoelectron microscopic studies have shown that immunoreactive prolactin (PRL) in rat pituitary can be detected not only in typical PRL cells, characterized by large secretory granules, but also in another type of cell, which contains small secretory granules. To determine whether or not these two cell types are involved in PRL biosynthesis, we developed a procedure to investigate PRL gene expression by using in situ hybridization at the ultrastructural level. Rat pituitary was fixed and vibratome sections were incubated with a PRL [35S]-cDNA probe and subsequently flat-embedded in Araldite. Semi-thin and ultra-thin sections were processed for autoradiography. The results indicate that only the two PRL cell types were labeled. When immunolabeling for PRL was applied to ultra-thin sections, only immunopositive cells were seen to contain silver grains. In these cells the silver grains were associated with the rough endoplasmic reticulum and nucleus. When a growth hormone (GH) [35S]-cDNA probe was used as a control, only GH-secreting cells were labeled. This study confirms that the two PRL cell types are involved in biosynthesis of PRL. Moreover, this simple in situ hybridization technique provides a new approach to accurately localize mRNA in complex tissue and to investigate the subcellular distribution of mRNA under differing experimental conditions.     

Somatostatin is targeted to the regulated secretory pathway of gonadotrophs in transgenic mice expressing a metallothionein-somatostatin gene

The pituitaries of transgenic mice that express a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin-14 exclusively in the gonadotrophic cells. The purpose of this study was to determine whether somatostatin expressed from the foreign fusion gene enters the normal secretory pathway within these cells. Immuno-gold labeling of serial thin sections localized somatostatin to the secretory granules of gonadotropin-producing cells. The gonadotroph-specific hypophysiotropic factor, luteinizing hormone-releasing hormone caused a dose-dependent secretion of somatostatin when applied to primary pituitary cultures from these mice. Growth hormone-releasing hormone, thyrotropin-releasing hormone, corticotropin releasing factor, and dopamine did not affect somatostatin secretion. These experiments demonstrate that a neurosecretory peptide encoded by a foreign gene can enter the regulated secretory pathway of pituitary cells from transgenic mice.