Choice of extraction procedure for estimation of anterior pituitary hormone content

Searching for the best procedure for simultaneous estimation of the anterior pituitary hormones, extraction efficiencies of various media, additives such as urea and triton X-100, and physical treatments such as freezing-thawing (F-T) and sonication, were examined by measuring prolactin (PRL), growth hormone (GH), lutropin (LH), follitropin (FSH), and thyrotropin (TSH) in the extracts. Ethanolic media (60% EtOH) gave high yields of PRL at neutral to alkaline pH, but poor extraction of GH accompanied by a marked loss of its immunoreactivity during storage. Ethanolic media also gave a poor yield of LH even at high pH. Aqueous media like PBS at various pH, 0.1 M acetic acid and distilled water were considerably effective in the extraction of GH, LH, FSH and TSH if they were coupled with F-T and sonication. However, high yields of PRL could not be obtained with these aqueous media even with F-T and sonication. Hartree's 40% EtOH-6% ammonium acetate, pH 5.1, solubilized considerable amounts of glycoprotein hormones, but yielded almost no GH and only a small amount of PRL. The addition of triton X-100 to PBS (pH 7) at 0.1% resulted in the maximum extraction of glycoprotein hormones with homogenization and F-T, but further sonication was necessary for GH and PRL. When the anterior pituitaries were homogenized and frozen-thawed in PBS (pH 7) containing 1 M urea, yields of PRL, GH, LH, FSH, and TSH were maximum, and sonication did not cause any additional extraction, indicating that this procedure, i.e. homogenization and F-T in 1 M urea-PBS, would be the best for the simultaneous estimation of these anterior pituitary hormones.     

Similarities and Differences Among Prolactins and Growth Hormones and Their Receptors

SYNOPSIS. Data from the literature and from our own studieson the receptors for prolactin (PRL) and growth hormone (GH)are reviewed and analyzed. Receptors for PRL have been studiedin a wider range of species and in a greater diversity of targetorgans than have the binding sites for GH. Although GHs arestructurally more highly conserved among the vertebrates thanare PRLs, the available data indicate that there is greaterdiversity among GH receptors than there is among PRL receptors.In general, GH receptors show greater species specificity butless hormone specificity than do PRL receptors. The reason forthe greater diversity among GH receptors as compared to PRLreceptors is unknown; it bears no relationship to phylogeny. Data on the binding of purified preparations of mammalian PRL,GH and placental lactogen (PL) to renal and hepatic receptorsfor PRL and GH, respectively, of several vertebrate speciesare reviewed. The species and hormone specificity of the bindingof the hormones to the two typesof receptors showed no consistentpattern. To explain this disarray, we propose that the receptorbinding domains of PRL and GH were present in their common ancestralgene and that they havebeen retained to variable degrees byall of the descendant members of the PRL-GH family. We furtherpropose that hormone and species specificity of binding is determinedby hindering features on the hormones and on the receptors,rather than by merely the presence or absence of the appropriatebinding determinants.     

生长激素和催乳素放射免疫测定法的建立与应用

目的：建立测定大鼠垂体和血浆中生长激素（GH）和催乳素（PRL）含量的高特异性、高灵敏度的双抗放射免疫测定（RIA）法;研究急性低氧对垂体激素GH和PRL的作用。方法：用氯胺－T法进行抗原放射性碘标记;采用平衡饱和加样程序的双抗RIA法测定。结果：用该方法测定急性低氧（0．5h）时血浆和垂体GH和PRL含量,7km低氧,垂体GH含量明显升高（P＜0．05）,血浆则相反;7km低氧,明显降低垂体和血浆PRL含量（P＜0．01）;而5km低氧对GH和PRL的作用与对照组比无统计学差异。结论：本双抗RIA法具有高特异性、高灵敏度及简便易行等特性;用该法测定提示急性低氧可抑制大鼠GH和PRL的分泌。     

Antibodies for Growth Hormone and Prolactin Using Multiple Antigen Peptide Immunogens

Antibodies elicited by novel synthetic peptide antigens derived from a highly conserved domain of the growth hormone (GH) and prolactin (PRL) of vertebrates were developed using the multiple antigen peptide approach. The sequence of the antigens is located near the carboxy-terminus in the D domain of the GH and PRL in a cluster of 11 and 10 conserved amino acids, respectively, within a sequence of 18 residues. The synthetic peptides were manually synthesized, purified by high-performance liquid chromatography, and the corresponding antibodies, elicited in rabbits, were cross-reacted with the GH and PRL of a variety of mammalian (human, bovine, ovine, pig, and equine) and nonmammalian (chicken, coho salmon, chum salmon, rainbow trout, catfish and striped bass) vertebrates. The cross-reactivity between the immunogen and its corresponding antigen was tested by immunobloting using either GH or PRL. The GH and PRL of the organisms tested cross-reacted specifically with the corresponding antibody. Chicken and fish GH and PRL showed stronger antibody cross-reactivity than that observed in mammalian sources. These results demonstrate the utility of peptide-derived polyclonal antibodies in the detection of native and recombinant GH and PRL of a variety of vertebrates. Received June 1, 1998; accepted November 13, 1998.     

Cloning of the cDNAs coding for cat growth hormone and prolactin

Characterization of the prolactin (PRL) amino acid (aa) or cDNA sequences has not been reported for any member of the Felidae family. We cloned cat growth hormone (cGH) and cat PRL (cPRL) cDNA sequences from a feline pituitary cDNA library. High homology between species allowed bovine PRL (bPRL) and bGH cDNA clones to be used to identify clones encoding the 229-aa cPRL and 216-aa cGH sequences. The cGH protein is most homologous to pig and dog GH. Similarly, cPRL shares the most aa identity to pig PRL (pPRL). Northern blot analysis revealed the mRNA size for cGH and cPRL to be approx. 1 and 1.1 kb, respectively. These results reveal that GH and PRL from the Felidae family are highly conserved to other families of GH and PRL.     

Rat ghrelin stimulates growth hormone and prolactin release in the tilapia,Oreochromis mossambicus

Recently, ghrelin (Ghr), a new peptide which specifically stimulates growth hormone (GH) release from the pituitary, was identified in the rat and human stomach. Ghrelin has been shown to stimulate GH release by acting through a growth hormone secretagogue (GHS) receptor in the rat. The present study describes the in vitro effect of rat Ghr on the release of GH and two forms of prolactin (PRL(177) and PRL(188)) in the tilapia, Oreochromis mossambicus. Rat Ghr stimulated the release of GH in a dose-related manner after 8 and 24 hr of incubation. Rat Ghr also significantly stimulated the release of PRL(177) and PRL(188) in a dose-related manner after 24 hr. Rat Ghr had no effect on the pituitary content of GH or PRL(188), but significantly increased PRL(177) content. These results show for the first time that rat Ghr significantly stimulates GH and PRL release in teleosts, and suggest that Ghr and a GHS receptor are present in fish.     

Elevated growth hormone levels in sera from breast cancer patients

The concentrations of growth hormone (GH) and prolactin (PRL) in the serum of 42 breast cancer patients were determined by radioimmunoassay (RIA). Forty percent of the patients had elevated GH levels while only 17% had elevated PRL levels. These findings suggest a relationship between GH and breast cancer; a weaker correlation exists between PRL and this malignancy. In addition, total lactogens in the serum were measured by a bioassay (BA). The BA/RIA (GH + PRL) ratio was greater in the breast cancer patients than the controls, indicating that variant forms of the hormones with higher than normal biological activity might be present.     

Simultaneous isolation of prolactin and growth hormone from "discarded acid pellet" obtained from buffalo pituitaries

An acid pellet, obtained as a side fraction from a conventional gonadotropin purification pathway, has been found to contain the bulk of the pituitary lactogenic hormones (growth hormone or GH and prolactin or PRL). This discarded side fraction has been utilized to obtain buffalo lactogenic hormones (buGH and buPRL), simultaneously, and in bulk. The immunoreactivities of the purified semi-pure buffalo GH and PRL (APECS and APP-I, respectively) preparations were compared by direct binding ELISA with semi-pure standard buGH and PRL (ECS and EP-I, respectively) and were found to be as pure as standard semi-pure buGH and buPRL. When checked by direct binding ELISA using buGH and buPRL antisera, it was observed that APECS and APP-I were not cross-immunoreactive. SDS-PAGE and western blot analysis of APECS and APP-I showed major bands located at the same positions as in the case of standard semi-pure preparations (20 kDa for APECS and 23 kDa for APP-I). The semi-purified buGH and buPRL (APECS and APP-I) were converted to a highly purified preparation by chromatographing them via Sephacryl S-200 gel-filtration chromatography.     

Growth hormone size variants: changes in the pituitary during development of the chicken

There is considerable evidence for the existence of structural variants of growth hormone (GH). The chicken is a useful model for investigating GH heterogeneity as both size and charge immunoreactive-(ir) variants have been observed in the pituitary and plasma. The present study examined the size distribution of ir-GH in the pituitary gland of chicken, from late embryogenesis through adulthood. Pituitaries were homogenized in the presence of protease inhibitor, and the GH size variants were separated by SDS-PAGE, transferred by Western blotting, immunostained with a specific antiserum to chicken GH, and quantitated by chemiluminescence followed by laser densitometry (chemiluminescent assay). Under nonreducing conditions ir-GH bands of 15, 22, 25, 44, 50, 66, 80, 98, 105 and >110 kDa were observed. Both the relative proportion of the GH size variants and the total pituitary content varied with developmental stage and age. The proportion of the 15-kDa fragment was greatest in the embryonic stage, and then it decreased. The proportion of the monomeric 22-kDa form was lowest at 18 days of embryogenesis (dE) and highest at 20 dE. In contrast, the high MW forms (>/=66 kDa) were lowest in embryos, and they increased (P < 0.05) after hatching. The 22-, 44-, 66-, and 80-kDa forms were assayed for activity by radioreceptor assay following isolation by semipreparative SDS-PAGE. Only the 22-kDa GH variant showed radioreceptor activity. Under reducing conditions for SDS-PAGE, ir-GH bands of 13, 15, 18, 23, 26, 36, 39, 44, 48, 59 and 72 kDa were oberved, but most of the high MW form disappeared. There was a concomitant increase in the proportion of the monomeric band and of several submonomeric forms. The present data indicate that the expression, processing, and/or release of some if not all size variants are under some differential control during growth and development of the chicken.     

Immunocytochemical and ultrastructural characterization of mammosomatotrope-, growth hormone-, and prolactin-cells from the gilthead sea bream (Sparus aurata l., Teleostei): an ontogenic study

Growth hormone (GH), prolactin (PRL), and mammosomatotrope (MS) cells of gilthead sea bream, Sparus aurata, a teleost fish, were studied in specimens from hatching to 15 months (adults) using conventional electron microscopy and an immunogold method using anti-tilapia GH sera and anti-chum salmon PRL serum. MS cells, immunoreactive to both anti-GH sera and anti-PRL sera, had been first identified in fish in a previous study in newly hatched larvae and in older larvae and juvenile specimens of Sparus aurata by light microscopic immunocytochemistry. In the present work, MS cells reacted positively to immunogold label only in older larvae and juveniles and their secretory granules immunoreacted with both GH and PRL antisera or with only one of them. MS cells were ultrastructurally similar to the PRL cells, with which they coincided in time. This is the first report on the ultrastructural characterization of MS cells in fish. In adults, the secretory granules of GH cells (immunoreactive to anti-GH serum) were mainly round, of variable size, and had a homogeneous, highly electron-dense content. Irregularly shaped secretory granules were also present. PRL cells (immunoreactive to anti-PRL serum) were usually observed in a follicular arrangement; they showed few, small, and mainly round secretory granules with a homogeneous and high or medium electron-dense content. Some oval or elongated secretory granules were also observed. GH and PRL cells that showed involutive features were also found. In newly hatched larvae, GH, PRL, and MS cells could not be distinguished either by their ultrastructure or by the immunogold labeling of the secretory granules. In 1-day-old larvae, presumptive GH and PRL cells were observed according to their position in the pituitary gland. In 2-day-old larvae, a few cells showed some of the ultrastructural features described for GH and PRL cells of adults. During development, the number, size, and shape of the secretory granules in both cell types clearly increased and the organelles developed gradually. Some GH cells were found undergoing mitosis.     

Partial purification and characterization of rhinoceros gonadotropins, growth hormone, and prolactin: comparison with the horse and sheep

The rhinoceros is an endangered species related to the horse family. Little is known of its reproductive endocrinology. The objectives of this study were to partially purify rhinoceros pituitary hormones, determine which assays could be used for their assessment, and to ascertain whether rhinoceros LH possesses the intrinsic FSH activity of equine LH. A single pituitary each from a White (1.3 g) and a Black (1.2 g) Rhinoceros was homogenized and extracted (pH 9.5), then subjected to pH and salt fractionation, and ion-exchange chromatography (DEAE and Sephadex SP-C50) to yield partially purified fractions of LH, FSH, growth hormone (GH), and prolactin (PRL). LH was readily measured by a rat Leydig cell assay (0.1-1% x equine LH) and an RIA using a monoclonal antibody to bovine LH (6-11% x equine LH). FSH activity detected in the LH by either an FSH RIA or a calf testis radioreceptor assay (RRA) was extremely low. No FSH activity could be detected in the White Rhinoceros pituitary "FSH" fraction, but was readily detected in the Black Rhinoceros fraction (RIA: 0.2% x equine FSH: RRA: 0.8% x equine FSH). The presence of GH and PRL was determined by SDS-PAGE and Western blots. Results showed a single immunoreactive GH band and multiple immunoreactive PRL bands. Adsorption with Concanavalin A-Sepharose indicated that some of the PRL bands are glycosylated.     

Immunocytochemical technique for detection of prolactin (PRL) and growth hormone (GH) in hyperplastic and neoplastic lesions of dog prostate and mammary gland.

An immunocytochemical technique was described to test for immunoreactive prolactin (PRL) and growth hormone (GH) in spontaneous and experimentally induced hyperplastic and neoplastic lesions of the prostate and mammary gland. The dog was used as an animal model. The specificity and validity of the immunocytochemical staining procedure and of the antisera to canine PRL and canine GH can be regarded as established for the demonstration of PRL- and GH-dependent staining respectively. In mammary and prostatic tissues, both endogenous PRL and GH as well as intracellular free binding sites (for exogenous PRL and GH) were detected immunocytochemically. The technique presented seems to be an important tool to localize putative target sites for pituitary hormones in hormone-dependent hyperplasia and neoplasia.     

A mutant lactogenic hormone binds, but does not activate, the prolactin receptor

We have generated mutations in mouse placental lactogen II, a hormone in the PRL/GH family that binds to the PRL receptor, to investigate the role of the conserved cysteine residues in hormone function. Disruption of the small C-terminal disulfide loop did not significantly alter hormone activity. Substitution of serine for cysteine-51, which prevents formation of the large disulfide loop, results in a protein equivalent to placental lactogen II in receptor-binding activity; however, this mutant protein is not mitogenic in an assay for lactogenic hormones. These results indicate that PRL receptor occupancy and activation are distinct events.     

Specific antisense RNA inhibition of growth hormone production in differentiated rat pituitary tumour cells.

An expression vector that carried an inverted 800 base pair insert of the rat growth hormone (rGH) cDNA downstream of the SV40 promotor was used to transfect two different growth hormone (GH) producing rat pituitary cell strains, GH12C1 and GH3. This resulted in a specific transient inhibition of growth hormone production up to 75 percent in the course of 72 hours. GH synthesis reduction occurred parallel to a decrease of GH cytoplasmic mRNA levels. Levels of beta-actin and guanine nucleotide-binding regulatory protein (G protein) mRNAs were unaltered, but PRL mRNA levels were increased. Transfection with a control vector did not affect GH production.     

Regulated expression of the prolactin gene in rat pituitary tumor cells

Prolactin (PRL) gene expression in three strains of GH cells (rat pituitary tumor cells) has been quantitated by measurement of: (a) intracellular and extracellular PRL, (b) cytoplasmic translatable PRL-specific mRNA (mRNAPRL), and (c) molecular hybridization of cytoplasmic poly(A) RNA to cDNAPRL (DNA complementary to mRNAPRL). Three GH cell lines utilized in this investigation were a PRL-producing (PRL+) strain, GH4C1, a PRL nonproducing 5-bromo-deoxyuridine resistnat (PRL- BrdUrdr) strain, F1BGH12C1, and a new strain, 928-9b, derived by fusion of PRL+ cells with a nuclear monolayer of the PRL-, BrdUrdr GH cell strain. PRL production is a characteristic of 928-9b cells, but the level of PRL production (2-4 micrograms/mg protein/24 h) is much lower than that of the PRL+ strain, GH4C1 (15-25 micrograms/mg protein/24 h). Levels of cytoplasmic translatable mRNAPRL and cytoplasmic PRL-RNA sequences quantitated with a cDNAPRL probe were also much lower in 928-9b as compared to the PRL+ parent. PRL-RNA sequences could not be detected in the PRL- strain. Thyrotopin-releasing hormone (TRH) stimulates PRL synthesis about threefold and inhibit a growth hormone (GH) synthesis 72% in the PRL+ strain. TRH has no effect on the synthesis of either PRL or GH in the 928-9b strain, although TRH receptors could be detected in these cells. Stimulation of PRL synthesis in the PRL+ strain by TRH could be correlated with increases in levels of cytoplasmic translatable mRNAPRL and increases in cytoplasmic PRL-RNA sequences. These results demonstrate that the graded expression of the PRL gene at the basal level, and in response to TRH, is caused by the regulated production of specific mRNA, i.e., mRNAPRL in these three GH cell strains.     

Effect of experimentally induced hyperprolactinemia on growth hormone secretion

In order to study the existence of possible interrelation-ships between prolactin (PRL) and growth hormone (GH) secretions, adult male rats bearing an anterior pituitary graft under the kidney capsule since day 90 of life and their sham-operated controls were submitted to a single i.p. administration of L-dopa (50 mg/kg weight) or saline 30 days after the operation. Plasma PRL and GH levels were measured by using specific RIA methods. Dopamine (DA) and norepinephrine (NE) contents in the hypothalamus and in the in situ anterior pituitary gland were measured by using a specific radioenzymatic assay. An increase in plasma PRL levels and a decrease in plasma GH levels were shown in grafted rats. Hypothalamic contents of DA and NE were increased in these animals, while the anterior pituitary content of DA was not modified as compared to controls. The administration of a single injection of L-dopa led to decreases of plasma PRL and GH levels in both grafted and control rats, but while marked increases in hypothalamic and anterior pituitary contents of DA were shown in both groups, the hypothalamic content of NE was only increased in control animals. These data suggest that PRL and GH secretions were closely related. Dopamine could be mediating the action of PRL on GH, while NE would be less involved.