Mammotroph autoregulation: intracellular fate of internalized prolactin

As part of a study concerning the mechanism of mammotroph autoregulation, the intracellular fate of [125I]iodo rat PRL, internalized by mammotrophs, was determined by quantitative electron microscope autoradiography. Simple grain density (GD) analysis of mammotroph autoradiograms showed that 5 cellular compartments (plasma membrane, Golgi apparatus, secretory granules, nucleus and mitochondria) were significantly labeled and that the labeling pattern in these compartments changed with time. Inclusion of a 25-fold excess of unlabeled PRL inhibited the binding of radiolabeled hormone to mammotrophs by approximately 85%. At no time were any grains found associated with lysosomes and there was no evidence of degradation of the radiolabeled PRL as judged by SDS-polyacrylamide gel electrophoresis or cold perchloric acid precipitation of cell extracts or media samples. Delivery of internalized PRL to a number of specific intracellular organelles suggests that these organelles may serve in the autoregulation of PRL secretion.     

Ultrastructural-immunocytochemical localization of growth hormone and prolactin in human pituitaries.

Immunocytochemical staining using the unlabeled antibody peroxidase-antiperoxidase method was undertaken to localize and characterize in ultrathin sections of human pituitaries the cells responsible for the secretion of GH and PRL. Somatotrophs in seven pituitaries stained with human (h) PRL-absorbed antiserum to hGH, were abundant, round to ovoid, densely granulated cells, whose mean (+/-SD) granule diameter was 368 +/- 60 nm. Lactotrophs immunostained with antiserum to hPRL were less numerous, angular or branching cells, with fewer round to ovoid granules, the mean diameter (+/-SD) of which was 185 +/- 35 nm in six pituitaries. The somewhat larger PRL granules (up to a mean diameter of 360 nm) seen in two of three additional pituitaries may have been related to the previous therapeutic administration of estrogen. Whereas the immunostained GH-secreting cells resemble the presumed somatotrophs identified in other studies on the basis of nonimmunological staining, the immunostained PRL-secreting cells differ considerably from the cells with large (600--1000 nm) granules designated as lactotrophs by several previous investigators. The hazards of ultrastructural identification of human pituitary cell types on purely morphological (as opposed to immunocytochemical) grounds are emphasized.     

Preferential release of newly synthesized prolactin granules is the result of functional heterogeneity among mammotrophs

Pituitary cells maintained in monolayer culture for 48 h were used for double isotope labeling to study the release of newly synthesized vs. old PRL. The intracellular pathway taken by newly synthesized PRL was studied by autoradiography. For double labeling the cells were first incubated in a 14C-labeled amino acid (4 h) and then in a 3H-labeled amino acid (1 h), each followed by a 1-h chase. Total PRL (RIA) and radiolabeled PRL (immunoprecipitation) were determined in both cells and media. Thre rate of release of PRL (RIA) was stable throughout the experimental period. In unstimulated cells the ratio of 3H- to 14C-labeled PRL in the medium at the end of a 1-h incubation following the second chase was twice that in the cells at the beginning of this incubation, indicating that newly synthesized [3H]PRL was preferentially released. Stimulation with TRH resulted in increased release of older [14C]PRL. The earliest that radiolabeled (14C or 3H) PRL could be detected in the medium was 15--30 min after exposure of the cells to isotope. For autoradiography, cells were given a 5-min pulse of [3H]leucine, followed by chase periods of 30--180 min. Grain counts indicated that mammotrophs maintained in culture for 48 h transported and packaged PRL with the same time course as those preparations studied previously. Analysis of the distribution of total grains per cell in mammotrophs revealed the presence of several functional subpopulations with different numbers of total grains per cell, indicating that some cells were manufacturing and secreting PRL at a very fast rate. It is concluded that 1) preferential release of newly synthesized PRL is due to preferential discharge of newly synthesized granules, since release occurs at a time when labeled hormone is already in the granules; 2) there is no evidence that the established secretory pathway is bypassed, since transport, concentration, and granule formation occur; and 3) preferential release of newly synthesized PRL is the result of functional heterogeneity within the mammotroph population. Functional heterogeneity is illustrated by major differences in both synthetic rates and TRH responsiveness.     

Adenohypophysial changes in mice transgenic for human growth hormone-releasing factor: a histological, immunocytochemical, and electron microscopic investigation

The effect of protracted GH-releasing factor (GRF) stimulation on adenohypophysial morphology was investigated in six mice transgenic for human GRF (hGRF). All animals had significantly higher plasma levels of GH and GRF and greater body weights than controls. Eight-month-old mice were killed, and the markedly enlarged pituitaries were studied by histology, immunocytochemistry, electron microscopy, and immunogold method, using double labeling at ultrastructural level. In all pituitaries, a massive hyperplasia, chiefly of mammosomatotrophs, was found. These bihormonal cells, containing GH and PRL, were demonstrated by light microscopy and ultrastructural immunocytochemistry. Electron microscopy revealed the presence of cells with characteristics of GH cells in three pituitaries and cells resembling human adenomatous mammosomatotrophs in the other three glands. All of these cells, regardless of their ultrastructural features, contained secretory granules heavily labeled for GH by immunogold technique; PRL labeling varied from cell to cell, with the predominance of a weak immunostaining and was colocalized with GH in secretory granules. These results indicate that chronic exposure to GRF excess leads to mammosomatotroph hyperplasia. It is suggested that GH cells proliferate and transform to mammosomatotrophs in response to GRF stimulation. Focal PRL cell hyperplasia noted in three pituitaries could also be due to a GRF effect. Longer exposure to GRF is needed to clarify whether GRF can cause adenoma.     

Analysis of mammosomatotropic cells in normal and neoplastic human pituitary tissues by the reverse hemolytic plaque assay and immunocytochemistry

The reverse hemolytic plaque assay (RHPA) was used to study hormone release from cultured normal and neoplastic human pituitary cells. The RHPA revealed a lower percentage of GH- and PRL-producing cells in normal and neoplastic pituitaries compared to the percentage of these cells revealed by immunocytochemical (ICC) staining for GH and PRL. Normal pituitary tissues as well as some PRL- or GH-producing adenomas contained large numbers of mammosomatotropic (MS) cells when analyzed by RHPA, combined RHPA-ICC, and ultrastructural immunohistochemistry with immunogold labeling. The percentage of GH and PRL cells in normal pituitaries ranged from 37-51% and 30-60%, respectively, by RHPA, while the percentage of MS cells ranged from 29-49%. The percentage of GH and PRL cells in normal pituitaries estimated by ICC ranged from 53-65% and 32-55%, respectively, while the percentage of MS cells estimated with this technique ranged from 26-50%. Double labeling with the immunogold technique detected GH and PRL in the same cells and within the same granules in both normal and neoplastic pituitary cells. These results indicate that MS cells are present in normal human pituitaries as well as in some pituitary adenomas, and in some pituitaries these two hormones are stored within the same secretory granules.     

Effects of growth hormone-releasing hormone (GHRH) on densely granulated somatotroph adenomas and sparsely granulated somatotroph adenomas in vitro: a morphological and functional investigation

The effects of growth hormone-releasing hormone (GHRH) were studied on densely granulated somatotroph adenoma cells and sparsely granulated somatotroph adenoma cells in culture by measuring release of growth hormone (GH) as well as ultrastructural morphometrical parameters and comparing them with those of control adenoma cells. Both types of adenoma cells cultured with GHRH showed similar increases of GH release into culture media and exhibited similar increases in cytoplasmic volume densities (CVD) of endoplasmic reticulum and Golgi apparatus and decreases in CVD of secretory granules and secretory granule diameter. These results indicate that (1) both types of somatotroph adenoma cells react similarly to GHRH stimulation, despite their morphologic differences, and (2) GHRH stimulates GH synthesis as well as GH release by somatotroph adenoma cells.     

Hormonal induction of secretory granules in a pituitary tumor cell line

GH4C1 cells are a rat pituitary tumor cell strain that secretes PRL and GH but contains almost no secretory granules. Treatment of GH4C1 cells with a combination of estradiol (1 nM), insulin (300 nM), and epidermal growth factor (10 nM) increased the cellular content of PRL by more than 30-fold above control levels but only increased PRL accumulation in the medium 6-fold. To determine whether the increase in intracellular PRL was accompanied by an increase in secretory granules, we compared the numbers of granules in ultrathin sections from untreated GH4C1 cells and from cells treated with the combined hormone regimen and found a nearly 50-fold increase in granule number. Only 75% of the granules stained for PRL by the protein-A gold technique; the other 25% stained for neither PRL nor GH. The occasional granules found in untreated GH4C1 cells stained for PRL. The data demonstrate that the number of granules in GH4C1 cells can be regulated by hormone treatment and that the increase in intracellular PRL is found in storage granules.     

Effects of hypophysiotropic factors on growth hormone and prolactin secretion from somatotroph adenomas in culture

In an attempt to characterize GH and PRL secretion in acromegaly, the effects of various stimuli on GH and PRL release by cultured pituitary adenoma cells derived from acromegalic patients were studied. In addition, the PRL responses of somatotroph adenoma cells were compared to those of prolactinoma cells. GH-releasing hormone-(1-44) (GHRH) consistently stimulated GH secretion in all 14 somatotroph adenomas studied in a dose-dependent manner. The sensitivity as well as the magnitude of the GH responses to GHRH were highly variable in individual tissues. Somatotroph adenomas that did not respond to dopamine were more sensitive and had greater GH responses to GHRH. In 8 of 9 somatotroph adenomas that concomitantly secreted PRL, the addition of GHRH likewise increased PRL release. Omission of extracellular Ca2+ blocked the stimulatory effect of GHRH on GH and PRL secretion. When cells were coincubated with 0.1 nM somatostatin, GH and PRL secretion induced by 10 nM GHRH were completely blocked in most adenomas. Similarly, coincubation of dopamine resulted in inhibition of GHRH-induced hormone secretion in some adenomas. Addition of TRH to the incubation medium, on the other hand, significantly stimulated GH secretion in 8 of 14 adenomas, while TRH stimulated PRL release in all of the adenomas. Vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH) produced an increase in GH and PRL secretion in other adenomas. In prolactinoma cells, somatostatin and dopamine unequivocally suppressed PRL secretion; however, other stimuli including GHRH, VIP, and CRF were ineffective. TRH induced a significant increase in PRL secretion in only one prolactinoma. These results suggest that responsiveness to GHRH and somatostatin is preserved in somatotroph adenomas; the responsiveness to GHRH is inversely correlated to that to dopamine; and PRL cells associated with somatotroph adenomas possess characteristics similar to those of GH cells. Further, the GH stimulatory actions of TRH and VIP are different.     

Cysteamine causes reduction of prolactin monomers followed by aggregation in the rat pituitary gland

Storage forms of PRL were studied in control and cysteamine-treated cultures of estradiol-induced tumors in Fischer 344 rats and in secretory granules isolated from these tumors to further investigate the mechanism of action of cysteamine on PRL. The two major bands visible when protein is stained after electrophoresis of isolated granules migrate to the position of PRL and GH monomers. Electrophoresis under reducing conditions changes the position, but does not noticeably increase the amount of each band. [3H]PRL in cells labeled for 8 h with [3H]leucine also exists predominantly as monomer. Immunoreactivity of PRL in cell lysates or isolated granules is not affected by incubation with reducing agents beta-mercaptoethanol or glutathione at concentrations up to 5 mM, but cysteamine decreases PRL immunoreactivity in isolated granules at concentrations of 3 mM and higher. Electrophoresis of isolated granules after incubation with 25 mM cysteamine for 1 h demonstrates that cysteamine converts PRL to the reduced form. After 4 h, or after dilution of the granules before solubilization, the amount of reduced monomer is decreased, and larger molecular weight species appear. The reduced monomer can be recovered by electrophoresis under reducing conditions. The fully immunoreactive form can be recovered by incubation for 1 h with dithiothreitol at concentrations of 0.3 mM-3 mM. These data indicate that: PRL exists predominantly in monomeric form in the rat pituitary gland, and cysteamine reduces PRL, and formation of disulfide-linked aggregates of PRL occurs subsequently under some conditions.     

Osmotic pressure regulation of prolactin and growth hormone release from bovine secretory granules

PRL and GH are stored in bovine pituitary secretory granules in part as intermolecular disulfide-linked multimers (as many as 50 monomers/oligomer). In vitro incubation of granules with reduced glutathione (GSH) results in the production of monomeric hormones and increased hormone release. We tested the hypothesis that increases in intragranular osmolality due to conversion of oligomers to lower mol wt forms are key to granule rupture. Hormone release was measured after granule incubations in medium made hyperosmotic by the addition of raffinose or sucrose. PRL release at pH 7.8 with 0.7 M raffinose was reduced to 43.5% of the control value, whereas GH release ranged from 70-100% of the control value. Augmented release stimulated by 2 mM GSH was also diminished by raffinose, to 17.9% (PRL) and 75.6% (GH) of control values. The IC50 for raffinose inhibition of PRL release was 0.62 M; this shifted to 0.38 M in the presence of 2 mM GSH. Raffinose also blunted the granule response to GSH at all other GSH concentrations tested and decreased stimulated release at pH 9 as well as at 7.8. Interference with the ability to maintain an osmotic gradient, a maneuver accomplished by partial disruption of granule membranes with 0.5% deoxycholate treatment, reduced or abolished the raffinose inhibition. Molecular sizing of granule fractions on Superose-6 (Pharmacia) indicated that PRL monomers remaining in the pelletable granule fraction increased with raffinose about 2-fold under both basal and GSH-stimulated conditions. These data support the concept that thiol:disulfide equilibria may be important in PRL and GH storage and secretion by their influence on intragranular osmolality. In exocytosis in vivo, osmotically mediated granule rupture may be triggered by thiols during granule:plasma membrane fusion.     

Prolactin (PRL)-releasing peptide stimulates PRL secretion from human fetal pituitary cultures and growth hormone release from cultured pituitary adenomas

The hypothalamic peptide PRL-releasing peptide (PrRP) has recently been cloned and identified as a ligand of an orphan pituitary receptor that stimulates in vitro PRL secretion. PrRP also induces PRL release in rats in vivo, especially in normal cycling females. However, no information on the effects of PrRP in the human is available. To elucidate the role of PrRP in regulating human anterior pituitary hormones, we used human PrRP-31 in primary cultures of human pituitary tissues, including fetal (20--27 weeks gestation) and normal adult pituitaries, as well as PRL- and GH-secreting adenomas. PrRP increased PRL secretion from human fetal pituitary cultures in a dose-dependent manner by up to 35% (maximal effect achieved with 10 nM), whereas TRH was slightly more potent for PRL release. Coincubation with estradiol resulted in enhanced fetal PRL response to PrRP, and GH release was only increased in the presence of estradiol. Although PRL secretion from PRL-cell adenomas was not affected by PrRP, PrRP induced PRL release from cultures of a GH-cell adenoma that cosecreted PRL. PrRP enhanced GH release in several GH-secreting adenomas studied by 25--27%, including GH stimulation in a mixed PRL-GH-cell tumor. These results show for the first time direct in vitro effects of PrRP-31 on human pituitary cells. PrRP is less potent than TRH in releasing PRL from human fetal lactotrophs and is unable to release PRL from PRL-cell adenomas in culture, but stimulated GH from several somatotroph adenomas. Thus, PrRP may participate in regulating GH, in addition to PRL, in the human pituitary.     

Studies on rat pituitary homografts. I. In vitro biosynthesis and release of growth hormone and prolactin.

Homologous anterior pituitaries grafted under the kidney capsule in hypophysectomized rats were studied 30 days after transplantation. Some cells maintained the ultrastructural features peculiar to the various cell types of normotopic glands, while the others were characterized by few, small, dense granules, spherical or polymorphic, located peripherally in the cytoplasm. This picture might be due to a functional adaptation which occurs in pituitary cells still producing different hormones, once removed from central nervous system control. The major change in polypeptide hormone composition of graft homogenates relative to normotopic pituitaries is the fall in GH and PRL concentration. The in vitro incorporation of L-[3H]leucine into the two hormones and the release of radioactive GH and PRL from L-[3H]leucine-prelabeled tissue fragments are also greatly decreased. The decrease in concentration, in vitro biosynthesis, and release of GH per mg tissue protein are approximately 87, 91, and 93%, respectively. These results might be due primarily to a decrease in the number of somatotrophs and/or in their secretory activity, with relatively minor changes in GH intracellular transport and turnover. In contrast, a clear-cut fall in in vitro turnover was detected for PRL, as shown by the fact that decreases in biosynthesis and release per mg tissue protein of this hormone (approximately -95% and -99%, respectively) by far exceed the decrease in the tissue concentration (-74%). These data indicate that in in vitro secretory activity of mammotrophs is greatly reduced in the grafts with respect to the normotopic glands. Thus, the high secretory activity previously reported in hypophysectomized rats bearing pituitary grafts should be attributed to the lack of the inhibitory control of the central nervous system rather than to an increase in secretory capacity under nonrestrained conditions.     

Subcellular localization of dopamine in the anterior pituitary gland of the rat: apparent association of dopamine with prolactin secretory granules.

The subcellular compartmentalization of endogenous dopamine in the anterior pituitary gland of the rat was investigated using continuous sucrose density gradient centrifugation. When anterior pituitary homogenates were layered on continuous sucrose density gradients (1.0--2.0 M) and centrifuged for 60 min at 40,000 X g, dopamine recovered from the gradients was associated with two sets of subcellular particles. The particles in one set were recovered near the top of the gradient, whereas those in the other set were recovered near the bottom of the gradient in the region where particles containing PRL were also found. In fact, these dense dopamine-containing particles could not be separated from those particles which contained PRL. These findings were suggestive that dopamine and PRL were present in the same particle, viz. the PRL secretory granule. This interpretation was further strengthened when it was established that the PRL-containing granules were separable on the gradient from granules which contained GH, LH, FSH, ACTH, and TSH. When [3H]dopamine was added to the solution in which the anterior lobes were homogenized, no radio-activity was found to be associated with the dense dopamine-containing particles. Also, the addition of a large excess of nonradiolabeled dopamine at the time of homogenization did not influence the amount of dopamine associated with the dense particles. Thus, the apparent association of dopamine with PRL secretory granules was not an artifact of the homogenization process per se. Therefore, it is concluded that an association exists between intracellular dopamine and the PRL secretory granule.     

Dopamine-induced physical barrier to inhibit prolactin release in mammotrophs disappears during resumption of prolactin secretion

After finding that dopamine can cause extensive changes in mammotroph ultrastructure within 2 min of administration, we further examined mammotrophs 20 and 40 min after dopamine injection when prolactin secretion had resumed. Dopamine (1,000 micrograms/kg) was injected into the right atrium of estradiol-primed male rats through indwelling cannulae. Rats were killed by decapitation 2, 20 and 40 min after dopamine treatment and their pituitaries were processed for electron microscopy. Within 2 min of administration, dopamine completely stopped prolactin release and significant changes in mammotroph ultrastructure were observed: peripheral relocation of rough endoplasmic reticulum (RER) ('dopamine barrier'); fewer secretory granules in close proximity to the cell membrane; fewer exocytoses, and increased numbers of 'intracellular bodies' associated with secretory granules (putative granule disposal system). 20 min following dopamine administration, when prolactin secretion had resumed, there appeared to be a breaking up of the 'RER barrier' in that more vesicular elements were seen and the amount of peripheral RER was reduced; more granules were adjacent to the cell membrane and exocytotic figures were more numerous. 'Intracellular bodies' decreased in number. By 40 min, mammotrophs appeared to resemble those of estradiol-primed controls with the RER stacked to one side of the cell and small numbers of 'intracellular bodies'. The correlation between ultrastructural changes in mammotrophs and prolactin release implies that the 'RER barrier' is part of a mechanism by which prolactin release is inhibited.     

The mechanism of action of cysteamine in depleting prolactin immunoreactivity

The thiol reagent cysteamine (CSH) depletes anterior pituitary cells of immunoreactive PRL both in vivo and in vitro. We examined the hypothesis that CSH affects either the solubility or immunoreactivity of PRL through a mechanism involving thiol-disulfide exchange. Adult female rats were treated with either CSH (300 mg/kg, sc) or an equimolar dose of ethanolamine as a control. Anterior pituitary glands were extracted in 0.1 M sodium borate buffer, pH 9.0. Treatment of pituitary extracts with beta-mercaptoethanol (BME) destroys the immunoreactivity of PRL. However, extraction in the presence of reduced glutathione or CSH of pituitaries of rats treated with CSH restores immunoreactive PRL to control levels. Extracts were also subjected to polyacrylamide gel electrophoresis (PAGE). On gels of pituitary extracts of CSH-treated rats, the band that comigrates with purified PRL is diminished compared to that in ethanolamine-treated controls. This is found regardless of whether the borate extracts are treated with BME. However, extraction of the pituitaries in sodium dodecyl sulfate-containing buffer followed by chemical reduction with BME restores the PRL band. Therefore, CSH acts on PRL through a thiol-related mechanism to yield a product that is poorly soluble in aqueous buffer at pH 9 and is poorly immunoreactive. Dispersed anterior pituitary cells in tissue culture were incubated with L-[35S]methionine to radiolabel newly synthesized peptides. These cultures were incubated in the presence of either CSH or ethanolamine. PAGE followed by autoradiography confirmed the above results obtained in vivo. Also, extracts of CSH-treated cultures were subjected to gel permeation chromatography. As determined by PAGE, at least some of the radiolabeled PRL can be recovered from void volume fractions by reduction with BME, indicating that CSH induces the formation, through disulfide exchange, of a high mol wt form of PRL, possibly PRL oligomers.     

Preferential interaction of [35S]cysteamine with pituitary secretory granule storage forms of prolactin

Cysteamine (CySH) inhibits the immunodetectability and bioactivity of prolactin (PRL), and we have proposed that it may act by impeding the conversion from secretory granule hormone storage forms to releasable and assayable hormone. This process appears to be dependent upon thiol:disulfide interchange reactions, which can be inhibited by the aminothiol. The present studies utilized [35S]CySH to determine whether preferential interactions could be demonstrated between CySH and bovine pituitary storage hormone forms as opposed to monomeric PRL. [35S]CySH was incubated with purified intact secretory granules, with granule 'core' preparations enriched in oligomeric forms by prior hypotonic exposure, with chromatographically isolated oligomers, and with monomeric PRL. Binding to granules was saturable and pH-dependent with greatest binding observed at pH 7.5-8.0. Binding to monomer was much less than binding to all other fractions, being 20% or less than that to any other form. HPLC studies of granules treated with [35S]CySH indicated that exposure to CySH was associated with a predominance of very high molecular weight oligomers. These forms were entrapped on the gel permeation columns, resulting in decreased protein and PRL recovery; as little as 6.6% of the PRL was eluted after 60 min of CySH exposure. CySH not only bound to storage forms of PRL but also to secretory granule membranes; whether the bioeffect is mediated through membrane modifications is unknown. Despite its relative ineffectiveness at altering growth hormone immunoactivity or secretion, CySH nonetheless also bound to growth hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent binding of growth hormone to surface receptors on rat adipocytes

GH specifically binds to receptors on the surface of adipocytes and produces a variety of biological effects in these cells. To gain insight into the nature of the GH receptors, [125I] human GH ([125I]hGH) was cross-linked to surface binding sites on intact rat adipocytes using the bifunctional reagent disuccinimidyl suberate. Plasma membranes were isolated, and after solubilization with sodium dodecyl sulfate (SDS), the proteins were subjected to electrophoresis on 5% or 7.5% polyacrylamide gel. Autoradiography of the 7.5% gels revealed three iodinated bands corresponding to apparent molecular weights of 56, 130, and more than 240 kilodaltons. The more than 240-kilodalton band contained approximately as much 125I as the 130-kilodalton species and about twice as much as the 56-kilodalton species. When run on the more porous 5% gel, the more than 240-kilodalton band resolved into two bands, corresponding to apparent molecular weights of 240 and 310 kilodaltons. Excess unlabeled human or bovine GH, but not ovine PRL, competed with [125I]hGH for binding and prevented the formation of all of the labeled bands. Treatment of the membranes and extracted proteins with dithiothreitol resulted in the generation of additional 130-kilodalton material at the expense of both the 310- and 240-kilodalton species, but failed to alter the amount of 125I that migrated with the 56-kilodalton species. The same pattern of labeling was seen regardless of whether protease inhibitors were present during isolation of membrane proteins or when membrane proteins were isolated under conditions that favored proteolysis, suggesting that the 56-kilodalton species is not a degradative product of the higher molecular weight species. When [125I]hGH was cross-linked to adipocytes in which total binding was decreased by hypophysectomy or starvation of the donor rats or by treatment of the cells with cycloheximide, there was a proportionate diminution in labeling of all species. It thus appears that the GH receptor contains a 130-kilodalton subunit, a portion of which is in disulfide linkage with higher molecular weight complexes and, in addition, contains a 56-kilodalton species. It cannot be determined from these studies if the various labeled protein complexes are components of a single or multiple classes of GH receptors in the adipocyte membrane.     

Morphological studies of prolactin-secreting cells in estrogen receptor alpha and estrogen receptor beta knockout mice

Estrogens play a major role in the regulation of prolactin (PRL) secretion through activation of pituitary and hypothalamic estrogen receptors (ERs). In order to evaluate the relative role of ERalpha and ERbeta in the control of PRL density in the pituitary gland, we performed immunocytochemical localization of PRL and ERs in pituitaries of wild-type (WT), ERalpha knockout (KO) and ERbetaKO mice. In WT and ERbetaKO anterior pituitaries, the vast majority of secretory cells contained ERalpha immunoreactivity, while no ERalpha immunostaining could be found in ERalphaKO pituitaries. No ERbeta immunoreactivity could be detected in pituitaries of WT, ERalphaKO or ERbetaKO mice. At the light microscopic level, a large number of cells staining for PRL were present in pituitaries of female WT, while in female ERalphaKO pituitaries, the density of PRL cells was much lower. In WT male pituitaries, the density of PRL cells was lower than observed in female WT, while PRL staining was markedly decreased in male ERalphaKO as compared to male WT. In ERbetaKO mice of both sexes, the results were identical to those observed in WT animals. At the electron microscopic level, in WT mice of both sexes, type 1 PRL cells exhibited a well-developed Golgi apparatus and a large number of strongly stained large mature and immature secretory granules. Type 2 PRL cells were also present in the pituitary. Type 2 PRL cells contain small poorly labelled granules. In ERalphaKO mice of both sexes, type 1 PRL cells were atrophied with poorly developed Golgi apparatus, and no type 2 PRL cells could be observed. In ERalphaKO pituitaries, typical gonadectomy cells were found. No ultrastructural changes were observed in PRL cells of ERbetaKO mice. The present data strongly suggest that the positive regulation of PRL expression at the pituitary level by estrogens is mediated by ERalpha and does not involve ERbeta activation.     

Purification, characterization, and bioassay of prolactin and growth hormone from temperate basses, genus Morone

Prolactin (PRL) and two variants of growth hormone (GH), purified from pituitaries of striped bass (Morone saxatilis) and its hybrid with white bass (M. saxatilis x M. chrysops) by gel filtration chromatography under alkaline conditions followed by reversed-phase high pressure liquid chromatography, appear similar between species. Both the minor (GH I) and the major (GH II) forms of purified GH appeared as single bands (M(r) approximately 23,000) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as did the purified PRL (M(r) approximately 24,000). The molecular weights of GH II and PRL determined by MALDI TOF mass spectroscopy were 21.2 and 21.3 kDa, respectively. In Western blotting experiments, an antiserum against tilapia (Oreochromis mossambicus) 24K PRL specifically recognized Morone PRL, while an antiserum against tilapia GH specifically recognized Morone GH I and II. Chemical identities of the putative PRL and GH I were further confirmed by N-terminal peptide sequencing, while internal sequence analysis was performed on GH II because it was blocked at its N-terminus. Over a stretch of 29 amino acids, Morone PRL was found to be 76% identical to tilapia 24K PRL, 72% identical to tilapia 20K PRL, 72% identical to chum salmon (Oncorhynchus keta) PRL I, and 69% identical to eel (Anguilla japonica) PRL I. Alignment of the hybrid striped bass GH sequences with those of several other advanced marine teleosts indicated 75-85% sequence identity for GH I (40 amino acids) and 95-98% identity for GH II (45 amino acids). Biological activity of striped bass GH II was confirmed using a heterologous in vitro assay of insulin-like growth factor I mRNA production by coho salmon (On. kisutch) hepatocytes. An in vivo bioassay, involving hypophysectomy of hybrid striped bass and treatment of the fish maintained in fresh water with homologous PRL, confirmed that the purified striped bass PRL was also bioactive.