In vitro and in vivo studies on cytodifferentiation of pituitary clonal cells derived from the epithelium of Rathke's pouch

Summary A clonal strain of anterior pituitary cells was derived from Rathke's pouch of the rat. These cells were shown to secrete ACTH, growth hormone and prolactin but no glycoprotein hormones, when grown in vitro. Cells from the 2A8 clone were implanted for one month under kidney capsules or into hypothalami of hypophysectomized female rats. Under the kidney capsule, prominent prolactin cells and poorly developed cells of other types were differentiated as seen in usual pituitary grafts. In hypophysiotrophic areas of the hypothalamus, the grafts were cytodifferentiated into various types of anterior pituitary cells with rich vascularization. These cells had the ultrastructural features indicative of hormone secretion. Increases in body and ovarian weights reflected the secretion of somatotrophic and gonadotrophic hormones. The results obtained indicate that implants of 2A8 clonal cells may differentiate into all types of anterior pituitary cells under the influence of hypothalamic hormones or perhaps some unknown factors present in the general systemic circulation of the rat.Supported by USPHS Grant AM 12583The authors wish to thank Mrs. Martha Castilleja and Mrs. Pauline Polette for their skillful technical assistance     

High-level expression of biologically active human prolactin from recombinant baculovirus in insect cells

We examined the feasibility of high-level production of recombinant human prolactin, a multifunctional protein hormone, in insect cells using a baculovirus expression system. The human prolactin cDNA with and without the secretory signal sequence was cloned into pFastBac1 baculovirus vector under the control of polyhedrin promoter. Prolactin was produced upon infection of either Sf9 or High-Five cells with the recombinant baculovirus containing the human prolactin cDNA. The production of recombinant prolactin varied from 20 to 40 mg/L of monolayer culture, depending on the cell types. The prolactin polypeptide with its own secretory signal was secreted into the medium. N-terminal amino acid sequence analysis of the recombinant polypeptide purified from the culture medium indicated that the protein was processed similar to human pituitary prolactin. Carbohydrate analysis of the purified protein indicated that a fraction of the recombinant prolactin made in insect cells appeared to be glycosylated. Also, both secreted and nonsecreted forms of the recombinant prolactin in insect cells were biologically equivalent to the native human prolactin (pituitary derived) in the Nb2 lymphoma cell proliferation assay.     

Prolactin receptor antagonism in mouse anterior pituitary: effects on cell turnover and prolactin receptor expression

Since anterior pituitary expresses prolactin receptors, prolactin secreted by lactotropes could exert autocrine or paracrine actions on anterior pituitary cells. In fact, it has been observed that prolactin inhibits its own expression by lactotropes. Our hypothesis is that prolactin participates in the control of anterior pituitary cell turnover. In the present study, we explored the action of prolactin on proliferation and apoptosis of anterior pituitary cells and its effect on the expression of the prolactin receptor. To determine the activity of endogenous prolactin, we evaluated the effect of the competitive prolactin receptor antagonist Δ1-9-G129R-hPRL in vivo, using transgenic mice that constitutively and systemically express this antagonist. The weight of the pituitary gland and the anterior pituitary proliferation index, determined by BrdU incorporation, were higher in transgenic mice expressing the antagonist than in wild-type littermates. In addition, blockade of prolactin receptor in vitro by Δ1-9-G129R-hPRL increased proliferation and inhibited apoptosis of somatolactotrope GH3 cells and of primary cultures of male rat anterior pituitary cells, including lactotropes. These results suggest that prolactin acts as an autocrine/paracrine antiproliferative and proapoptotic factor in the anterior pituitary gland. In addition, anterior pituitary expression of the long isoform of the prolactin receptor, measured by real-time PCR, increased about 10-fold in transgenic mice expressing the prolactin receptor antagonist, whereas only a modest increase in the S3 short-isoform expression was observed. These results suggest that endogenous prolactin may regulate its own biological actions in the anterior pituitary by inhibiting the expression of the long isoform of the prolactin receptor. In conclusion, our observations suggest that prolactin is involved in the maintenance of physiological cell renewal in the anterior pituitary. Alterations in this physiological role of prolactin could contribute to pituitary tumor development.     

N-terminal prolactin-derived fragments, vasoinhibins, are proapoptoptic and antiproliferative in the anterior pituitary

The anterior pituitary is under a constant cell turnover modulated by gonadal steroids. In the rat, an increase in the rate of apoptosis occurs at proestrus whereas a peak of proliferation takes place at estrus. At proestrus, concomitant with the maximum rate of apoptosis, a peak in circulating levels of prolactin is observed. Prolactin can be cleaved to different N-terminal fragments, vasoinhibins, which are proapoptotic and antiproliferative factors for endothelial cells. It was reported that a 16 kDa vasoinhibin is produced in the rat anterior pituitary by cathepsin D. In the present study we investigated the anterior pituitary production of N-terminal prolactin-derived fragments along the estrous cycle and the involvement of estrogens in this process. In addition, we studied the effects of a recombinant vasoinhibin, 16 kDa prolactin, on anterior pituitary apoptosis and proliferation. We observed by Western Blot that N-terminal prolactin-derived fragments production in the anterior pituitary was higher at proestrus with respect to diestrus and that the content and release of these prolactin forms from anterior pituitary cells in culture were increased by estradiol. A recombinant preparation of 16 kDa prolactin induced apoptosis (determined by TUNEL assay and flow cytometry) of cultured anterior pituitary cells and lactotropes from ovariectomized rats only in the presence of estradiol, as previously reported for other proapoptotic factors in the anterior pituitary. In addition, 16 kDa prolactin decreased forskolin-induced proliferation (evaluated by BrdU incorporation) of rat total anterior pituitary cells and lactotropes in culture and decreased the proportion of cells in S-phase of the cell cycle (determined by flow cytometry). In conclusion, our study indicates that the anterior pituitary production of 16 kDa prolactin is variable along the estrous cycle and increased by estrogens. The antiproliferative and estradiol-dependent proapoptotic actions of this vasoinhibin may be involved in the control of anterior pituitary cell renewal.     

Vinblastine-induced microtubular paracrystals in prolactin cells of anterior pituitary gland of lactating rats.

Vinblastine sulfate in physiological saline was injected directly into the pituitary glands of lactating rats. Injections were made through the ear canal using a syringe equipped with a 24-gauge needle. The animals were killed at 2, 4, or 6 hours after the injections. When the anterior pituitary glands were examined by electron microscopy, many microtubular paracrystalline deposits were seen in the prolactin and growth hormone cells. The usual cytoplasmic microtubules and microfilaments were not seen in the cytoplasm of these cells. Granular extrusion (exocytosis) was markedly depressed, and an accumulation of secretory granules was definitely observed in the prolactin cells after the administration of vinblastine.     

Prolactin Cells: Study of the Pituitary Gland of the Roach (Leuciscus rutilus) by Immuno-histochemical Methods

Fluorescein-labelled antibodies against ovine prolactin have been used to localize the prolactin-producing cells in the pituitary gland of the roach, Leuciscus rutilus. Two methods of protein tracing have been used: the direct and the indirect. In the direct method gammaglobulin fractions of rabbit-antisera were conjugated with fluorescein-isothiocyanate. Frozen sections of the pituitary gland were treated with conjugate. Cells with a content of prolactin gave off green fluorescence. Conjugate made from normal rabbit-serum failed to give this result. Blocking-experiments were also used to control the conjugate fluorescence. In the indirect method sheep-anti-rabbit-gammaglobulin was labelled with fluorescein. First the sections of the hypophysis were treated with antiserum from rabbits. After careful rinsing the sections were exposed to the dyemarked sheep-anti-rabbit-gammaglobulin. For orientation in the pituitary gland histological staining was carried out in parallel to that by fluorescence. The investigation has shown that the erythrosinophilic cells in the rostral pars distalis contained prolactin. Since no other cells in the gland were fluorescent, it can be concluded that the eta-cells are the prolactin cells.     

Molecular homology between prolactin and ovarian peptides: Evidence for physiologic modification of the parent molecule by the target

In recent years, prolactin has come to be recognized as an inhibitory gonadotrophin, with the ovarian follicular compartment as its target. In parallel with this, is evidence that granulosa cells give rise to substances secreted into follicular fluid that mimic the inhibitory actions of prolactin, but appear to be oligopeptides. The question that was examined here was whether these peptides are prolactin fragments. The endpoint was immunohistochemical reactivity in ovarian follicles of cycling rats. Antisera to homologous pituitary prolactin were used with and without modification by admixture either of highly purified rat pituitary prolactin or of follicular fluid enriched in prolactin-like bioactivities and depleted of peptides larger than 2000 MW. In growing follicles, immunohistochemical reactions, dependent upon unmodified anti-rat prolactins, occurred in all three follicular compartments but the microdistribution patterns and intensity of the reactions varied within follicles at different stages of development. These differences were compatible with the biological role of prolactin as an inhibitory gonadotrophin. Evidence that the reactions were dependent upon pituitary-like prolactin, present in the follicle, was obtained with highly purified rat prolactin which completely abolished reaction potential of the antisera in all follicles, at all stages. In contrast, the results with antisera to which oligopeptide follicular fluid had been added were end-point dependent: both partial and complete loss of antiserum potency, dependent upon cycle stage and sub-compartmentation of individual follicles were observed. These findings demonstrate molecular homology between pituitary prolactin and what appear to be small peptides of penultimate follicular origin and they lend support to the proposal that the mechanisms of action of prolactin involve modification of the parent molecule by the target.     

Effects of diethylstilbestrol on the cytogenesis of prolactin cells in the pars distalis of the pituitary gland of the mouse

This study was carried out to examine the developmental stage when prolactin cells differentiate in mice and to examine the effects of diethylstilbestrol on the development of prolactin cells in the fetal and neonatal pituitary glands. A small number of immunoreactive prolactin cells appeared first on embryonic day 15 in control (injected with oil) pituitary glands, whereas they did not increase in number until postnatal day 2. In diethylstilbestrol-treated mice (5 mg/kg body weight, 24 h before killing), a small number of immunoreactive prolactin cells were detectable as early as embryonic day 14, but not on day 13. They increased in number on embryonic days 15 and 16, and decreased markedly on days 17 and 18, followed by a rapid increase after birth. This transient reduction in the response to diethylstilbestrol was partially restored by treatment with metyrapone, a specific inhibitor of corticosteroid production. These results suggest that in the mouse: (1) differentiation of prolactin cells occurs between embryonic days 13 and 14, (2) prolactin gene expression is suppressed in the nascent prolactin cells presumably due to the presence of high levels of estrogen-binding protein, alpha-fetoprotein, and (3) prolactin gene expression is also suppressed by elevation of circulating glucocorticoids during the perinatal period. The present results suggest that, in the mouse, at least a proportion of prolactin cells are not derived from growth hormone cells, because the diethylstilbestrol-induced prolactin cells appear earlier than growth hormone gene expression.     

Immunocytochemistry of somatotrophs,gonadotrophs, prolactin and adrenocorticotropin cells in larval sea bream (Sparus auratus) pituitaries

Summary The chronological appearance of endocrine cells in the pituitary of sea-bream (Sparus auratus) larvae was studied using antisera against salmon prolactin, trout growth hormone, salmon gonadotropin and N-terminal human adrenocorticotropin. The larval pituitary (1–12 days after hatching) was oval in shape and was composed of a dense mass of cells with few neurohypophysial fibres. By 60 days after hatching it began to resemble the adult and was divisible into a distinct rostral pars distalis containing prolactin and adrenocorticotropin cells; a proximal pars distalis containing somatotrophs and gonadotrophs and a pars intermedia. Cells immunoreactive with antisera against growth hormone were observed immediately after hatching (2 days post-fertilization). Weakly staining prolactin cells were observed 2 days later in the region corresponding to the rostral pars distalis. Cells immunoreactive with anti-gonadotropin and anti-adrenocorticotropin sera were observed in the pituitary 6 and 8 days after hatching, respectively. All the cell-types studied were immunoreactive from the time they were first identified until the final samples 90 days after hatching.     

Phosphorylation of prolactin

Rat prolactin exhibits microheterogeneity when examined in electrophoretic systems, running as three isoforms having the same molecular weight but different net charges (prolactins 1, 2, and 3 with isoform 3 being the most acidic). As there is precedent for the phosphorylation of a pituitary hormone and phosphorylation is a common cause of microheterogeneity, we examined the possibility that rat prolactin existed in differentially phosphorylated forms. The investigation included examinations of rat prolactin phosphorylation both in vitro and in vivo. For the in vitro studies, purified rat prolactin was incubated with [gamma-32P]ATP and low levels of each of five purified protein kinases. Phosphorylated rat prolactin was identified by autoradiography of silver-stained one- and two-dimensional gels. For the in vivo studies, rat anterior pituitary cells in primary culture were incubated in the presence of H3 32PO4 for 2 or 12 h, after which time the proteins were extracted from the cells, cold acetone-precipitated, or immunoprecipitated and run on two-dimensional gels. We report the in vitro phosphorylation of rat prolactin by cAMP-dependent protein kinase, casein kinase I, protease-activated kinase I, and the calcium/phospholipid-dependent kinase, that phosphorylation with these kinases results in phosphate incorporation only into isoforms 2 and 3, and the phosphorylation of prolactin in rat pituitary cells in primary culture.     

Comparative ultrastructural studies on the prolactin cells in the pituitary of nine-spined sticklebacks,Pungitius pungitius L., with and without adenohypophyseal cysts

Summary The ultrastructure of the prolactin cells in nine-spined sticklebacks (Pungitius pungitius L.) with and without large pituitary cysts was compared. Prolactin cells from animals with cysts were less active as regards hormone synthesis and release than those from animals without cysts. Rough endoplasmic reticulum, mitochondria, Golgi apparatus and exocytotic figures were all more prominent in animals without cysts. Lysosomes and mature secretory granules were more plentiful in animals with cysts. The correlation between inactive prolactin cells and pituitary cysts argues against the hypothesis that cysts store prolactin released by holocrine secretion. It is more likely that the origin of cysts from degenerating cells signals extreme inactivity in the rostral pars distalis.     

Differentiation behavior of pituitary cells in normal and metamorphosis-arrested larvae of the salamander Hynobius retardatus.

When premetamorphic larvae of the salamander Hynobius retardatus were treated with potent goitrogens, or subjected to thyroidectomy, their metamorphosis was completely arrested. The pituitary gland of the arrested larvae consisted mostly of the hypertrophied Thyroid Stimulating Hormone (TSH) cells that are called "thyroidectomy cells". The development and dynamics of the TSH cells were studied by investigating uptake of BrdU into pituitary cell nuclei and by double-staining immunohistochemistry using anti-pituitary specific antibodies. The majority of the BrdU-positive cells expressed the TSHbeta antigen, suggesting that TSH cells increased in number by their extensive proliferation in the pituitary glands of the goitrogen-treated larvae. On the other hand, double-staining immunohistochemistry showed that several prolactin (PRL) immunoreactive cells coexpressed TSHbeta within single cells even in normal controls. Furthermore, pituitary cells coexpressing PRL and TSHbeta increased in number in the goitrogen-treated larvae. Whereas cells coexpressing GH and TSHbeta were not observed in normal controls, they appeared in the pituitary glands of the goitrogen-treated larvae. These results provide morphological evidence for considerable phenotypic plasticity in the pituitary cells of H. retardatus.     

Melanocyte-stimulating hormone and prolactin secretion in cell culture of estrogen-induced pituitary tumors in the hamster

Pituitary cells from hamsters bearing diethylstilbestrol induced renal adenocarcinomas were cultured in vitro. Dispersed cells in plastic dishes were viable for up to two weeks in Dulbecco's modified Eagle's medium supplemented with 17.5% of 6:1 horse serum to fetal calf serum. The secretion of alpha-melanocyte stimulating hormone and prolactin into the medium were measured by radioimmunoassay. The concentrations of both were elevated by day 3 in the medium from the hyperplastic pituitaries obtained from the estrogen treated, tumor bearing hamsters. Neither DES (10(-8)M) nor tamoxifen (10(-7)M) influenced the secretion of either hormone and neither altered either cell number or DNA synthetic activity as measured by thymidine incorporation. The secretion of hormones and the growth of the pituitary cells were, however, decreased by charcoal treatment of the serum. The results suggest that the elevation of serum alpha-MSH and prolactin observed in DES implanted hamsters is due to pituitary secretion of the hormones but that DES probably does not act directly on the pituitary to control the secretion.     

Origin of prolactin cells in the pituitary autografts in the renal capsule of young male and female rats.

Origin of the prolactin cells in the pituitary grafts autotransplanted into the renal capsule was electron microscopically investigated in young male and female rats. Prolactin cells may mainly originate from the completely degranulated acidophils after grafting in both sexes. The comprehensive degranulation of acidophils may be essential condition for the development of prolactin cells. Formation of initial prolactin granules is related to the Golgi apparatus in 3 and 6 days in the males. Even prolonged transplantation for the duration of 10 and 20 days failed to develop the typical prolactin cells storing large polymorphic granules in the grafts in the males. In the females, however, the population of prolactin cells became higher (20-30%) and their granulation was more rapidly advanced than in the male. Nine and 16 days after grafting quite numerous typical prolactin cells, characterized by the pronounced vesiculation of ER stored large polymorphic granules in the females. Progressive vesiculation of ER in degranulated acidophils may be the second necessary condition for establishing the prolactin cells. Thus, the entire course of cell conversion of acidophils into prolactin cells was observed in this study. All the graft cells including prolactin cells finally turned into the cells losing the respective individuality (10 days in the males; 32 days in the females). This fine structural similarity was striking in the males. We could not, however, deny the possibility of origination of prolactin cells from the follicular cells or their adjoining undifferentiated cells.     

Hormonal regulation of prolactin storage in a clonal strain of rat pituitary tumor cells

GH4C1 cells (GH cells) are a clonal strain of rat pituitary tumor cells which secrete prolactin. GH cells have been used to study hormone secretion, but they store relatively little prolactin compared to normal prolactin-secreting cells. They are not suitable, therefore, for studying some aspects of pituitary function. We have found that the amount of prolactin GH cells store can be regulated. When GH cells were plated at 10(6) cells/well and treated for six days with 180 nM insulin or 1 nM estradiol, there was a 60 percent increase in prolactin storage compared to control cells. Insulin and estradiol in combination acted synergistically to cause a 190 percent increase in prolactin storage. In contrast, they were additive in increasing extracellular prolactin; there was a 40 percent increase in extracellular prolactin after insulin, a 20 percent increase after estradiol, and a 50 percent increase after insulin plus estradiol. The increases in prolactin storage were always greater than the increases in extracellular prolactin. The increases in prolactin storage were dose-dependent and reached maximal levels after four days of treatment with 180 nM insulin plus 1 nM estradiol. Reducing the plating density to 10(3) cells/well increased the response to insulin and estradiol to nineteenfold. Epidermal growth factor (10 nM) acted synergistically with estradiol and insulin in combination to increase prolactin storage 27-fold. The insulin- and estradiol-induced increase in extracellular prolactin was caused by a specific increase in the rate of prolactin synthesis. The fractional increase in prolactin storage above the increase in prolactin production could not be explained by an increase in prolactin synthesis, an increase in intracellular transit time, or a change in the cell-cycle distribution of the population. Hormone storage can, therefore, be regulated independently from other processes which control hormone production. The prolactin stored in response to insulin and estradiol was releasable by potassium depolarization. Following depletion of intracellular prolactin by depolarization, the cells retained their increased capacity for prolactin storage. The ability to increase prolactin storage will make GH cells a more useful system in which to study pituitary function.     

Specific subclones derived from a multipotential clone of rat anterior pituitary cells.

Several functional subclones of rat anterior pituitary cells were established from our 2A8 clone which apparently contains a heterogenous population of committed and uncommitted cells. On the basis of the hormones secreted into the culture media, as measured by radioimmunoassay, these subclones were divided into four categories, i.e., subclones which secrete (1) ACTH only, (2) prolactin only, (3) prolactin and GH or (4) ACTH, prolactin and GH. None of the subclones produced detectable amounts of thyrotrophic or gonadotrophic hormones. Subclones which secrete a single hormone have shown no change in the type of hormone produced, indicating that these subclones were each derived from a committed cell. The cells of all subclones exhibit a normal diploid karyotype and show good growth characteristics. The cells of the different subclones can be classified by phase contrast microscopy into four categories. However, no clear-cut ultrastructural features have been observed which can be correlated with the different categories of subclones. On the basis of the results a hypothesis is proposed relative to the functional cytodifferentiation of anterior pituitary cells.     

Biosynthesis and degradation of prolactin in the rat anterior pituitary gland. Time course of incorporation of label in vitro and evidence for rapid degradation.

Biosynthesis of prolactin was studied in anterior pituitary glands from female rats, incubated in vitro. In this system [3H]leucine was incorporated into pituitary proteins, including somatotropin (growth hormone) and prolactin. The rate of uptake of label into prolactin (and to a lesser extent into total protein) slowed considerably during the first 2 h of incubation, although the rate of uptake into somatotropin was constant for 8 h. The most probable explanation for this apparent decrease in the rate of prolactin synthesis is degradation of prolactin in the gland. Degradation of this hormone was also demonstrated by incubating prelabelled pituitaries in unlabelled medium and following the content of labelled prolactin, and by studying the hormonal content of pituitary glands (by radioimmunoassay) before and after incubation. Degradation of prolactin appears to be much more rapid than that of somatotropin, and may represent a physiological mechanism whereby over-accumulation of prolactin is prevented when secretion of the hormone has been rapidly switched off.     

Dopaminergic reduction of intracellular calcium: the role of calcium influx

The effects of dopamine (DA) on 45Ca2+ ion movement and prolactin release in dispersed female rat anterior pituitary cells were studied to elucidate the mechanism for DA reduction of intracellular calcium levels. In 45Ca2+ prelabeled cells, DA inhibited fractional calcium efflux and prolactin release simultaneously and continuously in a concentration-dependent manner (IC50 20 nM DA). We then studied unidirectional calcium influx and observed haloperidol-reversible, concentration-dependent DA suppression of calcium influx into unlabeled cells. These data complement and extend reported fluorescent dye studies and suggest that dopamine primarily inhibits calcium influx, thereby reducing intracellular calcium levels, which leads to suppression of prolactin release and is manifest secondarily as a reduction in fractional 45Ca2+ efflux.     

Stereological and biochemical analysis of prolactin and growth hormone secreting rat pituitary cells in culture

Summary The secretion of prolactin is increased by treatment of prolactin producing rat pituitary cells with the hypothalamic tripeptide thyroliberin. To investigate the underlying mechanisms we used three closely related rat pituitary tumor cell strains (GH₁2C₁, GH₃ and GH₄C₁), which synthesize and spontaneously secrete prolactin and/or growth hormone. Growth hormone and prolactin released into the culture medium over a period of 24 h were measured by radioimmunoassay. Initial rates of synthesis were measured by immunoprecipitation of intracellular growth hormone and prolactin after incubation of cell cultures with ³H-leucine. The observed increase in prolactin synthesis and release was correlated with morphological effects of thyroliberin treatment. The volume density of Golgi complexes and the volume and surface densities of rough endoplasmic reticulum were compared in untreated cells and thyroliberin treated cells. As normal distribution could not be assumed, the non-parametric rank test of Wilcoxon was used whereby the densities calculated for each cell section were ranked. Alle three morphological parameters increased after thyroliberin treatment in cells secreting prolactin only (GH₄C₁), implying that the increase of prolactin secretion, at lest in part, is due to increased prolactin synthesis.