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.     

Culture of human pituitary prolactin and growth hormone cells

Summary Fragments of pituitary tissue obtained from a total of 37 patients with either breast cancer, diabetic retinopathy, galactorrhea, or acromegaly were dissociated into single cell suspensions prior to cell culture. Release of human growth hormone (hGH) and human prolactin (hPRL) into the culture medium was measured by radioimmunoassay. During a 3-week culture period, prolactin cells released 9–13 times the intracellular levels of hPRL at the time of seeding, whereas hGH release from growth hormone cells was only 1–2 times that of their initial intracellular level during this same time. Both growth hormone and prolactin cells retained distinctive ultrastructural features during culture. The prolactin cells responded to TRH stimulation by elevated release of PRL into the medium. No evidence for mitotic division of prolactin cells in vitro was found.This work was supported by NCI Contract NO 1-CB-23863     

Intracranial prolactin perfusion induces incubation behavior in turkey hens

Intracranial perfusion of ovine prolactin (oPrl) via osmotic pump in laying turkey hens caused a sudden onset in incubation behavior, defined as an increase in nest visits. The hens also displayed a gradual decrease in egg laying during the time they were receiving oPrl, another indicator of the onset of incubation. Circulating immunoreactive turkey Prl levels fell during the perfusion period, even though the hens were displaying persistent nesting activity and reduced egg laying. No effects on serum LH were noted. Perfusion of oPrl during the first 14 days of photostimulation delayed the onset of egg laying by several days. No effects on serum Prl or serum LH were noted. It is suggested that incubation behavior is facilitated by central levels of Prl.     

Ultrastructural immunocytochemical localization of prolactin and growth hormone in the porcine pituitary

Summary The immunocytochemical peroxidase-antiperoxidase technique was used to identify prolactin- and growth hormone-producing cells in the porcine pituitary at the ultrastructural level. The growth hormone-producing cells contain round secretory granules (300 nm to 500 nm in diameter). The prolactin-producing cells can be identified by their distinct round and ovoid secretory granules which vary in size. Most of these cells contain large granules (450 nm to 750 nm in diameter), but some prolactin-producing cells display smaller secretory granules (250 nm to 500 nm). The two hormones were localized exclusively in the secretory granules. Staining for prolactin was observed in round and ovoid granules, as well as in small and polymorphic granules within the Golgi complex. This study confirmed (i) that the two hormones are located in different cells, and (ii) that under normal physiological conditions no one cell can synthesize and store both hormones simultaneously.     

Developmental expression of growth hormone and prolactin genes in the bovine pituitary

Cell-free translation was used to initially characterize the major mRNA species present in the bovine anterior pituitary as a function of development. The only detectable change in translation products, which occurred during the transition from fetus to adult, was a reversal in the relative ratio of pituitary growth hormone and prolactin. Subsequent hybridization analysis with cloned growth hormone and prolactin cDNA probes indicated that growth hormone mRNA comprised over 40% of the total fetal mRNA and was 50- to 100-fold higher than prolactin mRNA. The steady state levels of growth hormone mRNA remained relatively constant throughout gestation. In contrast, prolactin mRNA levels, which were low early in gestation, increased during development to become the principal mRNA in the adult pituitary. Since growth hormone and prolactin are synthesized and secreted by specialized cells (somatotrophs and mammotrophs, respectively) immunochemical staining was used to determine whether the changes in the mRNA levels for these two hormones were a reflection of specific cell proliferation. For growth hormone, there was a close correlation between the number of somatotrophs and the relative levels of growth hormone mRNA. In contrast, the increase in prolactin mRNA exceeded the increase in the number of mammotrophs. Thus, the cellular concentration of growth hormone mRNA remains relatively constant during development, while the cellular concentration of prolactin mRNA increases by more than an order of magnitude.     

Immunocytochemical localisation of prolactin and growth hormone in the perinatal sheep pituitary

Summary The peroxidase anti-peroxidase immunocytochemical staining technique has been used to identify prolactin and growth hormone cells in pituitaries from fetal and neonatal sheep. The size of the secretory granules in these cell types has been measured using the image analysing computer Quantimet 720. The area size distributions of the fetal prolactin and growth hormone granules were compared with those in the neonate and the adult. It appears that the gestational age of the fetus may influence the size range of prolactin secretory granules.     

Variations in prolactin and growth hormone production during cellular growth in clonal strains of rat pituitary cells.

A permanent, clonal strain of rat pituitary tumor cells (GH3-cells) spontaneously synthesizes and secretes prolactin (rPRL) and growth hormone (rGH) into the culture medium. The rates of hormone production (microng extracellular hormone/mg cell protein/24 hours) and synthesis (vida infra) as well as the rate of [3H]thymidine incorporation into DNA (DNA synthesis) have been studied. During logarithmic growth rPRL and rGH production increased to 160 and 250% of the value at day 2 after plating, while during the plateau phase of cell growth hormone production decreased to initial values. The fluctuations in rPRL production could be fully explained by variations in the rate of rPRL synthesis: [3H]eucine incorporated into rPRL as measured with immunoprecipitation and polyacryl-amide gel electrophoresis. Also the rates of synthesis and production of rGH showed parallel changes during exponential and plateau phase of growth, but this hormone was probably degraded intracellularly. The relative reduction in the rate of synthesis of rPRL and rGH during the plateau of growth corresponded closely to the fall in the rate of DNA synthesis. The reduction in rPRL synthesis could not be explained through an inhibition by extra-cellular rPRL accumulation or by cell to cell interaction occurring in dense cultures. The intracellular concentrations of both hormones were unaltered during logarithmic growth, but rose to 500% for rPRL and 200% for rGH during the plateau phase. In spite of the marked variations in basal rPRL and rGH production the GH3 cultures of different ages were equally able to increase rPRL and decrease rGH production in response to thyrotropin releasing hormone (3 X 10(-7) M) and 17beta-estradiol (10(-8)M).     

Ultrastructural localization of prolactin,growth hormone and luteinizing hormone by immunocytochemical techniques in the bovine pituitary

Summary The technique of ultrastructural immunocytochemistry involving the unlabeled antibody and the soluble peroxidase-antiperoxidase complex was used to identify and describe the prolactin (P) cells, somatotropic (STH) cells and luteinizing hormone (LH) cells in the bovine anterior pituitary gland. This method was used to localize the three hormones at the electron microscopic level. Staining of varying intensity was found on the secretory granules and on the small granules and vesicles within the Golgi complex. No stain was found in nuclei, on mitochondria or in the endoplasmic reticulum.     

Characterization of pituitary IGF-I receptors: modulation of prolactin and growth hormone

There have been no studies in any vertebrate that have localized insulin-like growth factor (IGF)-I receptors in prolactin (PRL) cells or that have correlated pituitary binding to the potency of IGF-I in regulating both PRL and growth hormone (GH) secretion. We show that IGF-I binds with high affinity and specificity to the pituitary gland of hybrid striped bass (Morone saxatilis x M. chrysops). IGF-I and IGF-II were equipotent in inhibiting saturable (125)I-IGF-I binding, whereas insulin was ineffective. IGF-I binds with similar affinity to the rostral pars distalis (>95% PRL cells) as the whole pituitary gland and immunohistochemistry colocalizes IGF-I receptors and PRL in this same region. Des(1-3)IGF-I, a truncated analog of IGF-I that binds with high affinity to IGF-I receptors but weakly to IGF-I binding proteins (IGFBPs), showed a similar inhibition of saturable (125)I-IGF-I binding, but it was more potent than IGF-I in stimulating PRL and inhibiting GH release. These results are the first to localize IGF-I receptors to PRL cells, correlate IGF-I binding to its efficacy in regulating GH and PRL secretion, as well as demonstrate that IGFBPs may play a significant role in modulating the disparate actions of IGF-I on PRL and GH secretion.     

Monomeric pituitary growth hormone and prolactin variants in man characterized by immunoperoxidase electrophoresis

Immunoperoxidase electrophoresis, combining SDS--ME--PAGE and the 'double bridge' immunoperoxidase staining was applied to crude human pituitary homogenates. With anti-hGH and anti-hPL sera, 4 hGH-related monomers were characterized: a Mr 22 000 peptide corresponding to hGH; a Mr 20 000 peptide corresponding to the known hGH variant and two unknown hGH variants (Mr 65 000 and Mr 75 000). With anti-ovine, rat and human PRL sera, 4 PRL-related monomers were immunostained: one comigrated with purified hPRL (Mr 25 000), and 3 were unknown (Mr 29 000; Mr 45 000; Mr 16 000).     

Experience modulates emission of food calls in broody hens.

The aim of these experiments was to determine if previous experience of chicks' response to food calling influences subsequent propension of maternal hens to utter food calls. Seventeen broody hens were tested three times a day without their 3- or 4-day-old chicks. Hens were tested in two situations: chicks were returned either after each test or at the end of all the day's tests. As palatability influences food calling in maternal hens, experiments were conducted first with a highly preferred food item and then with the hens' usual feed. The chicks' capacity to respond regularly to their mother influences the hens' capacity to emit food calls. In fact, although the hens did not lose their maternal state, they uttered fewer food calls when their chicks were removed all day. These results suggest that the chicks' behaviour following food calling could be a social reinforcement for broody hens.     

Prevention of incubation behavior expression in turkey hens by active immunization against prolactin

The consequences of active immunization against prolactin on expression of incubation, reproductive performance and hormonal profiles were evaluated in turkey hens. Hens were injected weekly for 4 wk starting 8 wk before being submitted to a stimulatory photoperiod and 3 times thereafter at intervals of 4 to 5 wk. The hens were injected i.d. with 0.5 mL of a mixture diluted half in Freund's adjuvant. The mixture was prediluted in .9% saline and contained 100 micrograms of a fusion protein (GST-tPRL), GST, oPRL or vehicle. The results indicate that active immunizations with GST-tPRL or oPRL both induce production of specific prolactin antibodies. The onset of egg production was unaffected but higher egg production was observed for the GST-tPRL immunized hens. No GST-tPRL immunized hens expressed incubation behavior, whereas 20 to 30% of hens in the other experimental groups did so. Apparent hyperprolactinemia was detected by RIA for the GST-tPRL immunized groups starting before photostimulation and lasting until Week 10 of egg production but not in other groups. No significant differences were observed in either plasma LH or estradiol concentrations of immunized and nonimmunized turkey hens. In conclusion, both GST-tPRL or oPRL induced the production of antibodies against prolactin in turkey hens. However, only active immunization using GST-tPRL induced higher antibody titers as well as full prevention of incubation behavior expression. Such a pharmacological approach is of great practical interest, although its uses need to be carefully evaluated under commercial conditions.     

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.