Prolactin-producing Cells in Clupea harengus membras,Polypterus palmas and Calamoichthys calabaricus Studied by Immuno-histochemical Methods

The prolactin-producing cells in Clupea harengus membras, Polypterus palmas and Calamoichthys calabaricus have been localized by fluores-ceinlabelled antibodies to ovine prolactin. Pituitary sections were cut in a cryostat and both direct and indirect protein tracing methods have been used. As an additional method the paraffin embedding technique was carried out with Polypterus palmas. The prolactin-producing cells in Clupea harengus membras were localized to the follicle epithelium in the rostral pars distalis. In Polypterus palmas and Calamoichthys calabaricus the prolactin producing cells were scattered all over the pars distalis. A possible relationship between the erythrosinophilic prolactin cells and the epithelium cells in the buccohypophyseal canal is discussed.     

Histological,immuno- and enzyme-histochemical investigations on the adenohypophysis of the urodeles,Mertensiella caucasica and Triturus cristatus and the caecilian,Chthonerpeton indistinctum

Summary The pituitary glands of two urodelan species (Mertensiella caucasica, Triturus cristatus) and one caecilian species (Chthonerpeton indistinetum) were examined with histological (Alcian blue, Brookes' trichrome stain), enzyme histochemical (acid phosphatase, -naphthylacetate-esterase, acetylcholinesterase) and immunofluorescence techniques (anti-carp GTH, anti-ovine prolactin, anti-synthetic -MSH). In the pituitary gland of Mertensiella and Triturus six chromophilic cell types could be distinguished. A strong fluorescence was observed in the MSH-, GTH- and TSH-cells.In the pituitary gland of Chthonerpeton only five chromophilic cell types could be distinguished: in the rostral part of the pituitary gland the B₃-cell; in the basal region of the central area the B₂-cell; dorsocaudally the B₁-cell. The acidophilic cells were found in the central and caudal part of the pars distalis. The basophils of the pars intermedia could be observed in the dorsocaudal part of the pituitary gland surrounding the neurohypophysis. All acidophilic cells showed a strong immunofluorescence with anti-ovine prolactin (LTH).     

The identification of the thyrotropic cells in the adenohypophysis of the cichlid fish Cichlasoma biocellatum and the role of these cells and of the thyroid in osmoregulation

Summary In order to identify the thyrotropic cells in the pituitary gland of Cichlasoma biocellatum, adult animals were treated with thyroxine. Periodic acid Schiff (PAS)- and aldehyde fuchsin (AF)-positive, and Alcian blue (AB)-negative basophils in the meso-adenohypophysis as well as the thyroid were strongly inactivated by such treatment. Since other cell types in the adenohypophysis were not affected, it is concluded that the PAS-positive and AB-negative basophils in the meso-adenohypophysis are the thyrotrops. In normal animals the thyrotrops and the thyroid are very active. Propylthiouracil did not cause a further activation of TSH cells. Keeping the animals in 25% sea-water caused a strong inactivation of the prolactin cells, the thyrotrops and the thyroid. In deionized water the prolactin cells were stimulated, but the thyroid was less active than in controls. Injections of ovine prolactin did not enhance the activity of the thyroid in salt water animals. It is suggested that in Cichlasoma biocellatum prolactin does not have a direct stimulatory effect on the thyroid.Thanks are due to Prof. Dr. P. G. W. J. van Oordt for his continual interest and valuable suggestions, and to Dr. L. Boomgaart for correcting the english. We are indebted also to Messrs. H. van Kooten and E. van der Vlist for making the photographs. The National Institutes of Health, Bethesda, Maryland (U.S.A.) kindly presented the ovine prolactin.     

Immunohistochemical Detection of DNA Replication in Mouse Uterine Cells by Bromodeoxyuridine Labeling of Wax- and Resin-Embedded Tissue Sections

TO apply the bromodeoxyuridine (BrdU) labeling method using a monoclonal antibody to the study of cell proliferation in the mouse uterus, methods of fixation and embedding of tissues and of immunofluorescent staining were compared in terms of the rate of detection of labeled cells and specificity and stability of fluorescence obtained. BrdU was administered intravenously 2 hr before death and uterine blocks were embedded in polyester wax and Technovit resin after fixation in formalin and periodate-lysine-paraformaldehyde, respectively. The indirect method with anti-BrdU and fluorescein isothiocyanate (FITC) conjugated antimouse IgG antisera and the direct method with FITC conjugated anti-BrdU antibody were applied to both wax- and resin-embedded sections. Labeled and total cells were counted in luminal and glandular epithelia and stromata adjoining them. Counterstaining with hematoxylin for counting total cells produced intense fluorescence over the whole of resin sections and made counting of labeled cells impossible. On wax sections, on the other hand, the results were satisfactory, although the number of labeled cells detected was decreased slightly. In wax sections fluorescence due to nuclear incorporation of BrdU in the indirect method could be easily distinguished from the cytoplasmic or extracellular emission seen in some cells by its location and characteristic color. In resin sections, however, more careful observation was needed since the second antibody used in the indirect method cross-reacted with IgG in eosinophils and produced cyctoplasmic fluorescence of the same color. By the indirect method greater numbers of labeled cells were detected in wax sections than in resin sections. The difference was distinct in tissues with extensive cell proliferation. By the direct method the fluorescence obtained was weaker and apt to fade more quickly than that obtained by the indirect method; use of the direct method reduced the number of labeled cells detected in both wax- and resin-embedded sections.     

Immunohistochemical detection of DNA replication in mouse uterine cells by bromodeoxyuridine labeling of wax- and resin-embedded tissue sections.

To apply the bromodeoxyuridine (BrdU) labeling method using a monoclonal antibody to the study of cell proliferation in the mouse uterus, methods of fixation and embedding of tissues and of immunofluorescent staining were compared in terms of the rate of detection of labeled cells and specificity and stability of fluorescence obtained. BrdU was administered intravenously 2 hr before death and uterine blocks were embedded in polyester wax and Technovit resin after fixation in formalin and periodate-lysine-paraformaldehyde, respectively. The indirect method with anti-BrdU and fluorescein isothiocyanate (FITC) conjugated antimouse IgG antisera and the direct method with FITC conjugated anti-BrdU antibody were applied to both wax- and resin-embedded sections. Labeled and total cells were counted in luminal and glandular epithelia and stomata adjoining them. Counterstaining with hematoxylin for counting total cells produced intense fluorescence over the whole of resin sections and made counting of labeled cells impossible. On wax sections, on the other hand, the results were satisfactory, although the number of labeled cells detected was decreased slightly. In wax sections fluorescence due to nuclear incorporation of BrdU in the indirect method could be easily distinguished from the cytoplasmic or extracellular emission seen in some cells by its location and characteristic color. In resin sections, however, more careful observation was needed since the second antibody used in the indirect method cross-reacted with IgG in eosinophils and produced cytoplasmic fluorescence of the same color. By the indirect method greater numbers of labeled cells were detected in wax sections than in resin sections. The difference was distinct in tissues with extensive cell proliferation. By the direct method the fluorescence obtained was weaker and apt to fade more quickly than that obtained by the indirect method; use of the direct method reduced the number of labeled cells detected in both wax- and resin-embedded sections.     

THE INTRACELLULAR LOCALIZATION OF PITUITARY THYROTROPIC HORMONE

The intracellular localization of a bovine anterior pituitary preparation of thyroid-stimulating hormone (TSH) was studied in guinea pigs and dogs. The preparation was administered intravascularly or applied directly to tissue sections. TSH was detected by an indirect technique utilizing bovine TSH antiserum and fluorescein-labeled anti-rabbit globulin; the presence of TSH in the tissue was indicated by fluorescence when the tissue was examined under the microscope with an ultraviolet light source. After either intravascular administration or direct application of the TSH preparation, striking fluorescence was found in the nuclei of the thyroid cells and to a lesser degree in the nuclei of retro-orbital fat tissue and kidney tubules in both species studied. A little fluorescence was also seen in spleen tissue. No fluorescence was noted in comparable tissues removed from control animals injected with bovine albumin or globulin or when the tissues were treated with the fluorescein-labeled globulin alone. Fluorescence was also noted in the nuclei of adrenal cells treated with unabsorbed antiserum, but this was greatly diminished when antiserum absorbed with crystalline ACTH was used. The positive reactions were all markedly decreased when the tissues were treated with antisera absorbed with the original TSH preparation. Fluorescence was noted in the cytoplasm of pituitary tissue from both treated and control animals, suggesting a cross-reaction between the bovine pituitary antisera and guinea pig or dog hypophysis. The indirect technique seems to be highly satisfactory for demonstration of the pitiutary hormone within the cell. In addition, the demonstration of immunologically active anterior pituitary TSH bound to cell nuclei offers a clue to the site of action of this hormone.     

Gonadotroph-lactotroph associations and expression of prolactin receptors in the equine pituitary gland throughout the seasonal reproductive cycle

An interaction between gonadotroph and lactotroph cells of the pituitary gland has long been recognized in several species. The current study was conducted to investigate whether an association between gonadotrophs and lactotrophs occurs in mares and whether prolactin receptors are expressed within the pituitary gland of this species. The effects of both reproductive state and season on these variables were examined in pituitary glands obtained from sexually active mares in July (breeding season), sexually active mares in November (non-breeding season) and anoestrous mares in November. Pituitaries were dissected out immediately after death and immunofluorescent staining was carried out on 6 micrometer sections using specific antibodies to the LHbeta subunit, FSHbeta subunit, prolactin and prolactin receptor. Gonadotrophs were observed in both the pars distalis and pars tuberalis; although they appeared mostly as isolated cells, small groups of gonadotrophs were also identified in the pars distalis. In contrast, lactotrophs were observed only as clusters of cells exclusively in the pars distalis of sexually active and anoestrous mares in November and in most of the sexually active mares in July. A specific gonadotroph-lactotroph association was identified only between large isolated gonadotrophs and lactotroph clusters. Double immunofluorescent staining for FSHbeta and prolactin revealed a similar gonadotroph-lactotroph association to the one detected for LH gonadotrophs. No statistical difference in the gonadotroph:lactotroph ratio was observed as a result of changes in reproductive status or season. However, a tendency for a simultaneous decrease in the number of gonadotrophs and an increase in the number of lactotrophs was detected in anoestrous animals. Prolactin receptor immunoreactivity was found in the pars distalis, but not in the pars tuberalis, of sexually active (July and November) and anoestrous animals for both long and short forms of the receptor. No prolactin receptor co-localization for either form of the receptor was observed in LH or FSH gonadotrophs in either of the reproductive states examined during both summer and winter seasons. Furthermore, no significant difference was apparent in the proportion of cells expressing prolactin receptors between mares of different reproductive state or season. The specific anatomical association between gonadotroph and lactotroph cells and the expression of prolactin receptors in the equine pituitary gland indicate a potential role of prolactin in the regulation of gonadotrophin secretion. However, the absence of evidence for co-localization of prolactin receptors in LH or FSH cells does not support the hypothesis of a direct effect of prolactin on the gonadotroph as reported in a short day breeder. The results raise the possibility that, in horses, an intermediate regulatory cell may mediate the action of prolactin on gonadotroph function.     

Immunoreactive prolactin in the pituitary gland of cyprinodont fish at the time of hatching

In the developing pituitary gland of embryos of the annual fish Cynolebias whitei and the medaka, Oryzias latipes, prolactin cells have been identified before hatching by means of a light-microscopic immunocytochemical method with antiserum against ovine prolactin. At the time of hatching, changes in the intensity of the immunoperoxidase staining occur. Histological staining by Cleveland and Wolfe's trichrome shows differentiation of cell types in the adenohypophysis only later in ontogeny. Our results indicate that, in teleosts, differentiated prolactin cells are present before hatching and that prolactin may be involved in the endocrine control of the hatching process.     

Independent differentiation of mammotropes and somatotropes in the chicken embryonic pituitary gland

It has been reported that mammotropes in a rodent pituitary gland are derived from somatotropes via somatomammotropes (SMTs), cells that produce both growth hormone (GH) and prolactin (Prl). However, no studies have been done on the transdifferentiation of somatotropes in the chicken pituitary gland. In this study, in order to determine the origin of mammotropes, we studied detail property of appearance of chicken somatotropes, mammotropes and pit-1 cells and then evaluated the existence of SMTs in the chicken embryonic pituitary gland. Immunohistochemical analysis revealed that GH-immunopositive (GH-ip) cells appeared on embryonic day (E) 14 and were mainly distributed in the caudal lobe, while Prl-immunopositive (Prl-ip) cells appeared in the cephalic lobe of the pituitary gland on E16. In situ hybridization (ISH) and RT-PCR analysis showed that expression of GH and Prl mRNA starts at E12 in the caudal lobe and at E14 in the cephalic lobe respectively. Pit-1 mRNA was first detected on E5 by RT-PCR, and pit-1 mRNA-expressing cells were found in the cephalic lobe on E8. Then with the ontogeny of the chicken, these cells spread into both lobes. Using a double staining method with ISH and immunohistochemistry, we could not detect the existence of SMTs in the chicken embryonic pituitary gland even in the marginal region of either lobe. These results suggest that chicken somatotropes and mammotropes independently appear in different lobes of pituitary gland and that transdifferentiation from somatotropes to mammotropes is not the central route for differentiation of mammotropes in the embryonic chicken pituitary gland.     

Possible role of prolactin in growth regulation

To study the possible role of prolactin on growth regulation pituitary grafted rats of different ages and their sham-operated controls have been used. After the transplant operation of one pituitary gland from a litter-mate donor on day 5 or on day 30 of life, a marked prolactin increase has been observed. This increase has been immediate in 30 day-old rats and delayed in 5 day-old rats in which the elevation starts being significant on day 20 for females and on day 25 for male rats. Pituitary grafting on day 5 of life, with an adult donor gland, resulted in an immediate and marked increase of prolactin values in both sexes. Litter-mate donor pituitary grafting, on day 5 of life, resulted in an increase in body weight that could be directly correlated with the increase in prolactin levels. Adult pituitary grafting resulted in an increased body weight in females with no effects being detected in males. In 30 day-old grafted male and female rats, marked body weight increases were seen, over the whole studied period, together with an increase in nose-tail length (1 cm in female and 1.5 cm in males longer than the control animals). All these changes do not seem not be related to GH modifications in pituitary grafted rats, since GH changes were very slight with a final tendency to lower values in female rats but not in males. All these data could suggest that prolactin might exert a direct effect on growth both in male and female rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tachykinins and the control of prolactin secretion

Tachykinins are present in the pituitary gland and in brain areas involved in the control of the secretion of pituitary hormones. Tachykinins have been demonstrated to stimulate prolactin release acting directly on the anterior pituitary gland. These peptides have also been revealed to be able to act at the hypothalamic level, interacting with neurotransmitters and neuropeptides that have the potential to affect prolactin secretion. Tachykinins seem to act by stimulating or inhibiting the release of the factors that affect prolactin secretion. Among them, tachykinins have been demonstrated to stimulate oxytocin and vasopressin release, which in turn results in prolactin release. Tachykinins also potentiated the response to vasoactive intestinal peptide (VIP) and reinforced the action of glutamate, which in turn result in prolactin release. They have also been shown to interact with serotonin, a neurotransmitter involved in the control of prolactin secretion. In addition, tachykinins have been shown to inhibit GABA release, a neurotransmitter with prolactin-release inhibiting effect. This inhibition may result in an increased prolactin secretion by removal of the GABA inhibition. On the other hand, tachykinins have also been shown to stimulate dopamine release by the hypothalamus, an action that results in an inhibition of prolactin release. Dopamine is a well known inhibitor of prolactin secretion. In conclusion, although tachykinins have been shown to have a predominantly stimulatory effect on prolactin secretion, especially at the pituitary level, under some circumstances they may also exert an inhibitory influence on prolactin release, by stimulating dopamine release at the hypothalamic level.     

The Response of Prolactin,ACTH, and Growth Hormone Cells in the Pituitary Gland of the Three-Spined Stickleback,: Gasterosteus aculeatus L. form leiurus,to Increased Environmental Salinities

Benjamin, M. 1980. The response of prolactin, ACTH, and growth hormone cells in the pituitary gland of the three-spined stickleback, Gasterosteus aculeatus L. form leiurus, to increased environmental salinities. (Department of Anatomy, University College, Cardiff, Wales, U.K.) — Acta zool. (Stockh.) 61(1): 1–7. The time-sequence of response of the prolactin, ACTH and growth hormone cells in the pituitary gland of the leiurus form of the three-spined stickleback, Gasterosteus aculeatus L., to a transfer from freshwater to seawater, was studied by light microscopy. The appearance of the pituitary was compared with that of animals caught in brackish or sea water. The prolactin cells respond only slowly to seawater by cytological changes interpreted as signs of decreased secretory activity. It is thus suggested that prolactin is unlikely to be important for osmoregulation in this stickleback. The growth hormone cells are more active in all seawater-adapted animals, while the ACTH cells are less active—although only in laboratory experiments. The differing responses of the pituitaries of the leiurus and trachurus forms of G. aculeatus and Pungitius pungitius to high salinities are compared. Even in species whose pituitaries are virtually identical, the response to high salinites may differ widely.     

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.     

Combined use of in situ hybridisation and immunocytochemistry for the investigation of prolactin gene expression in immature,pubertal, pregnant,lactating and ovariectomised rats

Summary We have investigated the use of in situ hybridisation together with immunocytochemistry for the study of endocrine cell function, using as an example the expression of prolactin messenger RNA (mRNA) in pituitaries of rats under various endocrinological conditions. In situ hybridisation using a ³²P-labelled cRNA probe for rat prolactin was carried out on sections of 4% paraformaldehyde-fixed pituitaries from prepubertal, pubertal, pregnant, lactating and ovariectomised rats and adjacent sections were immunostained for prolactin. Northern gel analysis was performed on total RNA extracts of pregnant, lactating and control pituitaries. While in ovariectomised rat pituitaries both prolactin immunoreactivity and prolactin mRNA were decreased, no differences in prolactin immunostaining were seen between prepubertal, pubertal, pregnant or lactating rats and controls, even when the supra-optimal dilution technique was used. However, using in situ hybridisation, prolactin mRNA signal was increased in prepubertal rats, and with hybridisation and northern gel analysis the signal was reduced in pregnant rats and markedly increased in lactating rats. The combined use of in situ hybridisation and immunocytochemistry provides morphological information concerning endocrine gene expression and protein synthesis in the pituitary gland.     

Direct action of ethanol on pituitary prolactin secretion in vitro.

The effect of ethanol on prolactin release in vitro has been studied in order to investigate the direct action of ethanol on pituitary gland of the female rats. Animals were sacrificed in diestrus 2 and pituitary glands were incubated in TC-199 medium containing dopamine, noradrenaline, serotonin, TRH or cycloheximide with or without ethanol. The total amount of prolactin after the incubation period was calculated. Alcohol significantly increased the prolactin release in all groups. Cycloheximide and dopamine decreased the prolactin synthesis, but ethanol reduced the effect of dopamine. It is concluded that part of ethanol-induced hyperprolactinaemia, is due to a direct action of the alcohol on pituitary, affecting release and/or synthesis of prolactin.     

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.     

Presence of α-neo-endorphin-like immunoreactivity in the posterior lobe of the pituitary gland

α-neo-endorphin-like immunoreactivity was demonstrated in the nerve fibers and Herring's bodies in the posterior lobe of rat pituitary glands by an indirect immunoperoxidase method using α-neo-endorphin-antiserum. The number of α-neo-endorphin positive fibers and Herring's bodies did not decrease in the sections in which α-neo-endorphin-antisera pretreated with oxytocin, ADH and leu-enkephalin were used as primary antisera. In view of the reports that met-enkephalin, leu-enkephalin and dynorphin were present in the posterior lobe of the pituitary gland, this finding suggested that there were four kinds of opiate-like peptides in the posterior lobes of the pituitary gland. Furthermore, by staining alternately 3he serial sections of the rat pituitary glands with ADH and α-neo-endorphin-antisera, it was revealed that α-neo-endorphin-positive Herring's bodies were identical to a large number of ADH positive Herring's bodies. This finding, together with the observation that morphine injection caused ADH release, suggested that α-neo-endorphin may play an important role in the regulation of ADH release.