0013-7227/91/1291-0496$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 1 Printed in U.S.A.
Pituitary Prolactin Messenger Ribonucleic Acid Levels in Incubating and Laying Hens: Effects of Manipulating Plasma Levels of Vasoactive Intestinal Polypeptide RICHARD T. TALBOT, MARK C. HANKS, ROBERT J. STERLING, HELEN M. SANG, AND PETER J. SHARP Agricultural and Food Research Council Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Roslin, Midlothian EH25 9PS, United Kingdom
ABSTRACT. Pituitary PRL messenger RNA levels in hens, measured by dot-blot hybridization, correlated directly with concentrations of plasma PRL, being 3-fold higher in incubating than in laying birds. Nest deprivation of incubating hens for 24 h caused a rapid decrease in both plasma PRL and pituitary PRL mRNA, which remained depressed thereafter. A single injection of vasoactive intestinal polypeptide (VIP) in laying hens resulted in an increase (P < 0.05) in pituitary PRL mRNA whereas passive immunoneutralization of VIP in incubating hens resulted in a decrease (P < 0.001) in pituitary PRL mRNA. The rapid decrease in pituitary PRL mRNA after nest deprivation or passive immunoneutralization of VIP was associated with a significant increase in pituitary PRL content, presumably a consequence of the decreased PRL secretion. In situ hybridi-
zation showed PRL mRNA to be localized in the cephalic lobe of the anterior pituitary gland in which most PRL cells, identified immunocytochemically, were found. Northern blotting studies showed that the pituitary gland contains a single 860 base(s) mature PRL mRNA transcript irrespective of physiological state or VIP manipulation. Both in situ and Northern hybridization studies confirmed that the amount of pituitary PRL mRNA was related directly to the concentration of plasma PRL. These observations are consistent with the view that in incubating hens hypothalamic VIP, in addition to acting as a PRL releasing hormone, also plays a major role in the regulation of the amount of PRL mRNA in the anterior pituitary gland. (Endocrinology 129: 496-502, 1991)
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EVERAL hypothalamic neurotransmitters and neuropeptides are known to stimulate PRL release but their precise physiological functions are uncertain (1, 2). There is evidence that one of the neuropeptides, vasoactive intestinal polypeptide (VIP), is physiologically significant. Increased concentrations of VIP occur in rat hypophysial portal blood (3, 4), and the administration of anti-VIP serum inhibits PRL release induced by stress, serotonin, or suckling (5-8). In the lactating rat the inhibitory effect of anti-VIP serum on PRL release is transitory despite the presence of excess antibody (6). There is evidence that VIP is the major physiologically significant PRL releasing factor in birds. VIP acts directly on the avian anterior pituitary gland to release PRL specifically (9, 10) and is present in neurons in the basal hypothalamus with projections to the median eminence. These neurons show marked hyperplasia when plasma PRL levels are high in incubating birds (11-13). Passive immunization with anti-VIP serum depresses plasma PRL in incubating bantams (12) and in incubating ring
doves prevents the development of the crop sac (14) an organ which is used in a bioassay for PRL (15). If VIP plays a key role in the regulation of PRL secretion, it is possible that it is also involved in the control of PRL synthesis. Although several hormones and neurotransmitters are known to influence PRL gene expression (16), evidence that VIP plays such a role is limited to a study using GH3 cells, a rat pituitary tumor cell line (17). The availability of a complementary DNA probe for chicken PRL (18) now makes it possible to investigate the effects of VIP on pituitary PRL mRNA in the bantam hen. Earlier studies on this bird have shown that the increased concentrations of plasma PRL (19) and hypothalamic VIP observed during incubation (12) decrease rapidly after nest deprivation (20). This study was carried out to establish the dynamics of pituitary PRL and PRL mRNA content after nest deprivation and to obtain evidence that the predicted decrease in PRL mRNA might be a consequence of a decreased release of hypothalamic VIP.
Received March 25, 1991. Address requests for reprints to: Richard T. Talbot, Agricultural and Food Research Council Institute of Animal Physiology and Genetics Research, Roslin, Midlothian EH25 9PS, United Kingdom.
Animals and experimental design
Materials and Methods Laying bantam hens from the Institute's flock were maintained in battery cages exposed to 14 h light/day with food and
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VIP REGULATION OF PRL mRNA water freely available. Broodiness was induced by transferring hens, under the same lighting conditions, to deep litter floor pens containing nest boxes with five boiled eggs in each box. Incubating hens were allocated to experiments after they had been incubating eggs for 7-14 days. Blood samples (1 ml) were taken by direct venepuncture, and control and test substances were administered by the same route. Animals were killed by cervical dislocation in accordance with the UK Animals (Scientific Procedures) Act 1986. Anterior pituitary glands were collected in liquid nitrogen and stored at -70 C; plasma samples were stored at -20 C. Three sets of experiments were carried out. In the first the relationship between concentrations of plasma PRL and pituitary PRL and PRL mRNA content were examined in laying and incubating hens, and in incubating hens deprived of their nests for 1, 3, or 6 days. The second experiment investigated the effect of increasing or decreasing VIP in the peripheral circulation on the concentration of plasma PRL and on pituitary PRL and mRNA content. The plasma concentration of VIP was increased by iv injection of 75 ^g porcine (Peninsula Laboratories Ltd., Merseyside, U.K.) or chicken (12) VIP/kg into laying hens. This dose was chosen because it has been shown to cause a submaximal release of PRL (9). Control hens were injected with saline vehicle. Blood samples were taken immediately before and 10 min after injection; the hens were killed 90 min after injection to collect pituitary glands. Plasma VIP was immunoneutralized in incubating hens by injecting 1 ml of highly specific sheep antichicken VIP serum (code 6DL33/4) (12). Control injections were nonimmune sheep serum. Blood samples for PRL measurements were taken immediately before injection of sheep serum and 24 h later, at which time the hens were killed to collect pituitary glands. Pituitary PRL mRNA was assessed using dot blot and Northern hybridization. The final experiment was to establish whether PRL mRNA levels are increased in PRL cells in pituitary glands of incubating hens when compared with laying hens, using in situ hybridization. Observations were made on two laying and three incubating hens. Dot and Northern blotting The levels of PRL mRNA in the pituitary glands were measured using a cytoplasmic dot blot hybridization technique (21). A sample of liver from each experiment was used as the negative control. Pituitary gland cytosol extracts were diluted and applied in triplicate to a nylon membrane (Hybond N, Amersham International Ltd., Amersham, Bucks, U.K.) held in a dot blot apparatus (Gibco-BRL, Paisley, Strathclyde, U.K.). The RNA was cross-linked to the membrane with UV light, (U.V. Stratalinker, Stratagene, Cambridge, U.K.). The membranes were hybridized to the chicken PRL 101 probe (18). Subsequently they were washed in a boiling solution of 0.1% (wt/vol) sodium dodecyl sulfate for 30 min, to remove the PRL probe, then reprobed with oligo (dT) in order to assess RNA loadings (22). The level of hybridization was quantified by densitometric scanning of autoradiograms. For Northern transfer analyses total RNA was prepared from two pituitary glands from hens in the different treatment groups according to the method of Chomczynski and Sacchi (23). The RNA samples (4 Mg/lane) were electrophoresed
497
through 1.3% agarose formaldehyde denaturing gels (24). RNA size markers (Gibco-BRL) were included to determine the size of the PRL mRNA. The RNA was transferred to a nylon membrane (Hybond N, Amersham) using a vacuum blotting apparatus (Vacublot; Pharmacia, Milton Keynes, U.K.) with 1 M ammonium acetate, 0.02 M NaOH as the transfer solution. The RNA was cross-linked to the membrane using UV light and probed with PRL 101 as above. In situ hybridization and immunocytochemistry Pituitary glands with the adjacent basal hypothalamus attached were fixed for 3 h in Kryofix (British Drug Houses, Poole, Dorset, U.K.) and processed further by microwave irradiation (H2500 microwave processor, Bio-Rad, Hemel Hempstead, U.K.) (25). After clearing in toluene, the tissue was embedded in polystyrene (26) and cut at 2 fxm. The sections were mounted on slides coated with 2% (vol/vol) 3-aminopropyltriethoxysilane (Sigma Chemical Co., St. Louis, MO) in acetone. Two 33-mer oligonucleotides (Oswell DNA Services Edinburgh, Midlothian, U.K.) were constructed corresponding to the coding and noncoding strands of bases 545 to 578 (amino acids 170-180) of the chicken PRL cDNA sequence (18). The in situ hybridization protocol has been described previously (27) and was modified by omitting the use of intensifying screens for the autoradiography. Sections of pituitary glands adjacent to those used for in situ hybridization were selected to localize PRL-containing cells immunocytochemically using an avidin-biotin complex procedure (28). The primary antibody, raised in a rabbit against recombinant-derived chicken PRL (code 31/1) (see RIA) was used at a dilution of 1:40,000. The secondary antibody, biotinlyated goat-antirabbit immunoglobulin was used at a dilution of 1:150. Specificity was confirmed by showing that there was no reaction product in control sections treated the same way excepting that the primary antibody had been preincubated overnight at 4 C with recombinant-derived chicken PRL (500 pg/ml). Labeling of cDNA and oligonucleotide probes The PRL cDNA probe (PRL101) (18) was 32P labeled by an oligonucleotide priming method (29). Routinely specific activities of 1 x 109 cpm/pg were achieved. The oligonucleotide probes were end labeled with 32P using T4 polynucleotide kinase (Pharmacia, Milton Keynes, U.K.) (24). The (oligo) dT (12-18 mer) (Pharmacia) was labeled to a specific activity of 1 X 106 cpm//ug. The two (33 mer) oligonucleotides used in the in situ hybridization were routinely labeled to give specific activities of 2.5 X 108 cpm/jug. RIA A RIA specific for chicken PRL was developed to measure plasma and pituitary PRL concentrations. Recombinant PRL was purified to homogeneity, from Escherichia coli containing the pPRXlOOl plasmid (30) and used to raise an antibody (code 31/1) in a rabbit (31). The recombinant PRL was also used for standards and iodination using iodogen (1,3,4,6-tetra chloro3a,6a-diphenyl glycouril) as the oxidizing agent (32). The bound iodinated PRL was displaced by the recombinant PRL with a minimal detectable dose of 0.7 ± 0.3 ng/ml and 50% displacement of binding by 4.8 ± 0.4 ng/ml (Fig. 1). Dilutions
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VIP REGULATION OF PRL mRNA
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10 100 1000 Prolactin ( ng / tube) 1:10000
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FIG. 1. A RIA for recombinant-derived chicken PRL (cPrl) showing the displacement of 125I-labeled cPrl by the cPrl standard (•), pituitary gland homogenates from broody (•) and laying hens (O), and blood plasma samples from a broody (A) and laying (•) hens. No displacement of binding was observed with chicken LH, GH, or neural lobe extract or with plasma from a hypophysectomized hen (•). B/Bo refers to 125I label-bound (B) as a percentage of the label bound (Bo) in zero standard tubes in the absence of cPrl.
of plasmas and anterior pituitaries from broody and laying hens displaced the binding in a parallel fashion to the PRL standard. The binding was not displaced by chicken LH, GH and FSH, neural lobe extract, or plasma from a hypophysectomized hen (Fig. 1). The inter- and intraassay coefficients of variation were 7%-12%, respectively. Statistics Results were analyzed using analysis of variance and unpaired Student's t test.
Results Plasma and pituitary PRL and PRL mRNA in laying, incubating, and nest-deprived hens The concentration of plasma PRL was 20-fold greater in incubating than in laying hens: after nest deprivation PRL levels fell below those of laying hens (Fig. 2a). The pituitary content of PRL in incubating hens, which was about double (P < 0.05) that in laying hens, increased 3fold (P < 0.001) 24 h after nest deprivation and then decreased slowly (Fig. 2b). After 6 days nest deprivation the amount of PRL in the pituitary was not significantly different from that in either incubating or laying hens (Fig. 2b). The amount of PRL mRNA in the pituitaries of incubating hens was greater than in the pituitaries of laying hens (P < 0.001) (Fig. 2c). After 24 h nest deprivation pituitary PRL mRNA decreased to levels that were not significantly different from those in laying hens.
laying
incubating
24 h
72 h
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nest deprivation
FIG. 2. Changes in concentrations of plasma PRL (top), pituitary PRL content (middle), and pituitary PRL mRNA (bottom) content in laying and incubating bantam hens and in incubating bantam hens deprived of their nests for 24 h, 72 h, and 6 days. *, P < 0.05; **, P < 0.01; ***, P < 0.001; vs. laying hen values. The values are means ± SEM (n = 8).
Plasma PRL and pituitary PRL mRNA levels after injection of VIP or anti- VIP serum In laying hens plasma PRL concentrations were significantly increased 7-fold and 6-fold (P < 0.05) 10 min after injection of porcine or chicken VIP, respectively (Fig. 3a). The amounts of PRL mRNA in the pituitary gland were significantly higher 90 min after injection of porcine or chicken VIP than after injection of the saline vehicle (Fig. 3b). There was no significant difference in the response to either chicken or porcine VIP (Fig. 3b). In incubating hens plasma PRL concentrations fell to around the level of detection of the assay 24 h after injection of anti-VIP serum and remained high in control birds injected with nonimmune serum (Fig. 4a). Pituitary PRL content was 2.5-fold higher (Fig. 4b) while pituitary
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VIP REGULATION OF PRL mRNA
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FlG. 3. Effect of an injection of 75 ng porcine (p) or chicken (c) VIP/ kg or saline vehicle in laying bantam hens on a) concentrations of plasma PRL immediately before {solid box) and 10 min (open bar) after injection of VIP. *, P < 0.05; us. value immediately before injection and b) the amount of pituitary PRL mRNA 90 min after injection of VIP. *, P < 0.05; us. saline injection. The values are means ± SEM (n = 6).
mRNA was 3-fold lower (Fig. 4c) than in control pituitary glands 24 h after injection of anti-VIP serum. In situ hybridization In situ hybridization of the oligonucleotide complementary to PRL mRNA was observed primarily in the cephalic lobe of the anterior pituitary gland of incubating and laying hens (Fig. 5, a and b). No hybridization was observed when the sense strand was used. Hybridization occurred in the region of the anterior pituitary gland containing the PRL-producing cells as shown by immunocytochemical localization in adjacent sections (Fig. 5, c and d). No in situ hybridization was seen in the portion of the anterior pituitary gland which contained no immunoreactive PRL cells (Fig. 5, c and d). More hybridization occurred to sections of pituitary glands from incubating than from laying hens (Fig. 5, a and b). This observation was confirmed using pituitary glands from three incubating and two laying hens. Northern blot analysis Northern blot analyses with a chicken PRL probe of mRNA extracted from laying or incubating hens or hens treated with VIP or anti-VIP serum indicated the pres-
Normal Sheep Serum
Anti-VIP Serum
Normal Sheep Serum
Anti-VIP Serum
FlG. 4. Effect of iv injections (1 ml) of nonimmune sheep serum (control) or sheep anti-VIP serum (anti-VIP) on a) concentration of plasma PRL immediately before (solid bar) and 24 h after (open bar) injection. *,**, P < 0.001; us. sample immediately before injection and b) the amount of pituitary PRL and c) pituitary PRL mRNA 24 h after injection. ***, P < 0.001; us. nonimmune sheep values. The values are means ± SEM (n = 8).
ence of a single band of 860 bases (Fig. 6). The intensity of bands reflected the amount of PRL mRNA present, as measured in the dot blot assays, in pituitaries from all treatment groups. Discussion The objectives of this study were to analyze PRL and PRL mRNA levels in laying, incubating, and nest-deprived hens and to investigate the role of VIP in these different physiological states. The 20-fold difference in circulating levels of plasma PRL between laying and incubating hens was reflected by more modest increases in the amount of PRL and PRL mRNA in the pituitary gland. The absence of a large increase in stored pituitary PRL in incubating hens suggests either a small increase in PRL synthesis combined with an increase in the half-
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VIP REGULATION OF PRL mRNA
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E n d o • 1991 Vol 129 • No 1
In situ
FIG. 5. Morphological observations on anterior pituitary glands from incubating (left panels) and laying (rightpanels) bantam hens by in situ hybridization using a 33-mer [32P] oligonucleotide probe complementary to part of the sequence of chicken PRL cDNA (a and b), by immunocytochemistry, in adjacent sections using a primary antibody against chicken recombinant derived PRL (c and d), and by toluidene blue in adjacent sections to demonstrate the limits of the pituitary tissue (e and f). Scale bar, 1 mm; cp, cephalic lobe; cd, caudal lobe.
Immunocytochemistry
Toluidene blue
cp = cephalic lobe,
life of PRL, or a much larger increase in PRL synthesis which is not stored before release. In support of the latter hypothesis, when the stimulus for the release of PRL was removed either by nest deprivation or anti-VIP injection, the plasma PRL levels dropped rapidly (12) while pituitary PRL increased. The increase in PRL was presumably due to a combination of continued synthesis of PRL from the elevated levels of PRL mRNA and the absence of a stimulus for PRL release. The observation that differences in circulating levels of plasma PRL were correlated with levels of pituitary PRL mRNA and not with pituitary PRL content agrees with similar studies on the lactating rat (33). The state of hyperprolactinemia found in lactating rats is similar to that in incubating hens. Withdrawal of suckling pups results within 24 h in a rapid decrease in plasma PRL and pituitary PRL mRNA but no change in pituitary PRL content. In a similar study on lactating rats but not involving measurement of PRL mRNA, withdrawal of suckling pups for 24 h resulted in increased pituitary PRL in the mothers (34). This observation is analogous to that made in incubating hens deprived of nests for 24 h. Pituitary PRL mRNA increased after administration of either porcine or chicken VIP and decreased after circulating VIP was immunoneutralized. This observation suggests that in addition to serving as a physiological releasing factor (1-8), VIP also regulates PRL mRNA levels. The only other report that VIP stimulates pituitary PRL mRNA levels is derived from a study on GH3 cells (17). The data from this study have to be interpreted with caution because GH3 cells store less PRL than
cd = caudal lobe
normal lactotrophs (35) and, in addition to being transformed, they have been maintained for many years in the absence of hypothalamic control. It was not established whether VIP modulates PRL gene transcription or translation. Studies on GH3 tumor cells suggests that both modes of action are possible (36,37). VIP stimulates these cells to secrete PRL and to accumulate cAMP (36) while treatment with cAMP analogs or agents that increase intracellular cAMP rapidly stimulate transcription of the PRL gene (37). VIP may therefore stimulate PRL gene transcription in chicken PRL cells by activating adenylate cyclase. Indirect support for this view is provided by the finding that treatment of chicken pituitary glands in vitro with agents which increase intracellular cAMP, stimulates the secretion of PRL (38). An alternative mechanism of action is suggested by the observation that treatment of GH3 cells with TRH both increases the half-life on PRL mRNA and the rate of PRL gene transcription (39). It is therefore possible that VIP may exert a similar effect in PRL gene translation in chicken PRL cells. The direct correlation between plasma PRL and pituitary PRL mRNA in laying and incubating hens and incubating hens deprived of their nests may reflect changes in the release of VIP from the hypothalamus. The observation that a single injection of VIP failed to increase PRL mRNA levels to these observed in incubating hens indicates that this treatment failed to mimick this pattern of release in incubating hens. Conclusive evidence will require direct measurement of VIP release from the median eminence. Strong circumstantial evi-
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VIP REGULATION OF PRL mRNA •o
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in the anterior pituitary gland is greater in incubating than in laying hens. It also showed that PRL mRNA production in the hen is restricted to the cephalic lobe of the anterior pituitary gland, which contains the majority of immunoreactive PRL cells and that the pattern of hybridization is unaltered by the increased level of PRL on RNA. Northern blot analyses of PRL mRNA confirmed the presence of a mature mRNA of 860 base(s) (18) which was present in all treatment groups. The present study provides evidence that under physiological conditions VIP regulates circulating concentrations of PRL. This regulation is achieved by increasing the level of PRL mRNA in the pituitary and by increasing secretion of PRL. Since the incubating hen and suckling mammal represent analogous physiological states of hyperprolactinemia, it would be of interest to establish whether VIP is involved in the control of PRL mRNA levels in suckling mammals as it appears to be in incubating hens. References
0.24—
FIG. 6. Northern blots of pituitary mRNA probed with [32P] cDNA complementary to chicken PRL mRNA from a laying hen (lane 1), an incubating hen deprived of its nest for 24 h (lane 2), an incubating hen 24 h after being passively immunized with anti-VIP serum (lane 3), a laying hen 90 min after an iv injection of 75 ng VIP/kg (lane 4), and an incubating hen (lane 5). The sizes of the RNA markers are indicated by arrows.
dence is provided by the close correlation between plasma PRL levels and amounts of hypothalamic VIP and the degree of hypertrophy of hypothalamic VIP neurones in these different physiological states (11-13). Although the evidence that VIP is the major PRL releasing factor in incubating hens is now strengthened, more work is required to establish whether VIP is the major PRL releasing factor in other physiological states, i.e. during the onset of puberty (40) or during the development of photorefractoriness (41). It is probable that other hypothalamic factors and gonadal steroids may modulate the action of VIP on PRL release (42). The correlated in situ hybridization and immunocytochemical study confirmed that the level of PRL mRNA
1. Ben-Jonathan N, Arbogast LA, Hyde JF 1989 Neuroendocrine regulation of prolactin release. Prog Neurobiol 33:399-447 2. Lamberts SWJ, Macleod RM 1990 Regulation of prolactin secretion at the level of the lactotroph. Physiol Rev 70:279-318 3. Said SI, Porter JC 1979 Vasoactive intestinal polypeptide release into hypophysial portal blood. Life Sci 24:227-230 4. Shimatsu A, Kato Y, Matsushita N, Katakami N, Yainaihara N, Imura H 1981 Immunoreactive vasoactive intestinal polypeptide in rat hypophysial portal blood. Endocrinology 108:395-398 5. Shimatsu A, Kato Y, Ohta H, Tojo K, Kabayama Y, Inoue T, Yanaihara N, Imura H 1984 Involvement of hypothalamic vasoactive intestinal polypeptide (VIP) in prolactin secretion induced by serotonin in rats. Proc Soc Exp Biol Med 175:414-416 6. Abe H, Engler D, Molitch ME, Bollinger-Gruber J, Reichlin S 1985 Vasoactive intestinal peptide is a physiological mediator of prolactin release in the rat. Endocrinology 116:1383-1390 7. Kaji H, Chihara K, Abe H, Kita, T, Kashio Y, Okimura Y Fujita T 1985 Effect of passive immunization with antisera to vasoactive intestinal peptide and peptide histidine isoleucine amide on 5hydroxy-L-tryptophan induced prolactin release in rats. Endocrinology 117:1914-1919 8. Ohta H, Kato Y, Shimatsu A, Tojo K, Kabayama Y, Inoue T, Yanachara N, Imura H 1985 Inhibition by antiserum to vasoactive intestinal polypeptide (VIP) of prolactin secretion induced by serotonin in the rat. Eur J Pharmacol 109:409-412 9. Macnamee MC, Sharp PJ, Lea RW, Sterling RJ, Harvey S 1986 Evidence that vasoactive intestinal polypeptide is a physiological releasing factor in the bantam hen. Gen Comp Endocrinol 62:470478 10. Proudman J, Opel H 1988 Stimulation of prolactin secretion from turkey anterior pituitary cells. Proc Soc Exp Biol Med 187:448458 11. Mauro LJ, Elde RP, Youngren OM, Phillips RE, El Halawani ME 1989 Alterations in hypothalamic vasoactive intestinal peptide-like immuno-reactivity are associated with reproduction and prolactin release in the female turkey. Endocrinology 125:1795-1804 12. Sharp PJ, Sterling RJ, Talbot RT, Huskisson NS 1989 The role of hypothalamic vasoactive intestinal polypeptide in the maintenance of prolactin secretion in incubating bantam hens: observations using passive immunisation, radioimmunoassay and immunohistochemistry. J Endocrinol 22:5-13 13. Cloues R, Ramos C, Silver R 1990 VIP-like immunoreactivity during reproduction in doves: influence of experience and number
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of offspring. Horm Behav 33:399-447 14. Lea RW, Talbot RT, Sharp PJ, Passive immunization against chicken vasoactive intestinal polypeptide suppresses plasma prolactin and crop sac development in incubating ring doves. Horm Behav, in press 15. Nicoll CS 1967 Bioassay of prolactin. Analysis of the pigeon cropsac response to local prolactin injection by an objective quantitative method. Endocrinology 80:641-655 16. Shull J, Gorski J 1986 The hormone regulation of prolactin gene expression: an examination of mechanisms controlling prolactin synthesis and the possible relationships of estrogen to these mechanisms. Vitam Horm 43:197-249 17. Carrillo AJ, Pool TB, Sharp ZD 1985 Vasoactive intestinal peptide increases prolactin messenger ribonucleic acid content in GH3 cells. Endocrinology 116:202-206 18. Hanks MC, Alonzi JA, Sharp PJ, Sang HM 1989 Molecular cloning and sequence analysis of putative chicken prolactin cDNA. J Mol Endocrinol 2:21-30 19. Sharp PJ, Macnamee MC, Sterling RJ, Lea RW, Pederson HC 1988 Relationships between prolactin, LH and broody behaviour in bantam hens. J Endocrinol 118:279-286 20. Sterling RJ, Cheng WH, Sharp PJ 1990 Morphological changes in hypothalamic prolactin releasing hormone (VIP) neurones in bantam hens after nest deprivation. Neuroendocrinology 52[Suppl]: 165 (Abstract) 21. White BA, Bancroft T 1982 Cytoplasmic dot hybridisation. J Biol Chem 257:8569-8572 22. Harley CB 1987 Hybridisation of oligo (dT) to RNA on nitrocellulose. Gene Anal Tech 4:17-22 23. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 161:156-159 24. Sambrook J, Fritsch ET, Maniatis T 1989 Molecular Cloning. A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 25. Boon ME, Kok LP 1988 Microwave Cookbook of Pathology: The Art of Microscopic Visualization. Coulomb Press, Leyden, UK 26. Fragioni G, Borgioli G 1979 Polystyrene embedding: a new method for light and electron microscopy. Stain Technol 54:167-172 27. Bloch B, Popovici T, Le Guellec D, Normand E, Chouham S, Guitteny AT, Bohlen P 1986 In situ hybridization histochemistry for the analysis of gene expression in the endocrine and central nervous system tissues: a 3 year experience. J Neurosci Res 16:183200 28. Hsu SM, Rain EL, Tanger H 1981 The use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) proce-
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dures. J Histochem Cytochem 19:577-580 29. Feinberg AP, Vogelstein B 1983 A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 30. Hanks MC, Talbot RT, Sang HM 1990 Expression of biologically active recombinant-derived chicken prolactin in Escherichia coli. J Mol Endocrinol 3:15-21 31. Vaitukaitis J, Robbins JB, Nieschlag E, Ross GT 1971 A method for producing specific antisera with small doses of immunogen. J Clin Endocrinol Metab 33:988-991 32. Bolton AE 1985 Radioiodination Techniques, ed 2. Review 18 Amersham International PLC, Amersham, U.K. p 63 33. Lee LR, Haisenleder Marshall JC, Smith MS 1989 The role of the suckling stimulus in regulating pituitary prolactin mRNA in the rat. Mol Cell Endocrinol 64:245-249 34. Torres AI, Aoki A 1985 Subcellular compartmentation of prolactin in rat lactotrophs. J Endocrinol 105:219-225 35. Stachura ME 1982 Sequestration of an early-release pool of growth hormone and prolactin in GH3 rat pituitary tumor cells. Endocrinology 105:219-2258 36. Gourdji D, Bataille D, Vauclin N, Grouselle D, Rosselin C, TixierVidal A 1979 Vasoactive intestinal peptide (VIP) stimulates prolactin (PRL) release and cAMP production in a rat pituitary cell line (GH3/B6). Additive effects of VIP and TRH on PRL release. FEBS Lett 104:165-168 37. Murdoch G, Rosenfield M, Evans R 1982 Eukaryotic transcriptional regulation and chromatin associated protein phosphorylation by cyclic AMP. Science 218:1315-1317 38. Hall TR, Harvey S, Chadwick A 1985 Mechanisms of release of prolactin from fowl adenohypophyses incubated in vitro: effects of calcium and cyclic adenosine monophosphate. J Endocrinol 105:183-188 39. Laverriere JN, Morin A, Tixier-Vidal A, Truong AT, Gourdji D, Martial JA 1983 Inverse control of prolactin and growth hormone gene expression: effect of thyroliberin on transcription and RNA stabilization. EMBO J 2:1493-1499 40. Mikami SI, Yamada S 1984 Immunohistochemistry of hypothalamic neuropeptides and anterior pituitary cells in the Japanese Quail. J Exp Zool 232:405-419 41. Sterling RJ, Sharp PJ, Klandorf H, Harvey S, Lea RW 1984 Plasma concentrations of luteinizing hormone, follicle stimulating hormone, androgen, growth hormone, prolactin, thyroxine and triiodothyronine during growth and sexual development in the cockerel. Br Poult Sci 25:357-359 42. Nicholls TJ, Goldsmith AR, Dawson A 1988 Photorefractoriness in birds and mammals. Physiol Rev 68:133-176 43. Hall TR, Harvey S, Chadwick A 1986 Control of prolactin secretion in birds: a review. Gen Comp Endocrinol 62:171-184
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Vol. 129, No. 1 Printed in U.S.A.
Pituitary Prolactin Messenger Ribonucleic Acid Levels in Incubating and Laying Hens: Effects of Manipulating Plasma Levels of Vasoactive Intestinal Polypeptide RICHARD T. TALBOT, MARK C. HANKS, ROBERT J. STERLING, HELEN M. SANG, AND PETER J. SHARP Agricultural and Food Research Council Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Roslin, Midlothian EH25 9PS, United Kingdom
ABSTRACT. Pituitary PRL messenger RNA levels in hens, measured by dot-blot hybridization, correlated directly with concentrations of plasma PRL, being 3-fold higher in incubating than in laying birds. Nest deprivation of incubating hens for 24 h caused a rapid decrease in both plasma PRL and pituitary PRL mRNA, which remained depressed thereafter. A single injection of vasoactive intestinal polypeptide (VIP) in laying hens resulted in an increase (P < 0.05) in pituitary PRL mRNA whereas passive immunoneutralization of VIP in incubating hens resulted in a decrease (P < 0.001) in pituitary PRL mRNA. The rapid decrease in pituitary PRL mRNA after nest deprivation or passive immunoneutralization of VIP was associated with a significant increase in pituitary PRL content, presumably a consequence of the decreased PRL secretion. In situ hybridi-
zation showed PRL mRNA to be localized in the cephalic lobe of the anterior pituitary gland in which most PRL cells, identified immunocytochemically, were found. Northern blotting studies showed that the pituitary gland contains a single 860 base(s) mature PRL mRNA transcript irrespective of physiological state or VIP manipulation. Both in situ and Northern hybridization studies confirmed that the amount of pituitary PRL mRNA was related directly to the concentration of plasma PRL. These observations are consistent with the view that in incubating hens hypothalamic VIP, in addition to acting as a PRL releasing hormone, also plays a major role in the regulation of the amount of PRL mRNA in the anterior pituitary gland. (Endocrinology 129: 496-502, 1991)
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EVERAL hypothalamic neurotransmitters and neuropeptides are known to stimulate PRL release but their precise physiological functions are uncertain (1, 2). There is evidence that one of the neuropeptides, vasoactive intestinal polypeptide (VIP), is physiologically significant. Increased concentrations of VIP occur in rat hypophysial portal blood (3, 4), and the administration of anti-VIP serum inhibits PRL release induced by stress, serotonin, or suckling (5-8). In the lactating rat the inhibitory effect of anti-VIP serum on PRL release is transitory despite the presence of excess antibody (6). There is evidence that VIP is the major physiologically significant PRL releasing factor in birds. VIP acts directly on the avian anterior pituitary gland to release PRL specifically (9, 10) and is present in neurons in the basal hypothalamus with projections to the median eminence. These neurons show marked hyperplasia when plasma PRL levels are high in incubating birds (11-13). Passive immunization with anti-VIP serum depresses plasma PRL in incubating bantams (12) and in incubating ring
doves prevents the development of the crop sac (14) an organ which is used in a bioassay for PRL (15). If VIP plays a key role in the regulation of PRL secretion, it is possible that it is also involved in the control of PRL synthesis. Although several hormones and neurotransmitters are known to influence PRL gene expression (16), evidence that VIP plays such a role is limited to a study using GH3 cells, a rat pituitary tumor cell line (17). The availability of a complementary DNA probe for chicken PRL (18) now makes it possible to investigate the effects of VIP on pituitary PRL mRNA in the bantam hen. Earlier studies on this bird have shown that the increased concentrations of plasma PRL (19) and hypothalamic VIP observed during incubation (12) decrease rapidly after nest deprivation (20). This study was carried out to establish the dynamics of pituitary PRL and PRL mRNA content after nest deprivation and to obtain evidence that the predicted decrease in PRL mRNA might be a consequence of a decreased release of hypothalamic VIP.
Received March 25, 1991. Address requests for reprints to: Richard T. Talbot, Agricultural and Food Research Council Institute of Animal Physiology and Genetics Research, Roslin, Midlothian EH25 9PS, United Kingdom.
Animals and experimental design
Materials and Methods Laying bantam hens from the Institute's flock were maintained in battery cages exposed to 14 h light/day with food and
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VIP REGULATION OF PRL mRNA water freely available. Broodiness was induced by transferring hens, under the same lighting conditions, to deep litter floor pens containing nest boxes with five boiled eggs in each box. Incubating hens were allocated to experiments after they had been incubating eggs for 7-14 days. Blood samples (1 ml) were taken by direct venepuncture, and control and test substances were administered by the same route. Animals were killed by cervical dislocation in accordance with the UK Animals (Scientific Procedures) Act 1986. Anterior pituitary glands were collected in liquid nitrogen and stored at -70 C; plasma samples were stored at -20 C. Three sets of experiments were carried out. In the first the relationship between concentrations of plasma PRL and pituitary PRL and PRL mRNA content were examined in laying and incubating hens, and in incubating hens deprived of their nests for 1, 3, or 6 days. The second experiment investigated the effect of increasing or decreasing VIP in the peripheral circulation on the concentration of plasma PRL and on pituitary PRL and mRNA content. The plasma concentration of VIP was increased by iv injection of 75 ^g porcine (Peninsula Laboratories Ltd., Merseyside, U.K.) or chicken (12) VIP/kg into laying hens. This dose was chosen because it has been shown to cause a submaximal release of PRL (9). Control hens were injected with saline vehicle. Blood samples were taken immediately before and 10 min after injection; the hens were killed 90 min after injection to collect pituitary glands. Plasma VIP was immunoneutralized in incubating hens by injecting 1 ml of highly specific sheep antichicken VIP serum (code 6DL33/4) (12). Control injections were nonimmune sheep serum. Blood samples for PRL measurements were taken immediately before injection of sheep serum and 24 h later, at which time the hens were killed to collect pituitary glands. Pituitary PRL mRNA was assessed using dot blot and Northern hybridization. The final experiment was to establish whether PRL mRNA levels are increased in PRL cells in pituitary glands of incubating hens when compared with laying hens, using in situ hybridization. Observations were made on two laying and three incubating hens. Dot and Northern blotting The levels of PRL mRNA in the pituitary glands were measured using a cytoplasmic dot blot hybridization technique (21). A sample of liver from each experiment was used as the negative control. Pituitary gland cytosol extracts were diluted and applied in triplicate to a nylon membrane (Hybond N, Amersham International Ltd., Amersham, Bucks, U.K.) held in a dot blot apparatus (Gibco-BRL, Paisley, Strathclyde, U.K.). The RNA was cross-linked to the membrane with UV light, (U.V. Stratalinker, Stratagene, Cambridge, U.K.). The membranes were hybridized to the chicken PRL 101 probe (18). Subsequently they were washed in a boiling solution of 0.1% (wt/vol) sodium dodecyl sulfate for 30 min, to remove the PRL probe, then reprobed with oligo (dT) in order to assess RNA loadings (22). The level of hybridization was quantified by densitometric scanning of autoradiograms. For Northern transfer analyses total RNA was prepared from two pituitary glands from hens in the different treatment groups according to the method of Chomczynski and Sacchi (23). The RNA samples (4 Mg/lane) were electrophoresed
497
through 1.3% agarose formaldehyde denaturing gels (24). RNA size markers (Gibco-BRL) were included to determine the size of the PRL mRNA. The RNA was transferred to a nylon membrane (Hybond N, Amersham) using a vacuum blotting apparatus (Vacublot; Pharmacia, Milton Keynes, U.K.) with 1 M ammonium acetate, 0.02 M NaOH as the transfer solution. The RNA was cross-linked to the membrane using UV light and probed with PRL 101 as above. In situ hybridization and immunocytochemistry Pituitary glands with the adjacent basal hypothalamus attached were fixed for 3 h in Kryofix (British Drug Houses, Poole, Dorset, U.K.) and processed further by microwave irradiation (H2500 microwave processor, Bio-Rad, Hemel Hempstead, U.K.) (25). After clearing in toluene, the tissue was embedded in polystyrene (26) and cut at 2 fxm. The sections were mounted on slides coated with 2% (vol/vol) 3-aminopropyltriethoxysilane (Sigma Chemical Co., St. Louis, MO) in acetone. Two 33-mer oligonucleotides (Oswell DNA Services Edinburgh, Midlothian, U.K.) were constructed corresponding to the coding and noncoding strands of bases 545 to 578 (amino acids 170-180) of the chicken PRL cDNA sequence (18). The in situ hybridization protocol has been described previously (27) and was modified by omitting the use of intensifying screens for the autoradiography. Sections of pituitary glands adjacent to those used for in situ hybridization were selected to localize PRL-containing cells immunocytochemically using an avidin-biotin complex procedure (28). The primary antibody, raised in a rabbit against recombinant-derived chicken PRL (code 31/1) (see RIA) was used at a dilution of 1:40,000. The secondary antibody, biotinlyated goat-antirabbit immunoglobulin was used at a dilution of 1:150. Specificity was confirmed by showing that there was no reaction product in control sections treated the same way excepting that the primary antibody had been preincubated overnight at 4 C with recombinant-derived chicken PRL (500 pg/ml). Labeling of cDNA and oligonucleotide probes The PRL cDNA probe (PRL101) (18) was 32P labeled by an oligonucleotide priming method (29). Routinely specific activities of 1 x 109 cpm/pg were achieved. The oligonucleotide probes were end labeled with 32P using T4 polynucleotide kinase (Pharmacia, Milton Keynes, U.K.) (24). The (oligo) dT (12-18 mer) (Pharmacia) was labeled to a specific activity of 1 X 106 cpm//ug. The two (33 mer) oligonucleotides used in the in situ hybridization were routinely labeled to give specific activities of 2.5 X 108 cpm/jug. RIA A RIA specific for chicken PRL was developed to measure plasma and pituitary PRL concentrations. Recombinant PRL was purified to homogeneity, from Escherichia coli containing the pPRXlOOl plasmid (30) and used to raise an antibody (code 31/1) in a rabbit (31). The recombinant PRL was also used for standards and iodination using iodogen (1,3,4,6-tetra chloro3a,6a-diphenyl glycouril) as the oxidizing agent (32). The bound iodinated PRL was displaced by the recombinant PRL with a minimal detectable dose of 0.7 ± 0.3 ng/ml and 50% displacement of binding by 4.8 ± 0.4 ng/ml (Fig. 1). Dilutions
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VIP REGULATION OF PRL mRNA
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10 100 1000 Prolactin ( ng / tube) 1:10000
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FIG. 1. A RIA for recombinant-derived chicken PRL (cPrl) showing the displacement of 125I-labeled cPrl by the cPrl standard (•), pituitary gland homogenates from broody (•) and laying hens (O), and blood plasma samples from a broody (A) and laying (•) hens. No displacement of binding was observed with chicken LH, GH, or neural lobe extract or with plasma from a hypophysectomized hen (•). B/Bo refers to 125I label-bound (B) as a percentage of the label bound (Bo) in zero standard tubes in the absence of cPrl.
of plasmas and anterior pituitaries from broody and laying hens displaced the binding in a parallel fashion to the PRL standard. The binding was not displaced by chicken LH, GH and FSH, neural lobe extract, or plasma from a hypophysectomized hen (Fig. 1). The inter- and intraassay coefficients of variation were 7%-12%, respectively. Statistics Results were analyzed using analysis of variance and unpaired Student's t test.
Results Plasma and pituitary PRL and PRL mRNA in laying, incubating, and nest-deprived hens The concentration of plasma PRL was 20-fold greater in incubating than in laying hens: after nest deprivation PRL levels fell below those of laying hens (Fig. 2a). The pituitary content of PRL in incubating hens, which was about double (P < 0.05) that in laying hens, increased 3fold (P < 0.001) 24 h after nest deprivation and then decreased slowly (Fig. 2b). After 6 days nest deprivation the amount of PRL in the pituitary was not significantly different from that in either incubating or laying hens (Fig. 2b). The amount of PRL mRNA in the pituitaries of incubating hens was greater than in the pituitaries of laying hens (P < 0.001) (Fig. 2c). After 24 h nest deprivation pituitary PRL mRNA decreased to levels that were not significantly different from those in laying hens.
laying
incubating
24 h
72 h
144 h
nest deprivation
FIG. 2. Changes in concentrations of plasma PRL (top), pituitary PRL content (middle), and pituitary PRL mRNA (bottom) content in laying and incubating bantam hens and in incubating bantam hens deprived of their nests for 24 h, 72 h, and 6 days. *, P < 0.05; **, P < 0.01; ***, P < 0.001; vs. laying hen values. The values are means ± SEM (n = 8).
Plasma PRL and pituitary PRL mRNA levels after injection of VIP or anti- VIP serum In laying hens plasma PRL concentrations were significantly increased 7-fold and 6-fold (P < 0.05) 10 min after injection of porcine or chicken VIP, respectively (Fig. 3a). The amounts of PRL mRNA in the pituitary gland were significantly higher 90 min after injection of porcine or chicken VIP than after injection of the saline vehicle (Fig. 3b). There was no significant difference in the response to either chicken or porcine VIP (Fig. 3b). In incubating hens plasma PRL concentrations fell to around the level of detection of the assay 24 h after injection of anti-VIP serum and remained high in control birds injected with nonimmune serum (Fig. 4a). Pituitary PRL content was 2.5-fold higher (Fig. 4b) while pituitary
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VIP REGULATION OF PRL mRNA
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FlG. 3. Effect of an injection of 75 ng porcine (p) or chicken (c) VIP/ kg or saline vehicle in laying bantam hens on a) concentrations of plasma PRL immediately before {solid box) and 10 min (open bar) after injection of VIP. *, P < 0.05; us. value immediately before injection and b) the amount of pituitary PRL mRNA 90 min after injection of VIP. *, P < 0.05; us. saline injection. The values are means ± SEM (n = 6).
mRNA was 3-fold lower (Fig. 4c) than in control pituitary glands 24 h after injection of anti-VIP serum. In situ hybridization In situ hybridization of the oligonucleotide complementary to PRL mRNA was observed primarily in the cephalic lobe of the anterior pituitary gland of incubating and laying hens (Fig. 5, a and b). No hybridization was observed when the sense strand was used. Hybridization occurred in the region of the anterior pituitary gland containing the PRL-producing cells as shown by immunocytochemical localization in adjacent sections (Fig. 5, c and d). No in situ hybridization was seen in the portion of the anterior pituitary gland which contained no immunoreactive PRL cells (Fig. 5, c and d). More hybridization occurred to sections of pituitary glands from incubating than from laying hens (Fig. 5, a and b). This observation was confirmed using pituitary glands from three incubating and two laying hens. Northern blot analysis Northern blot analyses with a chicken PRL probe of mRNA extracted from laying or incubating hens or hens treated with VIP or anti-VIP serum indicated the pres-
Normal Sheep Serum
Anti-VIP Serum
Normal Sheep Serum
Anti-VIP Serum
FlG. 4. Effect of iv injections (1 ml) of nonimmune sheep serum (control) or sheep anti-VIP serum (anti-VIP) on a) concentration of plasma PRL immediately before (solid bar) and 24 h after (open bar) injection. *,**, P < 0.001; us. sample immediately before injection and b) the amount of pituitary PRL and c) pituitary PRL mRNA 24 h after injection. ***, P < 0.001; us. nonimmune sheep values. The values are means ± SEM (n = 8).
ence of a single band of 860 bases (Fig. 6). The intensity of bands reflected the amount of PRL mRNA present, as measured in the dot blot assays, in pituitaries from all treatment groups. Discussion The objectives of this study were to analyze PRL and PRL mRNA levels in laying, incubating, and nest-deprived hens and to investigate the role of VIP in these different physiological states. The 20-fold difference in circulating levels of plasma PRL between laying and incubating hens was reflected by more modest increases in the amount of PRL and PRL mRNA in the pituitary gland. The absence of a large increase in stored pituitary PRL in incubating hens suggests either a small increase in PRL synthesis combined with an increase in the half-
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VIP REGULATION OF PRL mRNA
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In situ
FIG. 5. Morphological observations on anterior pituitary glands from incubating (left panels) and laying (rightpanels) bantam hens by in situ hybridization using a 33-mer [32P] oligonucleotide probe complementary to part of the sequence of chicken PRL cDNA (a and b), by immunocytochemistry, in adjacent sections using a primary antibody against chicken recombinant derived PRL (c and d), and by toluidene blue in adjacent sections to demonstrate the limits of the pituitary tissue (e and f). Scale bar, 1 mm; cp, cephalic lobe; cd, caudal lobe.
Immunocytochemistry
Toluidene blue
cp = cephalic lobe,
life of PRL, or a much larger increase in PRL synthesis which is not stored before release. In support of the latter hypothesis, when the stimulus for the release of PRL was removed either by nest deprivation or anti-VIP injection, the plasma PRL levels dropped rapidly (12) while pituitary PRL increased. The increase in PRL was presumably due to a combination of continued synthesis of PRL from the elevated levels of PRL mRNA and the absence of a stimulus for PRL release. The observation that differences in circulating levels of plasma PRL were correlated with levels of pituitary PRL mRNA and not with pituitary PRL content agrees with similar studies on the lactating rat (33). The state of hyperprolactinemia found in lactating rats is similar to that in incubating hens. Withdrawal of suckling pups results within 24 h in a rapid decrease in plasma PRL and pituitary PRL mRNA but no change in pituitary PRL content. In a similar study on lactating rats but not involving measurement of PRL mRNA, withdrawal of suckling pups for 24 h resulted in increased pituitary PRL in the mothers (34). This observation is analogous to that made in incubating hens deprived of nests for 24 h. Pituitary PRL mRNA increased after administration of either porcine or chicken VIP and decreased after circulating VIP was immunoneutralized. This observation suggests that in addition to serving as a physiological releasing factor (1-8), VIP also regulates PRL mRNA levels. The only other report that VIP stimulates pituitary PRL mRNA levels is derived from a study on GH3 cells (17). The data from this study have to be interpreted with caution because GH3 cells store less PRL than
cd = caudal lobe
normal lactotrophs (35) and, in addition to being transformed, they have been maintained for many years in the absence of hypothalamic control. It was not established whether VIP modulates PRL gene transcription or translation. Studies on GH3 tumor cells suggests that both modes of action are possible (36,37). VIP stimulates these cells to secrete PRL and to accumulate cAMP (36) while treatment with cAMP analogs or agents that increase intracellular cAMP rapidly stimulate transcription of the PRL gene (37). VIP may therefore stimulate PRL gene transcription in chicken PRL cells by activating adenylate cyclase. Indirect support for this view is provided by the finding that treatment of chicken pituitary glands in vitro with agents which increase intracellular cAMP, stimulates the secretion of PRL (38). An alternative mechanism of action is suggested by the observation that treatment of GH3 cells with TRH both increases the half-life on PRL mRNA and the rate of PRL gene transcription (39). It is therefore possible that VIP may exert a similar effect in PRL gene translation in chicken PRL cells. The direct correlation between plasma PRL and pituitary PRL mRNA in laying and incubating hens and incubating hens deprived of their nests may reflect changes in the release of VIP from the hypothalamus. The observation that a single injection of VIP failed to increase PRL mRNA levels to these observed in incubating hens indicates that this treatment failed to mimick this pattern of release in incubating hens. Conclusive evidence will require direct measurement of VIP release from the median eminence. Strong circumstantial evi-
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VIP REGULATION OF PRL mRNA •o
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in the anterior pituitary gland is greater in incubating than in laying hens. It also showed that PRL mRNA production in the hen is restricted to the cephalic lobe of the anterior pituitary gland, which contains the majority of immunoreactive PRL cells and that the pattern of hybridization is unaltered by the increased level of PRL on RNA. Northern blot analyses of PRL mRNA confirmed the presence of a mature mRNA of 860 base(s) (18) which was present in all treatment groups. The present study provides evidence that under physiological conditions VIP regulates circulating concentrations of PRL. This regulation is achieved by increasing the level of PRL mRNA in the pituitary and by increasing secretion of PRL. Since the incubating hen and suckling mammal represent analogous physiological states of hyperprolactinemia, it would be of interest to establish whether VIP is involved in the control of PRL mRNA levels in suckling mammals as it appears to be in incubating hens. References
0.24—
FIG. 6. Northern blots of pituitary mRNA probed with [32P] cDNA complementary to chicken PRL mRNA from a laying hen (lane 1), an incubating hen deprived of its nest for 24 h (lane 2), an incubating hen 24 h after being passively immunized with anti-VIP serum (lane 3), a laying hen 90 min after an iv injection of 75 ng VIP/kg (lane 4), and an incubating hen (lane 5). The sizes of the RNA markers are indicated by arrows.
dence is provided by the close correlation between plasma PRL levels and amounts of hypothalamic VIP and the degree of hypertrophy of hypothalamic VIP neurones in these different physiological states (11-13). Although the evidence that VIP is the major PRL releasing factor in incubating hens is now strengthened, more work is required to establish whether VIP is the major PRL releasing factor in other physiological states, i.e. during the onset of puberty (40) or during the development of photorefractoriness (41). It is probable that other hypothalamic factors and gonadal steroids may modulate the action of VIP on PRL release (42). The correlated in situ hybridization and immunocytochemical study confirmed that the level of PRL mRNA
1. Ben-Jonathan N, Arbogast LA, Hyde JF 1989 Neuroendocrine regulation of prolactin release. Prog Neurobiol 33:399-447 2. Lamberts SWJ, Macleod RM 1990 Regulation of prolactin secretion at the level of the lactotroph. Physiol Rev 70:279-318 3. Said SI, Porter JC 1979 Vasoactive intestinal polypeptide release into hypophysial portal blood. Life Sci 24:227-230 4. Shimatsu A, Kato Y, Matsushita N, Katakami N, Yainaihara N, Imura H 1981 Immunoreactive vasoactive intestinal polypeptide in rat hypophysial portal blood. Endocrinology 108:395-398 5. Shimatsu A, Kato Y, Ohta H, Tojo K, Kabayama Y, Inoue T, Yanaihara N, Imura H 1984 Involvement of hypothalamic vasoactive intestinal polypeptide (VIP) in prolactin secretion induced by serotonin in rats. Proc Soc Exp Biol Med 175:414-416 6. Abe H, Engler D, Molitch ME, Bollinger-Gruber J, Reichlin S 1985 Vasoactive intestinal peptide is a physiological mediator of prolactin release in the rat. Endocrinology 116:1383-1390 7. Kaji H, Chihara K, Abe H, Kita, T, Kashio Y, Okimura Y Fujita T 1985 Effect of passive immunization with antisera to vasoactive intestinal peptide and peptide histidine isoleucine amide on 5hydroxy-L-tryptophan induced prolactin release in rats. Endocrinology 117:1914-1919 8. Ohta H, Kato Y, Shimatsu A, Tojo K, Kabayama Y, Inoue T, Yanachara N, Imura H 1985 Inhibition by antiserum to vasoactive intestinal polypeptide (VIP) of prolactin secretion induced by serotonin in the rat. Eur J Pharmacol 109:409-412 9. Macnamee MC, Sharp PJ, Lea RW, Sterling RJ, Harvey S 1986 Evidence that vasoactive intestinal polypeptide is a physiological releasing factor in the bantam hen. Gen Comp Endocrinol 62:470478 10. Proudman J, Opel H 1988 Stimulation of prolactin secretion from turkey anterior pituitary cells. Proc Soc Exp Biol Med 187:448458 11. Mauro LJ, Elde RP, Youngren OM, Phillips RE, El Halawani ME 1989 Alterations in hypothalamic vasoactive intestinal peptide-like immuno-reactivity are associated with reproduction and prolactin release in the female turkey. Endocrinology 125:1795-1804 12. Sharp PJ, Sterling RJ, Talbot RT, Huskisson NS 1989 The role of hypothalamic vasoactive intestinal polypeptide in the maintenance of prolactin secretion in incubating bantam hens: observations using passive immunisation, radioimmunoassay and immunohistochemistry. J Endocrinol 22:5-13 13. Cloues R, Ramos C, Silver R 1990 VIP-like immunoreactivity during reproduction in doves: influence of experience and number
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of offspring. Horm Behav 33:399-447 14. Lea RW, Talbot RT, Sharp PJ, Passive immunization against chicken vasoactive intestinal polypeptide suppresses plasma prolactin and crop sac development in incubating ring doves. Horm Behav, in press 15. Nicoll CS 1967 Bioassay of prolactin. Analysis of the pigeon cropsac response to local prolactin injection by an objective quantitative method. Endocrinology 80:641-655 16. Shull J, Gorski J 1986 The hormone regulation of prolactin gene expression: an examination of mechanisms controlling prolactin synthesis and the possible relationships of estrogen to these mechanisms. Vitam Horm 43:197-249 17. Carrillo AJ, Pool TB, Sharp ZD 1985 Vasoactive intestinal peptide increases prolactin messenger ribonucleic acid content in GH3 cells. Endocrinology 116:202-206 18. Hanks MC, Alonzi JA, Sharp PJ, Sang HM 1989 Molecular cloning and sequence analysis of putative chicken prolactin cDNA. J Mol Endocrinol 2:21-30 19. Sharp PJ, Macnamee MC, Sterling RJ, Lea RW, Pederson HC 1988 Relationships between prolactin, LH and broody behaviour in bantam hens. J Endocrinol 118:279-286 20. Sterling RJ, Cheng WH, Sharp PJ 1990 Morphological changes in hypothalamic prolactin releasing hormone (VIP) neurones in bantam hens after nest deprivation. Neuroendocrinology 52[Suppl]: 165 (Abstract) 21. White BA, Bancroft T 1982 Cytoplasmic dot hybridisation. J Biol Chem 257:8569-8572 22. Harley CB 1987 Hybridisation of oligo (dT) to RNA on nitrocellulose. Gene Anal Tech 4:17-22 23. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 161:156-159 24. Sambrook J, Fritsch ET, Maniatis T 1989 Molecular Cloning. A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 25. Boon ME, Kok LP 1988 Microwave Cookbook of Pathology: The Art of Microscopic Visualization. Coulomb Press, Leyden, UK 26. Fragioni G, Borgioli G 1979 Polystyrene embedding: a new method for light and electron microscopy. Stain Technol 54:167-172 27. Bloch B, Popovici T, Le Guellec D, Normand E, Chouham S, Guitteny AT, Bohlen P 1986 In situ hybridization histochemistry for the analysis of gene expression in the endocrine and central nervous system tissues: a 3 year experience. J Neurosci Res 16:183200 28. Hsu SM, Rain EL, Tanger H 1981 The use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) proce-
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dures. J Histochem Cytochem 19:577-580 29. Feinberg AP, Vogelstein B 1983 A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 30. Hanks MC, Talbot RT, Sang HM 1990 Expression of biologically active recombinant-derived chicken prolactin in Escherichia coli. J Mol Endocrinol 3:15-21 31. Vaitukaitis J, Robbins JB, Nieschlag E, Ross GT 1971 A method for producing specific antisera with small doses of immunogen. J Clin Endocrinol Metab 33:988-991 32. Bolton AE 1985 Radioiodination Techniques, ed 2. Review 18 Amersham International PLC, Amersham, U.K. p 63 33. Lee LR, Haisenleder Marshall JC, Smith MS 1989 The role of the suckling stimulus in regulating pituitary prolactin mRNA in the rat. Mol Cell Endocrinol 64:245-249 34. Torres AI, Aoki A 1985 Subcellular compartmentation of prolactin in rat lactotrophs. J Endocrinol 105:219-225 35. Stachura ME 1982 Sequestration of an early-release pool of growth hormone and prolactin in GH3 rat pituitary tumor cells. Endocrinology 105:219-2258 36. Gourdji D, Bataille D, Vauclin N, Grouselle D, Rosselin C, TixierVidal A 1979 Vasoactive intestinal peptide (VIP) stimulates prolactin (PRL) release and cAMP production in a rat pituitary cell line (GH3/B6). Additive effects of VIP and TRH on PRL release. FEBS Lett 104:165-168 37. Murdoch G, Rosenfield M, Evans R 1982 Eukaryotic transcriptional regulation and chromatin associated protein phosphorylation by cyclic AMP. Science 218:1315-1317 38. Hall TR, Harvey S, Chadwick A 1985 Mechanisms of release of prolactin from fowl adenohypophyses incubated in vitro: effects of calcium and cyclic adenosine monophosphate. J Endocrinol 105:183-188 39. Laverriere JN, Morin A, Tixier-Vidal A, Truong AT, Gourdji D, Martial JA 1983 Inverse control of prolactin and growth hormone gene expression: effect of thyroliberin on transcription and RNA stabilization. EMBO J 2:1493-1499 40. Mikami SI, Yamada S 1984 Immunohistochemistry of hypothalamic neuropeptides and anterior pituitary cells in the Japanese Quail. J Exp Zool 232:405-419 41. Sterling RJ, Sharp PJ, Klandorf H, Harvey S, Lea RW 1984 Plasma concentrations of luteinizing hormone, follicle stimulating hormone, androgen, growth hormone, prolactin, thyroxine and triiodothyronine during growth and sexual development in the cockerel. Br Poult Sci 25:357-359 42. Nicholls TJ, Goldsmith AR, Dawson A 1988 Photorefractoriness in birds and mammals. Physiol Rev 68:133-176 43. Hall TR, Harvey S, Chadwick A 1986 Control of prolactin secretion in birds: a review. Gen Comp Endocrinol 62:171-184
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