0013-7227/92/1301-0458$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
Vol. 130. No. 1 Society
Printed
Expression in the Rat*
and Secretion
M. E. VRONTAKIS,
I. C. SCHROEDTER,
Department
of
Physiology,
University
of Manitoba,
of Galanin H. COSBY,
Winnipeg,
AND H.
Manitoba,
ABSTRACT. The expression of galanin messenger RNA (mRNA) in the pituitary and conceptus of pregnant rats has been studied at various stages of gestation. Using Northern blot analysis and in situ hybridization we have found that high levels of mRNA coding for galanin were detected in the conceptus during early pregnancy. The level of expression in conceptuses increased until day 11-12 after which the levels decreased rapidly. In contrast, in the pituitary galanin mRNA continued to increase throughout pregnancy, especially in the latter half of pregnancy as serum estradiol levels has been reported to be increased. The size of the galanin transcript was the same in the conceptus and pituitary (0.9 kilobase). Expression of this mRNA was confined to the decidua and was first seen at day 5 of pregnancy at a time when implantation swellings were first observed. Galanin antisense probe hybridized strongly to the
G
ALANIN is a 29-amino acid neuropeptide first isolated from porcine intestinal extracts (1). Galaninlike immunoreactivity is widely distributed in the central nervous system of the rat with the highest concentrations in the median eminence (2). Although the physiological role of galanin remains to be determined, recent findings that intracerebroventricular injections of porcine galanin stimulate PRL and GH release and inhibit dopamine release (3-5) suggest a direct or indirect action on secretory events in the anterior pituitary. We have previously reported the isolation of a galanin complementary DNA (cDNA) clone from an estrogen-induced rat pituitary tumor cDNA library (6) and have shown that while galanin messenger RNA (mRNA) is undetectable in pituitary of normal animals, the message is very highly expressed in this tissue after E2 administration. We and others (7,8) have further shown by immunohistochemistry and in situ hybridization that galanin synthesis is induced greatly by estrogen in the anterior pituitary while no changes are observed in the posterior and intermediate pituitary lobes. Since galanin is an estrogen Received June 28, 1991. Address all correspondence and requests for reprints to: Dr. M. Vrontakis, Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3. *This study was supported by the Medical Research Council of Canada.
during
in U.S.A.
Pregnancy
G. FRIESEN
Canada R3E 0 W3
decidual cells that surround the implantation site. At day 11 of pregnancy the galanin mRNA was found in both the antimesometrial and the mesometrial tissue with the highest concentration in the region lateral to the antimesometrial cells and continued toward the mesometrial cells. Serum galanin levels measured by an RIA using synthetic rat galanin as standard and antisera raised against porcine galanin, exhibited a temporal pattern similar to the pattern of mRNA expression in decidua, with a 7.1-fold increase at day 12 of pregnancy followed by a decline. In summary, decidual cells may differentiate into an endocrine cell during pregnancy. The galanin secreted by these cells, in addition to acting locally in a paracrine/autocrine fashion, may function as a placental hormone with systemic effects on distal target tissues. (Endocrinology 130: 456-464, 1992)
regulated protein we have examined in the present study the expression of galanin during pregnancy, a circumstance when physiological changes lead to a marked increase in estrogen levels. Materials
and Methods
Animals
Timed pregnant Sprague-Dawleyrats were bred in the Central Animal Care Facility, University of Manitoba. All the experimental proceedingswere in accordancewith the guidelines of NIH on animal care and had the approval of the Institutional Committee on the use of Animals in Research. Day 1 was identified as the day when vaginal sperm were detected and the animalswere decapitated between day 3 and 19 of pregnancy. Pituitary and conceptuses(uterus-decidua, trophoblast-fetus) from 4-5 animals per group, on each day examined, wererapidly collectedand frozen on dry ice for RNA extraction. At later stages(day 15-day 19) the fetuses were dissected from the conceptuses.Anesthetized animals were perfused via the left ventricle at a rate of 15 ml/min with 100 ml of rinse [O.l M sodium phosphate (pH 7.4), 0.9% sodium chloride, 0.1 sodium nitrite, 10 p/ml heparin] and 400 ml of fixative [4% paraformaldehyde, 0.1 M phosphate buffer (pH 7.4)] on ice with chilled solutions. Tissueswere quickly removedand placedin the fixative at 4 C for 24-48 h. They were then transferred to 50% ethanol at 4 C. 458
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GALANIN RNA extraction
and Northern
SECRETION
DURING
blot analysis
RNA was prepared from frozen tissue by the guanidium isothiocyanate/cesiumchloride method (9). Total RNA (20 pg) was electrophoresedon a denaturing agaroseformaldehyde gel and the RNA was transferred to a Nitroplus 2000 membrane by blotting in 20 x SSC. The equivalent loading and transfer of RNA wasverified usingb-actin to reprobethe blots. Hybridization was performed at 42 C in 50% formamide using 10” dpm/ml of 32P-labeled rat preprogalanincDNA insert (6) which wasnick translated to a specific activity of 3-5 x 10’ dpm/Fg. Final washingconditions were0.1 x SSC, 0.1% sodiumdodecyl sulfate at 65 C for 30 min. Autoradiography wasdone usingXOmat AR film (EastmanKodak, Rochester,NY) and a lighting Plus enhancingscreenat -70 C for l-.7 days. In situ hybridization Tissues were processedto wax and blocked in Ameraffin (CanLab). The blocks were cut at 5 microns and floated onto aminoalkysilanetreated slides(10) and placed in 50 C oven for 12-18 h. In situ hybridization was performed following the protocol of Angerer et al. (11) with the following modifications. Briefly, the tissuewas permeabilizedwith 0.2 N HCl30 min at 20-24 C and then treated with proteinase K 1 Fg/ml at 37 C for 15 min. The slideswere acetylated and placed in a 50 C prehybridization solution which contained all the reagents of the hybridization mix minus the probe and the 2-mercaptoethanol for 2-4 h. The slideswerethen dehydrated in 100%ethanol and air dried. The probe used was a copy RNA labeled with [36S]uridine5’-triphosphate to a specific activity of 2-4 X lo” dpm/ng. We usedthe PromegaRiboprobe system with Pharmaciapolymerases. The hybridization mix contained 50% formamide, 0.3 M NaCl, 20 mM Tris HCl (pH 8.0), 1 mM EDTA, 1 X Denhardt’s (0.02% of each BSA, Ficoll 200,polyvinylpyrrolidone), 500 pg/ ml of salmontestesDNA, 50 fig/ml polyadenylic acid, 200 mM 2-mercaptoethanol.The probe wasaddedat a concentration of 0.1 ng/rl, usually 20 &ection under a 22-mm siliconized coverslip. The slideswere set up hot -60 C on a slide warmer and then submergedinto 50 C mineral oil. The slideswere incubated for lo-18 h. The stringent final washeswere 67 C with 50% formamide, 0.3 M NaCl20 mM Tris, 1 mM EDTA, 100mM P-mercaptoethanol for 30 min and 0.1% SSC at 67 C for 15 min. The tissues weretreated with RNAses “T 1” and “A”. The slides were dehydrated with 50, 70, and 90% ethanol containing 0.3 M ammonium acetate then dipped in Kodak NTBP emulsiondiluted 1:l with water, dried, and exposedat 4 C desiccated. RIA
PREGNANCY
IN THE RAT
4%
sera (Peninsula Laboratories) was used in a final dilution 1:50,000.Briefly, 100 ~1 standards or sampleswere incubated with 10 ~1 antisera and 100~1 tracer in phosphatebuffer with BSA (1%) for 48 h at 4 C. The boundpeptide wasseparatedby adding NRS and goat antirabbit y-globulins. Galanin levels were measuredin duplicate. There was parallel displacement with serum or pituitary extracts, using this assay. Screening with the antisera of fractions from sephedexG-50 chromatography of pituitary extract, revealedthat 85% of all immunoreactive materials eluted in the sameposition as synthetic rat galanin (data not shown). The recovery of the addedsynthetic galanin into serum was 90-95%. The sensitivity of the assay was 20 pg/tube and the total binding was usually 30-50% of the initial radioactivity added. The inter- and intraassay coef. ficient of variation was 11 and 5%, respectively. Data are expressedas the mean-CSE. Statistical analysiswasperformed by the two way analysisof variance.
Results Expression of galanin mRNA during pregnancy (time course of induction) To determine the levels of galanin mRNA in the pituitary and the conceptus at different stages of pregnancy, total RNA isolated from the conceptus and pituitaries of 4-5 Sprague-Dawley rats/group at defined stages of pregnancy (day 3-day 19) was fractionated on formaldehyde-agarose gels, transferred to nitrocellulose, and hybridized with the rat preprogalanin cDNA clone (6). Three independent experiments were performed. As seen in the Northern blot of Fig. 1, galanin mRNA was detected in the conceptus at day 5 of pregnancy. There was a decline on day 9 and then the level increased to a peak at day 11 then decreased gradually until day 16 and became undetectable at day 18. The size of the transcript is 0.9 kilobase, the same as in the pituitary. In the
28S18S-
b-actin
Serumblood sampleswere collected at the time of decapitation from control and timed pregnant rats and E2 treated as well. The serumwas collected on ice, centrifuged at 4 C (2000 rpm), and stored at -20 C till the assay. Synthetic rat galanin was iodinated using iodogen (Pierce, Rockford, IL) and was purified in a Sephadex G25-superfine column. Synthetic rat galanin (Peninsula Laboratories, Belmont, CA) wasusedas standards.Porcine galanin rabbit. anti-
3
5
7 Days
9
11 of
12
15
18
19
pregnancy
FIG. 1. Time course of galanin mRNA expression in rat conceptus. Total RNA (20 pg) isolated from conceptuses collected on days 3-19 of pregnancy were used in this Northern blot analysis with rat galanin cDNA probe. The position of the 18s and 28s ribosomal RNAs are indicated and the variability in RNA loading and transfer is shown in the her part of the figure using the b-actin probe to reprobe the blots.
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GALANIN
460
SECRETION
DURING
pituitary of the same animals (Fig. 2) galanin mRNA is detectable at day 3 of pregnancy, there is again a decline at day 9, and this is definitely increased by day 12 and remains elevated in the later half of pregnancy where estradiol levels have been reported to be increased (12). On the contrary, in pituitary RNA from nonpregnant, ovariectomized rats the galanin transcript is not detectable (data not shown). Densitometric analysis of the galanin mRNA transcript in the two tissues revealed that in the conceptus there is an M-fold increase at day 11 of pregnancy, while in the pituitary the highest expression was on day 15-18 (2.5fold increase).
b-actin
3
5
7 Days
9 of
11
12
15
18
19
pregnancy
FIG. 2. Expression
of galanin mRNA in rat pituitary during pregnancy. Total RNA (20 ag) isolated from the pituitaries of the same animals of Fig. 1 collected on days 3-19 of pregnancy were used in this Northern blot analysis. The explanation is as in Fig. 1.
PREGNANCY
IN THE
In situ hybridization
RAT
Endo. 1992 Vol130. No 1
of galanin in the conceptus
The specific cellular location of the synthesis of galanin mRNA was determined by in situ hybridization at days 7, 11, and 17. Figure 3A shows a darkfield photomicrograph of day 7 rat conceptus hybridized to the antisense rat galanin probe. There is a strong hybridization of the antimesometrial cells that surround the implantation site in the lumen which extends toward the mesometrial side as well. The brightfield of the same section is shown in Fig. 3C while a higher magnification of the implantation zone is shown in Fig. 3D. The control hybridization to the mRNA sense strand is shown in Fig. 3B. The dark and brightfield of the hybridization to the antisense rat galanin probe at day 11 is shown in Fig. 4, A and B. As is shown in Fig. 4, A (darkfield) and B (brightfield) at day 11 where both antimesometrial and mesometrial decidua are fully developed, galanin mRNA is found in both zones of decidua with the highest density of silver grains in the region lateral to the antimesometrial cells and extending toward the mesometrial cell. Higher magnification of this region is shown in Fig. 4, C (darkfield) and D (brightfield) which is the area where the placenta disc meets the uterine epithelium. As is shown in Fig. 4D not all decidual cells hybridize to the rat galanin antisense probe, although by light microscopy the morphological appearance of these cells, are indistinguishable from cells that hybridize. At day 17 decidual tissue is only a thin layer between the placenta and the undifferentiated myometrium (Fig. 5A). Although the rat galanin mRNA transcript is not
FIG. 3. Localization
of galanin mRNA in day 7 rat conceptus. ad, Antimesometrial decidua; L, lumen; mt, metrial gland. A, Darkfield photomicrograph of the conceptus hybridized to the antisense probe; B, darkfield of similar section hybridized to the control sense probe; C, brightfield photomicrograph of the same section as in (A) showing the structures that are present at the time. Hybridization is seen in the antimesometrial decidua surrounding the implantation site. D, Higher magnification of the area outlined in (A). Hybridization of the antisense probe is seen in the antimesometrial cells of decidua. Bar, low power = 500 pm; high power = 20
urn.
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GALANIN
SECRETION
DURING
PREGNANCY
IN THE
RAT
461
FIG. 4. Localization of rat galanin mRNA in day 11 conceptus. dc, Decidua capsularis; my, myometrium; pl, placenta, db, decidua basalis; mt, metrial gland, G, giant cells. A, Darkfield and B, brightfield photomicrographs of a cross-section hybridized to the rat galanin antisense probe. C, Darkfield of a higher magnification of the area outlined in (B). There is a strong hybridization of the antisense probe in the region lateral to antimesometrial cells and extending toward the mesometrial cell. D, Brightfield of a higher magnification of the area outlined in (C). Many but not all decidual cells hybridize to the rat galanin antisense probe mainly in the area where the placenta disc meet the uterine epithelium. Barlow power, 500 pm; medium power, 100 pm. High power, 50 pm.
detectable by Northern blot on this day, due to the very small contribution of decidual tissue to the whole conceptus, hybridization of the antisense rat galanin probe to the thin layer of decidual cells persisted. Figure 5A is a brightfield photomicrograph of hematoxylin and eosin staining of the whole conceptus at day 17. The boxed
area has been photographed at higher power in brightfield (D) and darkfield (Cl. It is obvious that a thin layer of decidual cells hybridizes to the rat galanin antisense probe and, at higher magnification, quite dense silver grains cover the layer of mesometrial decidual cells (Fig. 5B).
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462
GALANIN
SECRETION
DURING
PREGNANCY
IN THE
RAT
Endo. 1992 Vol 130.No 1
FIG. 6. Levels of galanin-like immunoreactivity in rat serum at different stages of pregnancy. For comparison the serum galanin concentrations in rat bearing 10 Kg implant of stilbestrol for 7 weeks are included on lane II; n = 5 for each group. Mean + SD are shown.
Discussion
FIG. 5. Localization of rat galanin mRNA in day 17 conceptus (after the removal of the embryo). mt, Metrial gland; pl, placenta, my, myometrium; db, decidua basalis; G, giant cells. A, Brightfield photomicrograph of hematoxylin and eosin staining. Decidua basalis is a thin layer between placental disc and the myometrium. C, Darkfield and D, brightfield of the same section hybridized to the antisense rat galanin clone. A thin layer of decidual cells with the outer part of decidual basalis hybridizes to the antisense probe. The arrows indicate the identical areas in bright and darkfield. B, Brightfield of higher magnification of the indicated area in (D). Bar-low power, 500 pm; medium power, 100 pm; higher power, 20 pm.
Detection of galanin-LI RIA
in the maternal
circulation
by
In nonpregnant rats serum galanin levels for galanin were 300 pg/ml f 90. At day 7 of pregnancy, serum levels start to rise with a peak at day 12 (2300 pg/ml) which represents almost 7.1-fold increase from the nonpregnant levels and then declines gradually with levels 2.5fold above nonpregnant levels at day 18 of pregnancy. Figure shows the serum levels at various stages of pregnancy. The second lane represents serum galanin levels in rats which had carried a lo-mg stilbestrol implant for 7 weeks, a condition known to greatly increase galanin mRNA in the pituitary (6, 8). The level of this group is almost similar to the levels of day 12 pregnant serum.
Northern blot analysis and in situ hybridization studies demonstrate that decidual tissue expresses galanin mRNA in the period immediately following implantation of the embryo. All available data suggest that decidual galanin, described in this paper, is identical to that expressed in the pituitary (6,8) including the observation that there is a similarly sized mRNA in both tissues, the fact that it appears to be a single galanin gene (our unpublished observation), and the preliminary data of nucleotide sequence analysis of a galanin cDNA clone isolated from a day 11 decidua library (our unpublished observation). Galanin mRNA levels peak at day 11 and then decline gradually to an undetectable level, at least by Northern at day 18, presumably due to the regression of decidual tissue. The reason for the reduction of the transcript at day 9 of pregnancy in both pituitary and conceptus is unclear. It is customary to consider that two basic stromal cell populations of fibroblast origin (13) constitute the decidual reaction, forming antimesometrial and mesometrial decidual cells (14). However, morphological variations of these cells do occur depending on their position in the uterus and the time during pregnancy when the cells are observed (15). The antimesometriai decidual cells appear first and are found beneath the epithelium on the lumen in the immediate vicinity of the implanting blastocyst. As shown by in situ hybridization in Fig. 3A at day 7 galanin mRNA expression occurs in this area, indicating localization of galanin mRNA in the antimesometrial decidua. These decidual cells reach a maximum cell number on day 10 and then enter a period of regression forming the decidua capsularis until there is virtually no trace of them by day 16 (16). Two days after the antimesometrial cells are formed, decidualization proceeds laterally to the mesometrial region, the site of trophoblastic invasion. Decidual cells in this region form the
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GALANIN
SECRETION
DURING
mesometrial decidua which becomes fully differentiated around day 11-12, when antimesometriai cells show signs of regression, and become progressively reduced in size forming the decidua basalis until it is a thin layer shed with the rest of the placenta at birth (16). The pattern of expression of galanin mRNA throughout pregnancy is correlated very well with the development of antimesometrial and mesometrial decidua with a decline around day 16-17 when only a thin layer of mesometrial cells persists as shown by hybridization with the rat galanin antisense probe (Fig. 5). At day 11, when both antimesometrial and mesometrial decidua are fully developed, galanin mRNA is found in both zones of decidua with the highest density of silver grains in the region lateral to the antimesometrial cells extending toward the mesometrial cells (Fig. 4C) in an area where the placenta disc meets the uterine epithelium. The exact role of galanin in this area is still unknown. Although the exact endocrine role of decidua has not been established, it would be expected that the process of differentiation of stromai fibroblast cells to decidual cells would be associated with an alt,eration in protein composition that would be consistent with the enormous growth occurring in this region and with evidence that new proteins appear in the decidualizing uterus. Umapathysivan and Jones (17) found a decidual specific protein in the rat at day 8 of pseudopregnancy with an approximate mol wt of 48,000, and Bell (18) has characterized a decidualization associated protein that appears to be specific for the mesometrial region t,hat may play a role in controlling tissue damage. Recently it has been suggested that it is identical with tu2-macroglobulin protein (19) a conclusion supported by the demonstration that its mRNA is also expressed in this region (20). During early antimesometrial decidualization, increased synthesis of a protein analogous to uterine estradiol induced protein has been reported (21) which has been suggested to be associated with cell division. The exact role of decidual galanin is not known but there is some evidence that could support the hypothesis that decidual galanin by a local aut.ocrine or paracrine effect could influence placent,al PRL or PRL-like hormone production. It is well established that intracerebroventricular injection of porcine galanin stimulates GH and PRL release and inhibits dopamine release (3-5) indicating a direct or indirect action on pituitary PRL and GH secretion. In a similar manner, decidual galanin could play a role in regulating decidual PRL-like hormone [e.g. decidual luteotropin (22) or even placental lactogens rPL-I, rPL-II (23, 24), or placental PRL-like peptides (rPL-A, rPL-B (2411. We have some preliminary data supporting the latest hypothesis. Another possibility, of course, is that decidual galanin could act in an autocrine manner as a growth factor for the growth of
PREGNANCY
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463
decidual tissue. The detection of galanin in the peripheral serum by RIA demonstrates that galanin is also secreted into the periphery circulation, confirming previous observations that galanin is secreted in the circulation (8, 25) Lopez et al. (25) concluded that 30% of serum galanin originated from the pituitary as serum galanin levels in hypox rats decreased by 70% compared to control rats. Since the levels in serum follow the pattern of induction of the mRNA levels in the decidua, it is possible that most of the galanin in serum originates from the decidua, indicating that besides a local autocrine or paracrine effect, galanin during pregnancy has an effect on distal organs. Further studies are required to establish the role of galanin during pregnancy. Acknowledgment The secretarial appreciated.
assistance
of Ms.
June
McDougald
is very
murh
References 1. Tatemoto K, Rokaeus A, Jornvall H, McDonald TJ, Mutt V 1983 Galanin--a novel biologically active peptide from porcine intestine. FEBS Lett 164:124-128 2. Skofitsch G, Jacobovitz DM 1986 Quantitative distribution of galanin-like immunoreactivity in the rat central nervous system. Peptides 7:609--6 I3 3< Ottlecz A, Snyder GD, McCann SM 1988 Regulatory role of galanin in control of hypothalamic-anterior pituitary function. Proc Nat1 Acad Sci USA 85:9861-9865 4. Koshiyama H, Shimatsu A, Kato Y, Assadian H, Hatton N, Ishikawa Y, Tanoh T, Yanaihara N, Imura H 1990 Galanin induced prolactin release in rats: pharmacological evidence for the involvement of alpha-andrenergic and opiodergic mechanisms. Brain Res 507:321--324 a. Nordstrom 0, Melander T, Hokfelt T, Bartfai T, Goldstein M 1987 Evidence for an inhibitory effect of the peptide galanin on dopamine release from the rat median eminence. Neurosci 1,et.t 73:2126 6. Vrontakis ME, Peden LM, Duckworth ML, Friesen HG 1987 Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA. J Biol Chem 262:16755-16758 7. Vrontakis ME, Yamamoto T, Schroedter IC, Nagy Jl, Friesen HG 1989 Estrogen induction of galanin synthesis in the rat anterior pituitary gland demonstrated by in situ hybridization and immunohistochemistry. Neurosci Lett. 100:59-64 8. Kaplan LM, Gabriel SM, Koenig JI, Sunday ME, Spindel ER, Martin JB, Chin WW 1988 Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary. Proc Natl Acad Sci USA 85:7408-~7412 9. Chirgwin ?JM, Przybyla aE, McDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonucleases. Biochemistry 185294-5301 10. Henderson C 1989 Aminoalkylsilane: an inexpensive, simple preparation for slide adhesion. J Histochem 12:123-124 11. Angerer LM, Staler MH, Angerer RC 1987, In situ hybridization with RNA probes: an annotated recipe. In: Valentino KL, Eberwine .JH, Barchas .JD (edsl In situ Hybridization: Applications to Neurobiology. Oxford Iiniversity Press, New York, pp 43-70
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12. Gibori G, Khan I, Warshaw ML, McLean MP, Puryear TK, Nelson S, Durkee TJ, Azhar S, Steinschneider A, Rao MC 1988 Placentalderived regulators and the complex control of luteal cell function. Recent Prog Horm Res 44:377-425 13. O’Shea JD, Kleinfeld RG, Morrow HA 1983 Ultrastructure of decidualization in the pseudopregnant rat. Am J Anat 166:271-298 14. Bell SC 1985, Comparative aspects of decidualization in rodents and humans: cell types, secreted products and associated function. In: Implantation of the Human Embryo. Academic Press, London, pp 71-122 15. Welsh AO, Enders AC 1985 Light and electron microscopic examination of the mature decidual cells of the rat with emphasis on the antimesometrial decidua and its degeneration. Am J Anat 172:1-29 16. Bell SC 1983 Decidualization: regional differentiation and associated function. In: Finn CA (ed) Oxford Reviews of Reproductive Biology. Oxford University Press, Oxford, pp 220-271 17. Umapathysivan K, Jones WR 1978 An investigation of decidual specific proteins in the rat. Int J Fertil 23:138-142 18. Bell SC 1979 Synthesis of decidualization associated protein in tissues of the rat uterus and placenta during pregnancy. J Reprod Fertil49:177-181
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19. Bell SC 1979 Immunochemical identity of decidualization associated protein and a2-acute phase macroglobulin in the pregnant rat. J Reprod Immunol 1:193-206 20. Fletcher S, Thomas T, Schreiber G, Heinrich PC, Yeoh GC 1988 The development of rat alpha 2-macroglobulin. Studies in uiuo and in cultured fetal rat hepatocytes. Eur J Biochem 171:703-709 21. Bell SC, Hamer J, Heald PJ 1980 Induced protein and deciduoma formation in rat uterus. Biol Reprod 23:935-940 22. Jayatilak PG, Puryear TK, Hem Z, Fazleabas A, Gibori G 1989 Protein secretion by mesometrial and antiomesometrial rat decidual tissue: evidence for a differential gene expression. Endocrinology 125:659-666 23. Robertson MC, Croze F, Schroedter IC, Friesen HG 1990 Molecular cloning and expression of rat placental lactogen-I cDNA. Endocrinology 127:702-710 24. Duckworth ML, Schroedter IC, Friesen HG 1990 Cellular localization of rat placental lactogen II and rat prolactin-like proteins A and B by in situ hybridization. Placenta 11:143-155 25. Lopez FJ, Meade EH, Negro-Vilar A 1990 Development and characterization of a specific and sensitive radioimmunoassay for rat galanin. Measurement in brain tissue, hypophyseal, portal and peripheral serum. Brain Res Bull 24:395-399
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Vol. 130. No. 1 Society
Printed
Expression in the Rat*
and Secretion
M. E. VRONTAKIS,
I. C. SCHROEDTER,
Department
of
Physiology,
University
of Manitoba,
of Galanin H. COSBY,
Winnipeg,
AND H.
Manitoba,
ABSTRACT. The expression of galanin messenger RNA (mRNA) in the pituitary and conceptus of pregnant rats has been studied at various stages of gestation. Using Northern blot analysis and in situ hybridization we have found that high levels of mRNA coding for galanin were detected in the conceptus during early pregnancy. The level of expression in conceptuses increased until day 11-12 after which the levels decreased rapidly. In contrast, in the pituitary galanin mRNA continued to increase throughout pregnancy, especially in the latter half of pregnancy as serum estradiol levels has been reported to be increased. The size of the galanin transcript was the same in the conceptus and pituitary (0.9 kilobase). Expression of this mRNA was confined to the decidua and was first seen at day 5 of pregnancy at a time when implantation swellings were first observed. Galanin antisense probe hybridized strongly to the
G
ALANIN is a 29-amino acid neuropeptide first isolated from porcine intestinal extracts (1). Galaninlike immunoreactivity is widely distributed in the central nervous system of the rat with the highest concentrations in the median eminence (2). Although the physiological role of galanin remains to be determined, recent findings that intracerebroventricular injections of porcine galanin stimulate PRL and GH release and inhibit dopamine release (3-5) suggest a direct or indirect action on secretory events in the anterior pituitary. We have previously reported the isolation of a galanin complementary DNA (cDNA) clone from an estrogen-induced rat pituitary tumor cDNA library (6) and have shown that while galanin messenger RNA (mRNA) is undetectable in pituitary of normal animals, the message is very highly expressed in this tissue after E2 administration. We and others (7,8) have further shown by immunohistochemistry and in situ hybridization that galanin synthesis is induced greatly by estrogen in the anterior pituitary while no changes are observed in the posterior and intermediate pituitary lobes. Since galanin is an estrogen Received June 28, 1991. Address all correspondence and requests for reprints to: Dr. M. Vrontakis, Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3. *This study was supported by the Medical Research Council of Canada.
during
in U.S.A.
Pregnancy
G. FRIESEN
Canada R3E 0 W3
decidual cells that surround the implantation site. At day 11 of pregnancy the galanin mRNA was found in both the antimesometrial and the mesometrial tissue with the highest concentration in the region lateral to the antimesometrial cells and continued toward the mesometrial cells. Serum galanin levels measured by an RIA using synthetic rat galanin as standard and antisera raised against porcine galanin, exhibited a temporal pattern similar to the pattern of mRNA expression in decidua, with a 7.1-fold increase at day 12 of pregnancy followed by a decline. In summary, decidual cells may differentiate into an endocrine cell during pregnancy. The galanin secreted by these cells, in addition to acting locally in a paracrine/autocrine fashion, may function as a placental hormone with systemic effects on distal target tissues. (Endocrinology 130: 456-464, 1992)
regulated protein we have examined in the present study the expression of galanin during pregnancy, a circumstance when physiological changes lead to a marked increase in estrogen levels. Materials
and Methods
Animals
Timed pregnant Sprague-Dawleyrats were bred in the Central Animal Care Facility, University of Manitoba. All the experimental proceedingswere in accordancewith the guidelines of NIH on animal care and had the approval of the Institutional Committee on the use of Animals in Research. Day 1 was identified as the day when vaginal sperm were detected and the animalswere decapitated between day 3 and 19 of pregnancy. Pituitary and conceptuses(uterus-decidua, trophoblast-fetus) from 4-5 animals per group, on each day examined, wererapidly collectedand frozen on dry ice for RNA extraction. At later stages(day 15-day 19) the fetuses were dissected from the conceptuses.Anesthetized animals were perfused via the left ventricle at a rate of 15 ml/min with 100 ml of rinse [O.l M sodium phosphate (pH 7.4), 0.9% sodium chloride, 0.1 sodium nitrite, 10 p/ml heparin] and 400 ml of fixative [4% paraformaldehyde, 0.1 M phosphate buffer (pH 7.4)] on ice with chilled solutions. Tissueswere quickly removedand placedin the fixative at 4 C for 24-48 h. They were then transferred to 50% ethanol at 4 C. 458
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GALANIN RNA extraction
and Northern
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blot analysis
RNA was prepared from frozen tissue by the guanidium isothiocyanate/cesiumchloride method (9). Total RNA (20 pg) was electrophoresedon a denaturing agaroseformaldehyde gel and the RNA was transferred to a Nitroplus 2000 membrane by blotting in 20 x SSC. The equivalent loading and transfer of RNA wasverified usingb-actin to reprobethe blots. Hybridization was performed at 42 C in 50% formamide using 10” dpm/ml of 32P-labeled rat preprogalanincDNA insert (6) which wasnick translated to a specific activity of 3-5 x 10’ dpm/Fg. Final washingconditions were0.1 x SSC, 0.1% sodiumdodecyl sulfate at 65 C for 30 min. Autoradiography wasdone usingXOmat AR film (EastmanKodak, Rochester,NY) and a lighting Plus enhancingscreenat -70 C for l-.7 days. In situ hybridization Tissues were processedto wax and blocked in Ameraffin (CanLab). The blocks were cut at 5 microns and floated onto aminoalkysilanetreated slides(10) and placed in 50 C oven for 12-18 h. In situ hybridization was performed following the protocol of Angerer et al. (11) with the following modifications. Briefly, the tissuewas permeabilizedwith 0.2 N HCl30 min at 20-24 C and then treated with proteinase K 1 Fg/ml at 37 C for 15 min. The slideswere acetylated and placed in a 50 C prehybridization solution which contained all the reagents of the hybridization mix minus the probe and the 2-mercaptoethanol for 2-4 h. The slideswerethen dehydrated in 100%ethanol and air dried. The probe used was a copy RNA labeled with [36S]uridine5’-triphosphate to a specific activity of 2-4 X lo” dpm/ng. We usedthe PromegaRiboprobe system with Pharmaciapolymerases. The hybridization mix contained 50% formamide, 0.3 M NaCl, 20 mM Tris HCl (pH 8.0), 1 mM EDTA, 1 X Denhardt’s (0.02% of each BSA, Ficoll 200,polyvinylpyrrolidone), 500 pg/ ml of salmontestesDNA, 50 fig/ml polyadenylic acid, 200 mM 2-mercaptoethanol.The probe wasaddedat a concentration of 0.1 ng/rl, usually 20 &ection under a 22-mm siliconized coverslip. The slideswere set up hot -60 C on a slide warmer and then submergedinto 50 C mineral oil. The slideswere incubated for lo-18 h. The stringent final washeswere 67 C with 50% formamide, 0.3 M NaCl20 mM Tris, 1 mM EDTA, 100mM P-mercaptoethanol for 30 min and 0.1% SSC at 67 C for 15 min. The tissues weretreated with RNAses “T 1” and “A”. The slides were dehydrated with 50, 70, and 90% ethanol containing 0.3 M ammonium acetate then dipped in Kodak NTBP emulsiondiluted 1:l with water, dried, and exposedat 4 C desiccated. RIA
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4%
sera (Peninsula Laboratories) was used in a final dilution 1:50,000.Briefly, 100 ~1 standards or sampleswere incubated with 10 ~1 antisera and 100~1 tracer in phosphatebuffer with BSA (1%) for 48 h at 4 C. The boundpeptide wasseparatedby adding NRS and goat antirabbit y-globulins. Galanin levels were measuredin duplicate. There was parallel displacement with serum or pituitary extracts, using this assay. Screening with the antisera of fractions from sephedexG-50 chromatography of pituitary extract, revealedthat 85% of all immunoreactive materials eluted in the sameposition as synthetic rat galanin (data not shown). The recovery of the addedsynthetic galanin into serum was 90-95%. The sensitivity of the assay was 20 pg/tube and the total binding was usually 30-50% of the initial radioactivity added. The inter- and intraassay coef. ficient of variation was 11 and 5%, respectively. Data are expressedas the mean-CSE. Statistical analysiswasperformed by the two way analysisof variance.
Results Expression of galanin mRNA during pregnancy (time course of induction) To determine the levels of galanin mRNA in the pituitary and the conceptus at different stages of pregnancy, total RNA isolated from the conceptus and pituitaries of 4-5 Sprague-Dawley rats/group at defined stages of pregnancy (day 3-day 19) was fractionated on formaldehyde-agarose gels, transferred to nitrocellulose, and hybridized with the rat preprogalanin cDNA clone (6). Three independent experiments were performed. As seen in the Northern blot of Fig. 1, galanin mRNA was detected in the conceptus at day 5 of pregnancy. There was a decline on day 9 and then the level increased to a peak at day 11 then decreased gradually until day 16 and became undetectable at day 18. The size of the transcript is 0.9 kilobase, the same as in the pituitary. In the
28S18S-
b-actin
Serumblood sampleswere collected at the time of decapitation from control and timed pregnant rats and E2 treated as well. The serumwas collected on ice, centrifuged at 4 C (2000 rpm), and stored at -20 C till the assay. Synthetic rat galanin was iodinated using iodogen (Pierce, Rockford, IL) and was purified in a Sephadex G25-superfine column. Synthetic rat galanin (Peninsula Laboratories, Belmont, CA) wasusedas standards.Porcine galanin rabbit. anti-
3
5
7 Days
9
11 of
12
15
18
19
pregnancy
FIG. 1. Time course of galanin mRNA expression in rat conceptus. Total RNA (20 pg) isolated from conceptuses collected on days 3-19 of pregnancy were used in this Northern blot analysis with rat galanin cDNA probe. The position of the 18s and 28s ribosomal RNAs are indicated and the variability in RNA loading and transfer is shown in the her part of the figure using the b-actin probe to reprobe the blots.
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460
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pituitary of the same animals (Fig. 2) galanin mRNA is detectable at day 3 of pregnancy, there is again a decline at day 9, and this is definitely increased by day 12 and remains elevated in the later half of pregnancy where estradiol levels have been reported to be increased (12). On the contrary, in pituitary RNA from nonpregnant, ovariectomized rats the galanin transcript is not detectable (data not shown). Densitometric analysis of the galanin mRNA transcript in the two tissues revealed that in the conceptus there is an M-fold increase at day 11 of pregnancy, while in the pituitary the highest expression was on day 15-18 (2.5fold increase).
b-actin
3
5
7 Days
9 of
11
12
15
18
19
pregnancy
FIG. 2. Expression
of galanin mRNA in rat pituitary during pregnancy. Total RNA (20 ag) isolated from the pituitaries of the same animals of Fig. 1 collected on days 3-19 of pregnancy were used in this Northern blot analysis. The explanation is as in Fig. 1.
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In situ hybridization
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Endo. 1992 Vol130. No 1
of galanin in the conceptus
The specific cellular location of the synthesis of galanin mRNA was determined by in situ hybridization at days 7, 11, and 17. Figure 3A shows a darkfield photomicrograph of day 7 rat conceptus hybridized to the antisense rat galanin probe. There is a strong hybridization of the antimesometrial cells that surround the implantation site in the lumen which extends toward the mesometrial side as well. The brightfield of the same section is shown in Fig. 3C while a higher magnification of the implantation zone is shown in Fig. 3D. The control hybridization to the mRNA sense strand is shown in Fig. 3B. The dark and brightfield of the hybridization to the antisense rat galanin probe at day 11 is shown in Fig. 4, A and B. As is shown in Fig. 4, A (darkfield) and B (brightfield) at day 11 where both antimesometrial and mesometrial decidua are fully developed, galanin mRNA is found in both zones of decidua with the highest density of silver grains in the region lateral to the antimesometrial cells and extending toward the mesometrial cell. Higher magnification of this region is shown in Fig. 4, C (darkfield) and D (brightfield) which is the area where the placenta disc meets the uterine epithelium. As is shown in Fig. 4D not all decidual cells hybridize to the rat galanin antisense probe, although by light microscopy the morphological appearance of these cells, are indistinguishable from cells that hybridize. At day 17 decidual tissue is only a thin layer between the placenta and the undifferentiated myometrium (Fig. 5A). Although the rat galanin mRNA transcript is not
FIG. 3. Localization
of galanin mRNA in day 7 rat conceptus. ad, Antimesometrial decidua; L, lumen; mt, metrial gland. A, Darkfield photomicrograph of the conceptus hybridized to the antisense probe; B, darkfield of similar section hybridized to the control sense probe; C, brightfield photomicrograph of the same section as in (A) showing the structures that are present at the time. Hybridization is seen in the antimesometrial decidua surrounding the implantation site. D, Higher magnification of the area outlined in (A). Hybridization of the antisense probe is seen in the antimesometrial cells of decidua. Bar, low power = 500 pm; high power = 20
urn.
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FIG. 4. Localization of rat galanin mRNA in day 11 conceptus. dc, Decidua capsularis; my, myometrium; pl, placenta, db, decidua basalis; mt, metrial gland, G, giant cells. A, Darkfield and B, brightfield photomicrographs of a cross-section hybridized to the rat galanin antisense probe. C, Darkfield of a higher magnification of the area outlined in (B). There is a strong hybridization of the antisense probe in the region lateral to antimesometrial cells and extending toward the mesometrial cell. D, Brightfield of a higher magnification of the area outlined in (C). Many but not all decidual cells hybridize to the rat galanin antisense probe mainly in the area where the placenta disc meet the uterine epithelium. Barlow power, 500 pm; medium power, 100 pm. High power, 50 pm.
detectable by Northern blot on this day, due to the very small contribution of decidual tissue to the whole conceptus, hybridization of the antisense rat galanin probe to the thin layer of decidual cells persisted. Figure 5A is a brightfield photomicrograph of hematoxylin and eosin staining of the whole conceptus at day 17. The boxed
area has been photographed at higher power in brightfield (D) and darkfield (Cl. It is obvious that a thin layer of decidual cells hybridizes to the rat galanin antisense probe and, at higher magnification, quite dense silver grains cover the layer of mesometrial decidual cells (Fig. 5B).
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FIG. 6. Levels of galanin-like immunoreactivity in rat serum at different stages of pregnancy. For comparison the serum galanin concentrations in rat bearing 10 Kg implant of stilbestrol for 7 weeks are included on lane II; n = 5 for each group. Mean + SD are shown.
Discussion
FIG. 5. Localization of rat galanin mRNA in day 17 conceptus (after the removal of the embryo). mt, Metrial gland; pl, placenta, my, myometrium; db, decidua basalis; G, giant cells. A, Brightfield photomicrograph of hematoxylin and eosin staining. Decidua basalis is a thin layer between placental disc and the myometrium. C, Darkfield and D, brightfield of the same section hybridized to the antisense rat galanin clone. A thin layer of decidual cells with the outer part of decidual basalis hybridizes to the antisense probe. The arrows indicate the identical areas in bright and darkfield. B, Brightfield of higher magnification of the indicated area in (D). Bar-low power, 500 pm; medium power, 100 pm; higher power, 20 pm.
Detection of galanin-LI RIA
in the maternal
circulation
by
In nonpregnant rats serum galanin levels for galanin were 300 pg/ml f 90. At day 7 of pregnancy, serum levels start to rise with a peak at day 12 (2300 pg/ml) which represents almost 7.1-fold increase from the nonpregnant levels and then declines gradually with levels 2.5fold above nonpregnant levels at day 18 of pregnancy. Figure shows the serum levels at various stages of pregnancy. The second lane represents serum galanin levels in rats which had carried a lo-mg stilbestrol implant for 7 weeks, a condition known to greatly increase galanin mRNA in the pituitary (6, 8). The level of this group is almost similar to the levels of day 12 pregnant serum.
Northern blot analysis and in situ hybridization studies demonstrate that decidual tissue expresses galanin mRNA in the period immediately following implantation of the embryo. All available data suggest that decidual galanin, described in this paper, is identical to that expressed in the pituitary (6,8) including the observation that there is a similarly sized mRNA in both tissues, the fact that it appears to be a single galanin gene (our unpublished observation), and the preliminary data of nucleotide sequence analysis of a galanin cDNA clone isolated from a day 11 decidua library (our unpublished observation). Galanin mRNA levels peak at day 11 and then decline gradually to an undetectable level, at least by Northern at day 18, presumably due to the regression of decidual tissue. The reason for the reduction of the transcript at day 9 of pregnancy in both pituitary and conceptus is unclear. It is customary to consider that two basic stromal cell populations of fibroblast origin (13) constitute the decidual reaction, forming antimesometrial and mesometrial decidual cells (14). However, morphological variations of these cells do occur depending on their position in the uterus and the time during pregnancy when the cells are observed (15). The antimesometriai decidual cells appear first and are found beneath the epithelium on the lumen in the immediate vicinity of the implanting blastocyst. As shown by in situ hybridization in Fig. 3A at day 7 galanin mRNA expression occurs in this area, indicating localization of galanin mRNA in the antimesometrial decidua. These decidual cells reach a maximum cell number on day 10 and then enter a period of regression forming the decidua capsularis until there is virtually no trace of them by day 16 (16). Two days after the antimesometrial cells are formed, decidualization proceeds laterally to the mesometrial region, the site of trophoblastic invasion. Decidual cells in this region form the
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GALANIN
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mesometrial decidua which becomes fully differentiated around day 11-12, when antimesometriai cells show signs of regression, and become progressively reduced in size forming the decidua basalis until it is a thin layer shed with the rest of the placenta at birth (16). The pattern of expression of galanin mRNA throughout pregnancy is correlated very well with the development of antimesometrial and mesometrial decidua with a decline around day 16-17 when only a thin layer of mesometrial cells persists as shown by hybridization with the rat galanin antisense probe (Fig. 5). At day 11, when both antimesometrial and mesometrial decidua are fully developed, galanin mRNA is found in both zones of decidua with the highest density of silver grains in the region lateral to the antimesometrial cells extending toward the mesometrial cells (Fig. 4C) in an area where the placenta disc meets the uterine epithelium. The exact role of galanin in this area is still unknown. Although the exact endocrine role of decidua has not been established, it would be expected that the process of differentiation of stromai fibroblast cells to decidual cells would be associated with an alt,eration in protein composition that would be consistent with the enormous growth occurring in this region and with evidence that new proteins appear in the decidualizing uterus. Umapathysivan and Jones (17) found a decidual specific protein in the rat at day 8 of pseudopregnancy with an approximate mol wt of 48,000, and Bell (18) has characterized a decidualization associated protein that appears to be specific for the mesometrial region t,hat may play a role in controlling tissue damage. Recently it has been suggested that it is identical with tu2-macroglobulin protein (19) a conclusion supported by the demonstration that its mRNA is also expressed in this region (20). During early antimesometrial decidualization, increased synthesis of a protein analogous to uterine estradiol induced protein has been reported (21) which has been suggested to be associated with cell division. The exact role of decidual galanin is not known but there is some evidence that could support the hypothesis that decidual galanin by a local aut.ocrine or paracrine effect could influence placent,al PRL or PRL-like hormone production. It is well established that intracerebroventricular injection of porcine galanin stimulates GH and PRL release and inhibits dopamine release (3-5) indicating a direct or indirect action on pituitary PRL and GH secretion. In a similar manner, decidual galanin could play a role in regulating decidual PRL-like hormone [e.g. decidual luteotropin (22) or even placental lactogens rPL-I, rPL-II (23, 24), or placental PRL-like peptides (rPL-A, rPL-B (2411. We have some preliminary data supporting the latest hypothesis. Another possibility, of course, is that decidual galanin could act in an autocrine manner as a growth factor for the growth of
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decidual tissue. The detection of galanin in the peripheral serum by RIA demonstrates that galanin is also secreted into the periphery circulation, confirming previous observations that galanin is secreted in the circulation (8, 25) Lopez et al. (25) concluded that 30% of serum galanin originated from the pituitary as serum galanin levels in hypox rats decreased by 70% compared to control rats. Since the levels in serum follow the pattern of induction of the mRNA levels in the decidua, it is possible that most of the galanin in serum originates from the decidua, indicating that besides a local autocrine or paracrine effect, galanin during pregnancy has an effect on distal organs. Further studies are required to establish the role of galanin during pregnancy. Acknowledgment The secretarial appreciated.
assistance
of Ms.
June
McDougald
is very
murh
References 1. Tatemoto K, Rokaeus A, Jornvall H, McDonald TJ, Mutt V 1983 Galanin--a novel biologically active peptide from porcine intestine. FEBS Lett 164:124-128 2. Skofitsch G, Jacobovitz DM 1986 Quantitative distribution of galanin-like immunoreactivity in the rat central nervous system. Peptides 7:609--6 I3 3< Ottlecz A, Snyder GD, McCann SM 1988 Regulatory role of galanin in control of hypothalamic-anterior pituitary function. Proc Nat1 Acad Sci USA 85:9861-9865 4. Koshiyama H, Shimatsu A, Kato Y, Assadian H, Hatton N, Ishikawa Y, Tanoh T, Yanaihara N, Imura H 1990 Galanin induced prolactin release in rats: pharmacological evidence for the involvement of alpha-andrenergic and opiodergic mechanisms. Brain Res 507:321--324 a. Nordstrom 0, Melander T, Hokfelt T, Bartfai T, Goldstein M 1987 Evidence for an inhibitory effect of the peptide galanin on dopamine release from the rat median eminence. Neurosci 1,et.t 73:2126 6. Vrontakis ME, Peden LM, Duckworth ML, Friesen HG 1987 Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA. J Biol Chem 262:16755-16758 7. Vrontakis ME, Yamamoto T, Schroedter IC, Nagy Jl, Friesen HG 1989 Estrogen induction of galanin synthesis in the rat anterior pituitary gland demonstrated by in situ hybridization and immunohistochemistry. Neurosci Lett. 100:59-64 8. Kaplan LM, Gabriel SM, Koenig JI, Sunday ME, Spindel ER, Martin JB, Chin WW 1988 Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary. Proc Natl Acad Sci USA 85:7408-~7412 9. Chirgwin ?JM, Przybyla aE, McDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonucleases. Biochemistry 185294-5301 10. Henderson C 1989 Aminoalkylsilane: an inexpensive, simple preparation for slide adhesion. J Histochem 12:123-124 11. Angerer LM, Staler MH, Angerer RC 1987, In situ hybridization with RNA probes: an annotated recipe. In: Valentino KL, Eberwine .JH, Barchas .JD (edsl In situ Hybridization: Applications to Neurobiology. Oxford Iiniversity Press, New York, pp 43-70
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12. Gibori G, Khan I, Warshaw ML, McLean MP, Puryear TK, Nelson S, Durkee TJ, Azhar S, Steinschneider A, Rao MC 1988 Placentalderived regulators and the complex control of luteal cell function. Recent Prog Horm Res 44:377-425 13. O’Shea JD, Kleinfeld RG, Morrow HA 1983 Ultrastructure of decidualization in the pseudopregnant rat. Am J Anat 166:271-298 14. Bell SC 1985, Comparative aspects of decidualization in rodents and humans: cell types, secreted products and associated function. In: Implantation of the Human Embryo. Academic Press, London, pp 71-122 15. Welsh AO, Enders AC 1985 Light and electron microscopic examination of the mature decidual cells of the rat with emphasis on the antimesometrial decidua and its degeneration. Am J Anat 172:1-29 16. Bell SC 1983 Decidualization: regional differentiation and associated function. In: Finn CA (ed) Oxford Reviews of Reproductive Biology. Oxford University Press, Oxford, pp 220-271 17. Umapathysivan K, Jones WR 1978 An investigation of decidual specific proteins in the rat. Int J Fertil 23:138-142 18. Bell SC 1979 Synthesis of decidualization associated protein in tissues of the rat uterus and placenta during pregnancy. J Reprod Fertil49:177-181
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19. Bell SC 1979 Immunochemical identity of decidualization associated protein and a2-acute phase macroglobulin in the pregnant rat. J Reprod Immunol 1:193-206 20. Fletcher S, Thomas T, Schreiber G, Heinrich PC, Yeoh GC 1988 The development of rat alpha 2-macroglobulin. Studies in uiuo and in cultured fetal rat hepatocytes. Eur J Biochem 171:703-709 21. Bell SC, Hamer J, Heald PJ 1980 Induced protein and deciduoma formation in rat uterus. Biol Reprod 23:935-940 22. Jayatilak PG, Puryear TK, Hem Z, Fazleabas A, Gibori G 1989 Protein secretion by mesometrial and antiomesometrial rat decidual tissue: evidence for a differential gene expression. Endocrinology 125:659-666 23. Robertson MC, Croze F, Schroedter IC, Friesen HG 1990 Molecular cloning and expression of rat placental lactogen-I cDNA. Endocrinology 127:702-710 24. Duckworth ML, Schroedter IC, Friesen HG 1990 Cellular localization of rat placental lactogen II and rat prolactin-like proteins A and B by in situ hybridization. Placenta 11:143-155 25. Lopez FJ, Meade EH, Negro-Vilar A 1990 Development and characterization of a specific and sensitive radioimmunoassay for rat galanin. Measurement in brain tissue, hypophyseal, portal and peripheral serum. Brain Res Bull 24:395-399
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