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141,279-291 (1990)

Ontogeny of Placental Lactogen-I and Placental Lactogen-II in the Developing Rat Placental TERESAN.

FARIA,*+~SANTANUDEB,*,~

SIMONC.

Expression

M. KWOK,~FRANKTALAMANTES,$ANDMICHAEL

J. SOARES***

Departments of *Physiology and fBiochem&ry, University of Kansas Medical Center, Kansas City, Kansas 66103; and $Departmennt of Biology, University of Califonia, Santa Cruz, CalZfornia 95064 Accepted May 29, 1990 The purpose of this investigation was to identify the cellular origin, and the temporal and regional characteristics of placental lactogen-I (PL-I) and placental lactogen-II (PL-II) expression during placental development in the rat. PL-I and PL-II mRNA expression were assessed by Northern blot analysis and in situ hybridization. PL-I and PL-II protein expression were determined by Western blot and immunocytochemical analyses. PL-I mRNA was first detected by in situ hybridization at Day 6 of gestation in mural trophoblast giant cells and a day later, PL-I protein was first detected by immunocytochemistry. PL-I immunostaining extended to the polar trophoblast giant cells as gestation advanced. Polar trophoblast giant cell staining for PL-I was not as intense as the mural trophoblast giant cell staining. Northern and Western blot analyses confirmed the asymmetric distribution of PL-I expression. PL-I mRNA migrated as a 1-kb species and PL-I protein migrated as 30- and 36-40-kDa forms. PL-I expression abruptly declined at Day 12, and by Day 13, PL-I was not detectable. PL-II protein was first detectable at Day 11 of gestation and was localized to trophoblast giant cells. PL-II mRNA could be detected at Day 10 of gestation. Northern and Western blot analyses indicated that PL-II expression significantly increased as gestation advanced and that PL-II expression was asymmetrically distributed similar to PL-I. PL-II mRNA migrated as a I-kb species and PL-II protein migrated as a 25-kDa species. Blastocysts recovered on Day 4 of gestation initially showed no detectable expression of PL-I or PL-II; however, after 2 days of culture PL-I protein expression was detectable. Biochemical characteristics of PL-I synthesized and secreted by blastocyst outgrowths were similar to PL-I synthesized and secreted by Day 10 placental explants. In summary, (1) PL-I and PL-II are produced by trophoblast giant cells of the developing placenta, (2) PL-I and PL-II exhibit distinct temporal and regional patterns of expression during placental morphogenesis, and (3) PL-I expression by blastocyst outgrowths can be induced in vitro, whereas a more complex array of signals appears necessary for induction of PL-II expresQ 19!30 Academic

Press. Inc.

INTRODUCTION

Placental tissues of the mouse and rat are sources of a number of proteins related to pituitary prolactin (see Duckworth et al., 1986b; Ogren and Talamantes, 1988 for reviews). Placental prolactins are produced by trophoblast cells with the potential to influence the behavior of maternal, fetal, and possibly placental cells (see Ogren and Talamantes, 1988 for a review). Early studies identified the presence of placental prolactins on the basis of their biological actions on the ovaries and mammary glands. Transplantation of trophoblast tissue, injections of placental extracts, or coculture experiments were all consistent with the presence of a placental prolactin (Astwood and Greep, 1938; Cerruti and Lyons, 1 Supported by grants from the National Institute of Child Health and Human Development, HD 22208 (M.J.S.), HD 20676 (M.J.S.), and HD 14966 (F.T.) ‘Recipient of a Fulbright predoctoral fellowship. ’ Recipient of a Wesley Foundation postdoctoral fellowship. ’ To whom all correspondence should be addressed at Department of Physiology, University of Kansas Medical Center, Kansas City, KS 66103.

1960; Matthies, 1974; Talamantes, 19’75a,b; Peters et aZ., 1977; Crister et ah, 1980). Some reports suggested that placental prolactin expression began relatively early in pregnancy. Prolactin-like activity could be detected from blastocysts transplanted beneath the kidney capsule (Zeilmaker, 1968; Beyer and Zeilmaker, 1974; Zeilmaker and Verhamme, 1978) or from blastocysts placed in culture (Glasser and McCormack, 1979). Placental prolactin production appeared to be associated with the presence of trophoblast giant cells (Kohmoto and Bern, 1970; Soares et al., 1985). The purification and characterization of two placental prolactins has provided the necessary tools to investigate the ontogeny of placental prolaetin expression. Placental lactogen-I (PL-I) was purified from midgestation mouse placentas (Colosi et aZ., 1987a), the cDNA for mouse PL-I cloned (Colosi et al, 1987b), and recombinant PL-I produced and characterized (Colosi et al., 1988). Placental lactogen-II (PL-II) was purified from late gestation mouse (Colosi et aZ., 1982) and rat (Robertson and Friesen, 1975) placentas, and cDNAs for mouse (Jackson et ak, 1986) and rat (Duckworth et al, 1986a) PL-II cloned. Radioimmunoassay analyses of circulat-

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0012-1606/90 $3.00 Copyright All rights

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

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cellulose was obtained from Schleicher and Schuell Inc. (Keene, NH). Restriction enzymes and polymerases were purchased from New England Biolabs (Beverly, MA). Reagents used for the synthesis of complementary RNA probes, DNase-I and dextran sulphate were purchased from Pharmacia Fine Chemicals (Piscataway, NJ). Reagents for sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis were purchased from Bio-Rad Chemicals (Richmond, CA). Radiolabeled nucleotides were obtained from DuPont-NEN (Boston, MA). Avidin-biotin immunoperoxidase kits for rabbit immunoglobulin G (IgG) were purchased from Vector Laboratories (Burlingame, CA). Unless otherwise noted all other chemicals and reagents were purchased from Sigma. FIG. 1. Schematic diagram of a midgestation rat conceptus. Modified from Anderson, (1959). CA, chorioallantoic placenta; CV, choriovitelline placenta; D, decidua; pTGC, polar trophoblast giant cells; mTGC, mural trophoblast giant cells; vys, visceral yolk sac; am, amnion. The black bars indicate the chorioallantoic and choriovitelline demarcations used in the dissection.

ing protein concentrations have indicated that PL-I predominates at midgestation (Ogren et al, 1989), whereas, PL-II is present during the latter half of gestation (Robertson and Friesen, 1981; Soares et ah, 1982). PL-II has been localized to trophoblast giant cells of the mouse and rat by immunocytochemical analysis (Hall and Talamantes, 1984; Lee et aZ., 1988; Campbell et aZ., 1989). The cell type(s) responsible for the production of PL-I are unknown. A PL-I to PL-II developmental switch is implied from studies of circulating PL concentrations (Robertson and Friesen, 1981; Soares et aZ., 1982; Robertson et al, 1982; Ogren et al, 1989) and the behavior of trophoblast tissues in vitro (Soares et aL, 1983, 1985); however, the timing and cell types involved in the PL-I to PL-II transition are yet to be determined. The purpose of this study was threefold: (1) to identify the cell type(s) responsible for PL-I production, (2) to determine the temporal and regional characteristics of PL-I and PL-II expression in the developing rat placenta, and (3) to investigate the biochemical nature of prolactin-like proteins expressed by blastocysts in vitro. MATERIALS

AND METHODS

Reagents cDNAs to mouse PL-I and rat PL-II were generously provided by Dr. D. Linzer (Northwestern University) and Drs. M. Duckworth and H. Friesen (University of Manitoba), respectively. pGEM DNA plasmid was purchased from Promega Biotech (Madison, WI) and nitro-

Animals

and Tissue Preparation

Holtzman rats were obtained from SASCO Breeders (Omaha, NE). The animals were housed in an environmentally controlled facility with lights on from 0600 to 2000 hr and allowed free access to food and water. Timed

Excess

Peptide

+

FIG. 2. Examination of the specificity of the Western blot analysis for PL-I. Equivalent amounts of Day 11 choriovitelline placental cytosolic proteins were separated by SDS gel electrophoresis in 12.5% polyacrylamide gels under reducing conditions and electrophoretitally transferred to nitrocellulose. Nitrocellulose membranes were probed with either antiserum to recombinant mouse PL-I (lane A) or antiserum saturated with recombinant PL-I (lane B). The PL-I antiserum specifically recognized 30- and 3%kDa species. Note the decreased immunoreactivity in lane B (arrows indicate where the 30- and 3%kDa species migrate). The migration of molecular weight standards [lysozyme, soybean trypsin inhibitor, carbonic anhydrase, ovalbumin, bovine serum albumin, and phosphorylase b (X10-‘)] is shown.

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PL Expression in Placenta

pregnancies were obtained by housing female rats with male rats and examining vaginal smears daily during the cohabitation. The presence of a copulatory plug or sperm in the vaginal smear was designated Day 0 of pregnancy. Conceptuses were removed from uteri on various days during the first half of gestation (Days 5 through 13), fixed in freshly prepared Bouin’s fluid and used for immunocytochemical analysis, or in paraformaldehyde (4%, w/v in phosphate-buffered saline, PBS; 10 mM sodium phosphate, pH 7.2, and 150 mMNaC1) and used for in situ hybridization analysis. Implantation sites on Days 5-7 of pregnancy were identified by intravenous injection of a 2% solution of Chicago Blue B (0.5 ml in the lateral tail vein) 10 min prior to autopsy. Rat trophoblast tissue from Days 10 through 13 was dissected into chorioallantoic and choriovitelline placentas (see Fig. 1). Decidual tissue and nonplacental extraembryonic membranes were mechanically removed from the chorioallantoic and choriovitelline placentas. Dissected trophoblast tissues were immediately frozen in liquid nitrogen and stored at -70°C until used for Northern and Western blot analyses. Blastocysts were isolated by flushing uterine horns on Day 4 of gestation, cultured in RPM1 1640 medium in a humidified incubator at 37°C with an atmosphere of 95% sir/5% CO*. The medium was supplemented with 100 units/ml of penicillin and 100 pg/ml streptomycin (Hazelton, Lenexa, KS), 1 mM sodium pyruvate, 50 PM 2-mercaptoethanol, and 20% fetal bovine serum (Hazelton). Blastocyst outgrowths were either fixed in Bouin’s fluid and used for immunocytochemical analysis of PL-I and PL-II expression, or metabolically labeled with radioactive methionine and used to evaluate the biochemical characteristics of newly synthesized PL-I and PL-II. Blastocysts used for immunocytochemical analysis were grown in Lab-Tek Tissue culture chamber slides (Miles Lab., Naperville, IL), while blastocysts used for metabolic labeling were grown in 10 X 35 mm plastic culture dishes (Be&on-Dickinson, Lincoln Park, NJ). Following isolation, some blastocysts were immediately adhered to glass slides by centrifugation using a Shandon cytospin apparatus (Selwick, PA), fixed, and processed for immunocytochemical analysis. Immunocytochemistry

Placental tissues fixed in Bouin’s fluid were dehydrated, cleared, embedded in paraffin, and sectioned at 7 pm. Blastocyst outgrowths cultured in chamber slides were fixed in Bouin’s fluid, dehydrated, and permeabilized with cold acetone. Placental tissue and blastocyst outgrowths were immunocytochemically stained for the

281

presence of either PL-I or PL-II using an avidin-biotin immunoperoxidase kit for rabbit IgG as previously described (Campbell et cc& 1989). PL-I expression was assessed with an antiserum to recombinant mouse PL-I (Colosi et al, 1988) and PL-II expression was assessed with an antiserum to amino acids 56-70 of rat PL-II (Deb et a& 1989). The immunostained tissues were counterstained with hematoxylin. Control sections were examined using antisera saturated with antigen. In Situ H$widization

PL-I and PL-II mRNA were detected in tissue sections according to a procedure modified from methods described by Angerer and co-workers (Angerer and Angerer, 1981; Angerer et a& 1984) and De et al. (1989). Tissues were fixed in 4% paraformaldehyde in PBS for 2 hr at 4”C, washed in PBS for 1 hr, dehydrated, cleared, and embedded. Sections were cut at 7 pm, mounted on poly-L-lysine-coated slides, and dried. Prior to hybridization, the slides were baked overnight at 37”C, deparaffinized, rehydrated, and acetylated. The slides were then rinsed in PBS and incubated for 10 min in PBS containing 5 mMMgCl,, 0.25 mMTris-0.1 Mglycine, pH 7.4, followed by an incubation in 2X SET (1X SET = 150 mM NaCl, 5 mlM ethylenediaminetetraacetic acid, 10 mM Tris-HCl, pH 8.0) containing 50% formamide at 37°C. The hybridization was performed for 4 hr at 42°C in a humidified chamber, with 100 pi/slide of buffer containing 2~ SET with 50% formamide, 10X Denhart’s (0.02% w/v of bovine serum albumin, 0.02% w/v of Fi~011, and 0.02% w/v of polyvinylpyrrolidone), 500 pg/ml yeast tRNA, 100 mMdithiothreito1 (DTT), 10% dextran sulfate, and approximately 0.04 pg/ml of ?S-labeled riboprobe. After 4 hr, slides were washed in 4~ SSC (IX SSC = 150 mMNaC1,15 mMsodium citrate, pH 7.0) and incubated in 50% formamide in 2X SET containing 10 mMDTT for 15 min at 60°C. Slides were then incubated in 3~ SET, containing 20 pg/ml RNase A and 100 kg/ml bovine serum albumin, for 30 min at 37°C. The RNase was washed away in a large volume of 1X SSC with moderate agitation. Finally, the slides were incubated in 0.2~ SET, containing 0.1% 2-mercaptoethanol at 50°C for 30 min. The slides were dehydrated, air dried, and dipped in Ilford K5 emulsion (Ilford, England) for autoradiography. After exposure (3-4 days) at 4°C the slides were developed and lightly stained with hematoxylin. PL-I and PL-II cDNAs (Duckworth et al, 1986a; Colosi et al, 1987b) subcloned into pGEM plasmids were used as templates for the synthesis of Y+labeled sense and antisense RNA probes (Melton et cd, 1984; De et cd, 1989).

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FIG. 3. Immunocytochemical analysis of PL-I within sections from conceptuses isolated for the presence of PL-I using an avidin-biotin immunoperoxidase kit for rabbit IgG. PL-I mouse PL-I. Micrograph a: Day 6 conceptus; micrograph b: Day 7 conceptus; micrograph c: the lack of PL-I-positive staining in micrograph a, and the more intense staining of mural

on Days 6 to 9 of gestation. The sections were stained was detected with a rabbit antiserum to recombinant Day 8 conceptus; micrograph d: Day 9 conceptus. Note trophoblast giant cells (arrows) when compared with

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Blot Analysis

Relative concentrations of PL-I and PL-II mRNA in the chorioallantoic and choriovitelline placentas on Days 10 through 13 of gestation were estimated by Northern blot analysis. Total RNA was isolated from tissues by SDS-phenol extraction (Andrews and Teng, 1979) and treated with 4 Mammonium acetate to precipitate RNA from soluble DNA (Palmiter, 1973). RNA samples (7 bg/lane) were fractionated by electrophoresis in 1% agarose gels containing 2.2 M formaldehyde (Lehrach et ah, 1977) and blotted to nitrocellulose (Thomas, 1980). The integrity of the RNA was monitored by acridine orange staining of gels. Molecular weight standards were run in parallel to the samples, to determine the size of the mRNAs. All hybridization and wash buffers were maintained at 66°C. The hybridization buffer included 10% dextran sulphate, 250 @g/ml tRNA, 3~ SET, 0.1% SDS, and either PL-I or PL-II =Plabeled probe. Prehybridization, hybridization, and posthybridization treatment of the filters were carried out as previously described (Andrews et al, 1984; Soares et al., 1987). Following hybridization, filters were dried and autoradiographed with Kodak X-Omat AR x-ray film at -70°C. PL-I and PL-II cDNAs (Duckworth et aa, 1986a; Colosi et aL, 1987b) subcloned into pGEM plasmids were used as templates for the synthesis of =P-labeled cRNA probes (Melton et ah, 1984). Western Blot Analysis Relative concentrations of PL-I and PL-II protein in the chorioallantoic and choriovitelline placentas on Days 10 through 13 of gestation were estimated by electrophoresis and immunoblotting. Cytosol preparations were generated by homogenization of tissues in cold ammonium bicarbonate buffer [lo0 mMNH,HCO, (pH 9.3), 100 mM NaCl, and 0.5 mM phenylmethylsulfonic fluoride]. The homogenates were further disrupted by sonication for 1 min, using a Microultrasonic Cell Disrupter (Kontes Instruments, Vineland, NJ). After sonication the homogenates were centrifuged at 12,000g for 15 min. Supernatants were centrifuged at 100,OOOg for 60 min. The final supernatant was referred to as the cytosol fraction. Protein concentrations of cytosol preparations were estimated by the method of Bradford (Bradford, 1976). Cytosol preparations containing equivalent amounts of protein were separated by SDS-polyacrylamide gel

electrophoresis in 12.5% gels under reducing conditions (Laemmli, 1970) and electrophoretically transferred to nitrocellulose (Towbin et al, 1979). .PL-I and PL-II proteins were detected with an antiserum to recombinant mouse PL-I (Colosi et aZ., 1988) and with an antiserum to amino acids 56-70 of rat PL-II (Deb et aC, 1989), respectively. The processing and development of the nitrocellulose membranes for reactivity with the antisera were performed as previously described (Soares et al., 1988). PL-I antiserum saturated with recombinant PL-I was used as a control. The specificity of the PL-II antipeptide antiserum has been described (Deb et aL, 1989).

In Vitro Synthesis of PLI

and PLII

The synthesis of PL-I and PL-II by blastocyst outgrowths and Day 10 choriovitelline placental explants was assessed by immunoprecipitation of [35S]methionine-labeled proteins, SDS gel electrophoresis and fluorography. Fifteen to twenty blastocysts were grown in 10 X 35 mm dishes for 5 days and then incubated for 24 hr with trams [YS]-label (ICN Biomedicals Inc., Irvine, CA; 70% [YSlmethionine, 1204 Ci/mmol) at a concentration of 100 &i/ml in methionine-free RPM1 1640 medium supplemented with 100 units/ml of penicillin and 100 pg/ml streptomycin, and 10% dialyzed fetal bovine serum. Trophoblast tissue was also isolated from pregnant rats on Day 10 of gestation and incubated for 24 hr under similar conditions. PL-I and PL-II were isolated from conditioned media by immunoprecipitation with antisera directed to either recombinant mouse PL-I (Colosi et aL, 1988) or to amino acids 56-70 of rat PL-II (Deb et aZ., 1989). Conditioned media (100 ~1) were incubated overnight at 4°C with immunoprecipitation buffer (200 ~1: 10 mM sodium phosphate buffer, pH 7.5, containing 150 mM NaCl, 10 mM L-methionine, 1% deoxycholic acid, and 2% Triton X-100) and antisera (10 ~1). After incubation, the antigen-antibody complexes were precipitated according to the double-antibody method (Comstock et a& 1984). The immunoprecipitates were solubilized in sample buffer (0.625 M Tris-HCl, pH 6.8,2% SDS, 10% glycerol, and 5% 2-mercaptoethanol) and heated at 90°C for 4 min. The samples were then centrifuged, and the supernatants recovered and electrophoretically separated by SDS-polyacrylamide gel electrophoresis in 12.5% gels under reducing conditions (Laemmli, 1970). The gels were stained with Coomassie blue, destained, rehydrated, incubated with 1 M sodium

polar trophoblast giant cells on micrographs h, c, and d. Magnification, 90X (micrographs a, b, and c), 40x (micrograph d). The conceptuses shown in the micrographs have the same orientation as the schematic diagram in Fig. 1. See the text for further information regarding the methodology.

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salicylate for 30 min, dried, and exposed to Kodak Omat AR x-ray film at -70% (Bonner, 1984).

X-

RESULTS

Characterization

of the PL-I

Antiserum

Antiserum directed against mouse PL-I specifically recognized proteins of 30 and 38 kDa in cytosolic preparations from Day 11 rat choriovitelline placenta (Fig. 2). The addition of excess PL-I effectively decreased the staining of the 30- and 38-kDa bands, demonstrating the specificity of the immunoreaction (Fig. 2). Temporal and Region& Expwssicm of PLI in the Developing Rat Placenta

and PL-II

Localization of PIrI and PLII. The immunocytochemical analyses for PL-I and PL-II were specific as demonstrated by cell-specific reactivity and the absence of staining with antigen-saturated antisera. In&u hybridization for PL-I and PL-II were specific as demonstrated by cell-specific hybridization and the absence of hybridization with sense probes. Detection of PL-I protein by immunocytochemistry was first demonstrated at Day 7 of gestation in mural trophoblast giant cells (Fig. 3). PL-I immunostaining extended to polar trophoblast giant cells as gestation advanced (Fig. 3) and eventually decreased below the limits of detection throughout the conceptus on Days 12 and 13 (Fig. 4). Polar trophoblast giant cell staining for PL-I was not as intense as the mural trophoblast giant cell staining. PL-I mRNA was first detected on Day 6 of gestation in mural trophoblast giant cells (Fig. 4). Differences in the onset of PL-I mRNA and protein expression may correspond to the relative sensitivities of the techniques used or to the maturation of additional regulatory controls from Day 6 to Day 7 of gestation. In situ localization of PL-I mRNA paralleled the immunolocalization of PL-I protein (Figs. 3,4, and 5). PL-II protein and mRNA were first detected on Day 11 of gestation in trophoblast giant cells and continued to be detected on Days 12 and 13 (Figs. 4 and 6). PL-II was localized to trophoblast giant cells and showed an asymmetric pattern of expression within the conceptus similar to that observed for PL-I. Biochemical analysis of the PLI to PGII transition. Consistent with the localization of PL-I and PL-II protein and mRNA during midgestation, Western and Northern blot analyses indicated that PL-I expression declined after Day 11 of gestation, while PL-II expression was initiated on Day 10 or 11 and increased as gestation advanced (Figs. 7 and 8). PL-II mRNA was first detectable in the choriovitelline placenta on Day 10 of

gestation whereas, PL-II protein was not detectable until a day later. Discrepancies in the appearance of PL-II mRNA and PL-II protein may reflect differences in the sensitivities of the techniques employed or in the maturation of additional regulatory processes. PL-I immunoreactive proteins had molecular weights approximating 30- and 36- to 40-kDa, whereas, PL-II migrated as a 25kDa species (Fig. 7). The 30-kDa PL-I species showed an abrupt decrease between Days 11 and 12, whereas the 36- to 40-kDa PL-I species appeared to persist, although at decreased concentrations (Fig. 7). PL-I and PL-II mRNA species principally migrated as 1-kb species (Fig. 8). Limited quantities of higher molecular weight PL-I and PL-II mRNAs were detectable (Fig. 8). The higher molecular weight PL-mRNAs may represent incompletely processed precursor mRNAs, similar to that reported for pituitary prolactin (Maurer et aL, 1980). The relative expression of PL-I and PL-II mRNA and protein were greater in the choriovitelline placenta than in the chorioallantoic placenta (Figs. 7 and 8). Characterization of PL Expression Blastocyst Outgrowths

by

PL-I protein was first immunocytochemically detected after 2 days of culture in giant cells growing out from the blastocysts (Fig. 9). PL-I expression continued for at least 8 days of culture; however, no evidence of PL-II expression was observed. In vitro incorporation of radiolabeled methionine into PL-I and PL-II by Day 10 choriovitelline placental explants and blastocyst outgrowths is shown in Fig. 10. Day 10 choriovitelline placental explants synthesized and secreted both PL-I and PL-II, whereas the blastocyst outgrowths only synthesized and secreted PL-I (Fig. 10). The biochemical characteristics of PL-I synthesized and secreted by the Day 10 choriovitelline placental explants and the blastocyst outgrowths were similar (36-40 kDa). The 30-kDa PL-I species detected by Western blot analysis of Day 10 cytosol was not observed in the fluorograms. DISCUSSION

In this report we have identified cell types responsible for the production of PL-I, determined the temporal and regional characteristics of PL-I and PL-II expression in the developing rat placenta, and ascertained the biochemical nature of prolactin-like proteins expressed by blastocysts grown in vitro. Cellular Localization

of PLI

Expression

In the present investigation, PL-I protein and mRNA were exclusively localized to trophoblast giant cells. Other members of the prolactin gene family have also

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FIG. 4. Immunocytochemical analysis of PL-I and PL-II within sections of conceptuses from Days 10 through 13 of gestation. Micrographs a and e: Day 10 conceptus; micrographs b and f: Day 11 conceptus; micrographs c and g: Day 12 conceptus; micrographs d and h: Day 13 conceptus. Micrographs a-d: stained for PL-I; micrographs e-h: stained for PL-II. Note the shift from PL-I immunostaining to PL-II immunostaining after Day 11 of pregnancy. Magnification, ‘73~ (a-c and e-g), 200X (d and h). The brown color in the trophoblast giant cell cytoplasm indicates a positive immunoreactive product. See Fig. 3 and the text for further information regarding the methodology.

been localized to trophoblast giant cells, including PLII, proliferin, and prolactin-like protein-A (Hall and Talamantes, 1984; Lee et a;& 1988; Campbell et al, 1989; present study). Trophoblast giant cells possess an amplified genome resulting from a process called endoreduplication (see Gardner, 1983; Ilgren, 1983; Rossant, 1986). The specific regulatory mechanisms responsible for trophoblast cell DNA amplification and how this process facilitates expression of PL-I and PL-II remain to be determined. Temporal and Regaimal Characteristics and PIrII Exlyression

of PLI

PL-I expression begins shortly after the initiation of blastocyst implantation into the uterine endometrium.

Blastocysts retrieved from the lumen of the uterus on Day 4 of pregnancy did not express PL-I (present study). By Day 6 of pregnancy, PL-I mRNA could be detected in trophoblast giant cells and a day later, PL-I protein was immunologically detectable; PL-I protein was detectable by Day 3 of blastocyst culture (present study). In viwo, PL-I expression was coupled to uterine invasion and the formation of trophoblast giant cells. In vitro, PL-I expression was associated with trophoblast cell outgrowth and giant cell formation. Blastocyst attachment, outgrowth, and giant cell formation are mediated by components of the extracellular matrix and do not require exposure to other serum factors (Armant et al, 1986; Carson et al, 1988). We hypothesize that attachment and outgrowth are the events responsible for the

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FIG. 9. Immunocytochemical analysis of PL-I within blastocyst outgrowths during in vitro culture. Micrograph a: Day 0 of culture; mierograph b: Day 2; micrograph c: Day 3; micrograph d: Day 4. Note that PL-I expression begins after 2 days of culture and is restricted to giant cells. Magnification, 148X. The brown color in the giant cell cytoplasm indicates a positive immunoreactive product. See Fig. 3 and the text for further information regarding the methodology.

induction of PL-I expression by trophoblast cells. The validity of this hypothesis is currently being tested. In the present study, PL-I expression abruptly decreased to low levels after Day 11 of gestation. The first appearance of PL-II preceded the decline of PL-I. Both PLs were expressed by the same cell types (trophoblast giant cells) on Day 11 of gestation (present study). The termination of PL-I expression appears to be linked with increased PL-II expression. PL-II possesses at least some of the same biological actions as PL-I and may be responsible for the decline in PL-I expression. PL-I has a markedly longer half-life in circulation than does PL-II (Kelly et aL, 1975; Voogt and Salamon, 1985; Pinon et ok, 1988) and thus, may provide the most biolog-

ically economic prolactin that can be produced given the limited number of trophoblast cells available shortly after implantation. The developmental switch from PLI to PL-II expression does not occur spontaneously in blastocyst cultures (present study), but instead appears to be associated with some as of yet unknown maturational event taking place in fetal, placental, or maternal compartments. Mural trophoblast giant cells of the choriovitelline placenta are the major sources of postimplantation PL-I production and midgestation PL-II production (present study). The high density of trophoblast giant cells in the choriovitelline placenta appears only partly responsible for the asymmetric expression of PLs. Regional differ-

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287

FIG. 5. Cellular localization of PL-I mRNA within conceptuses on Days 6, ‘7,11, and 13 of gestation. PL-I mRNA was hybridized in situ for 4 hr at 42°C with a [%lpL-I cRNA probe and RNase-resistant hybrids were detected autoradiographically. Sections were lightly stained with hematoxylin. Micrographs a and e: Day 6 conceptus; micrographs b and f: Day 7 conceptus; micrographs c and g: Day 11 conceptus; micrographs d and b: Day 13 conceptus. Micrographs a-d: bright field; micrographs e-h: dark field. Note the hybridization in mural trophoblast giant cells on Day 6 (arrows), the intense hybridization in trophoblast giant cells on Day 7 (arrows) and Day 11, and the absence of hybridization in trophoblast giant cells on Day 13. Dee, decidua; vys, visceral yolk sac; RM, Reichert’s membrane. Magnification, 150X. The orientation of the Day 6 and 7 conceptuses is the same as in Fig. 1. Micrographs depicting Day 11 and Day 13 conceptuses show a portion of the placenta containing a large number of trophoblast giant cells.

ences in the uterine environment may influence the regional pattern of PL expression. In support of this notion, differences in the types of proteins secreted by mesometrial and antimesometrial decidual tissues have recently been reported (Jayatilak et ab, 1989). Alternatively, trophoblast giant cell associations (polar) or lack of associations (mural) with other components of the chorioallantoic placenta may be responsible for the asymmetric pattern of PL expression. Expression of PL-I and PL-II represent specialized trophoblast cell functions necessary to accommodate the needs of the growing embryo. PL-I and PL-II are potent prolactin-like hormones. These hormones interact with prolactin receptors, stimulate rat Nb-2 lymphoma cell proliferation, and stimulate milk protein biosynthesis (Thordarson et al., 1986; Colosi et d, 1987a, 1988; Davis and Linzer, 1989). Prolactin receptors are located in a number of tissues required for successful pregnancy, including the ovary (MacLeod et ok, 1989) and uterus (Williams et aZ., 1978; Jayatilak and Gibori,

1986). During early postimplantation stages, PL-I is likely to behave in a paracrine role supporting the growth and differentiation of uterine decidual tissue. Additional systemic PL-I effects are probable by midgestation when circulating PL-I concentrations are elevated (Ogren et aL, 1989). PL-II is the predominant prolactin-like hormone in maternal and fetal circulation during the latter half of gestation (see Ogren and Talamantes, 1988), and is likely to have multiple actions, including effects on both maternal and fetal compartments. Biochemical

Nature

of Trophoblast

Cell PLS

PL-I is secreted as a 36- to 40-kDa glycosylated complex of proteins (Colosi et al., 1987a; present study), whereas, PL-II is secreted as a nonglycosylated 25-kDa species (present study). The major cytosolic PL-I species differed from those secreted by trophoblast cells in vitro. Cytosolic preparations isolated from trophoblast

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FIG. 6. Cellular localization of PL-II mRNA within conceptuses on Days 8 and 13 of gestation. Micrographs a and d: Day 8 conceptus; micrographs b, c, e, and f: Day 13 conceptus. Micrographs a, b, and c: bright fieId; micrographs d, e, and f: dark field. Note the absence of hybridization on Day 8, the absence of specific hybridization on sections hybridized with a sense probe for PL-II (micrographs c and f), and the hybridization with an antisense probe for PL-II on Day 13 of pregnancy (micrographs b and e). Dee, decidua; RM, Reichert’s membrane. Magnification, 190x. See Fig. 5 and the text for further information regarding the methodology.

tissue obtained on Days 10 or 11 of gestation contained predominantly a SO-kDa PL-I immunoreactive species with lesser amounts of the 36- to 40-kDa PL-I complex.

The 30-kDa PL-I species was previously purified (Colosi et aZ., 1987a); however, its relationship to the 36- to 40kDa PL-I species was unclear. The 30-kDa PL-I species

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B

C

D

E

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H

0

A

B

C

D

96 -

PL-I

67 43 -

./

PL-II Plecenta Day of Pregnancy

CA

CV

CA

CV

CA

~CA

12

11

1Q

CV

CV

31 (I*

13 21 -

FIG. 7. Western blot analysis of PL-I (top) and PL-II (bottom) expression in chorioallantoic (CA) and choriovitelline (CV) placentas. Lane A: Day 10 CA placenta; lane B: Day 10 CV placenta; lane C: Day 11 CA placenta; Lane D: Day 11 CV placenta; lane E: Day 12 CA placenta; lane F: Day 12 CV placenta; lane G: Day 13 CA placenta; lane H: Day 13 CV placenta. The PL-I antiserum specifically recognized 30and 36- to 40-kDa species, whereas the PL-II antiserum specifically recognized a 25-kDa species. Note the shift from PL-I expression to PL-II expression as gestation advances. See Fig. 2 and the text for further information regarding the methodology.

may be a stable intermediate in the production of the 36to 40-kDa PL-I species or a nonsecretory form of PL-I with intracellular actions. A~CDEFGH PL-I

14 AntIsera

ni323~

I

II

Day lo placenta

I

II

Blastocyst outgrowth

FIG. 10. In vitro incorporation of radiolabeled methionine into PL-I and PL-II by Day 10 choriovitelline placental explants and blastocyst outgrowths. Conditioned media from PQuethionine labeled Day 10 choriovitelline placental explants and blastocyst outgrowths were precipitated with antiserum to PL-I or PL-II. The precipitates were separated by SDS gel electrophoresis in 12.5% polyacrylamide gels under reducing conditions and subjected to fluorography. Lanes A and B: Day 10 choriovitelline placental explants; lanes C and D: blastocyst outgrowths. Lanes A and C: immunoprecipitated with PL-I antiserum; lanes B and D: immunoprecipitated with PL-II antiserum. Day 10 choriovitelline placental explants synthesized and secreted both PL-I and PL-II whereas, blastocyst outgrowths only synthesized and secreted PL-I. The secreted PL-I migrated as a 36- to IO-kDa complex and the secreted PL-II migrated as a 25-kDa species. The migration of molecular weight standards (X10-‘) is shown.

In conclusion, we have demonstrated that: (1) PL-I and PL-II are produced by trophoblast giant cells of the developing rat placenta, (2) PL-I and PL-II exhibit distinct temporal and regional patterns of expression during placental morphogenesis, and (3) PL-I expression can be induced from blastocysts in vitro, whereas a more complex array of signals appears necessary for induction of PL-II expression. Placenta Day of Pregnancy

AUNAU 10 11

12

13

FIG. 8. Northern blot analysis of PL-I (top) and PL-II (bottom) expression in chorioallantoic (CA) and choriovitelline (CV) placentas. Total RNA (7 fig/lane) was fractionated by electrophoresis and transferred to nitrocellulose prior to hybridization with [=P]PL-I and PL-II cRNA probes. Lane A: Day 10 CA placenta; lane B: Day 10 CV placenta; lane C: Day 11 CA placenta; lane D: Day 11 CV placenta; lane E: Day 12 CA placenta; lane F: Day 12 CV placenta; lane G: Day 13 CA placenta; lane H: Day 13 CV placenta. PL-I and PL-II mRNA species were detected in the appropriate size range (1 kb). Note the shift in expression from PL-I mRNA to PL-II mRNA as gestation advances. The migration of molecular weight standards (0.24- to 9.5-kb RNA ladder) was used to estimate the sizes of the mRNAs. The letters A and V on the bottom of the figure refer to chorioallantoic and choriovitelline, respectively.

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Ontogeny of placental lactogen-I and placental lactogen-II expression in the developing rat placenta.

The purpose of this investigation was to identify the cellular origin, and the temporal and regional characteristics of placental lactogen-I (PL-I) an...
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