00l3-7227/90/1265-2561$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 5 Printed in U.S.A.
S. OGILVIE, M. L. DUCKWORTH, L. H. LARKIN, W. C. BUHI, AND K. T. SHIVERICK Departments of Pharmacology and Therapeutics (S.O., K.T.S.), Anatomy and Cell Biology (L.H.L.), and Obstetrics and Gynecology (W.C.B.), University of Florida, Gainesville, Florida 32610; and the Department of Physiology, University of Manitoba (M.L.D.), Winnipeg, Manitoba, R3E 0W3 Canada
ABSTRACT. Mid- to late-gestation rat placenta expresses three PRL-related mRNAs, rat placental lactogen-II (rPL-II), rat PRL-like protein-A (rPLP-A), and rat PRL-like protein-B (rPLP-B). The protein product of rPL-II mRNA has been characterized, and the protein products of the rPLP-A mRNA were recently identified. The mol wt of a nonsecreted nonglycosylated rPLP-B protein would be 27,145 based on the mRNA sequence. The present study is the first to report the identification of the rPLP-B protein. Antiserum was generated against a chemically synthesized oligopeptide inferred from a specific region of the rPLP-B cDNA. Three or four distinct proteins synthesized and secreted by rat basal zone explants (day 15 gestation) showed cross-reactivity with the rPLP-B antiserum. The relative mol wt of these immunoreactive proteins is approximately 30,000, with a pi varying from 6.1-6.6. De novo synthesized rPLP-B proteins were not secreted by the explant tissue
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in the presence of tunicamycin, suggesting that the proteins are glycosylated. These data are consistent with the presence of one potential iV-glycosylation site derived from the rPLP-B mRNA sequence. The rPLP-B antiserum showed no cross-reactivity with proteins identified using antisera against rPLP-A, rPL-II, or human pregnancy-specific /Vglycoprotein. Immunocytochemical studies were carried out using paraffin sections from placentas of day 14 and 17 pregnant rats which were treated with anti-rPLP-B, followed by avidin-biotin-peroxidase complex. These experiments show perinuclear staining, which was localized in basophilic cytotrophoblast cells, confirming previous in situ mRNA hybridization studies. Although no physiological role has been established for rPLP-B, the synthesis and secretion of this protein by cells in contact only with maternal circulation suggest a hormonal role. (Endocrinology 126: 2561-2566, 1990)
HE PRL/GH gene family in the rat has recently expanded with the identification of a third PRLrelated mRNA in the mid- to late-gestation placenta (1). The predicted amino acid sequence of the new placental protein, rat PRL-like protein-B (rPLP-B), exhibits 44% homology to pituitary PRL, which in the rodent is secreted at very low levels from midpregnancy to term (2). The two previously identified placental family members, rat placental lactogen-II (rPL-II) and rat PRL-like protein-A (rPLP-A), show 52% and 45% homology, respectively, to rat PRL at the amino acid level (3, 4). On day 16 of gestation, the three histologically distinct cellular elements in the basal zone of the placenta (5, 6) include the small basophilic cytotrophoblast cells, glycogen cells, and giant trophoblastic cells. In situ hybridization studies have localized mRNA for rPL-II in the giant cells of the basal zone and in cells in the labyrinth region, while mRNA for rPLP-A was detected in the basophilic cytotrophoblast cells and the giant cells of the basal zone,
and mRNA for rPLP-B was detected in the basophilic cytotrophoblast cells only (7). In addition to different locations of the mRNAs of these PLPs, there appears to be different temporal expression of the mRNAs. The rPL-II mRNA is first detected on day 12, and its level remains high throughout gestation (3), whereas the mRNAs for rPLP-A and rPLP-B are first strongly expressed on day 14, 2 days later than rPL-II, and remain high until birth (1, 4). Of the three proteins, rPL-II has been purified and characterized (8, 9), while rPLP-A was only recently identified using an antipeptide antiserum (10). In this study we report the identification of proteins synthesized and secreted by rat basal zone explants which cross-react with an antiserum generated against a chemically synthesized oligopeptide inferred from a specific region of the rPLP-B cDNA. Identification of the rPLP-B gene product will lead to a better understanding of the function and temporal synthesis of PLPs in rat placenta.
Received October 6,1989. Address requests for reprints to: Dr. Kathleen T. Shiverick, Department of Pharmacology and Therapeutics, University of Florida, Box J267 JHMHC, Gainesville, Florida 32610. *This work was supported in part by NIH Grant ES-04435 (to K.T.S.).
Materials and Methods Materials Avidin-biotin-peroxidase complex for rabbit immunoglobulin G (IgG) was obtained from the Vectastain ABC kit (Vector,
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De Novo Synthesis and Secretion of Prolactin-Like Protein-B by Rat Placental Explants*
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Oligopeptide synthesis and antibody production The synthetic peptide Cys-Asn-Glu-Leu-Ala-Tyr-Leu-ArgSer-Pro-Asp-Glu-Glu-Arg-Arg-His was custom synthesized by the Institut Armand Frappier (Montreal, Quebec, Canada) and coupled to keyhole limpet hemocyanin. This peptide represents amino acids 186-200 in the deduced amino acid sequence (including the signal peptide) of rPLP-B (1), with an additional cysteine at the amino-terminus. This sequence was selected on the basis of its hydrophobic profile and its difference from sequences of other members of the rPRL gene family. New Zealand White rabbits were injected sc with 540 fig peptide conjugated to keyhole limpet hemocyanin in complete Freund's adjuvant and boosted sc with the same concentration of conjugated peptide in incomplete Freund's adjuvant on days 14 and 28 after the initial injection. The rabbits were bled 13 days after the final boost. The antiserum was able to detect 100 ng pure peptide at a 1:40,000 dilution by dot blot assay, using the alkaline phosphatase detection method. Rabbit polyclonal antiserum against rPLP-A was generated against an oligopeptide representing amino acids 160-176 of the deduced amino acid sequence (including signal peptide) of rPLP-A (4), with an additional cysteine at the amino-terminus, as described above. This oligopeptide is the same as the oligopeptide described by Deb et al. (10), representing amino acids 129-145 (excluding the signal peptide) of rPLP-A. The antiserum can detect 5-10 ng rPLP-A peptide at a 1:60,000 dilution by dot blot, using alkaline phosphatase as the substrate. A rabbit polyclonal antiserum against rPL-II was generated
against an oligopeptide representing amino acids 56-70 of rPLII; this was a generous gift from M. J. Soares (11). Placental explant culture Basal zone tissue was incubated under sterile conditions in Modified Eagle's Medium (175 mg tissue/5 ml) for 24 h at 37 C under 47.5% O2-2.5% CO2-50% N2. The culture medium was supplemented with 3 mg/ml glucose, penicillin (100 U/ml), streptomycin (100 Mg/ml), fungizone (0.25 Mg/ml), 1% (vol/vol) nonessential amino acids, and 1.5 fig/ml methionine (one tenth the normal concentration). In some experiments tunicamycin was added from a 10 mg/ml final concentration stock solution in dimethylsulfoxide at the initiation of culture. Dimethylsulfoxide alone was added to control explants. The radiolabel, [35S] methionine (40 /uCi) was added 16 h after the initiation of culture. After incubation, culture medium was dialyzed extensively (Mr cut-off, 3500), and aliquots were counted. Polyacrylamide gel electrophoresis and immunoblotting Two-dimensional SDS-polyacrylamide gel electrophoresis of the samples was performed according to methods described by Roberts et al. (12). Equal amounts of [35S]methionine-labeled protein (100,000 cpm) were loaded onto each gel. After electrophoresis, slab gels were fixed, prepared for fluorography using sodium salicylate (13), and dried. Fluorographs were prepared with Kodak XAR x-ray film (Eastman Kodak, Rochester, NY). Alternatively, gels were equilibrated for 30 min in electrophoretic transfer buffer containing 0.025 M Tris, 0.192 M glycine, and 20% (vol/vol) methanol, pH 8.3, and the proteins were transferred from gels to nitrocellulose membrane for immunoblot analysis (14). After transfer, rPLP-B, rPL-II, and rPLPA polypeptides were detected using a rabbit polyclonal antiserum against rPLP-B (1:1000), a rabbit polyclonal antiserum against rPL-II (1:500), or a rabbit polyclonal against rPLP-A (1:200), followed by affinity-purified goat anti-rabbit IgG horseradish peroxidase conjugate. Avidin-biotin-peroxidase immunocytochemistry Whole fetal-placental units were fixed by immersion in Bouin's solution, dehydrated with a graded series of alcohols, and embedded in paraffin. rPLP-B was localized in deparaffinized rehydrated 6-^m sections by the avidin-peroxidase complex technique as outlined by Renegar and Larkin (15), except that the peroxidase substrate was 0.04% diaminobenzidine in PBS, pH 7.2. The anti-rPLP-B antiserum and normal rabbit serum were used at a dilution of 1:200.
Results De nouo synthesis of rPLP-B in explant cultures The [35S]methionine-labeled proteins secreted by explants of rat basal zone tissue (day 15 of gestation) were characterized using two-dimensional SDS-polyacrylamide gel electrophoresis, as shown by a representative fluorograph in Fig. 1A. The total proteins secreted by explants of basal zone tissue shown in Fig. 1A were
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Burlingame, CA). Rabbit antiserum generated against pregnancy-specific jSrglycoprotein (PS/3G) purified from human pregnancy serum was made in Denmark by Dakopatts (Accurate Chemical Co., Westbury, NY). Blotting grade affinitypurified goat anti-rabbit IgG horseradish peroxidase conjugate and reagents for sodium dodecyl sulfate (SDS)-polyacrylamide gels (electrophoresis grade) were obtained from Bio-Rad (Richmond, CA). Blotting grade affinity-purified goat anti-rabbit IgG alkaline phosphatase conjugate was purchased from Promega Corp. (Madison, WI). Protein standards of known mol wt (Mr) were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of the best grade commercially available. Tissue culture supplies were purchased from Gibco (Grand Island, NY). L-[35S]Methionine (SA, 1045 Ci/mmol) was obtained from ICN Radiochemicals (Irvine, CA). Virgin female rats from the Holtzman Co. (Madison, WI) were housed in wire cages with a 12-h light cycle. Food and water were provided ad libitum. Animals were mated overnight and examined in the morning for the presence of a vaginal plug (day 0 of pregnancy). Two or more pregnant rats (days 14, 15, and 17 of gestation) were studied for each experimental condition. Placental tissue was removed from anesthetized rats aseptically, and either fixed for immunocytochemistry or dissected and cultured. The nomenclature of Davies and Glasser (5) was used to describe the respective zones of the rat placenta. The term basal zone refers to the zone between the labyrinth and decidua basalis, which is without fetal mesenchyme and in which only maternal vascular channels are found.
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transferred from gels to nitrocellulose paper, and the blot was probed with an antipeptide antiserum to rPLP-B. The antiserum was generated against an oligopeptide representing amino acids 186-200 of the deduced sequence of rPLP-B. Three or four proteins cross-reacted with the rPLP-B antiserum, which had a Mr of 30,000, with a pi varying from 6.1-6.6 (Fig. 1C). When normal rabbit serum (1:1,000) was substituted for rPLP-B antiserum, these proteins were not observed (data not shown). The continuous line seen in the rPLP-B immunoblot is also observed with some lots of nonimmune rabbit serum and is interpreted to be nonspecific staining. The specific immunoreactive rPLP-B proteins corresponded to a group of [35S]methionine-labeled spots with a similar weight and pi range, which are identified by an arrow in Fig. 1A. The multiple immunoreactive proteins observed on the blot most likely represent different isoelectric isoforms of the rPLP-B protein that can be easily visualized after two-dimensional SDS-poly-
Anti-PS/5G
acrylamide gel electrophoresis. Mr isoforms have been identified for two other rat PRL-related proteins, rPLII and rPLP-A (10, 16). Specificity of the rPLP-B antisera Insofar as the synthesis of other members of the rPRL gene family, rPL-II and rPLP-A, have been localized to the basal zone of the placenta (17), we further examined the specificity of the rPLP-B antipeptide antisera compared with antisera against rPL-II and rPLP-A. A rPLII antiserum generated against an oligopeptide representing amino acids 56-70 of the protein was used to probe Western blots of the total profile of proteins secreted by basal zone explants. The anti-rPL-II antisera recognized a group of proteins with a Mr of 25,000 and a pi of 6.2-6.8 (Fig. ID). A group of [35S]methioninelabeled proteins correspond to the same location as the rPL-II-immunoreactive proteins (Fig. 1A) and were
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FIG. 1. Fluorographs and immunoblots of two-dimensional SDS-polyacrylamide gel analysis of proteins secreted by explant cultures of rat basal zone. A, Fluorogram of total [35S]methionine-labeled proteins in culture medium. Proteins that cross-react with antiserum against rPLP-B (shown in C) are indicated by an arrow. B, Fluorogram of 3SS-labeled proteins secreted in the presence of tunicamycin. C, Immunoblot of secreted proteins with anti-rPLP-B (1:1000). D, Immunoblot of secreted proteins with anti-rPL-II (1:500). E, Immunoblot of secreted proteins with anti-rPLP-A (1:200). F, Immunoblot of secreted proteins with anti-human PS0G (1:200).
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Inhibition of rPLP-B secretion by tunicamycin The presence of tunicamycin, an inhibitor of JV-linked glycosylation, during the explant incubation period inhibited the secretion of a number of proteins synthesized de novo by the basal zone explants (Fig. IB). A comparison of the fluorographs in Fig. 1, A and B, with the immunoblots in Fig. 1, C and E, shows that rPLP-B and rPLP-A are among the [35S]methionine-labeled proteins that can no longer be visualized on the fluorograph of the proteins secreted from tunicamycin-treated cultures (Fig. IB). For rPLP-B, this observation is consistent with the reported presence of one potential glycosylation site derived from the mRNA sequence (1). Immunoblots of the conditioned medium from tunicamycin-treated cultures failed to detect any lower Mr rPLP-B products (data not shown), nor were any new smaller [35S]methionine-labeled proteins observed in the fluorograph of secretory products after tunicamycin treatment (Fig. IB). For rPLP-A, these data confirm the report of Deb et al. (10) that the 30,000 and 32,000 Mr rPLP-A proteins are glycosylated. In contrast, the de novo synthesis and
FIG. 2. Light micrographs of rat placenta stained with the avidinbiotin-peroxidase complex method. A, Day 14 of gestation, with antirPLP-B (1:200) as the primary antibody. The area of the placenta shown is the basal zone, and arrows indicate some of the stained basophilic cytotrophoblast cells. Magnification, X250. B, Day 14 of gestation, with nonimmune rabbit serum (1:200). Magnification, X250. C, Day 17 of gestation with anti rPLP-B (1:200) as the primary antibody. Arrows indicate some of the stained basophilic cytotrophoblast cells. Magnification, X250. gc, Glycogen cells.
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clearly different from the proteins recognized by the antirPLP-B antisernm. rPLP-A antiserum generated against an oligopeptide representing amino acids 160-176 of rPLP-A recognized a third distinct group of proteins with Mr of 30,000 and 32,000 and pi values of 7.9 and 7.6, respectively (Fig. IE). The corresponding [35S]methionine-labeled rPLP-A proteins were siginificant products of the basal zone tissue (Fig. 1A). The Mr values of the two proteins identified by the rPLP-A antiserum were similar to those recently reported by Deb et al. (10), using antisera generated against a similar oligopeptide sequence of rPLP-A. This is the first evidence of the very basic nature of these two proteins. Our recent studies have identified two proteins that cross-react with antiserum against human pregnancy-specific /Si-glycoprotein (PS/3G) that are synthesized de novo and secreted by explant cultures of rat basal zone tissue (18). Both the major 120,000 Mr and the minor 76,000 Mr pregnancy-specific /?i-glycoprotein species were detected on Western blots of the secretory products of the basal zone explants (Fig. IF). Thus, the rPLP-B antiserum crossreacted with a distinct group of proteins which was not recognized by the other three antisera.
E n d o • 1990 Vol 126 • No 5
PRL-LIKE PROTEIN-B secretion of rPL-II proteins were not inhibited in tunicamycin-treated explants (Fig. IB), which is consistent with the report that rPL-II is not a glycoprotein (8, 9).
Photomicrographs in Fig. 2 show sections of rat placenta on days 14 (Fig. 2A) and 17 (Fig. 2C) of gestation stained with rPLP-B antiserum. Specific staining was observed only in the basal zone of the placenta and was localized to the basophilic cytotrophoblast cells. The clear ovals noted within the stained basophilic trophoblast cells represent nuclear profiles that do not contain detectable rPLP-B. The perinuclear pattern of rPLP-B staining was observed not only on gestation days 14 and 17, but also on days 11, 12, 13, 15, and 16 (data not shown). The perinuclear pattern and intensity of staining of basophilic cytotrophoblast cells were similar despite the major morphological changes that occur in the basal zone during this period of gestation. As can be observed in Fig. 2, glycogen cells (Fig. 2C) replaced basophilic cytotrophoblast cells (Fig. 2A) as the predominate cell type in the basal zone between days 14-17. No specific staining was observed in rat placenta with normal rabbit serum on day 14 (Fig. 2B) or on any other day studied.
Discussion The rat placenta is a rich source of PRL-related mRNAs. The mRNAs for rPLP-A and rPLP-B have been recently described (1, 4) in addition to that for rPLII, which alone represents 0.7% of day 17 rat placental mRNA (3). The protein products of both the rPL-II and the rPLP-A mRNA have been identified and characterized (8-10). Our study is the first to identify the protein product of the rPLP-B gene. Immunocytochemical studies of day 14 and 17 placentas showed that the rPLP-B cross-reactive proteins are localized to the basophilic cytotrophoblast of the basal zone of the placenta, consistent with previous in situ mRNA hybridization studies (7). The pattern and intensity of staining in the basophilic cytotrophoblast cells appeared consistent through days 11-17 of gestation. We observed no rPLP-B immunostaining in giant trophoblast cells. Duckworth et al. (1) have reported that two rPLP-B mRNAs of approximately 0.9 and 1.2 kilobases are strongly expressed from day 13 to term, but rPLP-B mRNA expression can be seen at low levels as early as day 10. The two mRNAs differ in their 5'-untranslated region and presumably code for identical proteins. In comparison, recent studies by Campbell et al. (17) have shown that rPL-II protein and mRNA levels are highest in the junctional zone (basal zone) on day 13, declining to term, and rPL-II protein was localized to the cytoplasm of giant cells (17), in agreement with previously reported in situ hybridiza-
tion experiments (7). The rPL-II cytoplasmic immunostaining in giant cells was shown to be perinuclear (17), similar to the pattern of staining we observed with rPLPB in small basophilic cytotrophoblast cells. Campbell et al. (17) also reported that rat PLP-A mRNA increased from low levels on day 13 to higher levels by day 19 in both the junctional (basal) and labyrinth zones, while PLP-A proteins were detected only during the latter stages of gestation in giant trophoblast cells and spongiotrophoblast (basophilic cytotrophoblast) cells of the junctional (basal) zone (17). The antiserum used in the present study to identify rPLP-A on the Western blot confirms Mr values reported by other investigators (10), but this antiserum was not successful when used for immunocytochemistry. It may be significant that the anti-rPLP-A antiserum used by Campbell et al. (17) for immunocytochemistry was generated against a different oligopeptide than the anti-rPLP-A antiserum used in our study. Interestingly, a protein that exhibits cross-reactivity with antiserum to human pregnancy-specific /?i-glycoprotein has also been localized to the basophilic cytotrophoblast of the rat placenta during mid- to-late gestation (18, 19). In contrast to rPLP-B, a progressive gestational shift was observed in the localization of PSjSG-like proteins in the basal zone (18). The physiological function of PS/3G during pregnancy is presently unknown, although it has been suggested that PS/3G may have immunosuppressive activity (20, 21). Other proposed functions stem from shared sequence homology between PS/3G and carcinoembryonic antigen, both of which are oncofetal proteins (22-26). The function(s) of the PLPs remains to be elucidated, but certainly the specific temporal appearance and expression in different cell types suggest important and perhaps distinct roles for each of these proteins during feto-placental development. Their homology with pituitary PRL, levels of which are very low at this stage of pregnancy, might suggest a placental replacement for the pituitary hormone. This study is an initial step in the characterization of rPLP-B which should lead to a better understanding of the roles of PLPs in pregnancy. Acknowledgments The authors wish to thank Theresa Medrano, Stacie Cohen, and Ingo Schroedter for their technical assistance, and M. J. Soares for his gift of antiserum against rPL-II. We also thank Mrs. Judy Adams for manuscript preparation.
References 1. Duckworth ML, Peden LM, Friesen HG 1988 A third prolactinlike protein expressed by the developing rat placenta: complementary deoxyribonucleic acid sequence and partial structure of the gene. Mol Endocrinol 2:912 2. Shiu RP, Kelly PA, Friesen HG 1973 Radioreceptor assay for prolactin and other lactogenic hormones. Science 180:968
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Immunocytochemistry
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middle and late pregnancy. Biol Reprod 32:840 16. Soares MJ, De SK, Foster BA, Julian JA, Glasser SR 1988 Identification of multiple low molecular weight placental prolactin-like proteins produced by rat trophoblast cells. J Endocrinol 116:101 17. Campbell WJ, Deb A, Kwok SCM, Joslin JA, Soares MJ 1989 Differential expression of placental lactogen-II and prolactin-like protein-A in the rat chorioallantoic placenta. Endocrinology 125:1565 18. Ogilvie S, Kvello-Stenstrom AG, Hammond G, Buhi WC, Larkin LH, Shiverick KT 1989 Identification of proteins immunochemically related to pregnancy-specific ^-glycoprotein in the rat placenta. Endocrinology 125:287 19. Macpherson TA, Zheng SY, Kunz HW, Ghani A, Gill TJ 1985 The immunohistochemical localization of pregnancy-specific /Srglycoprotein in the postimplantation rat trophoblast. Placenta 6:427 20. Cerni C, Tatra G, Bohn H 1977 Immunosuppression by human placental lactogen (HPL) and pregnancy-specific /3i-glycoprotein (SPj). Arch Gynaekol 223:1 21. Tatarinov YS 1982 Pregnancy-specific /Jrglycoprotein, placentaspecific £*!- and a2-microglobulins. In: Grudzinskas JG, Teisner B, Seppala M (eds) Pregnancy Proteins. Academic Press, New York, pl79 22. Tatarinov YS 1978 Trophoblast-specific jSi-glycoprotein as a marker for pregnancy and malignancies. Gynecol Obstet Invest 9:65 23. Streydio C, Lacka K, Swillens S, Vassart G 1988 The human pregnancy-specific /^-glycoprotein (PS/3G) and the carcinoembryonic anigen (CEA) -related proteins are members of the same multigene family. Biochem Biophys Res Commun 154:130 24. Oikawa S, Inuzuka C, Kosaki G, Nadazato H 1988 Exon-intron organization of a gene for pregnancy-specific jSrglycoprotein, a subfamily of CEA family: implications for its characteristic repetitive domains and C-terminal sequences. Biochem Biophys Res Commun 156:68 25. Wattanabe S, Chou JY 1988 Human pregnancy-specific /Vglycoprotein: a new member of the carcinoembryonic antigen gene family. Biochem Biophys Res Commun 152:762 26. Chan WY, Borjigin FJ, Zheng QX, Shupert WL 1988 Characterization of cDNA encoding human pregnancy-specific /?! -glycoprotein from placental and extraplacental tissue and their comparsion with carcinoembryonic antigen. DNA 7:545
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3. Duckworth ML, Kirk KL, Friesen HG 1986 Isolation and identification of a cDNA clone of rat placental lactogen II. J Biol Chem 261:10871 4. Duckworth ML, Peden LM, Friesen HG 1986 Isolation of a novel prolactin-like cDNA clone from developing rat placenta. J Biol Chem 261:10879 5. Davies J, Glasser SR 1968 Histological and fine structural observations on the placenta of the rat. Acta Anat (Basel) 69:542 6. Jollie WP 1965 Fine structural changes in the junctional zone of the rat placenta with increasing gestational age. J Ultrastruct Res 12:420 7. Duckworth ML, Schroedter I, Friesen HG, Cellular localization of rat placental lactogen II and rat prolactin-like proteins A and B by in situ hybridization. Placenta, in press 8. Robertson MC, Friesen HG 1975 The purification and characterization of rat placental lactogen. Endocrinology 97:621 9. Robertson MC, Friesen HG 1981 Two forms of rat placental lactogen revealed by immunoassay. Endocrinology 108:2388 10. Deb S, Youngblood T, Rawitch AB, Soares MJ 1989 Placental prolactin-like protein A. Identification and characterization of two major glycoprotein species with antipeptide antibodies. J Biol Chem 264:14348 11. Deb S, Hashizume K, Boone K, Southard JN, Talamantes F, Rawitch A, Soares MJ 1989 Antipeptide antibodies reveal structural and functional characteristics of rat placental lactogen-II. Mol Cell Endocrinol 63:45 12. Roberts RM, Baumbach GA, Buhi WC, Denny JB, Fitzgerald LA, Babelyn SF, Horst, MN 1984 Analysis of membrane polypeptides by two dimensional polyacrylamide gel electrophoresis. In: Venter JC, Harrison LC (eds) Receptor Biochemistry and Methodology: Molecular and Chemical Characterization of Membrane Receptors. Liss, New York, vol 111:61 13. Chamberlain JP 1979 Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem 98:132 14. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350 15. Renegar RH, Larkin LH 1985 Relaxin concentrations in endometrial, placental and ovarian tissues and in sera from ewes during
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Vol. 126, No. 5 Printed in U.S.A.
S. OGILVIE, M. L. DUCKWORTH, L. H. LARKIN, W. C. BUHI, AND K. T. SHIVERICK Departments of Pharmacology and Therapeutics (S.O., K.T.S.), Anatomy and Cell Biology (L.H.L.), and Obstetrics and Gynecology (W.C.B.), University of Florida, Gainesville, Florida 32610; and the Department of Physiology, University of Manitoba (M.L.D.), Winnipeg, Manitoba, R3E 0W3 Canada
ABSTRACT. Mid- to late-gestation rat placenta expresses three PRL-related mRNAs, rat placental lactogen-II (rPL-II), rat PRL-like protein-A (rPLP-A), and rat PRL-like protein-B (rPLP-B). The protein product of rPL-II mRNA has been characterized, and the protein products of the rPLP-A mRNA were recently identified. The mol wt of a nonsecreted nonglycosylated rPLP-B protein would be 27,145 based on the mRNA sequence. The present study is the first to report the identification of the rPLP-B protein. Antiserum was generated against a chemically synthesized oligopeptide inferred from a specific region of the rPLP-B cDNA. Three or four distinct proteins synthesized and secreted by rat basal zone explants (day 15 gestation) showed cross-reactivity with the rPLP-B antiserum. The relative mol wt of these immunoreactive proteins is approximately 30,000, with a pi varying from 6.1-6.6. De novo synthesized rPLP-B proteins were not secreted by the explant tissue
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in the presence of tunicamycin, suggesting that the proteins are glycosylated. These data are consistent with the presence of one potential iV-glycosylation site derived from the rPLP-B mRNA sequence. The rPLP-B antiserum showed no cross-reactivity with proteins identified using antisera against rPLP-A, rPL-II, or human pregnancy-specific /Vglycoprotein. Immunocytochemical studies were carried out using paraffin sections from placentas of day 14 and 17 pregnant rats which were treated with anti-rPLP-B, followed by avidin-biotin-peroxidase complex. These experiments show perinuclear staining, which was localized in basophilic cytotrophoblast cells, confirming previous in situ mRNA hybridization studies. Although no physiological role has been established for rPLP-B, the synthesis and secretion of this protein by cells in contact only with maternal circulation suggest a hormonal role. (Endocrinology 126: 2561-2566, 1990)
HE PRL/GH gene family in the rat has recently expanded with the identification of a third PRLrelated mRNA in the mid- to late-gestation placenta (1). The predicted amino acid sequence of the new placental protein, rat PRL-like protein-B (rPLP-B), exhibits 44% homology to pituitary PRL, which in the rodent is secreted at very low levels from midpregnancy to term (2). The two previously identified placental family members, rat placental lactogen-II (rPL-II) and rat PRL-like protein-A (rPLP-A), show 52% and 45% homology, respectively, to rat PRL at the amino acid level (3, 4). On day 16 of gestation, the three histologically distinct cellular elements in the basal zone of the placenta (5, 6) include the small basophilic cytotrophoblast cells, glycogen cells, and giant trophoblastic cells. In situ hybridization studies have localized mRNA for rPL-II in the giant cells of the basal zone and in cells in the labyrinth region, while mRNA for rPLP-A was detected in the basophilic cytotrophoblast cells and the giant cells of the basal zone,
and mRNA for rPLP-B was detected in the basophilic cytotrophoblast cells only (7). In addition to different locations of the mRNAs of these PLPs, there appears to be different temporal expression of the mRNAs. The rPL-II mRNA is first detected on day 12, and its level remains high throughout gestation (3), whereas the mRNAs for rPLP-A and rPLP-B are first strongly expressed on day 14, 2 days later than rPL-II, and remain high until birth (1, 4). Of the three proteins, rPL-II has been purified and characterized (8, 9), while rPLP-A was only recently identified using an antipeptide antiserum (10). In this study we report the identification of proteins synthesized and secreted by rat basal zone explants which cross-react with an antiserum generated against a chemically synthesized oligopeptide inferred from a specific region of the rPLP-B cDNA. Identification of the rPLP-B gene product will lead to a better understanding of the function and temporal synthesis of PLPs in rat placenta.
Received October 6,1989. Address requests for reprints to: Dr. Kathleen T. Shiverick, Department of Pharmacology and Therapeutics, University of Florida, Box J267 JHMHC, Gainesville, Florida 32610. *This work was supported in part by NIH Grant ES-04435 (to K.T.S.).
Materials and Methods Materials Avidin-biotin-peroxidase complex for rabbit immunoglobulin G (IgG) was obtained from the Vectastain ABC kit (Vector,
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De Novo Synthesis and Secretion of Prolactin-Like Protein-B by Rat Placental Explants*
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Oligopeptide synthesis and antibody production The synthetic peptide Cys-Asn-Glu-Leu-Ala-Tyr-Leu-ArgSer-Pro-Asp-Glu-Glu-Arg-Arg-His was custom synthesized by the Institut Armand Frappier (Montreal, Quebec, Canada) and coupled to keyhole limpet hemocyanin. This peptide represents amino acids 186-200 in the deduced amino acid sequence (including the signal peptide) of rPLP-B (1), with an additional cysteine at the amino-terminus. This sequence was selected on the basis of its hydrophobic profile and its difference from sequences of other members of the rPRL gene family. New Zealand White rabbits were injected sc with 540 fig peptide conjugated to keyhole limpet hemocyanin in complete Freund's adjuvant and boosted sc with the same concentration of conjugated peptide in incomplete Freund's adjuvant on days 14 and 28 after the initial injection. The rabbits were bled 13 days after the final boost. The antiserum was able to detect 100 ng pure peptide at a 1:40,000 dilution by dot blot assay, using the alkaline phosphatase detection method. Rabbit polyclonal antiserum against rPLP-A was generated against an oligopeptide representing amino acids 160-176 of the deduced amino acid sequence (including signal peptide) of rPLP-A (4), with an additional cysteine at the amino-terminus, as described above. This oligopeptide is the same as the oligopeptide described by Deb et al. (10), representing amino acids 129-145 (excluding the signal peptide) of rPLP-A. The antiserum can detect 5-10 ng rPLP-A peptide at a 1:60,000 dilution by dot blot, using alkaline phosphatase as the substrate. A rabbit polyclonal antiserum against rPL-II was generated
against an oligopeptide representing amino acids 56-70 of rPLII; this was a generous gift from M. J. Soares (11). Placental explant culture Basal zone tissue was incubated under sterile conditions in Modified Eagle's Medium (175 mg tissue/5 ml) for 24 h at 37 C under 47.5% O2-2.5% CO2-50% N2. The culture medium was supplemented with 3 mg/ml glucose, penicillin (100 U/ml), streptomycin (100 Mg/ml), fungizone (0.25 Mg/ml), 1% (vol/vol) nonessential amino acids, and 1.5 fig/ml methionine (one tenth the normal concentration). In some experiments tunicamycin was added from a 10 mg/ml final concentration stock solution in dimethylsulfoxide at the initiation of culture. Dimethylsulfoxide alone was added to control explants. The radiolabel, [35S] methionine (40 /uCi) was added 16 h after the initiation of culture. After incubation, culture medium was dialyzed extensively (Mr cut-off, 3500), and aliquots were counted. Polyacrylamide gel electrophoresis and immunoblotting Two-dimensional SDS-polyacrylamide gel electrophoresis of the samples was performed according to methods described by Roberts et al. (12). Equal amounts of [35S]methionine-labeled protein (100,000 cpm) were loaded onto each gel. After electrophoresis, slab gels were fixed, prepared for fluorography using sodium salicylate (13), and dried. Fluorographs were prepared with Kodak XAR x-ray film (Eastman Kodak, Rochester, NY). Alternatively, gels were equilibrated for 30 min in electrophoretic transfer buffer containing 0.025 M Tris, 0.192 M glycine, and 20% (vol/vol) methanol, pH 8.3, and the proteins were transferred from gels to nitrocellulose membrane for immunoblot analysis (14). After transfer, rPLP-B, rPL-II, and rPLPA polypeptides were detected using a rabbit polyclonal antiserum against rPLP-B (1:1000), a rabbit polyclonal antiserum against rPL-II (1:500), or a rabbit polyclonal against rPLP-A (1:200), followed by affinity-purified goat anti-rabbit IgG horseradish peroxidase conjugate. Avidin-biotin-peroxidase immunocytochemistry Whole fetal-placental units were fixed by immersion in Bouin's solution, dehydrated with a graded series of alcohols, and embedded in paraffin. rPLP-B was localized in deparaffinized rehydrated 6-^m sections by the avidin-peroxidase complex technique as outlined by Renegar and Larkin (15), except that the peroxidase substrate was 0.04% diaminobenzidine in PBS, pH 7.2. The anti-rPLP-B antiserum and normal rabbit serum were used at a dilution of 1:200.
Results De nouo synthesis of rPLP-B in explant cultures The [35S]methionine-labeled proteins secreted by explants of rat basal zone tissue (day 15 of gestation) were characterized using two-dimensional SDS-polyacrylamide gel electrophoresis, as shown by a representative fluorograph in Fig. 1A. The total proteins secreted by explants of basal zone tissue shown in Fig. 1A were
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Burlingame, CA). Rabbit antiserum generated against pregnancy-specific jSrglycoprotein (PS/3G) purified from human pregnancy serum was made in Denmark by Dakopatts (Accurate Chemical Co., Westbury, NY). Blotting grade affinitypurified goat anti-rabbit IgG horseradish peroxidase conjugate and reagents for sodium dodecyl sulfate (SDS)-polyacrylamide gels (electrophoresis grade) were obtained from Bio-Rad (Richmond, CA). Blotting grade affinity-purified goat anti-rabbit IgG alkaline phosphatase conjugate was purchased from Promega Corp. (Madison, WI). Protein standards of known mol wt (Mr) were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of the best grade commercially available. Tissue culture supplies were purchased from Gibco (Grand Island, NY). L-[35S]Methionine (SA, 1045 Ci/mmol) was obtained from ICN Radiochemicals (Irvine, CA). Virgin female rats from the Holtzman Co. (Madison, WI) were housed in wire cages with a 12-h light cycle. Food and water were provided ad libitum. Animals were mated overnight and examined in the morning for the presence of a vaginal plug (day 0 of pregnancy). Two or more pregnant rats (days 14, 15, and 17 of gestation) were studied for each experimental condition. Placental tissue was removed from anesthetized rats aseptically, and either fixed for immunocytochemistry or dissected and cultured. The nomenclature of Davies and Glasser (5) was used to describe the respective zones of the rat placenta. The term basal zone refers to the zone between the labyrinth and decidua basalis, which is without fetal mesenchyme and in which only maternal vascular channels are found.
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PRL-LIKE PROTEIN-B 8
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transferred from gels to nitrocellulose paper, and the blot was probed with an antipeptide antiserum to rPLP-B. The antiserum was generated against an oligopeptide representing amino acids 186-200 of the deduced sequence of rPLP-B. Three or four proteins cross-reacted with the rPLP-B antiserum, which had a Mr of 30,000, with a pi varying from 6.1-6.6 (Fig. 1C). When normal rabbit serum (1:1,000) was substituted for rPLP-B antiserum, these proteins were not observed (data not shown). The continuous line seen in the rPLP-B immunoblot is also observed with some lots of nonimmune rabbit serum and is interpreted to be nonspecific staining. The specific immunoreactive rPLP-B proteins corresponded to a group of [35S]methionine-labeled spots with a similar weight and pi range, which are identified by an arrow in Fig. 1A. The multiple immunoreactive proteins observed on the blot most likely represent different isoelectric isoforms of the rPLP-B protein that can be easily visualized after two-dimensional SDS-poly-
Anti-PS/5G
acrylamide gel electrophoresis. Mr isoforms have been identified for two other rat PRL-related proteins, rPLII and rPLP-A (10, 16). Specificity of the rPLP-B antisera Insofar as the synthesis of other members of the rPRL gene family, rPL-II and rPLP-A, have been localized to the basal zone of the placenta (17), we further examined the specificity of the rPLP-B antipeptide antisera compared with antisera against rPL-II and rPLP-A. A rPLII antiserum generated against an oligopeptide representing amino acids 56-70 of the protein was used to probe Western blots of the total profile of proteins secreted by basal zone explants. The anti-rPL-II antisera recognized a group of proteins with a Mr of 25,000 and a pi of 6.2-6.8 (Fig. ID). A group of [35S]methioninelabeled proteins correspond to the same location as the rPL-II-immunoreactive proteins (Fig. 1A) and were
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FIG. 1. Fluorographs and immunoblots of two-dimensional SDS-polyacrylamide gel analysis of proteins secreted by explant cultures of rat basal zone. A, Fluorogram of total [35S]methionine-labeled proteins in culture medium. Proteins that cross-react with antiserum against rPLP-B (shown in C) are indicated by an arrow. B, Fluorogram of 3SS-labeled proteins secreted in the presence of tunicamycin. C, Immunoblot of secreted proteins with anti-rPLP-B (1:1000). D, Immunoblot of secreted proteins with anti-rPL-II (1:500). E, Immunoblot of secreted proteins with anti-rPLP-A (1:200). F, Immunoblot of secreted proteins with anti-human PS0G (1:200).
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Inhibition of rPLP-B secretion by tunicamycin The presence of tunicamycin, an inhibitor of JV-linked glycosylation, during the explant incubation period inhibited the secretion of a number of proteins synthesized de novo by the basal zone explants (Fig. IB). A comparison of the fluorographs in Fig. 1, A and B, with the immunoblots in Fig. 1, C and E, shows that rPLP-B and rPLP-A are among the [35S]methionine-labeled proteins that can no longer be visualized on the fluorograph of the proteins secreted from tunicamycin-treated cultures (Fig. IB). For rPLP-B, this observation is consistent with the reported presence of one potential glycosylation site derived from the mRNA sequence (1). Immunoblots of the conditioned medium from tunicamycin-treated cultures failed to detect any lower Mr rPLP-B products (data not shown), nor were any new smaller [35S]methionine-labeled proteins observed in the fluorograph of secretory products after tunicamycin treatment (Fig. IB). For rPLP-A, these data confirm the report of Deb et al. (10) that the 30,000 and 32,000 Mr rPLP-A proteins are glycosylated. In contrast, the de novo synthesis and
FIG. 2. Light micrographs of rat placenta stained with the avidinbiotin-peroxidase complex method. A, Day 14 of gestation, with antirPLP-B (1:200) as the primary antibody. The area of the placenta shown is the basal zone, and arrows indicate some of the stained basophilic cytotrophoblast cells. Magnification, X250. B, Day 14 of gestation, with nonimmune rabbit serum (1:200). Magnification, X250. C, Day 17 of gestation with anti rPLP-B (1:200) as the primary antibody. Arrows indicate some of the stained basophilic cytotrophoblast cells. Magnification, X250. gc, Glycogen cells.
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clearly different from the proteins recognized by the antirPLP-B antisernm. rPLP-A antiserum generated against an oligopeptide representing amino acids 160-176 of rPLP-A recognized a third distinct group of proteins with Mr of 30,000 and 32,000 and pi values of 7.9 and 7.6, respectively (Fig. IE). The corresponding [35S]methionine-labeled rPLP-A proteins were siginificant products of the basal zone tissue (Fig. 1A). The Mr values of the two proteins identified by the rPLP-A antiserum were similar to those recently reported by Deb et al. (10), using antisera generated against a similar oligopeptide sequence of rPLP-A. This is the first evidence of the very basic nature of these two proteins. Our recent studies have identified two proteins that cross-react with antiserum against human pregnancy-specific /Si-glycoprotein (PS/3G) that are synthesized de novo and secreted by explant cultures of rat basal zone tissue (18). Both the major 120,000 Mr and the minor 76,000 Mr pregnancy-specific /?i-glycoprotein species were detected on Western blots of the secretory products of the basal zone explants (Fig. IF). Thus, the rPLP-B antiserum crossreacted with a distinct group of proteins which was not recognized by the other three antisera.
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PRL-LIKE PROTEIN-B secretion of rPL-II proteins were not inhibited in tunicamycin-treated explants (Fig. IB), which is consistent with the report that rPL-II is not a glycoprotein (8, 9).
Photomicrographs in Fig. 2 show sections of rat placenta on days 14 (Fig. 2A) and 17 (Fig. 2C) of gestation stained with rPLP-B antiserum. Specific staining was observed only in the basal zone of the placenta and was localized to the basophilic cytotrophoblast cells. The clear ovals noted within the stained basophilic trophoblast cells represent nuclear profiles that do not contain detectable rPLP-B. The perinuclear pattern of rPLP-B staining was observed not only on gestation days 14 and 17, but also on days 11, 12, 13, 15, and 16 (data not shown). The perinuclear pattern and intensity of staining of basophilic cytotrophoblast cells were similar despite the major morphological changes that occur in the basal zone during this period of gestation. As can be observed in Fig. 2, glycogen cells (Fig. 2C) replaced basophilic cytotrophoblast cells (Fig. 2A) as the predominate cell type in the basal zone between days 14-17. No specific staining was observed in rat placenta with normal rabbit serum on day 14 (Fig. 2B) or on any other day studied.
Discussion The rat placenta is a rich source of PRL-related mRNAs. The mRNAs for rPLP-A and rPLP-B have been recently described (1, 4) in addition to that for rPLII, which alone represents 0.7% of day 17 rat placental mRNA (3). The protein products of both the rPL-II and the rPLP-A mRNA have been identified and characterized (8-10). Our study is the first to identify the protein product of the rPLP-B gene. Immunocytochemical studies of day 14 and 17 placentas showed that the rPLP-B cross-reactive proteins are localized to the basophilic cytotrophoblast of the basal zone of the placenta, consistent with previous in situ mRNA hybridization studies (7). The pattern and intensity of staining in the basophilic cytotrophoblast cells appeared consistent through days 11-17 of gestation. We observed no rPLP-B immunostaining in giant trophoblast cells. Duckworth et al. (1) have reported that two rPLP-B mRNAs of approximately 0.9 and 1.2 kilobases are strongly expressed from day 13 to term, but rPLP-B mRNA expression can be seen at low levels as early as day 10. The two mRNAs differ in their 5'-untranslated region and presumably code for identical proteins. In comparison, recent studies by Campbell et al. (17) have shown that rPL-II protein and mRNA levels are highest in the junctional zone (basal zone) on day 13, declining to term, and rPL-II protein was localized to the cytoplasm of giant cells (17), in agreement with previously reported in situ hybridiza-
tion experiments (7). The rPL-II cytoplasmic immunostaining in giant cells was shown to be perinuclear (17), similar to the pattern of staining we observed with rPLPB in small basophilic cytotrophoblast cells. Campbell et al. (17) also reported that rat PLP-A mRNA increased from low levels on day 13 to higher levels by day 19 in both the junctional (basal) and labyrinth zones, while PLP-A proteins were detected only during the latter stages of gestation in giant trophoblast cells and spongiotrophoblast (basophilic cytotrophoblast) cells of the junctional (basal) zone (17). The antiserum used in the present study to identify rPLP-A on the Western blot confirms Mr values reported by other investigators (10), but this antiserum was not successful when used for immunocytochemistry. It may be significant that the anti-rPLP-A antiserum used by Campbell et al. (17) for immunocytochemistry was generated against a different oligopeptide than the anti-rPLP-A antiserum used in our study. Interestingly, a protein that exhibits cross-reactivity with antiserum to human pregnancy-specific /?i-glycoprotein has also been localized to the basophilic cytotrophoblast of the rat placenta during mid- to-late gestation (18, 19). In contrast to rPLP-B, a progressive gestational shift was observed in the localization of PSjSG-like proteins in the basal zone (18). The physiological function of PS/3G during pregnancy is presently unknown, although it has been suggested that PS/3G may have immunosuppressive activity (20, 21). Other proposed functions stem from shared sequence homology between PS/3G and carcinoembryonic antigen, both of which are oncofetal proteins (22-26). The function(s) of the PLPs remains to be elucidated, but certainly the specific temporal appearance and expression in different cell types suggest important and perhaps distinct roles for each of these proteins during feto-placental development. Their homology with pituitary PRL, levels of which are very low at this stage of pregnancy, might suggest a placental replacement for the pituitary hormone. This study is an initial step in the characterization of rPLP-B which should lead to a better understanding of the roles of PLPs in pregnancy. Acknowledgments The authors wish to thank Theresa Medrano, Stacie Cohen, and Ingo Schroedter for their technical assistance, and M. J. Soares for his gift of antiserum against rPL-II. We also thank Mrs. Judy Adams for manuscript preparation.
References 1. Duckworth ML, Peden LM, Friesen HG 1988 A third prolactinlike protein expressed by the developing rat placenta: complementary deoxyribonucleic acid sequence and partial structure of the gene. Mol Endocrinol 2:912 2. Shiu RP, Kelly PA, Friesen HG 1973 Radioreceptor assay for prolactin and other lactogenic hormones. Science 180:968
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middle and late pregnancy. Biol Reprod 32:840 16. Soares MJ, De SK, Foster BA, Julian JA, Glasser SR 1988 Identification of multiple low molecular weight placental prolactin-like proteins produced by rat trophoblast cells. J Endocrinol 116:101 17. Campbell WJ, Deb A, Kwok SCM, Joslin JA, Soares MJ 1989 Differential expression of placental lactogen-II and prolactin-like protein-A in the rat chorioallantoic placenta. Endocrinology 125:1565 18. Ogilvie S, Kvello-Stenstrom AG, Hammond G, Buhi WC, Larkin LH, Shiverick KT 1989 Identification of proteins immunochemically related to pregnancy-specific ^-glycoprotein in the rat placenta. Endocrinology 125:287 19. Macpherson TA, Zheng SY, Kunz HW, Ghani A, Gill TJ 1985 The immunohistochemical localization of pregnancy-specific /Srglycoprotein in the postimplantation rat trophoblast. Placenta 6:427 20. Cerni C, Tatra G, Bohn H 1977 Immunosuppression by human placental lactogen (HPL) and pregnancy-specific /3i-glycoprotein (SPj). Arch Gynaekol 223:1 21. Tatarinov YS 1982 Pregnancy-specific /Jrglycoprotein, placentaspecific £*!- and a2-microglobulins. In: Grudzinskas JG, Teisner B, Seppala M (eds) Pregnancy Proteins. Academic Press, New York, pl79 22. Tatarinov YS 1978 Trophoblast-specific jSi-glycoprotein as a marker for pregnancy and malignancies. Gynecol Obstet Invest 9:65 23. Streydio C, Lacka K, Swillens S, Vassart G 1988 The human pregnancy-specific /^-glycoprotein (PS/3G) and the carcinoembryonic anigen (CEA) -related proteins are members of the same multigene family. Biochem Biophys Res Commun 154:130 24. Oikawa S, Inuzuka C, Kosaki G, Nadazato H 1988 Exon-intron organization of a gene for pregnancy-specific jSrglycoprotein, a subfamily of CEA family: implications for its characteristic repetitive domains and C-terminal sequences. Biochem Biophys Res Commun 156:68 25. Wattanabe S, Chou JY 1988 Human pregnancy-specific /Vglycoprotein: a new member of the carcinoembryonic antigen gene family. Biochem Biophys Res Commun 152:762 26. Chan WY, Borjigin FJ, Zheng QX, Shupert WL 1988 Characterization of cDNA encoding human pregnancy-specific /?! -glycoprotein from placental and extraplacental tissue and their comparsion with carcinoembryonic antigen. DNA 7:545
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3. Duckworth ML, Kirk KL, Friesen HG 1986 Isolation and identification of a cDNA clone of rat placental lactogen II. J Biol Chem 261:10871 4. Duckworth ML, Peden LM, Friesen HG 1986 Isolation of a novel prolactin-like cDNA clone from developing rat placenta. J Biol Chem 261:10879 5. Davies J, Glasser SR 1968 Histological and fine structural observations on the placenta of the rat. Acta Anat (Basel) 69:542 6. Jollie WP 1965 Fine structural changes in the junctional zone of the rat placenta with increasing gestational age. J Ultrastruct Res 12:420 7. Duckworth ML, Schroedter I, Friesen HG, Cellular localization of rat placental lactogen II and rat prolactin-like proteins A and B by in situ hybridization. Placenta, in press 8. Robertson MC, Friesen HG 1975 The purification and characterization of rat placental lactogen. Endocrinology 97:621 9. Robertson MC, Friesen HG 1981 Two forms of rat placental lactogen revealed by immunoassay. Endocrinology 108:2388 10. Deb S, Youngblood T, Rawitch AB, Soares MJ 1989 Placental prolactin-like protein A. Identification and characterization of two major glycoprotein species with antipeptide antibodies. J Biol Chem 264:14348 11. Deb S, Hashizume K, Boone K, Southard JN, Talamantes F, Rawitch A, Soares MJ 1989 Antipeptide antibodies reveal structural and functional characteristics of rat placental lactogen-II. Mol Cell Endocrinol 63:45 12. Roberts RM, Baumbach GA, Buhi WC, Denny JB, Fitzgerald LA, Babelyn SF, Horst, MN 1984 Analysis of membrane polypeptides by two dimensional polyacrylamide gel electrophoresis. In: Venter JC, Harrison LC (eds) Receptor Biochemistry and Methodology: Molecular and Chemical Characterization of Membrane Receptors. Liss, New York, vol 111:61 13. Chamberlain JP 1979 Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem 98:132 14. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350 15. Renegar RH, Larkin LH 1985 Relaxin concentrations in endometrial, placental and ovarian tissues and in sera from ewes during
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