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Analysis of rat placental plasma membrane proteins by two-dimensional gel electrophoresis
~lecularand C ellulrr
b
Endocrhbgy
ELSEVIER
Molecular and Cellular Endocrinology
I I5 (1995) l49- I59
Analysis of rat placental plasma membrane proteins by two-dimensional gel electrophoresis Ryuta
Ishimura,
Ken Noda,
Naka
Hattori,
Kunio
Shiota*,
Tomoya
Ogawa
Laboratory of’Cellular Biochemistry, Veterinary Medical Sciences/Animal Resource Sciences, University of Tokyo, I - 1- 1 YaJloi, Bunkyo-ku, Tokyo 113, Japan
Received 24 February 1995; accepted 6 September 1995
Abstract The placenta plays an essential role in fetal growth and the maintenance of pregnancy and its functions are strictly controlled in a stage-specific manner. To gain an insight into placental functions and their regulation, we analyzed the plasma membrane (2D/E). Plasma membrane fractions of the proteins of rat placenta by two-dimensional polyacrylamide gel electrophoresis
placenta obtained on days 12, 14, 16, 18 and 20 of pregnancy were purified by Percoll gradient centrifugation, and subjected to 2D/E analysis. After the proteins on the 2D/E gels had been visualized by silver staining, the patterns on the gels at different stages of pregnancy were compared using image analysis software. Proteins within an isoelectric point (pl) range of 4.0 to 7.0 and a molecular weight (M,) range of 20-100 kDa were analyzed in detail, and about 800 proteins on average were recognized on each gel. Of these, the expression of 150 proteins was found to change dramatically according to the stage of pregnancy. According to their expression patterns, these proteins were categorized into two groups, Group I and Group II. The proteins belonging to Group I showed a higher intensity of expression on day 12 and disappeared on day 20. They included 119 plasma membrane proteins and were divided into five subgroups. Group II. which consisted of three subgroups, included 31 proteins showing a low or negligible expression on day 12 and higher expression on day 20. Most of the other membrane proteins (about 600) were expressed constantly during pregnancy. On the basis of our data, we constructed a database for plasma membrane proteins
of the rat placenta.
Keywords:
Placenta; Plasma membrane
protein; Pregnancy; Rat; Two-dimensional
1. Introduction The placenta possesses multiple functions including regulation of the transport of nutrients and wastes (Morriss and Boyd, 1994) prevention of a directional
immune response between mother and fetus (Gill, 1994) and endocrine regulation to create and maintain the maternal physiological milieu by producing hormones such as placental lactogen (Albrecht and Pepe, 1990; Soares et al., 1991; Ogren and Talamantes, 1994; Shiota et al., 1994). Considering these multiple functions, not only functions that are required constantly but also pregnancy stage-specific events such as placental lactogen synthesis (Furuyama et al., 1991; Shiota et al., 1991; Hattori, et al., 1993; Hirosawa et al., 1994) the placenta is thought to express an abundance of biologi*Corresponding
author, Tel.: 81 3 3812 211 I, ext. 5372; Fax: 81 3
5689 7345. 0303-7207/95/$09.50 SSDI
8 1995 ~
0303-7207(95)03682-W
Elsevier Science Ireland
gel electrophoresis
tally active molecules. Therefore, plasma membrane proteins are thought to be involved in the transport of numerous materials and in various immunological and endocrinological processes. It is often the case that multiple intracellular signalling pathways are initiated at the cell surface by extracellular stimulation. The cellular composition of the placenta changes dramatically during pregnancy (Jollie, 1964a,b, 1965; Peel and Bulmer, 1977) and cell type-specific molecules have been identified, e.g., alkaline phosphatase is expressed on cytotrophoblast cells (Campbell et al., 1991) whereas Pa antigen, which is a class-I antigen, is expressed on spongiotrophoblast cells and trophoblast giant cells (TGCs) in the rat (Ho et al., 1987; KanbourShakir et al., 1993). It remains to be determined how many proteins are involved in placental functions and how they are regulated in a pregnancy stage-specific manner. Two-dimen-
Ltd. All rights reserved
150
R. Ishimuru
et ul.
; Molecular
und Cellular
sional polyacrylamide gel electrophoresis (2D/E) is a powerful method for analysis of plasma membrane proteins, because theoretically more than 1000 proteins including glycoproteins and phosphoproteins can be resolved on a single 2D/E gel (O’Farrell, 1975; Garrels, 1989; Garrels and Franza, 1989; Wirth et al., 1992; Latham et al., 1993; Guy et al., 1994). 2D/E analysis would provide information reflecting cellular composition as well as various cellular states, and it could be potentially useful for identifying the plasma membrane proteins involved in placental function. In the present study, in order to gain an insight into the regulation of placental functions, we analyzed placental plasma membrane proteins expressed on the rat placenta by 2D/E and constructed a protein database from their isoelectric point (pl) and molecular weight (M,) data. 2. Materials and Methods 2. I. Reagents and animals
Leupepsin and ethyleneglycol-bis-N,N,N’,N’-tetraacetic acid (EGTA) were purchased from Sigma (St. Louis, USA), Percoll was from LKB (Uppsala, Sweden), urea and protein quantifying reagents (Bio-Rad Protein Assay) were from Bio-Rad (Richmond, USA), nonidet P-40 (NP-40), acrylamide, ampholytes (pH 310) and dithiothreitol (DTT) were from Millipore (Bedford, USA), rabbit antiserum against rat transferrin was from Organon Teknika (Durham, USA) and phenylmethylsulfonyl fluoride (PMSF), silver staining reagents and other reagents were from Wako Pure Chemical Ind. (Osaka, Japan). Adult female Wistar-Imamichi rats were purchased from the Imamichi Institute for Animal Reproduction (Ibaraki, Japan). They were kept under a lighting regime of 14 h illumination and 10 h darkness (lights on between 05:OOh and 19:00 h) and allowed free access to food and water. Female rats were housed with a fertile male rat, and the day when sperm were observed in a vaginal smear was designated day 0 of pregnancy. 2.2. Isolation of plasma membrane fractions fLom placental tissue Three or four rats each on days 12, 14, 16, 18 and 20 of pregnancy were decapitated between 13:00 h and 14:00 h. The antimesometrial wall of the uterus was cut and the decidua was shelled out of the uterus with forceps. After removal of the embryos with forceps, the remaining placental tissue including decidual cells and trophoblasts was frozen quickly and stored at - 80°C until required for use. All subsequent procedures were carried out at 4°C. Isolation of plasma membrane fractions was performed as described previously using Percoll gradient centrifugation (Belsham et al., 1980). Briefly, the placental
Endwrinology
115 (1995)
149-159
tissue (1.0 g wet wt.) was cut into small pieces (each 2- 3 mm3) and homogenized in 5 ml of 10 mM TrisHCl (pH 7.5) containing 0.25 M sucrose, 2 mM EGTA, 0.5 mg/ml leupeptin and 1 mM PMSF (STE) with a teflon-glass homogenizer. After centrifugation at 150 x g (JAlO rotor: Beckman, Fullerton, USA) for 10 min, the supernatant was collected. The pellet was homogenized and centrifuged again. The supernatants were combined and centrifuged at 5000 x g (JAlO rotor) for 10 min. The precipitate was suspended in 3.0 ml of STE containing 2 mM MgCl, (STEM), and then mixed with 16 ml of iso-osmotic Percoll solution (Per~011:STEM: 80 mM Tris-HCl, pH 7.5, containing 2 M sucrose, 8 mM EGTA and 16 mM MgCl, = 7:32:1) followed by centrifugation at 10 000 x g for 1 h. The plasma membrane fractions floating just below the surface were removed in a total volume of about 1.0 ml. After washing twice by adding 6 ml of 1.0 mM TrisHCl (pH 7.5) containing 0.15 M NaCl followed by centrifugation at 17 000 x g for 10 min, the pellet was stored at - 80°C until analysis by 2D/E. 2.3. Two-dimensional gel electrophoresis The plasma membrane preparations from each rat were analyzed separately by the method of O’Farrell et al. (1975) with slight modifications. The purified plasma membrane (1 mg protein equivalent) was dissolved in 200 ~1 of 9.9 M urea, 4% (v/v) NP-40, 5.5% (v/v) ampholytes and 100 mM DTT. After centrifugation at 10 000 x g for 10 min, the protein concentration in the supernatant was determined by the method of Bradford (1976) using bovine serum albumin as the standard. Five samples were run simultaneously in one analysis. Sixteen microliters of each sample (80 ,ug protein when BSA) was loaded into an individual capillary tube (1.2 mm diameter x 260 mm length) containing 9.5 M urea, 4% (v/v) NP-40, 4.1% (w/v) acrylamide and 5.8% (v/v) ampholytes (pH 3- 10). Using a 2D/E system (Millipore, Bedford, USA), isoelectric focusing was carried out at room temperature at 1000 V for each capillary gel for 16.5 h, followed by 2000 V for 0.5 h. After the first electrophoresis, the gels were equilibrated in 3.0 ml of 0.3 M Tris-HCl (pH 6.8) containing 3.0% SDS, 50 mM DTT and 0.01% (w/v) bromophenol blue for 2 min. The gel was then loaded onto an SDS/polyacrylamide (10% w/v) gel (220 x 220 x 1 mm) and run for 3 h at 500 V at 20°C. After the second electrophoresis, the gels were fixed in 40% methanol and loo/o acetic acid solution. then the protein spots were visualized by silver staining according to the method of Morrissey (1981). To determine the M, and pZ of the proteins, hen egg white conalbumin type I (M, 76.0 kDa, pZ 6.0, 6.3, 6.6) (76.0 kDa/6.0, 6.3, 6.6), bovine serum albumin (66.2 kDa/5.4, 5.5, 5.6), bovine muscle actin (43.0 kDa/S.O, 5.1), rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (36.0 kDa/8.3-8.5), bovine
R. Ishimura et al. 1 Molecular and Cellular Endocrinology 115 (1995) 149-159
carbonic anhydrase (31.0 kDa/5.9, 6.0) soybean trypsin inhibitor (21.5 kDa/4.5) and equine myoglobin (17.5 kDa/7.0) (2-D SDS-PAGE Standards, Bio-Rad, Richmond, USA) were run in parallel as standards. Transferrin ( # 120 in Fig. 3B) was identified by immunoblotting using a polyclonal antibody against rat transferrin according to the method described previously (Miura et al., 1992). The intensities of the protein spots were evaluated quantitatively and compared among the 2D/E gels using computer software (Image Master, Ver. 1.0, Pharmacia LKB, Uppsala, Sweden) on a ProLinea 4/66 (Compaq) and NIH image analysis software (Ver.1.4.7.) on a Macintosh LC475 (Apple). Reproducibility was confirmed by three repeated experiments for three independent pooled samples as indicated in Fig. 2. 3. Results 3.1. Two -dimensional gel electrophoresis of plasma membrane proteins of the rat placenta at various stages of pregnancy Plasma membrane fractions prepared from the placenta on days 12, 14, 16, 18 and 20 of pregnancy were subjected to 2D/E, and the spots on the gels were visualized by silver staining (Fig. lA-E). Images of the 2D/E gels were prepared by subtraction of non-specific background staining and construction of spot images using Image Master software, and a typical gel image for day 12 of pregnancy is shown in Fig. 1F. The expected sensitivity for selective spots was 0.042 ng/ mm2 for bovine serum albumin, 0.083 ng/mm* for ovalbumin and 0.17 ng/mm2 for cytochrome c (Morrissey, 1981). In the present study, the proteins within a pZ range of 4.0-7.0 and an M, range of 20-100 kDa were analyzed in detail, and 800 protein spots per gel were separable on average. All M, and pZ values are expressed as (M,/pZ) in the text, unless otherwise indicated. 3.2. Changes in plasma membrane proteins during pregnancy:
class@cation of the membrane proteins
The intensity of expression of plasma membrane proteins was compared among the various stages of pregnancy. Protein spots were evaluated quantitatively using NIH image software and data are expressed as mean + SE in Fig. 2. Protein i (50.8 kDa/4.4) and plasma membrane-associated housekeeping proteins such as actin (42.0 kDa/5.2) showed constant expression during pregnancy (Figs. lA-E and 2). In contrast, other spots increased or decreased according to the stage of pregnancy. For example, protein b(72.8 kDa/ 4.5) was highly expressed on day 12 and had decreased in intensity by day 14 of pregnancy, and protein a (46.5 kDa/5.9) decreased gradually as pregnancy advanced. Protein f (79.8 kDa/6.4) showed higher expression on day 20 than on day 12 of pregnancy.
151
Based on the changing pattern of intensity shown in Fig. 2, these placental plasma membrane proteins were categorized as follows.
3.3. Classification of Group Z membrane proteins The protein spots with identification numbers (Fig. 3A-H) indicate the proteins whose expression changed during pregnancy. M, and pZ values for each of the proteins with the identification numbers shown in Fig. 3A-H are shown in Table 1. Plasma membrane proteins that showed higher expression on day 12 of pregnancy (mid-pregnancy) than on day 20 (late-pregnancy) were classified into Group I, and were further divided into five subgroups (G-I-l -5) (Fig. 4). Membrane proteins belonging to G-I-l decreased gradually from day 12 to day 20 of pregnancy. This subgroup comprised 85 proteins (spot # l-85, Figs. 3A and 4). Some membrane proteins were arranged side by side on the gel image. For example, (45.3-46.0 kDa/4.2-4.5, # 20-25) and (65.0-66.5 kDa/5.6-5.8, # 92-95) proteins were considered to be isoforms of each other arising from some modification such as glycosylation or phosphorylation. Subgroup G-I-2 included 17 proteins, which decreased abruptly on day 14 of pregnancy ( # 86- 102, Figs. 3B and 4). These proteins (66.0-74.4 kDa/6.06.2, # 88-91 and 65.0-66.5 kDa/5.6-5.8, # 92-95) were also considered to be isoforms of each other. Subgroup G-I-3 inclued five proteins, which decreased between days 14 and 16 of pregnancy ( # 103107, Figs. 3C and 4). Three proteins belonging to G-I-4 decreased on day 18 of pregnancy ( # 108- 110, Figs. 3D and 4) and nine proteins belonging to G-I-5 decreased on day 20 of pregnancy ( # 1 11- 119, Figs. 3E and 4).
3.4. Classljication of Group ZZmembrane proteins The proteins with low or negligible expression on day 12 and an increase of expression on day 20 were categorized into Group II, and further divided into three subgroups (G-11-1-3) (Fig. 4). Membrane proteins belonging to G-II-l (28 proteins; # 120- 147, Figs. 3F and 4) increased before day 16 of pregnancy. Four proteins with identical pZ values (37.4-39.5 kDa/5.8, # 131-134) were thought to be isoforms. Similarly, three other proteins (37.3-38.3 kDa/5.9, # 135-137) may have been isoforms of each other because of their similar spot forms. Proteins belonging to G-II-2 increased after day 16 of pregnancy ( # 148 and # 149, Figs. 3G and 4) and only one protein increased after day 18 of pregnancy (G-11-3) ( # 150, Figs. 3H and 4). About 600 spots showed no marked change and were categorized as Group III.
152
R. Ishimura et al. / Molecular and Cellular Endocrinology 1 IS (1995) 149- 1.59
490
k
+80 k d-70 k +60
k
+50k t-40 k
+30
k
4-90 k 4-80 k 4-70 k M-60 k t-50 k 440
k
+30
k
MW
Fig. 1. Two-dimensional electrophoretic analysis of rat placental plasma membrane proteins from days 12, 14, 16, 18 and 20 of pregnancy visualized by silver staining. The ampholytes (pH 3- 10) and 10% polyacrylamide were used as described in the text. Some proteins distributed widely in the gel were selected and shown (A, day 12; B, day 14; C, day 16; D, day 18; E, day 20) (proteins a, h, c, d, e,f; g, h and i). (F) For determination of M, and pl values and comparison of expression of the protein spots among the gels, the original data shown in (A-E) were processed by Image Master software (Ver.l.O). The gel image of a pregnancy day 12 preparation is shown as an example.
R. Ishimura et al. / Molecular and Cellular Endocrinology I15 (1995) 149-159
153
480
90 k k
d-70 k 460
k
450k 440
k
+30k
+90 k 4-80 k 470 k 4
-60k
4
-50k
440k
4-30 k MW
Fig. l(C) and (D)
4. Discussion
The placenta was found to express various kinds of
plasma membrane proteins: 119 proteins were dominant in mid-pregnancy (Group I), 31 were dominant in late-pregnancy (Group II), and about 600 spots were
154
90 k 80 k 70 k 60 k 50 k
4-40
k
4-30
k
MW
Fig. l(E) and (F).
stably expressed throughout pregnancy (Group III). Therefore, about 20% (total 150 proteins) of the placental plasma membrane proteins, those which belonged to Groups I and II, changed dramatically during pregnancy. These results are in accord with
the concept that the placenta possesses multiple functions. The plasma membrane proteins belonging to Groups I and II were divided into eight classes according to their expression pattern. The rat placenta is composed
R. Ishimuru
et al. :’Moleculur
Prot. a
and Crllulur
Endocrinology
,Prot. b
,
I I5 (1995)
149-159
155
Prot. c
0.40 0.20 0.00W 12 14 16 18 20
Prot. e
12 14 16 18 20
.
Prot. f
12 14 16 18 20
Day of Pregnancy
Fig. 2. Typical expression pattern of plasma membrane proteins in the placenta. Protein spots indicated in Fig. I (A-E, proteins a-i) were evaluated quantitatively by NIH imaging software (Ver. I .4.7.). The relative density of each spot was measured in relation to that of actin, which was considered to have a value of 1.0. Vertical line indicates the mean value of the density with SE for three independent gels.
of at least six different cell types: trophoblast stem cells, trophoblast giant cells (TGCs), cytotrophoblast cells, spongiotrophoblast cells, syncytial trophoblast cells and glycogen cells (Davies and Glasser, 1968). The cellular composition of the placenta changes ,markedly during pregnancy through the growth, degeneration, or differentiation of these cells (Jollie, 1964a,b, 1965; Peel and Bulmer, 1977). The rodent possesses two placental structures, the choriovitelline placenta and the chorioallantoic placenta (Davies and Glasser, 1968). The choriovitelline placenta entirely disappears by day 14 of pregnancy and the chorioallantoic placenta begins to develop before this stage (Davies and Glasser, 1968; Peel and Bulmer, 1977). The placental tissue also contains decidual cells, which are dominant on day 12 and minimal in number on day 20 (Davies and Glasser, 1968). The reciprocal relationship between Groups I and II proteins may be explained by the changes in the cellular composition in the placenta. Alternatively, the expression of some of these proteins may influence the processes of differentiation of the cells. Subgroup G-I-1 accounted for more than 70% of Group I proteins. Mid-pregnancy is an important period because this is when fetal growth is most evident (Brown and Fabro, 1981) natural fetal death may occur (Shiota et al., 1993) and the responsible organ for
the control of ovarian progesterone secretion is switched from the anterior pituitary gland to the placenta (Morishige and Rothchild, 1974). In addition to G-I-l proteins, the proteins belonging to G-I-2 (17 proteins) and G-I-3 (five proteins), which are highly expressed at mid-pregnancy, may be involved in these events. It has been reported that HLA-G is expressed in the early placenta in humans and that its level declines as pregnancy advances (Claman, 1993). Equine class-I antigen is also expressed during mid-pregnancy in the placenta and its level decreases in late-pregnancy (Donaldson et al., 1992). MHC class-I antigen, which is a heterodimer protein consisting of an cxchain (45 kDa) and µglobulin (12 kDa), is expressed in spongiotrophoblast cells in the rodent (Jenkinson and Owen, 1980; Billington and Burrows, 1986). In the present study, the proteins with an M, range of 40-55 kDa belonged to G-I- 1. The proteins with an M, of approximately 45 kDa were: # 8 (45.7 kDa/4.9), # 9 (45.2 kDa/4.8), # 10 (44.0 kDa/4.7), # 11 (44.2 kDa/4.7), # 16 (45.6 kDa/S.O), # 17 (45.1 kDa/4.9), # 18 (44.1 kDa/4.9), # 20 (46.0 kDa/4.2), # 21 (45.5 kDa/4.3), # 22 (45.4 kDa/4.3), # 23 (45.3 kDa/4.4), # 24 (45.7 kDa / 4.4) # 25 (45.5 kDa/4.5). Proteins with a similar M, were absent in Group II. All of these proteins are
156
A
PI
4.6 4.85.05.2 5.45.65.86.06.26.4 t9Ok t80k e70k t60k t50k t40k
t30k
MW
QI
4.6 4.85.05.2 5.45.65.86.06.26.4
4
-9Ok -8Ok -7Ok
4
-6Ok
4
-5Ok
4
-4Ok
4
-3Ok
4 4
MW Fig. 3. Plasma membrane proteins whose expression changed during pregnancy are marked. (A-E) Membrane proteins on day 12. These proteins marked in the figure (85 spots in A, 17 spots in B, five spots in C, three spots in D, and nine spots in E) are recognized on day 12 and their expression has a tendency to decrease with the advance of pregnancy. (F- H) Membrane proteins on day 20. Expression of these membrane proteins (28 spots in F, two spots in G and one spot in H) was minimal on day 12 and became high on day 20.
R. lshitnura et al. / Molecular and Cellular Endocrinology 115 (1995) 149- 159 Table I The h4, and pl values of the placental indicated in Fig. 3)
plasma
membrane
proteins
categorized
as Groups
ELI
No.
No.
K., &Da)
PI
No.
K
44 45 46 47 48 49 50 51 52 53 54 55 56 5-l 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 14 15 76 77 78 79 80 81 82 83 84 85 119
36.9 36.2 36.6 36.8 35.9 34.1 34.4 34.3 34.2 34.2 34.0 33.5 31.7 31.8 32.5 32.6 31.8 31.1 30.5 30.5 30.6 28.8 28.1 29.9 29.6 29.4 28.5 21.9 26.4 26.4 25.6 25.1 24.7 24.0 24. I 23. I 22.6 22.3 22.5 23.3 25.0 24. I 25.8
6.0 6.3 6.4 6.4 6.5 5.9 5.9 5.9 5.6 5.1 5.9 5.1 5.2 5.4 5.6 5.7 5.8 5.1 5.5 5.1 5.0 5.5 5.5 5.8 5.9 5.8 5.8 5.8 5.1 5.7 5.2 4.1 4.1 4.8 4.9 4.9 4.9 5.2 5.5 5.1 5.9 6.1 5.8
86 87 88 89 90 91 92 93 94 95 96 91 98 99 100 101 102
53.4 52.6 50.7 50.8 47.1 47.1 46.0 45.7 45.2 44.0 44.2 46.5 46.5 46.5 47.1 45.6 45. I 44.1 42.5 46.0 45.5 45.4 45.3 45.1 45.5 43.3 43.0 43.0 41.9 41.5 41.3 41.7 41.4 41.1 38.9 39.6 39.0 39.4 37.3 37.1 38.3 38.1 31.6
5.2 4.7 4.9 5.1 4.8 4.9 4.9 4.9 4.8 4.1 4.1 5.8 5.9 5.9 5.0 5.0 4.9 4.9 4.9 4.2 4.3 4.3 4.4 4.4 4.5 4.6 4.6 4.8 4.9 4.9 4.6 5.8 5.8 5.6 5.4 4.9 4.9 4.8 4.1 4.7 6.0 5.6 5.6
I and II (each of the spot numbers
G-I-2
acidic ones, and the class-I molecule appears to be included in subgroup G-I-l. Group III proteins are expressed constantly during pregnancy. This includes cytoskeltal components, which seem to migrate into the plasma membrane fraction as they are located on the inner surface of the plasma membrane. In the present study, actin (42.0 kDa/5.2) was constantly recognized by 2D/E throughout pregnancy. Intermediate filaments of rat trophoblast giant cells have been investigated using 2D/E by Glasser and Julian (1986) who identified six different proteins. These six intermediate filaments (54 kDa (pZ 5.6, 5.7),
No.
K
72.8 65.5 14.4 72.2 69.7 66.0 66.5 66.1 66.0 65.0 35.2 31.2 30.8 30.0 27.8 21.3 22.0
4.5 4.6 6.0 6.0 6.1 6.2 5.6 5.1 5.8 5.8 5.2 6.4 6.0 5.2 4.7 4.7 5.9
71.1 47.2 31.2 34.3 28.2
5.1 4.5 4.8 4.6 5.6
120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 I40 141 142 143 I44 I45 I46 147
79.8 53.0 53.0 53.0 49.0 40.4 38.3 38.4 38.1 38.2 37.4 39.5 38.8 38.1 31.4 38.3 38.0 31.3 34.2 33.3 31.0 21.4 21.5 27.0 21.4 22.1 22.5 21.5
6.4 5.5 5.6 5.7 5.8 5.1 5.1 5.3 5.3 5.5 5.5 5.8 5.8 5.8 5.8 5.9 5.9 5.9 5.0 4.6 5.5 4.9 5.6 5.6 5.4 5.4 6.2 5.9
39.3 27.1 24.8
5.1 5.8 6.8
I48 149 C-II-3
36.2 35.9
5.3 5.3
I50
21.0
5.1
G-I-4 108 109 110 C-l-5 Ill II2 II3 II4 II5 II6 II7 II8
to those
PI
&Da)
G-I-3 103 104 105 106 107
are identical
G-II-1
G-I-l
I 2 3 4 5 6 7 8 9 IO II I2 I3 I4 I5 I6 I7 18 I9 20 21 22 23 24 25 26 21 28 29 30 31 32 33 34 35 36 31 38 39 40 41 42 43
151
76.4 68.4 50.1 44.2 40.7 35.1 29.4 27.3
5.8 5.5 5.7 5.6 5.3 5.3 5.2 5.8
&Da)
PI
G-U-2
52 kDa (pZ 5.5, 5.6), 46 kDa (pZ 5.1, 5.1, 5.2), 45 kDa (pZ 5.0, 5.1, 5.1), 43 kDa (pZ 5.0), 40 kDa (pZ 5.1, 5.1, 5.2)) were confirmed in all of the 2D/E gels obtained on days 12, 14, 16, 18 and 20 of pregnancy. These intermediate filament proteins seem to be specific to trophoblast giant cells, uterine epithelium and pancreatic islets (Glasser and Julian, 1986). Except for one intermediate filament, expression of the five others (M, 54, 52,46,45 and 43 kDa) did not change during pregnancy in the present study. The 40-kDa intermediate filament protein consisted of three isoforms (pZ 5.11, 5.14, 5.21). Of these, two with pZ values of 5.11
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and Cellular
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Group I
decrease between decrease between decrease between
L-J total 119
Group II G-II- 1 G-II-2 G-II-3
la
increase between day 12 and day 16 12 16 20 increase between Q. day 16 and day 20 12 16 La 12 1620
increase between day18andday20
28
#120-#147
f
2
#148-#149
g
1
#150
h
total
Group III El
did not change 12 16 20 during pregnancy
31
About 600
Fig. 4. The placental plasma membrane proteins were categorized into three major groups (Groups patterns. Group I included membrane proteins which were recognized on day 12 and had a tendency (119 proteins), and these proteins were further categorized into five subgroups (G-I-l -5); Group 11 (31 their expression as pregnancy advanced (31 proteins) and these were categorized into three subgroups membrane proteins that were constantly expressed during pregnancy
Actin i I, II and III) according to their expression to decrease with the advance of pregnancy proteins) included proteins which increased (G-II-1 -3); Group 111 included about 600
and 5.21 were expressed constantly during pregnancy, while the elrpression of one intermediate filament protein (~1 5.14) was relatively low on day 12 and then
Group III, although the expression creased in late-pregnancy.
increased gradually until day 16. Accordingly, the 40kDa intermediate filament isoform with pl 5.14 was classified into G-II-1 ( # 125) in this study. The expression of transferrin receptor has been demostrated in the labyrinth and junctional zone of the rat placenta by immunohistochemistry (Hunt and Soares, 1988). Human transferrin receptor (M, 90 kDa) is reported to be localized close to transferrin on 2D/E gels (Wada et al., 1979; Webb et al., 1985; Truman and Ford, 1986). Since protein # 120 (G-II-l, M, 79.8 kDa) was identified as transferrin by Western blotting in this experiment, the protein (M, 90 kDa, p/ 5.5) might be transferrin receptor although further experiment will be needed. This protein did not change markedly during pregnancy and was categorized into
Acknowledgements
of transferrin
in-
We are grateful to Dr. D. B. Douglas for proofreading the manuscript. This work was supported in part by a grant from the Ministry of Education, Science and Culture, Japan. References Albrecht, E.D. and Pepe, G.J. (1990) Endocrinol. Rev. 1I, 124- 150. Belsham, G.J., Denton, R.M. and Tanner, M.J.A. (1980) Biochem. J. 192, 457- 467. Billington, W.D. and Burrows, F.J. (1986) J. Reprod. Immunol. 9, 1555160. Bradford, M. (1976) Anal. Biochem., 72, 248-254 Brown, N.A. and Fabro. S. (1981) TerdtoIogy 24, 65-78.
R. Ishimura et al. / Molecular and Cellular Endocrinology 115 (1995) 149-159
Campbell, W.J., Larsen, D., Deb, S., Kwok, S.C.M. and Soares, M.J. (1991) Placenta 12, 227-237 Claman, H.N. (1993) The Immunology of Human Pregnancy, pp. 51-79, Humana Press, New Jersey. Davies, J. and Glasser, S.R. (1968) Acta Anat. 69, 542-608. Donaldson, W.L., Oriol, J.G., Plavin, A. and Antczak, D.F. (1992) Differentiation 52, 69-78. Furuyama, N., Shiota, K. and Takahashi, M. (1991) Endocrinol. Japon. 38, 533-540. Garrets, J.I. (1989) J. Biol. Chem. 264, 5269-5282. Garrels, J.I. and Franza, B.R. Jr. (1989) J. Biol. Chem. 264, 5299-5312. Gill, T.J.111. (1994) in The Physiology of Reproduction, Second Edition, Vol. 2 (Knobil, E. and Neil], J.D., eds.), pp. 783-812, Raven Press, New York. Glasser, S.R. and Julian, J. (1986) Dev. Biol. 113, 356-363. Guy, G.R., Philip, R. and Tan, Y.H. (1994) Electrophoresis 15, 417-440. Hattori, N., Wakimasu, M., Takahashi, M., Hirosawa, M., Shiota, K. and Ogawa, T. (1993) Endocrine J. 40, 727-735. Hirosawa, M., Miura, R., Min, K., Hattori, N., Shiota, K. and Ogawa, T. (1994) Endocrine J. 41, 387-397. Ho, H., Macpherson, T.A., Kunz, H.W. and Gill, T.J. III (1987) Am. J. Reprod. Immunol. Microbial. 13, 51-61. Hunt, J.S. and Soares, M.J. (1988) Placenta 9, 1599171. Jenkinson, E.J. and Owen, V. (1980) J. Reprod. Immunol. 2, 173-181. Jollie, W.P. (1964a) J. Ultrastruct. Res. 10, 27-47. Jollie, W.P. (1964b) Am. J. Anat. 114, 161-171. Jollie, W.P. (1965) J. Ultrastruct. Res. 12, 420-438. Kanbour-Shakir, A., Kunz, H.W. and Gill, T.J. III. (1993) Biol. Reprod. 48, 977-986.
159
Latham, K.E., Beddington, R.S.P., Solter, D. and Garrels, J.I. (1993) Mol. Reprod. Dev. 35, 140-150. Miura, R., Noda, K., Shiota, K. and Takahashi, M. (1992) J. Reprod. Dev. 38, 249-254 Morishige, W.K. and Rothchild, I. (1974) Endocrionology 95, 260-273. Morriss, F.H. Jr., Boyd, R.D.H. and Mahendran, D. (1994) in The Physiology of Reproduction, Second Edition, Vol. 2 (Knobil, E. and Neill, J.D., eds.), pp. 813-861, Raven Press, New York. Morrissey, J.H. (1981) Anal. Biochem. 117, 307-310. O’Farrell, P.H. (1975) J. Biol. Chem. 250, 4007-4021. Ogren, L. and Talamantes, F. (1994) in The Physiology of Reproduction, Second Edition, Vol. 2 (Knob& E. and Neil], J.D., eds.), pp. 875-945, Raven Press, New York. Peel, S. and Bulmer, D. (1977) J. Anat. 124, 675-687. Shiota. K., Furuyama, N. and Takahashi, M. (1991) Endocrinol. Japon. 38, 541-549. Shiota, K., Seong, H., Noda, K., Hattori, N., Ikeda, A., Ogura, A., Itagaki, S., Takahashi, M. and Ogawa, T. (1993) Endocrine J. 40, 673-681. Shiota, K., Hirosawa, M., Hattori, N., Itonori, S., Miura, R., Noda, K., Takahashi, M. and Ogawa, T. (1994) Endocrine J. 41, S43-S56. Soares, M.J., Faria, T.N., Roby, K.F. and Deb, S. (1991) Endocrinol. Rev. 12, 402-423. Truman, P. and Ford, H.C. (1986) Placenta 7, 1I I-120. Wada, H.G., Hass, P.E. and Sussman, H.H. (1979) J. Biol. Chem. 254, 12629- 12635. Webb, P.D., Evans, P.W., Molloy, C.M. and Johnson, P.M. (1985) Am. J. Reprod. Immunol. Microbial. 8, 113-l 19. Wirth, P.J., Luo, L., Fujimoto, Y. and Bisgaard, H.C. (1992) Electrophoresis 13, 305-320.
b
Endocrhbgy
ELSEVIER
Molecular and Cellular Endocrinology
I I5 (1995) l49- I59
Analysis of rat placental plasma membrane proteins by two-dimensional gel electrophoresis Ryuta
Ishimura,
Ken Noda,
Naka
Hattori,
Kunio
Shiota*,
Tomoya
Ogawa
Laboratory of’Cellular Biochemistry, Veterinary Medical Sciences/Animal Resource Sciences, University of Tokyo, I - 1- 1 YaJloi, Bunkyo-ku, Tokyo 113, Japan
Received 24 February 1995; accepted 6 September 1995
Abstract The placenta plays an essential role in fetal growth and the maintenance of pregnancy and its functions are strictly controlled in a stage-specific manner. To gain an insight into placental functions and their regulation, we analyzed the plasma membrane (2D/E). Plasma membrane fractions of the proteins of rat placenta by two-dimensional polyacrylamide gel electrophoresis
placenta obtained on days 12, 14, 16, 18 and 20 of pregnancy were purified by Percoll gradient centrifugation, and subjected to 2D/E analysis. After the proteins on the 2D/E gels had been visualized by silver staining, the patterns on the gels at different stages of pregnancy were compared using image analysis software. Proteins within an isoelectric point (pl) range of 4.0 to 7.0 and a molecular weight (M,) range of 20-100 kDa were analyzed in detail, and about 800 proteins on average were recognized on each gel. Of these, the expression of 150 proteins was found to change dramatically according to the stage of pregnancy. According to their expression patterns, these proteins were categorized into two groups, Group I and Group II. The proteins belonging to Group I showed a higher intensity of expression on day 12 and disappeared on day 20. They included 119 plasma membrane proteins and were divided into five subgroups. Group II. which consisted of three subgroups, included 31 proteins showing a low or negligible expression on day 12 and higher expression on day 20. Most of the other membrane proteins (about 600) were expressed constantly during pregnancy. On the basis of our data, we constructed a database for plasma membrane proteins
of the rat placenta.
Keywords:
Placenta; Plasma membrane
protein; Pregnancy; Rat; Two-dimensional
1. Introduction The placenta possesses multiple functions including regulation of the transport of nutrients and wastes (Morriss and Boyd, 1994) prevention of a directional
immune response between mother and fetus (Gill, 1994) and endocrine regulation to create and maintain the maternal physiological milieu by producing hormones such as placental lactogen (Albrecht and Pepe, 1990; Soares et al., 1991; Ogren and Talamantes, 1994; Shiota et al., 1994). Considering these multiple functions, not only functions that are required constantly but also pregnancy stage-specific events such as placental lactogen synthesis (Furuyama et al., 1991; Shiota et al., 1991; Hattori, et al., 1993; Hirosawa et al., 1994) the placenta is thought to express an abundance of biologi*Corresponding
author, Tel.: 81 3 3812 211 I, ext. 5372; Fax: 81 3
5689 7345. 0303-7207/95/$09.50 SSDI
8 1995 ~
0303-7207(95)03682-W
Elsevier Science Ireland
gel electrophoresis
tally active molecules. Therefore, plasma membrane proteins are thought to be involved in the transport of numerous materials and in various immunological and endocrinological processes. It is often the case that multiple intracellular signalling pathways are initiated at the cell surface by extracellular stimulation. The cellular composition of the placenta changes dramatically during pregnancy (Jollie, 1964a,b, 1965; Peel and Bulmer, 1977) and cell type-specific molecules have been identified, e.g., alkaline phosphatase is expressed on cytotrophoblast cells (Campbell et al., 1991) whereas Pa antigen, which is a class-I antigen, is expressed on spongiotrophoblast cells and trophoblast giant cells (TGCs) in the rat (Ho et al., 1987; KanbourShakir et al., 1993). It remains to be determined how many proteins are involved in placental functions and how they are regulated in a pregnancy stage-specific manner. Two-dimen-
Ltd. All rights reserved
150
R. Ishimuru
et ul.
; Molecular
und Cellular
sional polyacrylamide gel electrophoresis (2D/E) is a powerful method for analysis of plasma membrane proteins, because theoretically more than 1000 proteins including glycoproteins and phosphoproteins can be resolved on a single 2D/E gel (O’Farrell, 1975; Garrels, 1989; Garrels and Franza, 1989; Wirth et al., 1992; Latham et al., 1993; Guy et al., 1994). 2D/E analysis would provide information reflecting cellular composition as well as various cellular states, and it could be potentially useful for identifying the plasma membrane proteins involved in placental function. In the present study, in order to gain an insight into the regulation of placental functions, we analyzed placental plasma membrane proteins expressed on the rat placenta by 2D/E and constructed a protein database from their isoelectric point (pl) and molecular weight (M,) data. 2. Materials and Methods 2. I. Reagents and animals
Leupepsin and ethyleneglycol-bis-N,N,N’,N’-tetraacetic acid (EGTA) were purchased from Sigma (St. Louis, USA), Percoll was from LKB (Uppsala, Sweden), urea and protein quantifying reagents (Bio-Rad Protein Assay) were from Bio-Rad (Richmond, USA), nonidet P-40 (NP-40), acrylamide, ampholytes (pH 310) and dithiothreitol (DTT) were from Millipore (Bedford, USA), rabbit antiserum against rat transferrin was from Organon Teknika (Durham, USA) and phenylmethylsulfonyl fluoride (PMSF), silver staining reagents and other reagents were from Wako Pure Chemical Ind. (Osaka, Japan). Adult female Wistar-Imamichi rats were purchased from the Imamichi Institute for Animal Reproduction (Ibaraki, Japan). They were kept under a lighting regime of 14 h illumination and 10 h darkness (lights on between 05:OOh and 19:00 h) and allowed free access to food and water. Female rats were housed with a fertile male rat, and the day when sperm were observed in a vaginal smear was designated day 0 of pregnancy. 2.2. Isolation of plasma membrane fractions fLom placental tissue Three or four rats each on days 12, 14, 16, 18 and 20 of pregnancy were decapitated between 13:00 h and 14:00 h. The antimesometrial wall of the uterus was cut and the decidua was shelled out of the uterus with forceps. After removal of the embryos with forceps, the remaining placental tissue including decidual cells and trophoblasts was frozen quickly and stored at - 80°C until required for use. All subsequent procedures were carried out at 4°C. Isolation of plasma membrane fractions was performed as described previously using Percoll gradient centrifugation (Belsham et al., 1980). Briefly, the placental
Endwrinology
115 (1995)
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tissue (1.0 g wet wt.) was cut into small pieces (each 2- 3 mm3) and homogenized in 5 ml of 10 mM TrisHCl (pH 7.5) containing 0.25 M sucrose, 2 mM EGTA, 0.5 mg/ml leupeptin and 1 mM PMSF (STE) with a teflon-glass homogenizer. After centrifugation at 150 x g (JAlO rotor: Beckman, Fullerton, USA) for 10 min, the supernatant was collected. The pellet was homogenized and centrifuged again. The supernatants were combined and centrifuged at 5000 x g (JAlO rotor) for 10 min. The precipitate was suspended in 3.0 ml of STE containing 2 mM MgCl, (STEM), and then mixed with 16 ml of iso-osmotic Percoll solution (Per~011:STEM: 80 mM Tris-HCl, pH 7.5, containing 2 M sucrose, 8 mM EGTA and 16 mM MgCl, = 7:32:1) followed by centrifugation at 10 000 x g for 1 h. The plasma membrane fractions floating just below the surface were removed in a total volume of about 1.0 ml. After washing twice by adding 6 ml of 1.0 mM TrisHCl (pH 7.5) containing 0.15 M NaCl followed by centrifugation at 17 000 x g for 10 min, the pellet was stored at - 80°C until analysis by 2D/E. 2.3. Two-dimensional gel electrophoresis The plasma membrane preparations from each rat were analyzed separately by the method of O’Farrell et al. (1975) with slight modifications. The purified plasma membrane (1 mg protein equivalent) was dissolved in 200 ~1 of 9.9 M urea, 4% (v/v) NP-40, 5.5% (v/v) ampholytes and 100 mM DTT. After centrifugation at 10 000 x g for 10 min, the protein concentration in the supernatant was determined by the method of Bradford (1976) using bovine serum albumin as the standard. Five samples were run simultaneously in one analysis. Sixteen microliters of each sample (80 ,ug protein when BSA) was loaded into an individual capillary tube (1.2 mm diameter x 260 mm length) containing 9.5 M urea, 4% (v/v) NP-40, 4.1% (w/v) acrylamide and 5.8% (v/v) ampholytes (pH 3- 10). Using a 2D/E system (Millipore, Bedford, USA), isoelectric focusing was carried out at room temperature at 1000 V for each capillary gel for 16.5 h, followed by 2000 V for 0.5 h. After the first electrophoresis, the gels were equilibrated in 3.0 ml of 0.3 M Tris-HCl (pH 6.8) containing 3.0% SDS, 50 mM DTT and 0.01% (w/v) bromophenol blue for 2 min. The gel was then loaded onto an SDS/polyacrylamide (10% w/v) gel (220 x 220 x 1 mm) and run for 3 h at 500 V at 20°C. After the second electrophoresis, the gels were fixed in 40% methanol and loo/o acetic acid solution. then the protein spots were visualized by silver staining according to the method of Morrissey (1981). To determine the M, and pZ of the proteins, hen egg white conalbumin type I (M, 76.0 kDa, pZ 6.0, 6.3, 6.6) (76.0 kDa/6.0, 6.3, 6.6), bovine serum albumin (66.2 kDa/5.4, 5.5, 5.6), bovine muscle actin (43.0 kDa/S.O, 5.1), rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (36.0 kDa/8.3-8.5), bovine
R. Ishimura et al. 1 Molecular and Cellular Endocrinology 115 (1995) 149-159
carbonic anhydrase (31.0 kDa/5.9, 6.0) soybean trypsin inhibitor (21.5 kDa/4.5) and equine myoglobin (17.5 kDa/7.0) (2-D SDS-PAGE Standards, Bio-Rad, Richmond, USA) were run in parallel as standards. Transferrin ( # 120 in Fig. 3B) was identified by immunoblotting using a polyclonal antibody against rat transferrin according to the method described previously (Miura et al., 1992). The intensities of the protein spots were evaluated quantitatively and compared among the 2D/E gels using computer software (Image Master, Ver. 1.0, Pharmacia LKB, Uppsala, Sweden) on a ProLinea 4/66 (Compaq) and NIH image analysis software (Ver.1.4.7.) on a Macintosh LC475 (Apple). Reproducibility was confirmed by three repeated experiments for three independent pooled samples as indicated in Fig. 2. 3. Results 3.1. Two -dimensional gel electrophoresis of plasma membrane proteins of the rat placenta at various stages of pregnancy Plasma membrane fractions prepared from the placenta on days 12, 14, 16, 18 and 20 of pregnancy were subjected to 2D/E, and the spots on the gels were visualized by silver staining (Fig. lA-E). Images of the 2D/E gels were prepared by subtraction of non-specific background staining and construction of spot images using Image Master software, and a typical gel image for day 12 of pregnancy is shown in Fig. 1F. The expected sensitivity for selective spots was 0.042 ng/ mm2 for bovine serum albumin, 0.083 ng/mm* for ovalbumin and 0.17 ng/mm2 for cytochrome c (Morrissey, 1981). In the present study, the proteins within a pZ range of 4.0-7.0 and an M, range of 20-100 kDa were analyzed in detail, and 800 protein spots per gel were separable on average. All M, and pZ values are expressed as (M,/pZ) in the text, unless otherwise indicated. 3.2. Changes in plasma membrane proteins during pregnancy:
class@cation of the membrane proteins
The intensity of expression of plasma membrane proteins was compared among the various stages of pregnancy. Protein spots were evaluated quantitatively using NIH image software and data are expressed as mean + SE in Fig. 2. Protein i (50.8 kDa/4.4) and plasma membrane-associated housekeeping proteins such as actin (42.0 kDa/5.2) showed constant expression during pregnancy (Figs. lA-E and 2). In contrast, other spots increased or decreased according to the stage of pregnancy. For example, protein b(72.8 kDa/ 4.5) was highly expressed on day 12 and had decreased in intensity by day 14 of pregnancy, and protein a (46.5 kDa/5.9) decreased gradually as pregnancy advanced. Protein f (79.8 kDa/6.4) showed higher expression on day 20 than on day 12 of pregnancy.
151
Based on the changing pattern of intensity shown in Fig. 2, these placental plasma membrane proteins were categorized as follows.
3.3. Classification of Group Z membrane proteins The protein spots with identification numbers (Fig. 3A-H) indicate the proteins whose expression changed during pregnancy. M, and pZ values for each of the proteins with the identification numbers shown in Fig. 3A-H are shown in Table 1. Plasma membrane proteins that showed higher expression on day 12 of pregnancy (mid-pregnancy) than on day 20 (late-pregnancy) were classified into Group I, and were further divided into five subgroups (G-I-l -5) (Fig. 4). Membrane proteins belonging to G-I-l decreased gradually from day 12 to day 20 of pregnancy. This subgroup comprised 85 proteins (spot # l-85, Figs. 3A and 4). Some membrane proteins were arranged side by side on the gel image. For example, (45.3-46.0 kDa/4.2-4.5, # 20-25) and (65.0-66.5 kDa/5.6-5.8, # 92-95) proteins were considered to be isoforms of each other arising from some modification such as glycosylation or phosphorylation. Subgroup G-I-2 included 17 proteins, which decreased abruptly on day 14 of pregnancy ( # 86- 102, Figs. 3B and 4). These proteins (66.0-74.4 kDa/6.06.2, # 88-91 and 65.0-66.5 kDa/5.6-5.8, # 92-95) were also considered to be isoforms of each other. Subgroup G-I-3 inclued five proteins, which decreased between days 14 and 16 of pregnancy ( # 103107, Figs. 3C and 4). Three proteins belonging to G-I-4 decreased on day 18 of pregnancy ( # 108- 110, Figs. 3D and 4) and nine proteins belonging to G-I-5 decreased on day 20 of pregnancy ( # 1 11- 119, Figs. 3E and 4).
3.4. Classljication of Group ZZmembrane proteins The proteins with low or negligible expression on day 12 and an increase of expression on day 20 were categorized into Group II, and further divided into three subgroups (G-11-1-3) (Fig. 4). Membrane proteins belonging to G-II-l (28 proteins; # 120- 147, Figs. 3F and 4) increased before day 16 of pregnancy. Four proteins with identical pZ values (37.4-39.5 kDa/5.8, # 131-134) were thought to be isoforms. Similarly, three other proteins (37.3-38.3 kDa/5.9, # 135-137) may have been isoforms of each other because of their similar spot forms. Proteins belonging to G-II-2 increased after day 16 of pregnancy ( # 148 and # 149, Figs. 3G and 4) and only one protein increased after day 18 of pregnancy (G-11-3) ( # 150, Figs. 3H and 4). About 600 spots showed no marked change and were categorized as Group III.
152
R. Ishimura et al. / Molecular and Cellular Endocrinology 1 IS (1995) 149- 1.59
490
k
+80 k d-70 k +60
k
+50k t-40 k
+30
k
4-90 k 4-80 k 4-70 k M-60 k t-50 k 440
k
+30
k
MW
Fig. 1. Two-dimensional electrophoretic analysis of rat placental plasma membrane proteins from days 12, 14, 16, 18 and 20 of pregnancy visualized by silver staining. The ampholytes (pH 3- 10) and 10% polyacrylamide were used as described in the text. Some proteins distributed widely in the gel were selected and shown (A, day 12; B, day 14; C, day 16; D, day 18; E, day 20) (proteins a, h, c, d, e,f; g, h and i). (F) For determination of M, and pl values and comparison of expression of the protein spots among the gels, the original data shown in (A-E) were processed by Image Master software (Ver.l.O). The gel image of a pregnancy day 12 preparation is shown as an example.
R. Ishimura et al. / Molecular and Cellular Endocrinology I15 (1995) 149-159
153
480
90 k k
d-70 k 460
k
450k 440
k
+30k
+90 k 4-80 k 470 k 4
-60k
4
-50k
440k
4-30 k MW
Fig. l(C) and (D)
4. Discussion
The placenta was found to express various kinds of
plasma membrane proteins: 119 proteins were dominant in mid-pregnancy (Group I), 31 were dominant in late-pregnancy (Group II), and about 600 spots were
154
90 k 80 k 70 k 60 k 50 k
4-40
k
4-30
k
MW
Fig. l(E) and (F).
stably expressed throughout pregnancy (Group III). Therefore, about 20% (total 150 proteins) of the placental plasma membrane proteins, those which belonged to Groups I and II, changed dramatically during pregnancy. These results are in accord with
the concept that the placenta possesses multiple functions. The plasma membrane proteins belonging to Groups I and II were divided into eight classes according to their expression pattern. The rat placenta is composed
R. Ishimuru
et al. :’Moleculur
Prot. a
and Crllulur
Endocrinology
,Prot. b
,
I I5 (1995)
149-159
155
Prot. c
0.40 0.20 0.00W 12 14 16 18 20
Prot. e
12 14 16 18 20
.
Prot. f
12 14 16 18 20
Day of Pregnancy
Fig. 2. Typical expression pattern of plasma membrane proteins in the placenta. Protein spots indicated in Fig. I (A-E, proteins a-i) were evaluated quantitatively by NIH imaging software (Ver. I .4.7.). The relative density of each spot was measured in relation to that of actin, which was considered to have a value of 1.0. Vertical line indicates the mean value of the density with SE for three independent gels.
of at least six different cell types: trophoblast stem cells, trophoblast giant cells (TGCs), cytotrophoblast cells, spongiotrophoblast cells, syncytial trophoblast cells and glycogen cells (Davies and Glasser, 1968). The cellular composition of the placenta changes ,markedly during pregnancy through the growth, degeneration, or differentiation of these cells (Jollie, 1964a,b, 1965; Peel and Bulmer, 1977). The rodent possesses two placental structures, the choriovitelline placenta and the chorioallantoic placenta (Davies and Glasser, 1968). The choriovitelline placenta entirely disappears by day 14 of pregnancy and the chorioallantoic placenta begins to develop before this stage (Davies and Glasser, 1968; Peel and Bulmer, 1977). The placental tissue also contains decidual cells, which are dominant on day 12 and minimal in number on day 20 (Davies and Glasser, 1968). The reciprocal relationship between Groups I and II proteins may be explained by the changes in the cellular composition in the placenta. Alternatively, the expression of some of these proteins may influence the processes of differentiation of the cells. Subgroup G-I-1 accounted for more than 70% of Group I proteins. Mid-pregnancy is an important period because this is when fetal growth is most evident (Brown and Fabro, 1981) natural fetal death may occur (Shiota et al., 1993) and the responsible organ for
the control of ovarian progesterone secretion is switched from the anterior pituitary gland to the placenta (Morishige and Rothchild, 1974). In addition to G-I-l proteins, the proteins belonging to G-I-2 (17 proteins) and G-I-3 (five proteins), which are highly expressed at mid-pregnancy, may be involved in these events. It has been reported that HLA-G is expressed in the early placenta in humans and that its level declines as pregnancy advances (Claman, 1993). Equine class-I antigen is also expressed during mid-pregnancy in the placenta and its level decreases in late-pregnancy (Donaldson et al., 1992). MHC class-I antigen, which is a heterodimer protein consisting of an cxchain (45 kDa) and µglobulin (12 kDa), is expressed in spongiotrophoblast cells in the rodent (Jenkinson and Owen, 1980; Billington and Burrows, 1986). In the present study, the proteins with an M, range of 40-55 kDa belonged to G-I- 1. The proteins with an M, of approximately 45 kDa were: # 8 (45.7 kDa/4.9), # 9 (45.2 kDa/4.8), # 10 (44.0 kDa/4.7), # 11 (44.2 kDa/4.7), # 16 (45.6 kDa/S.O), # 17 (45.1 kDa/4.9), # 18 (44.1 kDa/4.9), # 20 (46.0 kDa/4.2), # 21 (45.5 kDa/4.3), # 22 (45.4 kDa/4.3), # 23 (45.3 kDa/4.4), # 24 (45.7 kDa / 4.4) # 25 (45.5 kDa/4.5). Proteins with a similar M, were absent in Group II. All of these proteins are
156
A
PI
4.6 4.85.05.2 5.45.65.86.06.26.4 t9Ok t80k e70k t60k t50k t40k
t30k
MW
QI
4.6 4.85.05.2 5.45.65.86.06.26.4
4
-9Ok -8Ok -7Ok
4
-6Ok
4
-5Ok
4
-4Ok
4
-3Ok
4 4
MW Fig. 3. Plasma membrane proteins whose expression changed during pregnancy are marked. (A-E) Membrane proteins on day 12. These proteins marked in the figure (85 spots in A, 17 spots in B, five spots in C, three spots in D, and nine spots in E) are recognized on day 12 and their expression has a tendency to decrease with the advance of pregnancy. (F- H) Membrane proteins on day 20. Expression of these membrane proteins (28 spots in F, two spots in G and one spot in H) was minimal on day 12 and became high on day 20.
R. lshitnura et al. / Molecular and Cellular Endocrinology 115 (1995) 149- 159 Table I The h4, and pl values of the placental indicated in Fig. 3)
plasma
membrane
proteins
categorized
as Groups
ELI
No.
No.
K., &Da)
PI
No.
K
44 45 46 47 48 49 50 51 52 53 54 55 56 5-l 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 14 15 76 77 78 79 80 81 82 83 84 85 119
36.9 36.2 36.6 36.8 35.9 34.1 34.4 34.3 34.2 34.2 34.0 33.5 31.7 31.8 32.5 32.6 31.8 31.1 30.5 30.5 30.6 28.8 28.1 29.9 29.6 29.4 28.5 21.9 26.4 26.4 25.6 25.1 24.7 24.0 24. I 23. I 22.6 22.3 22.5 23.3 25.0 24. I 25.8
6.0 6.3 6.4 6.4 6.5 5.9 5.9 5.9 5.6 5.1 5.9 5.1 5.2 5.4 5.6 5.7 5.8 5.1 5.5 5.1 5.0 5.5 5.5 5.8 5.9 5.8 5.8 5.8 5.1 5.7 5.2 4.1 4.1 4.8 4.9 4.9 4.9 5.2 5.5 5.1 5.9 6.1 5.8
86 87 88 89 90 91 92 93 94 95 96 91 98 99 100 101 102
53.4 52.6 50.7 50.8 47.1 47.1 46.0 45.7 45.2 44.0 44.2 46.5 46.5 46.5 47.1 45.6 45. I 44.1 42.5 46.0 45.5 45.4 45.3 45.1 45.5 43.3 43.0 43.0 41.9 41.5 41.3 41.7 41.4 41.1 38.9 39.6 39.0 39.4 37.3 37.1 38.3 38.1 31.6
5.2 4.7 4.9 5.1 4.8 4.9 4.9 4.9 4.8 4.1 4.1 5.8 5.9 5.9 5.0 5.0 4.9 4.9 4.9 4.2 4.3 4.3 4.4 4.4 4.5 4.6 4.6 4.8 4.9 4.9 4.6 5.8 5.8 5.6 5.4 4.9 4.9 4.8 4.1 4.7 6.0 5.6 5.6
I and II (each of the spot numbers
G-I-2
acidic ones, and the class-I molecule appears to be included in subgroup G-I-l. Group III proteins are expressed constantly during pregnancy. This includes cytoskeltal components, which seem to migrate into the plasma membrane fraction as they are located on the inner surface of the plasma membrane. In the present study, actin (42.0 kDa/5.2) was constantly recognized by 2D/E throughout pregnancy. Intermediate filaments of rat trophoblast giant cells have been investigated using 2D/E by Glasser and Julian (1986) who identified six different proteins. These six intermediate filaments (54 kDa (pZ 5.6, 5.7),
No.
K
72.8 65.5 14.4 72.2 69.7 66.0 66.5 66.1 66.0 65.0 35.2 31.2 30.8 30.0 27.8 21.3 22.0
4.5 4.6 6.0 6.0 6.1 6.2 5.6 5.1 5.8 5.8 5.2 6.4 6.0 5.2 4.7 4.7 5.9
71.1 47.2 31.2 34.3 28.2
5.1 4.5 4.8 4.6 5.6
120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 I40 141 142 143 I44 I45 I46 147
79.8 53.0 53.0 53.0 49.0 40.4 38.3 38.4 38.1 38.2 37.4 39.5 38.8 38.1 31.4 38.3 38.0 31.3 34.2 33.3 31.0 21.4 21.5 27.0 21.4 22.1 22.5 21.5
6.4 5.5 5.6 5.7 5.8 5.1 5.1 5.3 5.3 5.5 5.5 5.8 5.8 5.8 5.8 5.9 5.9 5.9 5.0 4.6 5.5 4.9 5.6 5.6 5.4 5.4 6.2 5.9
39.3 27.1 24.8
5.1 5.8 6.8
I48 149 C-II-3
36.2 35.9
5.3 5.3
I50
21.0
5.1
G-I-4 108 109 110 C-l-5 Ill II2 II3 II4 II5 II6 II7 II8
to those
PI
&Da)
G-I-3 103 104 105 106 107
are identical
G-II-1
G-I-l
I 2 3 4 5 6 7 8 9 IO II I2 I3 I4 I5 I6 I7 18 I9 20 21 22 23 24 25 26 21 28 29 30 31 32 33 34 35 36 31 38 39 40 41 42 43
151
76.4 68.4 50.1 44.2 40.7 35.1 29.4 27.3
5.8 5.5 5.7 5.6 5.3 5.3 5.2 5.8
&Da)
PI
G-U-2
52 kDa (pZ 5.5, 5.6), 46 kDa (pZ 5.1, 5.1, 5.2), 45 kDa (pZ 5.0, 5.1, 5.1), 43 kDa (pZ 5.0), 40 kDa (pZ 5.1, 5.1, 5.2)) were confirmed in all of the 2D/E gels obtained on days 12, 14, 16, 18 and 20 of pregnancy. These intermediate filament proteins seem to be specific to trophoblast giant cells, uterine epithelium and pancreatic islets (Glasser and Julian, 1986). Except for one intermediate filament, expression of the five others (M, 54, 52,46,45 and 43 kDa) did not change during pregnancy in the present study. The 40-kDa intermediate filament protein consisted of three isoforms (pZ 5.11, 5.14, 5.21). Of these, two with pZ values of 5.11
158
R. Ishimura
et al. / Molecular
and Cellular
Endocrinology
I15 (1995) 149-159
Group I
decrease between decrease between decrease between
L-J total 119
Group II G-II- 1 G-II-2 G-II-3
la
increase between day 12 and day 16 12 16 20 increase between Q. day 16 and day 20 12 16 La 12 1620
increase between day18andday20
28
#120-#147
f
2
#148-#149
g
1
#150
h
total
Group III El
did not change 12 16 20 during pregnancy
31
About 600
Fig. 4. The placental plasma membrane proteins were categorized into three major groups (Groups patterns. Group I included membrane proteins which were recognized on day 12 and had a tendency (119 proteins), and these proteins were further categorized into five subgroups (G-I-l -5); Group 11 (31 their expression as pregnancy advanced (31 proteins) and these were categorized into three subgroups membrane proteins that were constantly expressed during pregnancy
Actin i I, II and III) according to their expression to decrease with the advance of pregnancy proteins) included proteins which increased (G-II-1 -3); Group 111 included about 600
and 5.21 were expressed constantly during pregnancy, while the elrpression of one intermediate filament protein (~1 5.14) was relatively low on day 12 and then
Group III, although the expression creased in late-pregnancy.
increased gradually until day 16. Accordingly, the 40kDa intermediate filament isoform with pl 5.14 was classified into G-II-1 ( # 125) in this study. The expression of transferrin receptor has been demostrated in the labyrinth and junctional zone of the rat placenta by immunohistochemistry (Hunt and Soares, 1988). Human transferrin receptor (M, 90 kDa) is reported to be localized close to transferrin on 2D/E gels (Wada et al., 1979; Webb et al., 1985; Truman and Ford, 1986). Since protein # 120 (G-II-l, M, 79.8 kDa) was identified as transferrin by Western blotting in this experiment, the protein (M, 90 kDa, p/ 5.5) might be transferrin receptor although further experiment will be needed. This protein did not change markedly during pregnancy and was categorized into
Acknowledgements
of transferrin
in-
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Latham, K.E., Beddington, R.S.P., Solter, D. and Garrels, J.I. (1993) Mol. Reprod. Dev. 35, 140-150. Miura, R., Noda, K., Shiota, K. and Takahashi, M. (1992) J. Reprod. Dev. 38, 249-254 Morishige, W.K. and Rothchild, I. (1974) Endocrionology 95, 260-273. Morriss, F.H. Jr., Boyd, R.D.H. and Mahendran, D. (1994) in The Physiology of Reproduction, Second Edition, Vol. 2 (Knobil, E. and Neill, J.D., eds.), pp. 813-861, Raven Press, New York. Morrissey, J.H. (1981) Anal. Biochem. 117, 307-310. O’Farrell, P.H. (1975) J. Biol. Chem. 250, 4007-4021. Ogren, L. and Talamantes, F. (1994) in The Physiology of Reproduction, Second Edition, Vol. 2 (Knob& E. and Neil], J.D., eds.), pp. 875-945, Raven Press, New York. Peel, S. and Bulmer, D. (1977) J. Anat. 124, 675-687. Shiota. K., Furuyama, N. and Takahashi, M. (1991) Endocrinol. Japon. 38, 541-549. Shiota, K., Seong, H., Noda, K., Hattori, N., Ikeda, A., Ogura, A., Itagaki, S., Takahashi, M. and Ogawa, T. (1993) Endocrine J. 40, 673-681. Shiota, K., Hirosawa, M., Hattori, N., Itonori, S., Miura, R., Noda, K., Takahashi, M. and Ogawa, T. (1994) Endocrine J. 41, S43-S56. Soares, M.J., Faria, T.N., Roby, K.F. and Deb, S. (1991) Endocrinol. Rev. 12, 402-423. Truman, P. and Ford, H.C. (1986) Placenta 7, 1I I-120. Wada, H.G., Hass, P.E. and Sussman, H.H. (1979) J. Biol. Chem. 254, 12629- 12635. Webb, P.D., Evans, P.W., Molloy, C.M. and Johnson, P.M. (1985) Am. J. Reprod. Immunol. Microbial. 8, 113-l 19. Wirth, P.J., Luo, L., Fujimoto, Y. and Bisgaard, H.C. (1992) Electrophoresis 13, 305-320.