Serological analysis of early mouse embryo with rat monoclonal antibodies produced against mouse teratocarcinoma cells

Differentiation

Differentiation (1985) 28 :260-267

I(:,

Springer-Verlag1985

Serological analysis of early mouse embryo with rat monoclonal antibodies produced against mouse teratocarcinoma cells Noriyuki Hamasima', Masao &to2, Takashi Momoi3, and Toshitada Takahashi'

' Laboratory of Cell Biology and

Laboratory of Experimental Pathology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464,Japan Division or Chemical Pathology, National Center for Nervous, Mental and Muscular Disorders, Kodaira, Tokyo 187, Japan

Abstract. Rat-mouse hybridoma antibodies were produced against mouse teratocarcinoma F9 or PCC4 azal cells, and four clones were established. Both the F11 (IgM) and F20 (IgG2c) antibodies showed a similar specificity, reacting only with nullipotential teratocarcinoma cells. They were also found to agglutinate sheep red blood cells. Solid-phase enzyme-linked immunofluoresccnce assay showed that, among the neutral glycolipids studied, they only reacted with the Forssman antigen. P2 antibody (IgG2b) reacted with the undifferentiated-type and embryonal endodermtype teratocarcinoma cells. During the preimplantation stage, this antibody did not stain mouse embryos, but it reacted very weakly with the inner cell mass of blastocysts cultured in vitro. In the 5th-day embryo, the embryonic ectoderm as well as the visceral and parietal endoderm were positive, but the extraembryonic ectoderm was not. Mesoderm of the 7.5th-day embryo also reacted with this antibody. However, P2 antigen was not observed in the 16thday embryo or in adult tissues. F2 antibody (IgG2a), which was reactive with all of the cultured cell lines tested, showed an immunoreaction with mouse embryos throughout the preimplantation stage. However, in the 75th-day embryo, the presence of F2 was limited to the cells forming the parietal endoderm. This antigen was present in some epithelial tissues of the 16th-day embryo and adult mouse. Of these antigens, P2 and F2 are probably novel differentiation antigens of the early mouse embryo. Together with the Forssman antigen, these will be important markers for analyzing cell-surface antigens of mouse teratocarcinoma cells as well as embryos.

Introduction Cell-surface molecules probably play an important role in the cell-differentiation process of a single multipotential cell into numerous types of tissue during embryogenesis. As one of the approaches to characterize these molecules, antisera have been produced against early mouse embryos or embryonal carcinoma (EC) cells [13, 371. Among these, F9 antigen, which was detected by the antisera produced by immunizing syngeneic 129 mice with F9 teratocarcinoma cells [3], has provided a useful marker for investigating mouse embryogenesis. However, the biochemical properties and the function of F9 antigen still remain to be elucidated. With the advent of the hybridoma technique [19], mono-

clonal antibodies to dissect the cell surface of the mouse embryo have been produced in several laboratories. Among these, SSEA-1 antibody, which detects fucosylated N-acetyllactosamine units [lo] in high-molecular-weight glycopeptides [26], first appears on the cell surface of preimplantational embryos at the eightcell stage and then on the inner cell mass of blastocysts during the preimplantation stage [31]. M1122.25 antibody detects the Forssman antigen, which is expressed as a differentiation antigen from the morula stage onwards and is present on the inner cell mass [38]. Recently, Sato et al. [28] have demonstrated that C-9-1 monoclonal antibody, which was raised against human nullcell-type acute lymphocytic leukemia in our laboratory [36], also reacts with mouse teratocarcinoma cells as well as selected regions of the developing mouse embryo. Some of these monoclonal antibodies produced against nullipotential teratocarcinoma cells or mouse preimplantational embryos detect carbohydrate moieties of glycoproteins or glycolipids, which are found to undergo significant alterations during the early stages of mouse embryogenesis [12, 21, 321. In contrast to nullipotential teratocarcinoma cells, multipotential teratocarcinoma cells retain most of the properties of the original tumor, i.e., the capacity to differentiate in vivo and in vitro into derivatives of the three germ layers [22]. Immunization with multipotential teratocarcinoma cells such as PCC4 may serve to reveal new surface antigens on the mouse embryo. In the present study, rat monoclonal antibodies were produced against nullipotential F9 or multipotential PCC4azal teratocarcinoma cells. Serological analysis of the four antibodies produced demonstrated that two detect a specificity of the Forssman antigen, and the other two define two different novel differentiation antigens of the mouse embryo. Methods

Cells. The characteristics of the mouse teratoma cell lines used for the present study are as follows: F9 [4], Nulli-SCC. 1 [23], PCC4 azal [14], and OTT6050 [34] are of the undifferentiated type and are generally referred to as ECs. The first two are thought to lose their potential to differentiate in vitro or in vivo, unless chemical inducers, such as retinoic acid, are used ([15] ; undifferentiated nullipotential type), but the other two are able to differentiate into several tissues in vitro ([22] ; undifferentiated multipotential type). PSA-

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5E [2], PYS-2 [20], and PYS-4 [20] are of the differentiated type that express embryonal endoderm-like characteristics (embryonal endoderm type). PCD-1 [5] has features characteristic of myocardial myoblast cells, whereas OTT6050f [31] is a fibroblast cell line (mesodermally differentiated type). Besides mouse teratoma cells, cultured skin fibroblasts and lymphocytes from 129 mice were included in the panel cells for the screening of monoclonal antibodies. Tera 2 [8] and NEC 8 [40] from human teratocarcinoma and other human cell lines were also used. All mouse cell lines (except OTT6050) and the human cell lines derived from solid tumors were maintained as a monolayer in Eagle's minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) in 5% COz in humidified air at 37" C. The human hematopoietic cell lines, NS-1mouse myeloma cell line, and hybridomas were maintained in RPMI-1640 supplemented with 10% FCS. The F9, PCC4 azal, PSA-SE, PYS-4, and PCD-1 cell lines were kindly provided by Dr. K. Artzt (Memorial Sloan-Kettering Cancer Center, NY, USA). PYS-2 and OTT6050f were given by Dr. K. Ajiro (this Institute). OTT6050, which was provided by Dr. T. Muramatsu (Kagoshima University School of Medicine, Kagoshima, Japan), was intraperitoneally transplanted in 129 mice. Immunization and cell fusion. Wistar-King-A rats were immunized three times by subcutaneous injection of 5 x lo6 F9 or PCC4 azal cells. Four days after the booster immunization, the spleen cells were obtained and fused with NS-1 according to the method of Kiihler and Milstein [19] with a slight modification [29]. The antibody activity of the supernatants was examined by a rat mixed hemadsorption assay (MHA [6]) against the immunizing cells. The positive supernatants were further tested against the panel cells consisting of other mouse teratoma cell lines and several human cell lines which have been reported to be positive with SSEA-1 antibody [31]. Enzyme-linked immunofluorescence assay. The reactivity of antibodies with neutral glycolipids was examined using a solid-phase enzyme-linked immunofluorescence assay (ELFA) according to a previously described method [41]

with a slight modification. Microelisa plates (Dynatech) were coated by evaporation with 10 p1 each glycolipid (10 pg/ml) dissolved in Dulbecco's phosphate-buffered saline without C a + + and Mg' [PBS(-)] containing 0.1% sodium deoxycholate. After 45 min of incubation with antibodies at 37" c , the plates were washed three times in PBS (-) containing 0.05% Tween 20. Alkaline phosphataseconjugated anti-rat IgG (Cappel) was distributed into each well at a dilution of 1 : 100 and then incubated for 45 min at 37" C. After three washings in PBS( -) containing Tween 20,50 pl M 4-methylumbelliferyl phosphate in 50 mM carbonate buffer (pH9.8) was added to each well as the substrate. After 30 min of incubation at 37" C, the resulting fluorescence was measured in arbitrary units. +

Immunohistological study. Preimplantational embryos were obtained from ICR mice by hormonal treatment as previously described [l11 and then examined by indirect membrane immunofluorescence (IF) assay. The day when vaginal plugs were observed was designated as day 0 of pregnancy. Since it is difficult to obtain frozen sections of implantational embryo, blastocysts without zonae pellucidae were cultured in MEM with 20% FCS in Lab-Tek chamber slides for up to 48 h as described elsewhere [l 11. The preimplantational and implantational embryos were examined by membrane IF. Postimplantational embryos with maternal decidual tissues were dissected from the uteri, embedded in OCT compound (Lab-Tek), and sectioned using a cryostat. The specimens were then air-dried, fixed in cold acetone for 10 min, and examined by indirect IF.

Results Production of rat monoclonal antibodies against F9 and PCC4 azal mouse teratocarcinoma cells

The spleen cells from rats immunized with either nullipotential F9 or multipotential PCC4 azal EC cells were fused with NS-1 cells, and four clones were established. Three (F11,F20, and F2) were from immunization with F9 cells, while the other (P2) was from immunization with PCC4 azal cells.

Table 1. Reactivity of the four monoclonal antibodies tested against mouse teratocarcinoma cell lines by mixed hemadsorption assay ~~~~~

Cell line

Target cells

Titer of monoclonal antibody x lo-"

Type

Potentiality

EC EC EC EC Dif' Dif Dif Dif Dif

Nullipotent Nullipotent Multipotent Multipotent Primitive endoderm Parietal yolk sac Parietal yolk sac Myocardial myoblast Fibroblast

Fl l(IgM)

F20(IgG2c)

P2(IgG2b)

F2(IgG2a)

~

F9 Nulli-SCC.1 PCC4 azal

OTT6050' PSA-SE PYS-2 PYS-4 PCD-1 OTT6050f a

In vivo tumors Embryonal carcinoma cell Differentiatedtype of teratocarcinoma cells

100 100 100

I00 10 100 100 100 100

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25 50 100 200 400 Reciprocal of dilution

800

Fig. 1. Reactivity of F11 antibody tested against neutral glycolipids by solid-phase enzyme-linked immunofluorescence assay. The amount of fluorescence was measured in arbitrary unity (excitation at 365 nm and fluorescence at 450 nm). Among the various neutral glycolipids tested, the Forssman antigen (-0-0-0-) reacted with F11 antibody. The following neutral glycolipids (-m-m-m-)were unreactive : glycosyl ceramide, galactosyl ceramide, kactosyl ceramide, globoside, and asialo GMl

bodies secreted by the hybridomas were absorbed in vivo with the corresponding antigens in adult mice. Both antibodies agglutinated sheep red blood cells (SRBC), but not human red blood cells (RBC), suggesting that the antigen detected is related to the Forssman antigen. P2 (IgG2b) antibody showed immunoreactivity to the undifferentiated type of teratocarcinoma cells (F9, Nulli-SCC.l, PCC4 azal, and OTT6050) as well as to the embryonal endoderm type of teratocarcinoma cells (PSA-SE, PYS-2, and PYS-4) at dilutions of up to 1/10' to 1/106. F2 (IgG2a) antibody was reactive with most of the mouse cell lines, including the B6-3A-SF (Rous-sarcoma virus-transformed cells) and BALB/3T6 fibroblast cell lines (Table 1). Thymocytes and spleen cells as well as spermatogenic cells were tested with these antibodies, but were not found to be reactive. None of the four antibodies produced was reactive with the human teratocarcinoma cell lines, Tera 2 and NEC 8, or with the human panel cells, including HepG2, SW1116, and HL-60,which have been reported to react with SSEA-1 antibody [31]. Other human lymphoid cell lines (CCRF-SB, CCRF-CEM, and K562) were also unreactive with these antibodies. Neither P2 nor F2 was reactive with human fetal or adult RBC.

Serological reactivities against teratocarcinoma cell lines

Biochemical characterization of the antigen detected

Both F11 (IgM) and F20 (IgG2c) showed a quite similar specificity, reacting only with the undifferentiated nullipotential type of teratocarcinoma cells (F9 and Nulli-SCC.l); however, the titers of ascites from hybridoma bearing mice were relatively low (1/103), probably because these anti-

As already mentioned, the relationship of F11 and F20 with the Forssman antigen was suspected due to the reactivity of these antibodies against SRBC. Accordingly, further analyses were carried out. First, an absorption experiment showed that the antigens on SRBC were heat stable for

Fig. 2 A-D. Indirect-immunofluorescence staining of embryoid bodies from the ascitic OTT6050 teratocarcinoma with F2, P2, and F11 antibodies. The entire surface of the cell mass was reactive with both the F2 (A) and P2 (B) antibodies. In a cryostat section, P2 antibody reacted with the surrounding tissues (arrow) more intensely than with the inner core (C). However, F11 antibody showcd no membrane fluorescence (D). A, B, D x 200; C x 100

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Fig. 3A-F. Indirect-immunofluorescence staining of preimplantational embryos with monoclonal antibodies. A fertilized egg (A) and a twocell-stage embryo (B) reacted with F2 antibody. However, P2 antibody was not reactive with embryos at the two-cell, eight-cell, or blastocyst stage (C). D Phase-contrast microscopy of the same embryo. E A morula-stage embryo was unreactive with F11 antibody. F Phase-contrast microscopy of the same embryo. A, B, E, F x 200; C, D x 100

5 min at 100" C (data not shown). Second, the reactivity

of the antibodies against neutral glycolipids was examined by solid-phase ELFA, and it was found that both antibodies reacted specifically with the purified Forssman antigen as shown in Fig. 1 (data with F20 not shown). For immunohistological study, F11 antibody was predominantly used. The reactivity of the P2 and F2 antibodies against neutral glycolipids was also tested, but not significant reactions were observed.

of the inner core as well as the surrounding tissues; P2 antibody reacted more strongly with the latter than with the former. In contrast, F11 was found to be negative (Fig. 2).

Immunohistological localization of the antigen detected

Mouse embryos during preimplantational development. The cell surface of a fertilized egg showed no reactivity with the P2 or F11 antibodies, whereas F2 antibody showed a strong reactivity. The presence of F2 was observed throughout the preimplantation stage, while the P2 and F11 antigens remained negative during this period (Fig. 3).

OTT6050 embryoid bodies. Both the F2 and P2 antibodies reacted with the entire surface of OTT6050 embryoid bodies by membrane IF. Examination of the frozen sections demonstrated that both antibodies reacted with the cell surface

Blastocysts grown in vitro. Blastocysts on day 3 of pregnancy were cultured for up t o 48 h before study, because it is difficult to obtain frozen sections of the embryo at this stage. F2 antibody was still reactive with these cultured

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with the 5th-day embryo. The visceral and parietal endoderm was stained more strongly than the embryonic ectoderm. The trophoblastic and ectoplacental cone cells were weakly positive. The cells forming the extraembryonic ectoderm were, however, negative. The immunoreactivity of P2 antibody to the 73th-day embryo was similar to its immunoreactivity to the 5th-day embryo, but in the former, it reacted with the cells of the mesoderm as well. F I I was present on the cells forming the visceral and parietal endoderm of the 5th- and 73th-day embryos (Fig. 5). Sixteenth-day embryo and adult mouse. The immunoreactions of the 16th-day embryo and adult mouse were quite different from those of early stage embryos. F2 antibody stained some of the tissues, e.g., the epidermal tissues and the underlying basement membrane of the 16th-day embryo. In adult mice, the cells forming the proximal tubules of the kidney as well as the inner lining of the oviduct were intensely positive with this antibody. On the other hand, P2 antibody showed no reactivity with the 16th-day embryo or the adult tissues tested. In the 16thday embryo, F I 1 antibody showed strong reactivity with certain cell types in the mesenchyme, but these were difficult to identify. The reaction with adult tissues was limited to a few cell types. Thus, some of the cells forming the interstitial cells in the testis showed a positive reaction with this antibody.

Discussion

Fig. 4A-C. Blastocysts on day 3 of pregnancy grown in vitro for 48 h stained by membrane immunofluorescence with monoclonal antibodies. The F2 (A) and F11 (C) antibodies were strongly reactivc with the embryo at this stage. P2 (B) antibody was weakly reactive, predominantly with the inner cell mass (arrow). x 100

blastocysts. P2 antibody was also weakly positive on the cell surface of the inner cell mass and some of the cells of the trophectoderm. F11 antibody was found to become strongly positive on the embryo at this stage (Fig. 4). FijUi- and 7.51h-duyembryos. These were studied by preparing frozen sections. On the 5th-day embryo, F2 antigen was no longer detected, but the decidual cells of the maternal uterus reacted strongly with this antibody. However, F2 antibody showed a weak reaction with the parietal endoderm cells of the 7.5th-day embryo, and the visceral endoderm seemed to be very weakly positive as well. P2 antibody, which showed an immunoreactivity with the embryonal endoderm type of teratocacinomas, showed a reactivity

From analysis of rat-mouse hybridoma antibodies against F9 or PCC4 azal teratocarcinoma cells, two different novel differentiation antigens, P2 and F2, of the mouse embryo, as well as the Forssman antigen, were defined. In 1978, Stern et al. [32] reported that the Forssman antigen is predominantly present on mouse EC cells and selected cells of the developing mouse embryo [35]. The distribution of F11 antigen was in general accordance with that reported by Willison and Stern [38]. However, this antibody did not react with the embryo at the morula or blastocyst stages. These minor differences in antigen distribution between M1122.25 and F11 may reflect differences in the detined epitopes, as demonstrated in the human suppressor/ cytotoxic T-cell molecule, Tp32 [24]. Thus, antibody-binding inhibition assays are now in progress to analyze each epitope. P2 antigen was absent from the mouse embryo during the preimplantation stage, but it appeared in the embryonic ectoderm as well as in the visceral and parietal endoderm of the 5th- and 75th-day embryos. This antigen is probably different from SSEA-1 antigen, because SSEA-1 has been reported to be present on the embryo from the eightcell stage [31]. The expression of antigens, such as S E A - 1 191, I [16], C-9-1 [28], AS, and C6 [7] is restricted primarily to the luminal surface of the proamniotic cavity of the egg cylinder, whereas P2 seems to be present in the whole embryonic ectoderm. It is notable that P2 was no longer observed in the 16th-day embryo and adult mouse. The fact that the titers of P2 antibody in nude mouse ascites are high - in contrast to those of the F11 and F20 antibodies against the Forssman antigen - shows that this antibody is not absorbed in adult mice, thus indicating that there are not so many cells with P2 antigen in adult mice. Accordingly, P2 is a good differentiation marker for cells mainly derived from the inner cell mass at the egg-cylinder stage.

265

Fig. 5A-F. lmmunofluorescence of Sthday and 7.5thday embryos with monoclonal antibodies. The 5th-day embryo seemed to be unreactive with F2 antibody, while the maternal decidual tissue was strongly positive (A). The parietal endoderm cells of the 73th-day embryo were weakly positive with F2 antibody, and the visceral endoderm also seemed to be slightly positive (B).P2 antibody reacted strongly with the embryonic ectoderm of the Sthday embryo (C). In the 7.5th-day embryo (D), the cells forming the mesoderm (arrows) were stained with P2 antibody, as were the embryonic ectoderm and the visceral and parietal endoderm. Fl1 antibody reacted with the visceral and parietal endoderm cells of both the Sthday (E)and 7.5thday 0 embryos. x 100

In contrast to P2, F2 antigen appeared as early as on the one-cell-stage embryo. However, this antigen was not detected in the 5th-day embryo and was observed again on the parietal and visceral endoderm of the 7.5th-day embryo. Of the monoclonal antibodies so far reported to be reactive against preimplantational embryo, such as ECMA 2 and 3 [17], I [18], SSEA-3 [30], 2C5 [27], and 5D4 [33], none have the same reactivity as F2 antibody after implantation. The distribution of F2 on the 75thand 16th-dayembryos suggested a similarity to that of typeIV collagen [l], entactin, or laminin [39]; however, these extracellular matrix components are not detected serologically on embryos during the early preimplantation stage, indicating that these are different from F2 antigen. Serological analysis of the P2 and F2 antibodies sug-

gested that these antigens are probably novel differentiation antigens of the mouse embryo. However, it is very important to characterize the biochemical nature of these antigens in order to compare them with those reported by various investigators. Thus, the following preliminary experiments are now in progress: solid-phase ELFA of these antibodies against acidic and neutral glycolipids from PCC4-azal or F9 cells, modified Farr’s assay [25] of these antibodies with 3H-galactose-labeled high-molecular-weight glycopeptides prepared from F9 cells [26], and binding-inhibition experiments with various carbohydrates. However, no significant results have been obtained to date. These experiments will be continued in order to investigate and define the nature of the P2 and F2 antigens. One of the aims of producing antibodies against mouse

266

embryos or teratocarcinomas is to characterize surface molecules which may have a functional role in mouse embryogenesis. For this purpose, the screening of the antibody produced by hybridoma clones should be carried out with a functional assay, e.g., to test whether the supernatants are able to prevent induction by retinoic acid. The production and analysis of many mom antibodies using a functional assay will provide valuable reagents for studying mouse embryogenesis. Acktion.fedgetnents. We are most grateful to Dr. R. Ueda (Laboratory of Chemothcrapy of this Institute) and Dr. T. Muramatsu (Kagoshima University School of Medicinc, Kagoshima, Japan) for stimulating discussions throughout this study and also for providing cultured cell lines. We also thank Dr. K. Artzt (Memorial Sloan-Kettering Cancer Center, New York, USA) and Dr. K. Ajiro (this Institute) for their gift of teratocarcinoma cell lines. This work was supported in part by the Ministries of Education, Science and Culture, and Health and Welfare (Comprehensive 10-YearStratcgy for Cancer Control), Japan, and by the Cancer Research Institute, Ncw York, USA.

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Received August 1984 / Accepted in revised form November 1984