DEVELOPMENTAL
BIOLOGY
47, 407-418
(1975)
The Effects of Anti-Embryo Sera and Their Localization on the Cell Surface during Mouse Preimplantation Development LYNN Departments
of Anatomy
M. WILEY’
and Pediatrics,
AND
PATRICIA
University
G.
of California,
CALARCO San Francisco,
California
94143
Accepted July 16, 1975 We have developed two rabbit antisera, one against mouse blastocysta and a second against mouse placentae. After absorption of these antisera with adult mouse tissues and extensive dialysis, results from indirect immunofluorescence, cytotoxicity, and culturing experiments lead us to two major conclusions.‘First, anti-blastocyst serum detects a group of cell surface molecules whose expression is embryo-specific, stage-specific, and whose unaltered presence is required for preimplantation development in vitro. Second, anti-placenta serum detects a different group of cell surface molecules that are present before fertilization and become segregated to the syncytiotrophoblast, appear unessential for preimplantation development in vitro, and may function in trophoblast differentiation, and/or in protection of the fetus from maternal immunologic attack. INTRODUCTION
ing early sea urchin development (Krach 1974). The cell surfaces of mouse preimplantation embryos possess molecules that exhibit stage differences in their glycoproteins (Pinsker and Mintz, 1973). Other surface moieties are transitorily expressed during cleavage : Antiserum produced against mouse primitive teratocarcinoma cells reveals the presence of surface molecules maximally expressed on mouse &cell cleavage embryos (Artzt et al., 1973) and an antiserum to human chorionic gonadotropin (hCG) detects hCG-like molecules whose expression peaks on mouse morulae (Wiley, 1974). During preimplantation development, transitorily expressed cell surface molecules may be associated with specific intracellular events and could also mediate interactions between cells during blastocyst formation. This report describes an immunologic approach used to study the appearance and function of surface molecules on mouse preimplantation embryos. Two heterologous antisera, made separately against mouse placentae and mouse blastocysts, are shown by indirect immunofluorescence and cytotoxicity to interact with
et al.,
Many changes in an animal cell’s physiologic or developmental state are accompanied by changes in the cell surface. Cell cycle changes (Smets, 1973; Kerbel’ and Doer&off, 19741, oncogenic transformation (Bosenblith et al., 1973; Barnett et al., 1974a,b; Edelman et al., 1973; Yahara and Edelman, 19731, cell differentiation in culture (Akeson et al., 1974a,b), tissue-specific cell recognition (Goldschneider and Moscona, 1972) species-specific cell recognition (Moscona, 19681, and temporal, or time-dependent, tissue-specific cell recognition (Gottlieb et al., 1974) are associated with cell surface alterations. Temporal expression of antigenic molecules occurs as early as cleavage and gastrulation in sea urchin embryos (Westin et al., 1967). However, in these studies, the embryos were homogenized before analysis and it was not determined if any of these molecules were external plasma membrane components. More recently, specific changes in carbohydrate-containing surface molecules were detected duri In partial fulfillment of the requirements for the degree of Doctor of Philosophy, with the Department of Anatomy, University of California, San Francisco. 407 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
408
DEVELOPMENTALBIOLOGY
embryonic cell surfaces. Effects of both antisera on mouse preimplantation development in vitro are also described.
VOLUME 47, 1975
mately 200 blastocysts per weekly injection and 800 blastocysts per booster injection. Beginning 1 week after booster injecMATERIALS AND METHODS tions, rabbits were bled at weekly interAntisera Preparation vals and sera were heat-inactivated (56”C!, Anti-plncenta serum (A-PL). A modified 30 min), filter-sterilized, and frozen until method (Kometani and Behrman, 1971) needed. Before use, thawed aliquots of was used to produce the A-PL serum. Pla- sera were absorbed in the following way. A centae from 16- to 18-day gestation SWR crude membrane preparation was obmice were dissected free of decidual tissue tained by mincing SWR mouse livers, kidand fetal membranes, minced, and rinsed neys and spleen (LKS) in PBS and homogeseveral times with cold phosphate-buffered nizing minced tissues with a Dounce hosaline (PBS). Minced placentae were then mogenizer. The homogenate was centrihomogenized with a Dounce homogenizer, fuged at low speed (lOOO-12OOg) and the and 0.5 ml of the homogenate was emulsipellets washed with PBS until the supernafied with 0.5 ml Freund’s incomplete adju- tant was clear. Pellets were resuspended vant. A male New Zealand white rabbit in an equal volume of antiserum and alreceived 1.0 ml of the placental antilowed to stand at 4°C for 45 min before gen/Freund’s incomplete adjuvant prepararecovering the serum by centrifugation. tion suprascapularly, subcutaneously, Absorptions were repeated two more times once weekly for 4 consecutive weeks, 1 so that each aliquot of serum was exposed to a volume of packed “membranes” three week following an injection of Freund’s complete adjuvant. This initial injection of times the volume of the serum. complete adjuvant is thought to nonspecifiAbsorbed antisera were dialyzed in cold PBS for 5 days and filter-sterilized into lcally stimulate the rabbit’s immunologic ml rubber-capped vials. Nondialyzed antisystem, enhancing the response to the antiserum was found to be toxic in a nonspegen (Dov Michaeli, personal communication). The rabbit was allowed to “rest” for 4 cific way to mouse embryos in vitro. weeks and was then boosted with an addiNormal rabbit control serum was treated as described above for immune tional injection. sera. Anti-blastocyst serum (A-BL). Blastocysts were harvested and freed from their zonae pellucidae by mouth pipet (Tar- Embryos kowsky, 1961) in culture medium which All embryos came from either SWR or had 0.5 g/100 ml medium Ficoll (Brinster, mice. Superovulation 1965) in place of 0.3 g/100 ml medium bo- ICR superovulated vine albumin (BSA). Pronase was not used (Gates, 1971) and recovery of embryos (Rafdeto remove zonae because it alters cell sur- ferty, 1970) were done as previously face molecules (Wallach, 1972). After re- scribed. A modified Bigger’s (Biggers et moving the zonae from the injection prepaal., 1971) ovum culture medium (Spindle, was used for culration, the blastocysts, in 0.1 ml PBS or personal communication) Ficoll-containing medium were emulsified turing, indirect immunofluorescence aswith 0.1 ml Freund’s incomplete adjuvant. says with live embryos and cytotoxicity tests. Follicular cells were removed from Two male New Zealand white rabbits were immunized with this preparation using unfertilized oocytes and l-cell embryos the same injection protocol followed for the with hyaluronidase (180 IU/O.5 ml culture A-PL serum. Each rabbit received approximedium).
WILEY
Indirect
AND
CALARCO
Immunofluorescence
Cell Surface
Assays (ZIF)
Livepreimplantation embryos. These assays were performed under humidified 5% CO, in air at 37°C. Embryos were incubated for 30 min in immune serum diluted 1:l with ovum culture medium, rinsed with two 5-min changes of ovum culture medium, incubated for 10 min in goat anti-rabbit IgG conjugated with fluorescein (Antibodies, Inc.) diluted 1:4 with PBS, and rinsed with two 5min changes of ovum culture medium to remove unbound conjugate as described previously (Wiley, 1974). In some cases the zona pellucida was removed by mouth pipet. We found that embryos with zonae were much easier to handle than zona-free embryos and, therefore, suggest that zona removal be done following the final rinse. Blastocyst outgrowths. Assays on outgrowths were also performed at 37°C under humidified 5% CO, in air using the same IIF protocol as for the preimplanted embryos. Paraffin-embedded, sectioned material. IIF was done with slides of paraffin-embedded mouse 16-18day gestation placentae, fetus, and mouse ovary using methods previously described (Glass, 1971). Sections of mouse blastocysts were similarly treated after first being placed in ant pupae cases (Mintz, 1971). Carnoy’s fixative (Humason, 1962) was used with all tissues except for the blastocysts, which were fixed in Tellyesniczky’s fixative (Humason, 19621. IIF samples were viewed with a Zeiss fluorescence microscope and photographed using Agfa 500 daylight color slide film, lmin exposures. Cytotoxicity
Tests
A fine-bore pipet was used to remove the zona before cytotoxicity was assessed. Zona-free 2-cell and 8-cell embryos were incubated in serum diluted 1:l with ovum culture medium for 45 min at 37°C under humidified 5% CO, in air. Sera were taken from the same batches used in the IIF
in Mouse Preimplantation
Embryo
409
assays and culturing experiments. Embryos were next transferred to a mixture of 50% fresh guinea pig serum (complement), 25% antiserum, and 25% ovum culture medium, and incubated as before. Viability was determined by placing the embryos in 0.5% trypan blue in 0.85% NaCl for 5 min and counting the number of blue embryos (2-cell stage) or blastomeres (8cell stage). Embryo Culture The effect of immune sera in preimplantation development was determined by culturing 2-cell embryos in dilutions of serum using the microdrop culture technique (Rafferty, 1970). Culturing experiments were carried out in duplicate, and each experiment was repeated two or more times. Each dilution of antiserum was tested on a total of at least 100 embryos. After 65-72 hr in culture, embryos were scored for blastocyst formation, All embryos containing blastocoels were scored as blastocysts regardless of whether or not expansion had occurred. Blastocyst outgrowths were produced by a method previously described (Spindle and Pedersen, 1973). Briefly, expanded blastocysts were placed into the chambers of tissue culture slides (Lab. Tek., Catalog No. 4808) containing 0.3 ml Eagle’s minimal essential medium (MEM) fortified with amino acids (Spindle and Pedersen, 1973) and 10% fetal calf serum. After 4-5 days of culture the outgrowths were used for IIF assays. RESULTS
Indirect
Immunofluorescence
Assays (II%)
IIF is far more sensitive in detecting antigen-antibody binding than immunodiffusion or cytotoxicity. We have defined the specificity of A-PL and A-BL sera in terms of IIF data and have also used IIF to monitor completeness of absorption. Anti-placenta serum (A-PL). Unabsorbed A-PL serum reacts strongly in IIF assays with all mouse tissues tested (see
410
DEVELOPMENTAL BIOLOGY
below). After absorption only the syncytiotrophoblast of sectioned mouse placenta is positive. None of the fetal (fetal red blood cells, connective tissue components of the chorionic villi) or maternal (mature erythrocytes in the intervillous lakes) elements of the placenta fluoresce (Fig. 1). All cell types in sections of mouse ovary and all tissues of sagittal sections of 16-day mouse fetus treated with absorbed A-PL serum resemble their counterpart NRS controls, i.e., they are negative. These observations lead us to conclude that antigens reacting with A-PL serum are not present on fetal or maternal cells, or they are present in levels too low for detection by our methods in these tissues. A-PL serum localizes on the surfaces of ovarian and ovulated oocytes (Figs. 2a,b), and mouse embryos throughout preimplantation development (Figs. 2c-g). While there is no appreciable difference in the intensity of fluorescence among the oocytes and preimplantation stages, the pattern of membrane fluorescence changes with the onset of cleavage. Surfaces of ovarian and ovulated oocytes and l-cell embryos display patches of membrane fluorescence which disperse with the first cleavage (Figs. 2c and d) to yield a diffuse, even membrane fluorescence which persists throughout cleavage and blastocyst formation. On sectioned blastocysts, A-PL
VOLUME 47, 1975
serum does not differentiate betwen trophoblast and ICM. For blastocyst outgrowths induced by a change in medium (Spindle and Pedersen, 1973) both the inner cell mass (ICM) and the trophoblast sheet show a diffuse, even reaction with APL serum (Fig. 2h). As the outgrowths continue to develop, however, membrane fluorescence appears to decrease on portions of the ICM and persists over the trophoblast sheet. Although other workers have cultured mouse embryos to early somite stages in similar culture systems (Hsu et al ., 1974; Spindle et al., unpublished results), we emphasize that this is an in vitro system and may not resemble in uiuo development. Positive fluorescence of A-PL serum with mouse placenta and preimplantation embryos is removed by absorption with cell membranes isolated from homogenized 16-Wday mouse placentae. Pretreating mouse unfertilized oocytes or preimplantation embryos with either 0.5% Pronase (in Ficoll-containing ovum culture medium, for 5 min), or trypsin (Lin and Florence, 1970) abolishes A-PL membrane fluorescence. Some degree of A-PL serum binding is regained after 4 hr incubation in culture medium. Anti-blastocyst serum (A-BL). When tested on preimplantation embryos, absorbed A-BL serum produces little mem-
FIG. 1. Indirect immunofiruorescence: Anti-placenta serum, mouse placenta. In paraflin-embedded, sectioned mouse placenta only the trophoblast (T) is positive with A-PL serum (a). Compare with the NFW control (b), which shows only a low level of orange autofluorescence. Magnification, 500 x
WILEY
AND
CALARCO
Cell Surface
brane fluorescence until the 4-cell stage (Figs. 3a-c). Reactivity towards A-BL serum is low with unfertilized oocytes and lcell embryos, increases with cleavage, and peaks on 8-12-cell embryos (Figs. 3d and e). Thereafter, fluorescence diminishes (Figs. 3f and g> and disappears completely on blastocyst outgrowths (Fig. 3h). The pattern of membrane fluorescence is always diffuse. A very low level of fluorescence results when sectioned blastocysts are treated with A-BL serum. No consistent differences are noted between trophoblast and ICM cells. Sections of mouse ovary, fetus, and placenta show no reac-
in Mouse Preimplantation
tion whatsoever rum. Cytotoxicity
with
Embryo
absorbed A-BL
411 se-
Tests
To verify that these sera were detecting a surface antigen, cytotoxicity tests were performed (Table 1). The A-PL serum was cytotoxic (diluted 1:l with culture medium) to 2-cell (i.e., 29 lysed/43 total No. embryos) and El-cell (27/80) embryos, while A-BL serum (diluted 1:l with culture medium) was not cytotoxic for 2-cell embryos (O/23) but did lyse 75% of &cell blastomeres (SO/SO). Serial dilutions of antisera were not tested.
FIG. 2. Indirect immunofluorescence: Anti-placenta serum, mouse preimplantation embryos. The intensity of membrane fluorescence is constant on (a) ovarian oocytes, (b) ovulated ova, and all of the preimplantation stages l(c), zygote; (d), 2-cell; (e), 8-cell; (0, morula; and (g), blastecystl. However the pattern of fluorescence changes with the first cleavage. This is evident where the lower l-cell (c) and 2-cell (d) embryos are compared, where the cell surfaces are in focus. In all the other micrographs the cell rims are in focus. Membrane fluorescence is even and diffuse over the trophoblast sheet (T) and ICM (I) of blastocyst outgrowths (h). NRS control embryos: blastocyst (i), and 2-cell (i). Magnification, 500 X, except the outgrowth that is 300 x.
412
DEVELOPMENTAL
BIOLOGY
VOLUME
47, 1975
FIG. 3. Indirect immunofluorescence: Anti-blustocyst serum, mouse preimplantation embryos (zona-free). What little membrane fluorescence is seen on l-cell (a) and 2-cell (b) embryos increases on the 4-cell (c) and peaks around the g-cell (d) to 12-cell stages (e). Thereafter membrane fluorescence diminishes on morulae (f~ and blastocysts (g). This blastocyst appears to have transported fluorescein conjugate into its blastocoel and also contains a filled, fluorescing blasmmere (due to cell death). No fluorescence is seen on the trophoblast sheet (T) or ICM (I) of blastocyst outgrowths (h). NBS control embryos: 2-cell (i) and 8-cell (j) stages. Magnification: 500 x , except for the outgrowth that is 300 x .
TABLE CYTOTOXICITY
OF ANTISERA
Stage
1
TO PREIMPLANTATION
A-PL Serum No.
2-Cell embryos Lysed or blue embryos/total No. embryos g-Cell embryos Lysed or blue blastomeres/total No. blastomeres B Cytotoxicity b A different
Culturing
%
23/25 6/18@ 27/80b
92 33 34
was based on the lysis or the tilling of blastomeres batch of antiserum was used in these tests.
Experiments
Extensive dialysis of sera used in culturing was found necessary to eliminate nonspecific toxicity (see Materials and Meth-
EMBRYOS”
A-BL Serum No. 0123 60/80
with trypan
NBS
%
No.
%
0
o/21
0
75
5140
12.5
blue.
ads). Embryos cultured in insut&iently dialyzed sera progressively turned grainy and darkened with time. Although they generally cleaved, these embryos often failed to blastulate. After extensive di-
WILEY
AND
CALARCO
Cell Surface
alysis, however, the ability of 2-cell embryos to form blastocysts in l-10% A-PL serum was comparable to that of embryos cultured in the same dilutions of NRS (Fig. 4). The A-BL serum, however, continued to adversely affect cleavage in vitro and at concentrations of 4% or more prevented development (Fig. 4). Embryos cultured in A-BL serum differed in appearance from those nonspecifically killed by insufficiently dialyzed serum. In higher concentrations of A-BL serum (2.54%) very little, if any cleavage occurred and almost all embryos shrank and darkened overnight. An occasional embryo arrested and remained at the 2-cell stage the entire culture period. As the concentration of ABL serum decreased an increasing number of embryos consisted of a mixture of degenerate and “normal” cleaving blastomeres. Also the number of degenerate blastomeres per embryo went down as the concentration of A-BL serum was lowered. As IN VITRO EMBRYOS
in Mouse Preimplantation
Embryo
413
serum concentrations continued to diminish an increasing percentage of embryos with damaged blastomeres formed blastocoels. Preliminary data from culturing embryos in concentrations of A-BL immunoglobulin fractions indicate that immune antibodies are responsible for (at least some of) the effects A-BL serum has in culture. DISCUSSION
Our data probably do not result from antibodies recognizing “universal” mouse cell surface molecules because not only were our antisera absorbed with adult mouse liver, kidney, spleen homogenates, but they were also negative in IIF assays for all tissue types in sagittal sections of whole mouse 16- to l&day fetus and in sections of adult mouse ovary. It is unlikely that H-2 antigens are responsible for out results because (i) embryos and placentae from random-bred mice were used as
CULTURE OF MOUSE PREIMPLANTAT ION IN THE PRESENCE OF IMMUNE ANTIBODIES
FIG. 4. Culturing experiments. Two-cell embryos, after 65-72 b in microdrop culture, are scored for blaatocyst formation. All embryos having a blastocoel are counted as blastocysts, regardless of whether or not expansion has occurred. Each point represents the average value from at least two separate experiments and, for each experiment, each dilution is done in duplicate drops. Between 20 and 25 embryos are placed in each drop, so that each point represents the response of at least 100 embryos.
414
DEVELOPMENTAL BIOLOGY
immunogens, (ii) exhaustive absorptions were done with pooled random-bred LKS homogenates, and (iii) H-2 antigens are not detectable by IIF before implantation (Heyner et al., 1969; Palm et al., 1971; and Heyner, 1973) and may not appear on embryonic cells before day 6 or 7 of gestation mouse (Edidin et al ., 1971). Although preimplantation embryonic surfaces crossreact weakly with non H-2 histocompatibility antigens (Palm et al., 1971), these antigens are probably not responsible for our results for reasons (i) and (ii) above.
Anti-Placenta Serum (A-PL) Our A-PL serum detects surface molecules present on oocytes, preimplantation embryos and trophoblast derivatives following implantation. An A-PL serum developed in another laboratory (Kometani et al., 1973) differs from ours in IIF-defined specificities by not recognizing unfertilized ova or zygotes. Further, when tested on sectioned blastocysts and egg cylinders, the embryonic part, rather than the trophoblast appears to have a greater affinity for their serum, although their serum is cytotoxic for trophoblast cells cultured from e&placental cones. These workers hypothesize their A-PL serum might contain “fetal-specific” antibodies although they do not mention any tests done on sectioned fetal tissues. Our A-PL serum, however, is positive for unfertilized oocytes, all other preimplantation stages, and syncytiotrophoblast, negative for fetal and adult tissues, and does not differentiate between trophoblast and embryonic portions of IIFassayed, sectioned blastocysts. Our A-PL serum could differ from their A-PL serum for the following reasons: (i) genetic variability among rabbits; response to the same immunogen by nonsyngeneic animals will differ because the response will depend on the animal’s genotype; (ii) the way in which an immunogen is administered can determine the immune response, and slight differences in immunization procedures could result in differing antisera. In addition to its well-documented roles
VOLUME 47,
1915
in fetal nutrition and hormone production, additional evidence suggests that the trophoblast (Simmons and Russell, 1962, 1966; Vandeputte and Sobis, 1972; Stevens, 1968), its “fibrinoid coat” (Kirby et al., 1964, 1966; Kirby, 1968) or, in particular, the maternal interface of the syncytiotrophoblast (Martin et al., 1974) may fence off the fetus from maternal immunologic attack. Our IIF data suggest that maternal-embryonic interfaces are, from the very beginning of development, continuously supplied with A-PL-specific molecules. Some of these A-PL-specific molecules may, then, function in isolating the fetus from immunologic attack. Alternatively, these molecules may participate in differentiation, function, trophoblast and/or maintenance.
Anti-Blastocyst Serum (A-BL) Our IIF, cytotoxicity, and culturing data all indicate that absorbed A-PL and A-BL sera are different (Table 2). In particular, they appear to detect two separate populations of surface antigens. A-PL serum-detected antigens are present before fertilization and become segregated to syncytiotrophoblast. A-BL serum recognizes surface antigens whose detection (by our methods) is confined to preimplantation development and is maximal on 8+-cell embryos. To support these conclusions, we propose that these two separate groups of molecules differ in the times during development when they are immunogenic (can elicit an immune response) and differ in times when they are antigenic (accessible to, and recognized by, antibodies) (Campbell et al., 1964). Antigenicity and immunogenicity are different attributes and can occur independently (Campbell et al., 1964). At the blastocyst stage, for example, A-PL detected surface molecules are not yet immunogenic but are antigenic (i.e., detectable by IIF); while A-BL detected molecules are both immunogenic (induce antibody formation) and antigenic (detectable by IIF). Furthermore, the constant expression of
WILEY AND CALARCO
Cell
Surfacein TABLE
Mouse Preimplantation
415
Embryo
2
CHARACTERISTICS OF ABSORBED ANTISERA Procedure Indirect immunofluorescence
Anti-placenta
Anti-blastocyst
(A-PL)
Positive for all ova, preimplantation embryos, blastocyst outgrowths, and trophoblast of sectioned mouse placenta; negative for sectioned mouse ovary and fetus Fluorescence is patchy on ova and lcell embryos, diffuse on all other stages
Cytotoxicity
Cytotoxic
for 2-cell and g-cell embryos
Culturing
No effect on preimplantation ment
A-PL-specific molecules is initially controlled by the maternal genome. In contrast, the temporal expression of A-BLspecific molecules is most likely a result of embryonic genome activity and protein synthesis. A major group of proteins (MW 53,000-57,000) is synthesized primarily between the 2- and 4-8-cell stage (Epstein and Smith, 19741, while a large shift in the nature of surface glycoproteins occurs between the 4-8-cell stage and the blastocyst stage (Pinsker and Mintz, 1973). Some of these proteins may be components of the A-BL-specific surface molecules maximally expressed on 8 +-cell embryos, while the shift in surface glycoproteins may be related to the diminishing expression of ABL-specific molecules after the 8+-cell stage. The peak expression of A-BL-detected surface molecules on mouse embryos coincides very closely with the time of peak expression of a surface moiety(ies) reportedly controlled by an allele at the T locus (Artzt et al., 1974). The T locus has a number of recessive alleles, one of which, the t12 allele is lethal in homozygous embryos at the morula to early blastocyst stage (Mintz, 1964). Recently, an antiserum to mouse primitive teratocarcinoma cells (APTC) was shown to be cytotoxic to mouse
develop-
(A-BL)
Negative for all ova, weak on l-cell and 2-cell embryos increases on 4-cell stage, peaks on 8-12 cell stage, diminishes thereafter to disappear on blastocyst outgrowths; negative for all sectioned mouse tissues Fluorescence is diffuse when present
Noncytotoxic for 2 cell embryos, cytotoxic for g-cell embryos
and is
Impairs development at a concentration of 2.5% serum, stops development altogether at 4% serum
sperm and to contain antibodies recognizing cell surface molecules maximally expressed on normal 8-cell mouse embryos when (Artzt et al., 1973). Interestingly, used for quantitative absorptions, sperm from TIP mice were only half as effective as sperm from normal mice at removing activity from the A-PTC serum (Artzt et al., 1974). Their inference is that, on both P sperm and t12/t12embryos, molecules on the cell surface normally detected by APTC serum may be missing or altered. Another antiserum to a mouse teratoma cell line is cytotoxic to mouse unfertilized eggs and to several transformed mouse cell lines (Edidin et al., 1971; Cooding and Edidin, 1974). IIF with pre- and postimplantation mouse embryos shows that their antiteratoma serum localizes on cells believed destined to form tissues expressing H-2 antigens in the adult; normal adult mouse cells do not react with anti-teratoma serum. These authors suggest that their anti-teratoma serum is detecting H-2 antiwhich are, therefore, gen precursors, found only on cells which will later acquire transplantation antigens. It is unlikely, however, that our A-BL serum is detecting these “H-2 antigen precursors” because ABL serum is negative for all cells in postimplantation mouse embryos in IIF assays.
416
DEVELOPMENTAL BIOLO~,Y
Since the complexing of A-BL-specific molecules with antibodies impairs development, we assume that the A-BL-specific molecules are required for development to proceed. These molecules might be involved in such processes as cell recognition, cell adhesion, the transitory appearance of a new, embryo-specific substrate carrier or could function as receptor molecules which trigger intracellular event(s) characterizing the 8 +-cell embryo. Intriguing to us is the possibility that A-BL-detected molecules participate, by whatever process, in separating ICM from trophoblast during blastocyst formation. We are presently investigating the mobility, spatial distribution, density, and cross-reactivity of A-BL and A-PL specific molecules on preimplantation embryos. Other studies on the obvious contraceptive potential of these antisera are contemplated. In conclusion, anti-blastocyst serum shows that mouse preimplantation embryos possess immunogenic surface molecules which are embryo-specific, stage-specific and whose presence is required for development. Anti-placenta serum reveals the presence of a different group of surface molecules that, when bound with antibody, have no effect on preimplantation development, and appear to segregate to the trophoblast; they may, then, function in trophoblast differentiation, in maintenance, or in protection of the fetus from maternal immunologic attack. The authors are grateful for the expert technical assistance of Peg Siebert, Jean Hanson, Sandra Smith, and Estrella Lamella and for the photographic talents provided by Mr. Dave Akers. Special thanks are given to Dr. Akiko Spindle and Dr. Roger A. Pedersen for their assistance in culturing blastocyst outgrowths. We thank Dr. Dov Michaeli and Dr. Robert P. Erickson for assistance with the immunologic aspects of this work and Dr. Charles J. Epstein, Dr. Dov. Michaeli, Dr. William J. Rutter, and Dr. Laurel E. Glass for help in preparing the manuscript. This work was supported by the Department of Anatomy and by grants, U.S.P.H.S. No. GM-19527 and l-ROl-CA-16493-01.
VOLUME 47, 1975 REFERENCES
AKESON, R., and HERSCHMAN, H. R. (1974a). Modulation of cell-surface antigens of a murine neuroblastoma. Proc. Nut. Acad. Sci. USA 71, 187-191. AKESON, R., and HERSCHMAN, H. R. (1974b). Neural antigens of morphologically differentiated neuroblastoma cells. Nature (London) 249, 620-623. ARTZT, K., DUBOIS, P., BENNETT, D., CONDAMINE, H., BABINET, C., and JACOB, F. (1973). Surface antigens common to mouse cleavage embryos and primitive teratocarcinoma cells in culture. PFOC. Nat. Acad. Sci. USA 70, 2988-2992. ARTZT, K., BENNETT, D., andJAcoB, F. (1974). Primitive teratocarcinoma cells express a differentiation antigen specified by a gene at the T-locus in the mouse. Proc. Nat. Acad. Sci. USA 71, 811814. BARNETT, R. E., FURCHT, L. T., and SCOTT, R. E. (1974a). Differences in membrane fluidity and structure in contact-inhibited and transformed cells. Proc. Nut. Acad. Sci. USA 71, 1992-1994. BARNETT, R. E., SCOTT, R. E., FURCHT, L. T., and KERSEY, J. H. (1974b). Evidence that mitogenic lectins induce changes in lymphocyte membrane fluidity. Nature (London) 149, 465-466. BIGGERS, J. D., WHITTEN, W. K., and WHITTINGHAM, D. G. (1971). The culture of mouse embryos in vitro. In “Methods in Mammalian Embryology” (J. C. Daniel, Jr., ed.), pp. 86-116. W. H. Freeman, San Francisco. BRINSTER, R. L. (1965). Studies on the development of mouse embryos in vitro. III. The effect of fixednitrogen source. J. Exp. 2001. 158, 69-78. CAMPBELL, D. H., GARVEY, J. S., CREMER, N. E., and SUSSDORF,D. H. (1964). “Methods in Immunology.” W. A. Benjamin, New York. EDELMAN, G. M., YAHARA, I., and WANG, J. L. (1973). Receptor mobility and receptor-cytoplasmic interactions in lymphocytes. Proc. Nat. Acad. Sci. USA 70, 1442-1446. EDIDIN, M., PATTHEY, H. L., MCGIJIRE, E. J., and SHEFFIELD, W. D. (1971). An antiserum to “embryoid body” tumor cells that reacts with normal mouse embryos. In “Conference and Workshop on Embryonic and Fetal Antigens in Cancer, Oak Ridge National Laboratories” (N. G. Anderson, and J. H. Coggin, Jr., eds.), pp. 239-248. Oak Ridge, Tenn. EPSTEIN, C. J., and SMITH, S. A. (1974). Electrophoretie analysis of proteins synthesized by preimplantation mouse embryos. Develop. Biol. 40,233244. GATES, A. H. (1971). Maximizing yield and developmental uniformity of eggs. In “Methods in Mammalian Embryology” (J. C. Daniel, Jr., ed.), pp. 64-75. W. H. Freeman, San Francisco. GLASS, L. E. (1971). Fluorescent antibody methods
WILEY
AND CALARCO
Cell
Surfa :e in Mouse Preimpluntation
applicable to studies of mammalian embryos. In “Methods in Mammalian Embryology” (J. C. Daniel, Jr., ed.), pp, 355-377. W. H. Freeman, San Francisco. GOLDSCHNEIDER, I., and MOSCONA, A. A. (1972). Tissue-specific cell-surface antigens in embryonic cells. J. Cell Biol. 53, 435-449. GOADING, L. R., and EDIDIN, M. (19741. Cell surface antigens of a mouse testicular teratoma. J. Exp. Med. 140.61-78. GOTTLIEB, D. I., MERRELL, R., and GLASER, L. (1974). Temporal changes in embryonal cell surface recognition. Proc. Nut. Acad. Sci. USA 71, 1800-1802. HEYNER, S., BRINSTER, R. L., and PALM, J. (1969). Effect of iso-antibody on preimplantation mouse embryos. Nature (London) 222, 783-784. HEYNER, S. (1973). Detection of H-2 antigens on the cells of the early mouse embryo. Transplantation 16, 675-677. HUMASON, G. L. (1962). “Animal Tissue Techniques.” W. H. Freeman, San Francisco. Hsu, Y-C., BASKAR, J., STEVENS, L. C., and RASH, J. E. (1974). Development in vitro of mouse embryos from the two-cell egg stage to the early somite stage. J. Embryol. Exp. Morphol. 31, 235-245. KERBEL, R. S., and DOENHOFF, M. J. (1974). Resistance of mitotic B lymphocytes to cytotoxic effects of anti-Ig serum. Nature (London) 250,342-344. KIRBY, D. R. S., BILLINGTON, W. D., BRADBURY, S., and GOLDSTEIN, D. J. (1964). Antigen barrier of the mouse placenta. Nature (London) 204, 548549. KIRBY, D. R. S., BILLINGTON, W. D., and JAMES, D. A. (1966). Transplantation of eggs to the kidney and uterus of immunized mice. Transplantation 4, 713-718. KIRBY, D. R. S. (1968). The immunological consequences of extrauterine development of allogeneic mouse blastocysts. Transplantation 6, 1005-1009. KOMETANI, K., and BEHRMAN, S. J. (1971). The time of onset of placental susceptibility in mice to heterologous anti-mouse placental serum. Znt. J. Pert. 16, 139-143. KOMETANI, K., PAINE, P., COSSMAN, J., and BEHRMAN, S. J. (1973). Detection of antigens similar to placental antigens in mouse fertilized eggs by immunofluorescence. Amer. J. Obstet. Gynecol. 116, 351-357. KRACH, S. W., GREEN, A., NICOLSON, G. L., and OPPENHEIMER, S. B. (1974). Cell surface changes occurring during sea urchin embryonic development monitored by quantitative agglutination with plant lectins. Exp. Cell Res. 84, 191-198. LIN, T. P., and FLCIRENCE, J. (1970). Aggregation of dissociated mouse blastomeres. Exp. Cell Res. 63, 220-224. MARTIN, B. J., SPICER, S. S., and SMYTH, N. M. (1974). Cytochemical studies of the maternal sur-
Embryo
417
face of the syncytiotrophoblast of human early and term placenta. Anut. Rec. 178, 769-786. MINTZ, B. (1966). Gene expression in the morula stage of mouse embryos, as observed during development of t’2/t’2 lethal mutants in vitro. J. Exp. Zool. 157, 267-272. MINTZ, B. (1971). Allophenic mice of multi-embryo origin. In “Methods in Mammalian Embryology” (J. C. Daniel, Jr., ed.), pp. 186-214. W. H. Freeman, San Francisco. MOSCONA, A. A. (19681. Cell aggregation: Properties of specific cell-ligands and their role in the formation of multicellular systems. Develop. Biol. 18, 250-277. PALM, J., HEYNER, S., and BRINSTER, R. L. (1971). Differential immunofluorescence of fertilized mouse eggs with H-2 and non-H-2 antibody. J. Exp. Med. 133, 1282-1293. PINSKER, M. C., and MINTZ, B. (1973). Change in cell-surface glycoproteins of mouse embryos before implantation. Proc. Nat. Acud. Sci. USA 70, 1645-1648. RAFFERTY, K. A., JR. (1970). “Methods in Experimental Embryology of the Mouse,” pp. 24-34. Johns Hopkins Press, Baltimore. ROSENBLITH, J. Z., UKENA, T. E., YIN, H. H., BERLIN, R. D., and KARNOVSKY, M. J. (1973). A comparative evaluation of the distribution of concanavalin A-binding sites on the surfaces of normal, virally-transformed, and protease-treated fibroblasts. Proc. Nut. Acud. Sci. USA 70, 1625-1629. SIMMONS, R. L., and RUSSELL, P. S. (19621. The antigenicity of mouse trophoblast. Ann. N. Y. Acad. Sci. 99, 717-732. SIMMONS, R. L., and RUSSELL, P. S. (1966). The hi&compatibility antigens of fertilized mouse eggs and trophoblast. Ann. N. Y. Acud. Sci. 129, 35-45. SPINDLE, A. I., and PEDERSEN, R. A. (1973). Hatching, attachment, and outgrowth of mouse blastocysts in vitro: Fixed nitrogen requirements. J. Exp. Zool. 186, 305-318. SMETS, L. A. (1973). Agglutination with Con-A dependent on cell cycle. Nature New Biol. 245, 113115. STEVENS, L. C. (1968). The development of teratomas from intratesticular grafts of tubal mouse eggs. J. Embryol. Exp. Morphol. 20, 329-341. TARKOWSKI, A. K. (1961). Mouse chimaeras developed from eggs. Nature (London) 190, 857-860. VANDEPUTTE, M., and SOBIS, H. (1972). Histocompatibility antigens on mouse blastocysts and ectoplacental cones. Transplantation 14, 331-338. WALLACH, D. F. H. (1972). The dispositions of proteins in the plasma membranes of animal cells: Analytical approaches using controlled peptidolysis and protein labels. Biochim. Biophys. Actu. 265, 61-83.
418
DEVELOPMENTAL BIOLQGY
WESTIN, M., PERLMANN, H., and PERLMANN, P. (1967). Immunological studies of protein synthesis during sea urchin development. J. Exp Zool. 166, 331-346. WILEY, L. D. (1974). Presence of a gonadotropin on
VOLUME 47, 1975
the surface of preimplanted mouse embryos. Nuture &mm!on) 252, 715-716. YAHAIZA, I., and EDELMAN, G. M. (1973). The effects of concanavalin A on the mobility of lymphocyte surface receptors. Exp. Cell Res. 81, 143-155.
BIOLOGY
47, 407-418
(1975)
The Effects of Anti-Embryo Sera and Their Localization on the Cell Surface during Mouse Preimplantation Development LYNN Departments
of Anatomy
M. WILEY’
and Pediatrics,
AND
PATRICIA
University
G.
of California,
CALARCO San Francisco,
California
94143
Accepted July 16, 1975 We have developed two rabbit antisera, one against mouse blastocysta and a second against mouse placentae. After absorption of these antisera with adult mouse tissues and extensive dialysis, results from indirect immunofluorescence, cytotoxicity, and culturing experiments lead us to two major conclusions.‘First, anti-blastocyst serum detects a group of cell surface molecules whose expression is embryo-specific, stage-specific, and whose unaltered presence is required for preimplantation development in vitro. Second, anti-placenta serum detects a different group of cell surface molecules that are present before fertilization and become segregated to the syncytiotrophoblast, appear unessential for preimplantation development in vitro, and may function in trophoblast differentiation, and/or in protection of the fetus from maternal immunologic attack. INTRODUCTION
ing early sea urchin development (Krach 1974). The cell surfaces of mouse preimplantation embryos possess molecules that exhibit stage differences in their glycoproteins (Pinsker and Mintz, 1973). Other surface moieties are transitorily expressed during cleavage : Antiserum produced against mouse primitive teratocarcinoma cells reveals the presence of surface molecules maximally expressed on mouse &cell cleavage embryos (Artzt et al., 1973) and an antiserum to human chorionic gonadotropin (hCG) detects hCG-like molecules whose expression peaks on mouse morulae (Wiley, 1974). During preimplantation development, transitorily expressed cell surface molecules may be associated with specific intracellular events and could also mediate interactions between cells during blastocyst formation. This report describes an immunologic approach used to study the appearance and function of surface molecules on mouse preimplantation embryos. Two heterologous antisera, made separately against mouse placentae and mouse blastocysts, are shown by indirect immunofluorescence and cytotoxicity to interact with
et al.,
Many changes in an animal cell’s physiologic or developmental state are accompanied by changes in the cell surface. Cell cycle changes (Smets, 1973; Kerbel’ and Doer&off, 19741, oncogenic transformation (Bosenblith et al., 1973; Barnett et al., 1974a,b; Edelman et al., 1973; Yahara and Edelman, 19731, cell differentiation in culture (Akeson et al., 1974a,b), tissue-specific cell recognition (Goldschneider and Moscona, 1972) species-specific cell recognition (Moscona, 19681, and temporal, or time-dependent, tissue-specific cell recognition (Gottlieb et al., 1974) are associated with cell surface alterations. Temporal expression of antigenic molecules occurs as early as cleavage and gastrulation in sea urchin embryos (Westin et al., 1967). However, in these studies, the embryos were homogenized before analysis and it was not determined if any of these molecules were external plasma membrane components. More recently, specific changes in carbohydrate-containing surface molecules were detected duri In partial fulfillment of the requirements for the degree of Doctor of Philosophy, with the Department of Anatomy, University of California, San Francisco. 407 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
408
DEVELOPMENTALBIOLOGY
embryonic cell surfaces. Effects of both antisera on mouse preimplantation development in vitro are also described.
VOLUME 47, 1975
mately 200 blastocysts per weekly injection and 800 blastocysts per booster injection. Beginning 1 week after booster injecMATERIALS AND METHODS tions, rabbits were bled at weekly interAntisera Preparation vals and sera were heat-inactivated (56”C!, Anti-plncenta serum (A-PL). A modified 30 min), filter-sterilized, and frozen until method (Kometani and Behrman, 1971) needed. Before use, thawed aliquots of was used to produce the A-PL serum. Pla- sera were absorbed in the following way. A centae from 16- to 18-day gestation SWR crude membrane preparation was obmice were dissected free of decidual tissue tained by mincing SWR mouse livers, kidand fetal membranes, minced, and rinsed neys and spleen (LKS) in PBS and homogeseveral times with cold phosphate-buffered nizing minced tissues with a Dounce hosaline (PBS). Minced placentae were then mogenizer. The homogenate was centrihomogenized with a Dounce homogenizer, fuged at low speed (lOOO-12OOg) and the and 0.5 ml of the homogenate was emulsipellets washed with PBS until the supernafied with 0.5 ml Freund’s incomplete adju- tant was clear. Pellets were resuspended vant. A male New Zealand white rabbit in an equal volume of antiserum and alreceived 1.0 ml of the placental antilowed to stand at 4°C for 45 min before gen/Freund’s incomplete adjuvant prepararecovering the serum by centrifugation. tion suprascapularly, subcutaneously, Absorptions were repeated two more times once weekly for 4 consecutive weeks, 1 so that each aliquot of serum was exposed to a volume of packed “membranes” three week following an injection of Freund’s complete adjuvant. This initial injection of times the volume of the serum. complete adjuvant is thought to nonspecifiAbsorbed antisera were dialyzed in cold PBS for 5 days and filter-sterilized into lcally stimulate the rabbit’s immunologic ml rubber-capped vials. Nondialyzed antisystem, enhancing the response to the antiserum was found to be toxic in a nonspegen (Dov Michaeli, personal communication). The rabbit was allowed to “rest” for 4 cific way to mouse embryos in vitro. weeks and was then boosted with an addiNormal rabbit control serum was treated as described above for immune tional injection. sera. Anti-blastocyst serum (A-BL). Blastocysts were harvested and freed from their zonae pellucidae by mouth pipet (Tar- Embryos kowsky, 1961) in culture medium which All embryos came from either SWR or had 0.5 g/100 ml medium Ficoll (Brinster, mice. Superovulation 1965) in place of 0.3 g/100 ml medium bo- ICR superovulated vine albumin (BSA). Pronase was not used (Gates, 1971) and recovery of embryos (Rafdeto remove zonae because it alters cell sur- ferty, 1970) were done as previously face molecules (Wallach, 1972). After re- scribed. A modified Bigger’s (Biggers et moving the zonae from the injection prepaal., 1971) ovum culture medium (Spindle, was used for culration, the blastocysts, in 0.1 ml PBS or personal communication) Ficoll-containing medium were emulsified turing, indirect immunofluorescence aswith 0.1 ml Freund’s incomplete adjuvant. says with live embryos and cytotoxicity tests. Follicular cells were removed from Two male New Zealand white rabbits were immunized with this preparation using unfertilized oocytes and l-cell embryos the same injection protocol followed for the with hyaluronidase (180 IU/O.5 ml culture A-PL serum. Each rabbit received approximedium).
WILEY
Indirect
AND
CALARCO
Immunofluorescence
Cell Surface
Assays (ZIF)
Livepreimplantation embryos. These assays were performed under humidified 5% CO, in air at 37°C. Embryos were incubated for 30 min in immune serum diluted 1:l with ovum culture medium, rinsed with two 5-min changes of ovum culture medium, incubated for 10 min in goat anti-rabbit IgG conjugated with fluorescein (Antibodies, Inc.) diluted 1:4 with PBS, and rinsed with two 5min changes of ovum culture medium to remove unbound conjugate as described previously (Wiley, 1974). In some cases the zona pellucida was removed by mouth pipet. We found that embryos with zonae were much easier to handle than zona-free embryos and, therefore, suggest that zona removal be done following the final rinse. Blastocyst outgrowths. Assays on outgrowths were also performed at 37°C under humidified 5% CO, in air using the same IIF protocol as for the preimplanted embryos. Paraffin-embedded, sectioned material. IIF was done with slides of paraffin-embedded mouse 16-18day gestation placentae, fetus, and mouse ovary using methods previously described (Glass, 1971). Sections of mouse blastocysts were similarly treated after first being placed in ant pupae cases (Mintz, 1971). Carnoy’s fixative (Humason, 1962) was used with all tissues except for the blastocysts, which were fixed in Tellyesniczky’s fixative (Humason, 19621. IIF samples were viewed with a Zeiss fluorescence microscope and photographed using Agfa 500 daylight color slide film, lmin exposures. Cytotoxicity
Tests
A fine-bore pipet was used to remove the zona before cytotoxicity was assessed. Zona-free 2-cell and 8-cell embryos were incubated in serum diluted 1:l with ovum culture medium for 45 min at 37°C under humidified 5% CO, in air. Sera were taken from the same batches used in the IIF
in Mouse Preimplantation
Embryo
409
assays and culturing experiments. Embryos were next transferred to a mixture of 50% fresh guinea pig serum (complement), 25% antiserum, and 25% ovum culture medium, and incubated as before. Viability was determined by placing the embryos in 0.5% trypan blue in 0.85% NaCl for 5 min and counting the number of blue embryos (2-cell stage) or blastomeres (8cell stage). Embryo Culture The effect of immune sera in preimplantation development was determined by culturing 2-cell embryos in dilutions of serum using the microdrop culture technique (Rafferty, 1970). Culturing experiments were carried out in duplicate, and each experiment was repeated two or more times. Each dilution of antiserum was tested on a total of at least 100 embryos. After 65-72 hr in culture, embryos were scored for blastocyst formation, All embryos containing blastocoels were scored as blastocysts regardless of whether or not expansion had occurred. Blastocyst outgrowths were produced by a method previously described (Spindle and Pedersen, 1973). Briefly, expanded blastocysts were placed into the chambers of tissue culture slides (Lab. Tek., Catalog No. 4808) containing 0.3 ml Eagle’s minimal essential medium (MEM) fortified with amino acids (Spindle and Pedersen, 1973) and 10% fetal calf serum. After 4-5 days of culture the outgrowths were used for IIF assays. RESULTS
Indirect
Immunofluorescence
Assays (II%)
IIF is far more sensitive in detecting antigen-antibody binding than immunodiffusion or cytotoxicity. We have defined the specificity of A-PL and A-BL sera in terms of IIF data and have also used IIF to monitor completeness of absorption. Anti-placenta serum (A-PL). Unabsorbed A-PL serum reacts strongly in IIF assays with all mouse tissues tested (see
410
DEVELOPMENTAL BIOLOGY
below). After absorption only the syncytiotrophoblast of sectioned mouse placenta is positive. None of the fetal (fetal red blood cells, connective tissue components of the chorionic villi) or maternal (mature erythrocytes in the intervillous lakes) elements of the placenta fluoresce (Fig. 1). All cell types in sections of mouse ovary and all tissues of sagittal sections of 16-day mouse fetus treated with absorbed A-PL serum resemble their counterpart NRS controls, i.e., they are negative. These observations lead us to conclude that antigens reacting with A-PL serum are not present on fetal or maternal cells, or they are present in levels too low for detection by our methods in these tissues. A-PL serum localizes on the surfaces of ovarian and ovulated oocytes (Figs. 2a,b), and mouse embryos throughout preimplantation development (Figs. 2c-g). While there is no appreciable difference in the intensity of fluorescence among the oocytes and preimplantation stages, the pattern of membrane fluorescence changes with the onset of cleavage. Surfaces of ovarian and ovulated oocytes and l-cell embryos display patches of membrane fluorescence which disperse with the first cleavage (Figs. 2c and d) to yield a diffuse, even membrane fluorescence which persists throughout cleavage and blastocyst formation. On sectioned blastocysts, A-PL
VOLUME 47, 1975
serum does not differentiate betwen trophoblast and ICM. For blastocyst outgrowths induced by a change in medium (Spindle and Pedersen, 1973) both the inner cell mass (ICM) and the trophoblast sheet show a diffuse, even reaction with APL serum (Fig. 2h). As the outgrowths continue to develop, however, membrane fluorescence appears to decrease on portions of the ICM and persists over the trophoblast sheet. Although other workers have cultured mouse embryos to early somite stages in similar culture systems (Hsu et al ., 1974; Spindle et al., unpublished results), we emphasize that this is an in vitro system and may not resemble in uiuo development. Positive fluorescence of A-PL serum with mouse placenta and preimplantation embryos is removed by absorption with cell membranes isolated from homogenized 16-Wday mouse placentae. Pretreating mouse unfertilized oocytes or preimplantation embryos with either 0.5% Pronase (in Ficoll-containing ovum culture medium, for 5 min), or trypsin (Lin and Florence, 1970) abolishes A-PL membrane fluorescence. Some degree of A-PL serum binding is regained after 4 hr incubation in culture medium. Anti-blastocyst serum (A-BL). When tested on preimplantation embryos, absorbed A-BL serum produces little mem-
FIG. 1. Indirect immunofiruorescence: Anti-placenta serum, mouse placenta. In paraflin-embedded, sectioned mouse placenta only the trophoblast (T) is positive with A-PL serum (a). Compare with the NFW control (b), which shows only a low level of orange autofluorescence. Magnification, 500 x
WILEY
AND
CALARCO
Cell Surface
brane fluorescence until the 4-cell stage (Figs. 3a-c). Reactivity towards A-BL serum is low with unfertilized oocytes and lcell embryos, increases with cleavage, and peaks on 8-12-cell embryos (Figs. 3d and e). Thereafter, fluorescence diminishes (Figs. 3f and g> and disappears completely on blastocyst outgrowths (Fig. 3h). The pattern of membrane fluorescence is always diffuse. A very low level of fluorescence results when sectioned blastocysts are treated with A-BL serum. No consistent differences are noted between trophoblast and ICM cells. Sections of mouse ovary, fetus, and placenta show no reac-
in Mouse Preimplantation
tion whatsoever rum. Cytotoxicity
with
Embryo
absorbed A-BL
411 se-
Tests
To verify that these sera were detecting a surface antigen, cytotoxicity tests were performed (Table 1). The A-PL serum was cytotoxic (diluted 1:l with culture medium) to 2-cell (i.e., 29 lysed/43 total No. embryos) and El-cell (27/80) embryos, while A-BL serum (diluted 1:l with culture medium) was not cytotoxic for 2-cell embryos (O/23) but did lyse 75% of &cell blastomeres (SO/SO). Serial dilutions of antisera were not tested.
FIG. 2. Indirect immunofluorescence: Anti-placenta serum, mouse preimplantation embryos. The intensity of membrane fluorescence is constant on (a) ovarian oocytes, (b) ovulated ova, and all of the preimplantation stages l(c), zygote; (d), 2-cell; (e), 8-cell; (0, morula; and (g), blastecystl. However the pattern of fluorescence changes with the first cleavage. This is evident where the lower l-cell (c) and 2-cell (d) embryos are compared, where the cell surfaces are in focus. In all the other micrographs the cell rims are in focus. Membrane fluorescence is even and diffuse over the trophoblast sheet (T) and ICM (I) of blastocyst outgrowths (h). NRS control embryos: blastocyst (i), and 2-cell (i). Magnification, 500 X, except the outgrowth that is 300 x.
412
DEVELOPMENTAL
BIOLOGY
VOLUME
47, 1975
FIG. 3. Indirect immunofluorescence: Anti-blustocyst serum, mouse preimplantation embryos (zona-free). What little membrane fluorescence is seen on l-cell (a) and 2-cell (b) embryos increases on the 4-cell (c) and peaks around the g-cell (d) to 12-cell stages (e). Thereafter membrane fluorescence diminishes on morulae (f~ and blastocysts (g). This blastocyst appears to have transported fluorescein conjugate into its blastocoel and also contains a filled, fluorescing blasmmere (due to cell death). No fluorescence is seen on the trophoblast sheet (T) or ICM (I) of blastocyst outgrowths (h). NBS control embryos: 2-cell (i) and 8-cell (j) stages. Magnification: 500 x , except for the outgrowth that is 300 x .
TABLE CYTOTOXICITY
OF ANTISERA
Stage
1
TO PREIMPLANTATION
A-PL Serum No.
2-Cell embryos Lysed or blue embryos/total No. embryos g-Cell embryos Lysed or blue blastomeres/total No. blastomeres B Cytotoxicity b A different
Culturing
%
23/25 6/18@ 27/80b
92 33 34
was based on the lysis or the tilling of blastomeres batch of antiserum was used in these tests.
Experiments
Extensive dialysis of sera used in culturing was found necessary to eliminate nonspecific toxicity (see Materials and Meth-
EMBRYOS”
A-BL Serum No. 0123 60/80
with trypan
NBS
%
No.
%
0
o/21
0
75
5140
12.5
blue.
ads). Embryos cultured in insut&iently dialyzed sera progressively turned grainy and darkened with time. Although they generally cleaved, these embryos often failed to blastulate. After extensive di-
WILEY
AND
CALARCO
Cell Surface
alysis, however, the ability of 2-cell embryos to form blastocysts in l-10% A-PL serum was comparable to that of embryos cultured in the same dilutions of NRS (Fig. 4). The A-BL serum, however, continued to adversely affect cleavage in vitro and at concentrations of 4% or more prevented development (Fig. 4). Embryos cultured in A-BL serum differed in appearance from those nonspecifically killed by insufficiently dialyzed serum. In higher concentrations of A-BL serum (2.54%) very little, if any cleavage occurred and almost all embryos shrank and darkened overnight. An occasional embryo arrested and remained at the 2-cell stage the entire culture period. As the concentration of ABL serum decreased an increasing number of embryos consisted of a mixture of degenerate and “normal” cleaving blastomeres. Also the number of degenerate blastomeres per embryo went down as the concentration of A-BL serum was lowered. As IN VITRO EMBRYOS
in Mouse Preimplantation
Embryo
413
serum concentrations continued to diminish an increasing percentage of embryos with damaged blastomeres formed blastocoels. Preliminary data from culturing embryos in concentrations of A-BL immunoglobulin fractions indicate that immune antibodies are responsible for (at least some of) the effects A-BL serum has in culture. DISCUSSION
Our data probably do not result from antibodies recognizing “universal” mouse cell surface molecules because not only were our antisera absorbed with adult mouse liver, kidney, spleen homogenates, but they were also negative in IIF assays for all tissue types in sagittal sections of whole mouse 16- to l&day fetus and in sections of adult mouse ovary. It is unlikely that H-2 antigens are responsible for out results because (i) embryos and placentae from random-bred mice were used as
CULTURE OF MOUSE PREIMPLANTAT ION IN THE PRESENCE OF IMMUNE ANTIBODIES
FIG. 4. Culturing experiments. Two-cell embryos, after 65-72 b in microdrop culture, are scored for blaatocyst formation. All embryos having a blastocoel are counted as blastocysts, regardless of whether or not expansion has occurred. Each point represents the average value from at least two separate experiments and, for each experiment, each dilution is done in duplicate drops. Between 20 and 25 embryos are placed in each drop, so that each point represents the response of at least 100 embryos.
414
DEVELOPMENTAL BIOLOGY
immunogens, (ii) exhaustive absorptions were done with pooled random-bred LKS homogenates, and (iii) H-2 antigens are not detectable by IIF before implantation (Heyner et al., 1969; Palm et al., 1971; and Heyner, 1973) and may not appear on embryonic cells before day 6 or 7 of gestation mouse (Edidin et al ., 1971). Although preimplantation embryonic surfaces crossreact weakly with non H-2 histocompatibility antigens (Palm et al., 1971), these antigens are probably not responsible for our results for reasons (i) and (ii) above.
Anti-Placenta Serum (A-PL) Our A-PL serum detects surface molecules present on oocytes, preimplantation embryos and trophoblast derivatives following implantation. An A-PL serum developed in another laboratory (Kometani et al., 1973) differs from ours in IIF-defined specificities by not recognizing unfertilized ova or zygotes. Further, when tested on sectioned blastocysts and egg cylinders, the embryonic part, rather than the trophoblast appears to have a greater affinity for their serum, although their serum is cytotoxic for trophoblast cells cultured from e&placental cones. These workers hypothesize their A-PL serum might contain “fetal-specific” antibodies although they do not mention any tests done on sectioned fetal tissues. Our A-PL serum, however, is positive for unfertilized oocytes, all other preimplantation stages, and syncytiotrophoblast, negative for fetal and adult tissues, and does not differentiate between trophoblast and embryonic portions of IIFassayed, sectioned blastocysts. Our A-PL serum could differ from their A-PL serum for the following reasons: (i) genetic variability among rabbits; response to the same immunogen by nonsyngeneic animals will differ because the response will depend on the animal’s genotype; (ii) the way in which an immunogen is administered can determine the immune response, and slight differences in immunization procedures could result in differing antisera. In addition to its well-documented roles
VOLUME 47,
1915
in fetal nutrition and hormone production, additional evidence suggests that the trophoblast (Simmons and Russell, 1962, 1966; Vandeputte and Sobis, 1972; Stevens, 1968), its “fibrinoid coat” (Kirby et al., 1964, 1966; Kirby, 1968) or, in particular, the maternal interface of the syncytiotrophoblast (Martin et al., 1974) may fence off the fetus from maternal immunologic attack. Our IIF data suggest that maternal-embryonic interfaces are, from the very beginning of development, continuously supplied with A-PL-specific molecules. Some of these A-PL-specific molecules may, then, function in isolating the fetus from immunologic attack. Alternatively, these molecules may participate in differentiation, function, trophoblast and/or maintenance.
Anti-Blastocyst Serum (A-BL) Our IIF, cytotoxicity, and culturing data all indicate that absorbed A-PL and A-BL sera are different (Table 2). In particular, they appear to detect two separate populations of surface antigens. A-PL serum-detected antigens are present before fertilization and become segregated to syncytiotrophoblast. A-BL serum recognizes surface antigens whose detection (by our methods) is confined to preimplantation development and is maximal on 8+-cell embryos. To support these conclusions, we propose that these two separate groups of molecules differ in the times during development when they are immunogenic (can elicit an immune response) and differ in times when they are antigenic (accessible to, and recognized by, antibodies) (Campbell et al., 1964). Antigenicity and immunogenicity are different attributes and can occur independently (Campbell et al., 1964). At the blastocyst stage, for example, A-PL detected surface molecules are not yet immunogenic but are antigenic (i.e., detectable by IIF); while A-BL detected molecules are both immunogenic (induce antibody formation) and antigenic (detectable by IIF). Furthermore, the constant expression of
WILEY AND CALARCO
Cell
Surfacein TABLE
Mouse Preimplantation
415
Embryo
2
CHARACTERISTICS OF ABSORBED ANTISERA Procedure Indirect immunofluorescence
Anti-placenta
Anti-blastocyst
(A-PL)
Positive for all ova, preimplantation embryos, blastocyst outgrowths, and trophoblast of sectioned mouse placenta; negative for sectioned mouse ovary and fetus Fluorescence is patchy on ova and lcell embryos, diffuse on all other stages
Cytotoxicity
Cytotoxic
for 2-cell and g-cell embryos
Culturing
No effect on preimplantation ment
A-PL-specific molecules is initially controlled by the maternal genome. In contrast, the temporal expression of A-BLspecific molecules is most likely a result of embryonic genome activity and protein synthesis. A major group of proteins (MW 53,000-57,000) is synthesized primarily between the 2- and 4-8-cell stage (Epstein and Smith, 19741, while a large shift in the nature of surface glycoproteins occurs between the 4-8-cell stage and the blastocyst stage (Pinsker and Mintz, 1973). Some of these proteins may be components of the A-BL-specific surface molecules maximally expressed on 8 +-cell embryos, while the shift in surface glycoproteins may be related to the diminishing expression of ABL-specific molecules after the 8+-cell stage. The peak expression of A-BL-detected surface molecules on mouse embryos coincides very closely with the time of peak expression of a surface moiety(ies) reportedly controlled by an allele at the T locus (Artzt et al., 1974). The T locus has a number of recessive alleles, one of which, the t12 allele is lethal in homozygous embryos at the morula to early blastocyst stage (Mintz, 1964). Recently, an antiserum to mouse primitive teratocarcinoma cells (APTC) was shown to be cytotoxic to mouse
develop-
(A-BL)
Negative for all ova, weak on l-cell and 2-cell embryos increases on 4-cell stage, peaks on 8-12 cell stage, diminishes thereafter to disappear on blastocyst outgrowths; negative for all sectioned mouse tissues Fluorescence is diffuse when present
Noncytotoxic for 2 cell embryos, cytotoxic for g-cell embryos
and is
Impairs development at a concentration of 2.5% serum, stops development altogether at 4% serum
sperm and to contain antibodies recognizing cell surface molecules maximally expressed on normal 8-cell mouse embryos when (Artzt et al., 1973). Interestingly, used for quantitative absorptions, sperm from TIP mice were only half as effective as sperm from normal mice at removing activity from the A-PTC serum (Artzt et al., 1974). Their inference is that, on both P sperm and t12/t12embryos, molecules on the cell surface normally detected by APTC serum may be missing or altered. Another antiserum to a mouse teratoma cell line is cytotoxic to mouse unfertilized eggs and to several transformed mouse cell lines (Edidin et al., 1971; Cooding and Edidin, 1974). IIF with pre- and postimplantation mouse embryos shows that their antiteratoma serum localizes on cells believed destined to form tissues expressing H-2 antigens in the adult; normal adult mouse cells do not react with anti-teratoma serum. These authors suggest that their anti-teratoma serum is detecting H-2 antiwhich are, therefore, gen precursors, found only on cells which will later acquire transplantation antigens. It is unlikely, however, that our A-BL serum is detecting these “H-2 antigen precursors” because ABL serum is negative for all cells in postimplantation mouse embryos in IIF assays.
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DEVELOPMENTAL BIOLO~,Y
Since the complexing of A-BL-specific molecules with antibodies impairs development, we assume that the A-BL-specific molecules are required for development to proceed. These molecules might be involved in such processes as cell recognition, cell adhesion, the transitory appearance of a new, embryo-specific substrate carrier or could function as receptor molecules which trigger intracellular event(s) characterizing the 8 +-cell embryo. Intriguing to us is the possibility that A-BL-detected molecules participate, by whatever process, in separating ICM from trophoblast during blastocyst formation. We are presently investigating the mobility, spatial distribution, density, and cross-reactivity of A-BL and A-PL specific molecules on preimplantation embryos. Other studies on the obvious contraceptive potential of these antisera are contemplated. In conclusion, anti-blastocyst serum shows that mouse preimplantation embryos possess immunogenic surface molecules which are embryo-specific, stage-specific and whose presence is required for development. Anti-placenta serum reveals the presence of a different group of surface molecules that, when bound with antibody, have no effect on preimplantation development, and appear to segregate to the trophoblast; they may, then, function in trophoblast differentiation, in maintenance, or in protection of the fetus from maternal immunologic attack. The authors are grateful for the expert technical assistance of Peg Siebert, Jean Hanson, Sandra Smith, and Estrella Lamella and for the photographic talents provided by Mr. Dave Akers. Special thanks are given to Dr. Akiko Spindle and Dr. Roger A. Pedersen for their assistance in culturing blastocyst outgrowths. We thank Dr. Dov Michaeli and Dr. Robert P. Erickson for assistance with the immunologic aspects of this work and Dr. Charles J. Epstein, Dr. Dov. Michaeli, Dr. William J. Rutter, and Dr. Laurel E. Glass for help in preparing the manuscript. This work was supported by the Department of Anatomy and by grants, U.S.P.H.S. No. GM-19527 and l-ROl-CA-16493-01.
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