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Production of congenital malformations using tissue antisera
DEVELOPMENTAL
BIOLOGY 42, 1-12
Production
(19%)
of Congenital Malformations Antisera
XV. Reichert’s
Membrane
and Visceral
Using Tissue
Yolk Sac Antisera
MARCELA JENSEN, THOMAS R. KOSZALKA, AND ROBERT L. BRENT Stein Research Center, Thomas Jefferson
University,
Philadelphia,
Pennsylvania
19107
Accepted August 26, 1974 Rabbit anti-rat Reichert’s membrane (RM) serum, when injected into pregnant rats on the ninth day of gestation, produced embryolethality and stunting but a surprisingly low incidence of malformations. Immunofluorescent studies revealed that the maternally injected RM antiserum localizes in RM and in the basement membrane of some maternal kidney tubules but not in the maternal glomerulus or in the cytoplasm of the visceral yolk sac (VYS) cells as had teratogenic VYS and kidney antisera. When antiserum against rat RM was injected into mice, localization occurred in the glomerular basement membranes, thus confirming the findings of Pierce and co-workers. When teratogenic term VYS antiserum is adsorbed with RM, it still localizes in the VYS, does not localize in RM, and is still teratogenic. When this same antiserum is adsorbed with VYS, the antiserum is no longer teratogenic. It appears that the localization of antibodies in the VYS cells is the major contributor to the production of congenital malformations because localization of antisera in RM alone was associated with a very low incidence of teratogenesis. Since both structures participate in a multitude of biochemical processes, which include adsorption, transport, catabolism, and anabolism of nutrients, nonnutrients, and waste products, there are many plausible hypotheses to explain the nature of the parietal and visceral yolk sac dysfunction.
INTRODUCTION
Dobrowolski (1903) observed that specific anti-placental serum interrupted pregnancy in guinea pigs and rabbits. Seegal and Loeb (1940) reported that rabbit anti-rat placental serum was abortogenic and nephrotoxic. They did not report an increase in the incidence of congenital malformations, in spite of the fact that pregnant rats were injected on the ninth day of gestation. Pressman and Korngold (1957) found that cross-reacting antibodies were present in Seegal’s antiplacental and antikidney sera. Brent et al. (1961) first reported that heterologous kidney antiserum is teratogenic when injected into pregnant rats. This phenomenon was described in detail in a series of papers which included the spectrum and incidence of malformations following the administration of kidney antiserum to pregnant rats (Brent, 1964), some experiments testing Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
various hypotheses pertaining to the mechanism of teratogenesis (Brent, 1966b), the cross specificity of teratogenic antisera in mouse and rat (Brent, 1968), the biologic and biochemical properties of teratogenic antisera (Brent, 1966a; Bragonier et al., 1970) and several reviews of this subject (Brent, 1970; Brent, 1971a). Some of these findings were confirmed and extended by other investigators (David et al., 1966; Mercier-Parot et al., 1963; Mikhailov, 1967; Eyguem et al., 1968; Gebhardt et al., 1970; Barrow and Taylor, 1971). The teratogenic factor was demonstrated to be in the IgG fraction and reduction, and alkylation or preparation of the F(ab)” fragment of IgG from sheep anti-rat kidney teratogenic serum did not interfere with the potency of the teratogenic IgG (Bragonier et al., 1970; Brent, 1970). Thus, complement fixation was not an essential step in the teratogenic process. It was also
2
DEVELOPMENTALBIOLOGY
reported that lz51-labeled teratogenic gamma globulin and fluorescent-labeled teratogenic gamma globulin localized in the maternal glomerular basement membrane, in the Reichert’s membrane (RM), and in the cytoplasm of the visceral yolk sac (VYS) endodermal cells (Slotnick and Brent, 1966). However, no specific antibody localization was demonstrated in the embryo. Teratogenic kidney antiserum localized in the developing yolk sac structures and, therefore, it seemed logical to determine whether antigens in the visceral or parietal yolk sac were capable of stimulating teratogenic antibodies. Sheep antiterm whole-visceral rat yolk sac serum proved to be teratogenic (Brent et al., 1971b). Since localization of this antiserum also occurred in the maternal glomerular basement membrane, RM and in the cytoplasm of the VYS, it was not possible to determine whether localization in RM, VYS cells or both were primarily involved in the teratogenic process. If an antiserum which localized in one or the other of these structures could be prepared, then it might be possible to determine whether localizations in either the visceral or parietal yolk sac is associated with teratogenesis. Since RM is an epithelial basement membrane, it seemed logical as an initial step to study the localization, cross-reactivity, and embryotoxicity of antisera made against this epithelial basement membrane. MATERIALS
AND
VOLUME 42, 1975
structure. The trophoblastic layer can be removed from the PYS by simple stripping, leaving RM and the associated cells (Fig. 1). If the embryonic site is frozen and thawed before dissecting the uterine wall, the trophoblastic layer and the PYS cells can be removed by simply shaking the PYS in distilled water (Fig. 2). For each immunization six RM were homogenized in 2 ml of distilled water and emulsified with an equal volume of Freund’s incomplete adjuvant. Rabbits were injected at weekly intervals for 7 weeks before the first bleeding. The preparation of antisera with whole rat kidney and whole term rat VYS have been previously described (Brent et al., 1961; Brent et al., 1971b).
METHODS
Reichert’s membrane was prepared using the following protocol. Embryonic sites obtained from Wistar rats on the 15th day of pregnancy were placed in cold phosphate buffered saline at pH 7.2. The uterine musculature was carefully dissected from the surface of the extraembryonic membranes in order to prevent rupture of the parietal yolk sac (PYS). Recognition and dissection of the PYS is enhanced by the nonglossy appearance of this
FIG. 1. Fresh preparation of 15th day parietal yolk sacs immediately after dissection from the embryonic site. Reichert’s membrane (RM) was dissected free of the trophoblastic cells and then fixed in Bouin’s solution and embedded in paraffin. Sections cut at 5 pm and stained with hematoxylin and eosin. Note the RM and associated parietal yolk sac cells. x 125.
JENSEN, KOSZALKA AND BRENT
Reichert’s Membrane Antiserum
Adsorption
FIG. 2. Similar preparation to Fig. 1, except that the embryonic site was frozen at -60°C for at least 24 hr before the parietal yolk sac was dissected. Note the relatively pure preparation of RM. x 125.
Testing for Embryotoxicity Pregnant Wistar rats were obtained by mating rats from 4 PM to the following 9 AM. If sperm were detected in the vaginal smear, then the pregnancy was considered to be 0 hr and 0 days old at 9 AM, or the beginning of the first day of pregnancy. On the ninth day of pregnancy the rats were anesthetized with sodium pentobarbital (30 mg/kg) and a laparotomy was performed in order to determine the number and condition of the embryonic sites. After closing the incision, the various adsorbed and unadsorbed antisera were injected ip into the pregnant rat. On the 22nd day of pregnancy, the rats were sacrificed and the incidence of resorption and malformations was determined as well as the extent of fetal growth retardation (Tables 1 and 2).
3
Studies
Fresh splenic tissue was obtained from 12- to la-week-old female BALB/c mice weighing 18-25 g (Jackson Laboratory). The splenic tissue was homogenized in 0.85% saline to a final concentration of 10%. The cell suspension was disrupted by sonication for 15-30 min and the insoluble fraction collected by centrifugation at 1400 g for 30 min at 4°C. The precipitate was washed in distilled water and lyophilized. The insoluble splenic residue contained splenic reticulum and vascular basement membranes. Specific antisera were adsorbed by shaking a suspension of splenic residue in the conjugate at 4°C for 24 hr, centrifuging, and repeating the adsorption process twice (Pierce et al., 1963). Adsorption of teratogenic term VYS antiserum with 15th day RM and 15th day whole VYS was performed by combining 10 mg of lyophilized antigenic material per milliliter of antiserum at 4°C for 18 hr, centrifuging, and repeating the adsorption process three times. The adsorbed antisera were then utilized for localization and embryopathic studies as described in the sections dealing with immunofluorescent localization and embryotoxicity testing (Tables 1 and 2). Immunofluorescent
Localization
After the injection of rabbit anti-rat RM serum into pregnant rats on the ninth day of pregnancy, the rats were sacrificed 48 hr later. Frozen sections of adult kidney and embryonic tissue were cut at 4 pm. The direct fluorescent antibody technique was used for localization of the antiserum. The 7S fraction of goat anti-rabbit gamma globulin (Nutritional Biochemical Corp., Cleveland, Ohio) was conjugated with fluorescein isothiocyanate (FITC) in borate buffer at pH 9.0 for 3 hr at 4°C (Albrecht, 1965). Unreacted FITC was removed by adsorption with charcoal for 15 min (20 mg/ml of conjugate) and by adsorption of
4
DEVELOPMENTALBIOLOGY
VOLUME 42, 1975
TABLE
1
COMPARISON
OF THE EMBRYOPATHIC EFFECTS OF ANTISERUM PREPARED AGAINST (1) ~&DAY-OLD (15TH RM, (2) ADULT RAT KIDNEY, (3) ABSORBED AND UNAESORBEDTERM WHOLE-RAT VYS
DAY)
Tissue antiserum injected on the ninth day of pregnancy
Dose (mg/kg)
Number of pregnant rats
Total number of embryos on ninth day
Total number of embryos at term
Control sheep serum RM RM RM RM RM Whole kidney Term VYS Term VYS adsorbed with 15th day RM Term VYS adsorbed with 15th day RM Term VYS adsorbed with 15th day RM Term VYS adsorbed with 15th day yolk sac Term VYS adsorbed with 15th day yolk sac Term VYS adsorbed with 15th day yolk sac
350 50 70-100 110-150 160-400 240-600 175 100 70-100
9 2 6 14 9 2 4 5 3
94 22 65 169 105 24 50 65 34
84 22 49 91 20 0 20 47 30
0.0 24.6 46.1 81.0 100.0 60 27.6 12.3
4.68 4.91 4.26 4.19 3.12 3.89 3.90 4.02
1.2 0 0 0
125
5
58
20
41.2
3.35
100.0
140
2
30
7
70.0
2.16
100.0
100-120
3
34
29
14.7
4.89
0.0
140
2
23
18
21.7
4.37
0.0
160
2
26
8
69.2
4.12
0.0
(o/o) Resorbtions 10.6
Term fetal weight (4
(WI Malformations
100.0
TABLE 2 INTENSITY
OF TMMUNOFLUORESCENT
Antiseraprepared against
Intensity Maternal rat glomerular )asement membrane
Control: sheep serum 15th day embryonic rat Reichert’s membrane Whole adult rat kidney Term whole rat visceral yolk sac Term whole rat visceral yolk sac adsorbed with 15th day Reichert’s membrane Term whole rat visceral yolk sac absorbed with 15th day visceral yolk sac
LOCALIZATION AND EMBRYOTOXICITY EMBRYOTOXIC ANTISERA of immunofluorescent
Mousr maternal glomerular basement membrane
Maternal rat kidney tubular basement membrane
+++
Rat Reichert’s membrane
+++
+++
it+
++
tt+
+++
tt
++t+
++
+
ABSORBED AND UNABSORBED
localization
Cytoplasm of maternal rat kidney tubular cells
-
OF VARIOUS
+
Embryonic lethality
Embryonic growth retardation
+t++
++t+
*
+++
+ttt
+t+t
+++t
+tt
++tt
+tt+
++++
Cytoplasm of rat visceral wJJl;=
Embryonic malformations
t+
+++
+++
++++
i
t
*
*
JENSEN, KOSZALKA AND BRENT
the conjugate on acetone dried rat liver powder (100 mg/ml of conjugate). The conjugate was finally adsorbed with a 15% suspension of the particular tissue to be stained. Adsorption was performed for 1 hr at room temperature and overnight at 4°C. The adsorbed conjugate was then centrifuged and used for staining. Tissue sections that had been exposed to various teratogenic and nonteratogenic antisera prepared in the rabbit were fixed for 10 minutes in acetone and then stained for 30 min with goat anti-rabbit gamma globulin conjugated with FITC. After staining they were rinsed in three changes of phosphate-buffered saline. Sections were mounted in 1% Tris(hydroxymethyl)aminomethane in glycerine (Mrenova, and Albrecht, 1966). Examinations were made using a Leitz fluorescent microscope with an HBO 200 ultraviolet light source, using a dark-field condenser, BG 12 exciting filter, and OG 1 eyepiece filter. Photographs were made with ASA 3000 Polaroid roll film.
5
Reichert’s Membrane Antiserum
tions in surviving fetuses from a litter exposed to an LD,, dose of either whole rat kidney antiserum (Brent, 1966) or whole term rat VYS antiserum (Brent et al., 1971b) is greater than 50% (Table 3). In Vivo Localization
of Various Adsorbed
and Unadsorbed Antisera
In vivo localization of RM antiserum within the embryonic site revealed a linear staining along RM (Fig. 3, Table 2), but there was no localization in the cytoplasm of the VYS cells as has been observed with teratogenic antisera prepared against term whole VYS (Brent et al., 1971b) or whole rat kidney (Slotnick and Brent, 1966). Reichert’s membrane antiserum did not localize in the maternal glomerular basement membrane but did localize in the basement membrane of some kidney tubules adjacent to the glomeruli (Fig. 4). When rabbit anti-15th day rat embryonic RM serum is injected into the BALB/c goat anti-rabbit mouse, FITC-labeled gamma globulin does stain the glomerular basement membrane (Fig. 5). FurtherTesting for Nephrotoxicity more, if this antiserum is adsorbed with Male Wistar strain rats weighing insoluble splenic residue containing reticu230-270 g were injected intravenously with lum and vascular basement membrane and 200 mg/kg of pooled RM antiserum. Daily is then injected into the mouse, the glomerurine specimens were collected for 3 weeks ular masangium still localized the RM and tested for protein according to the antisera. Thus, rabbit anti-rat RM serum procedure of Kingsburg et al. (1926). After does not localize in the adult rat glomerurats were sacrificed, the kidneys were stud- lus but does localize in the mouse glomeruied for localization of antisera by the im- lus. munofluorescent technique described in TABLE 3 the previous section. RELATIONSHIP OF THE EMBRYONIC LD,, DOSE OF RESULTS
Embryotoxic
Effects of Various Antisera
Rabbit anti-rat RM serum when injected ip into pregnant rats on the ninth day of pregnancy produced embryolethality and fetal growth retardation (Table 1). The incidence of congenital malformations was very low (Tables l-3). This was unexpected since the incidence of malforma-
VARIOUS ANTISERA AND THE INCIDENCE OF MALFORMATIONS OBSERVED IN THE SURVIVING RAT FETUSES AT TERM LD,, dose injected on the ninth day of gestation Adult kidney antiserum Term chorioplacental antiserum Whole term yolk sac antiserum 15th day Reichert’s membrane and antiserum
Percent malformations in survivors 80 58 66 <8
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DEVELOPMENTALBIOLOGY
VOLUME 42, 1975
bryonic rat RM are different from the embryotoxic effects of antisera prepared against whole rat kidney and whole term rat VYS. While the latter two antisera produce a high incidence of malformations in the survivors at term following an embryonic LD,, dose, the RM antiserum produced a very low incidence of malformations, in spite of the fact that embryonic lethality and severe growth retardation were observed (Table 3). None of these antisera, when administered t,o the mother, localized in the developing rat embryo, as demonstrated by the in uivo immunofluorescence studies during the embryonic and early fetal stages of development. Similarly, the antisera do not localize in the basement membrane of
FIG. 3. Frozen section of 11th day embryonic site from a rat injected with rabbit anti-rat RM serum on the ninth day of gestation. Stained with FITC-labeled goat anti-rabbit IgG as described in the Materials and Methods section. Note that the VYS is unstained and therefore not visible. Only RM fluoresces (arrow) having localized RM antisera. x 50.
Adsorption of teratogenic term VYS antiserum with 15th day RM did not remove the teratogenic properties of the antiserum, but did eliminate its localization in RM (Table 2, Fig. 6). Adsorbtion of teratogenic term VYS antiserum with 15th day whole VYS eliminated malformations, although some resorptions and growth retardation did occur (Table 1 and 2). When RM antiserum was injected into eight adult rats and 24-hr urines were tested for proteinuria over a period of 3 weeks, the daily urinary protein content ranged between 2 and 15 mg, which is the normal range of 24-hr protein for rats. DISCUSSION
The embryotoxic effects of heterologous antiserum prepared against 15th day em-
FIG. 4. Frozen section of kidney from a pregnant rat injected with rabbit anti-rat RM serum on the ninth day of pregnancy and sacrificed 48 hr later. Stained with FITC-labeled goat anti IgG serum as described in the Materials and Methods section. No staining of the glomeruli was observed, but the basement membrane of some cortical tubules located near glomeruli were stained. x 450.
JENSEN, KOSZALKA AND BRENT
Reichert’s
Membrane
Antiserum
7
body localization in the PYS or VYS can result in varying degrees of embryotoxicity, although localization in the VYS cytoplasm seems to have a greater embryotoxic effect. Since the yolk sac placenta is the major placental structure on the ninth, lOth, and 11th days of gestation, one could propose a number of mechanisms to explain the resulting embryopathology. Following fixation of antibody on RM, PYS dysfunction could result from the following: 1. Interference with transport of metabolic and catabolic products between mother and embryo following structural alterations in RM due to antibody localiza-
FIG. 5. Frozen section of mouse kidney obtained from a mouse injected with rat RM antiserum and sacrificed 6 days later. There is specific staining of glomerular basement membrane and renal tubular basement membrane. x 200.
the endodermal epithelium or mesothelial portion of the developing VYS within the first 48 hr of administration. Fixation of antibodies only in RM (antiserum against 15th day embryonic RM) results in embryonic death and fetal growth retardation in survivors (Table 2). In contrast, fixation of antibodies only in the cytoplasm of the VYS endodermal cells results in the triad of malformations, embryonic death, and fetal growth retardation (Table 2). It is of interest that the triad can be produced by antisera that localize in both the VYS endodermal cells and RM simultaneously (antisera against term whole rat VYS or adult rat kidney) or by antisera that localize only in the cytoplasm of the VYS endodermal cells (teratogenic antiserum against term whole rat VYS adsorbed with RM (Table 2)). Thus, anti-
FIG. 6. Four-micron frozen section of 11th day embryonic site from a pregnant rat injected with antiserum on the ninth day of pregnancy. The antiserum injected was teratogenic sheep anti-rat whole serum adsorbed with RM. The indirect fluorescent technique was utilized in this preparation (Brent et al., 1971b). Only the cytoplasm of the VYS cells stains with the VYS antiserum adsorbed with RM, yet this adsorbed antiserum is still teratogenic. x 210.
8
DEVELOPMENTALBIOLOGY
tion on RM or in the cytoplasm of the PYS cell. 2. Interference with the synthesis and growth of RM following localization of antibody on the developing RM or in the cytoplasm of the PYS cell. Jollie (1968) studied PYS permeability in the rat to ferritin and thorotrast by electron microscopy. He suggested that the overall function of the PYS is to regulate the passage of materials between maternal blood and yolk sac cavity. Reichert’s membrane and its associated cells apparently serve as the only barrier to this interchange through the 17th day of pregnancy. It was observed by Everett (1935) that trypan blue and toluidine blue readily penetrated RM but iron ammonium citrate did not. It has also been demonstrated that lithium carmine particles traversed the membrane, but lampblack did not (Al-Abbass and Schultz, 1966). Jollie’s observations suggested that, although ferritin is transferred readily across RM, thorotrast is not. Robertson et al. (1971) found that in term murine placenta, horseradish peroxidase (HRP) rapidly passes across RM. Padykula et al. (1966) showed that vitamin B,, labeled with 58Cois taken up by endoderma1 cells of the VYS but not by the PYS. It is difficult to explain why RM antiserum produced embryonic growth retardation and embryolethality but only results in a low incidence of malformations. Alterations of permeability and function of the PYS could conceivably interfere with growth and survival and not necessarily produce a high incidence of malformations. On the other hand, the mechanical restrictions produced by the retarded growth and development of RM could result in lethality and/or growth retardation without necessarily producing a high incidence of malformation in the surviving fetuses. The PYS consists of two cell layers, trophoblast and endodermal epithelium, which are separated by RM. Reichert’s membrane is an exceedingly thick base-
VOLUME 42, 1975
ment membrane which appears between the fifth and sixth days in the rat embryo (Pierce, 1970). Its fine structure reveals a uniform fine fibrillar or lamellar appearance throughout its width in the rat (Wislocki and Dempsey, 1955) and in the mouse (Pierce et al., 1962). Reichert’s membrane was thickened following exposure of the embryos on the ninth day of gestation to RM antiserum and teratogenic term VYS antiserum adsorbed with RM (Fig. 8). The number and distribution of the PYS cells was also abnormal (Fig. 8) compared to the control embryos at the same stage (Fig. 7). Reichert’s membrane antibody does not localize in the trophoblastic layer, the outer cellular layer on the maternal side of RM, and there appear to be some pathological changes in the appearance of these trophoblastic cells following exposure to RM antiserum. Antibody localization in the cytoplasm of the VYS endodermal cell may result in
FIG. 7. Histological section of a 15th day rat embryonic site. Note the normal thinness of RM. x 150.
JENSEN, KOSZALKA AND BRENT
Reichert’s Membrane Antiserum
9
processes occur in the VYS cell that do not occur in the PYS cell (Padykula et al., 1966). Studies of yolk sac function at term or in the latter stages of gestation may be quite inappropriate if one wishes to learn about yolk sac function during the period of organogenesis. There is some evidence to indicate that the antibody transport increases during the later stages of the yolk sac development (Brambell, 1970). Thus, it is important to perform those studies dealing with the teratogenic effects of altered yolk sac function during early organogenesis, since teratogenic kidney and visceral yolk sac antisera do not produce malformations later in gestation (Jensh et al., 1973). Transport studies, ultrastructural studies, and metabolic studies of the altered yolk sac placenta should be performed before the chorioplacenta becomes functional. The previous discussion has indicated that the visceral and parietal yolk sac FIG. 8. Fifteenth day rat embryonic site from the placenta are the primary sites of action of pregnant rats injected on the ninth day of pregnancy these embryotoxic antisera. None of these with rabbit anti-rat RM serum. This histological antisera localize in the embryo (Slotnick preparation exhibits significant thickening of RM when compared to control embryos the same age. x and Brent, 1966; Brent, 1970). Further150. more, there is no localization in the developing ectoplacental cone or in the chorio(1) interference with lysosomal function placenta following in uiuo administration and, therefore, embryotrophic function of of these embryotoxic antisera (Brent et al., the yolk sac as described by Beck and 1971b). Even when cultured rat embryos Lloyd (1968) following trypan blue adminare grown in antiserum against term whole istration, (2) interference with anabolic, rat yolk sac, the ectoplacental cone is catabolic, or transport functions of the unaffected while the VYS and embryo are VYS placenta secondary to antibody fixa- severely stunted (New and Brent, 1972). It tion by cytoplasmic constituents of the is well known that the chorioplacenta, VYS VYS endoderm, and (3) interference with placenta, and kidney contain some antigrowth and development of the VYS pla- gens in common (Pressman and Korngold, centa. 1957; Pierce et al., 1962; Pierce et al., 1964; It is not clear why specific antibody Slotnick and Brent, 1966; Brent and Johnlocalization in the VYS results in a much son, 1967; Gebhardt et al., 1970). Obvihigher incidence of malformations than ously, the antigenic sites of the chorioplaspecific antibody localization in RM. The centa are not accessible to the antisera structural complexity of the VYS would when it is administered in vivo. indicate that a larger number of processes Pierce and Nakane (1967) have reported are carried out by the cells, but they would the relationship among human, rat, mouse, only indicate that some functions and and other mammalian basement mem-
10
DEVELOPMENTALBIOLOGY
branes in an interesting and important series of experiments. This report confirms their findings that antiserum against rat RM localizes in RM but not in the maternal glomerular basement membrane of the rat. Furthermore, embryotoxic anti-rat RM serum does not produce proteinuria in the rat. Yet, rabbit anti-rat RM serum does localize in the adult mouse glomerulus and absorption with splenic endothelial basement materials does not remove all the antibodies which localize in the mouse glomerulus. These data, which confirm the findings of Pierce and co-workers, were the basis of their hypothesis that the mouse glomerular basement membrane is synthesized by both endothelial and epithelial cells, while in the rat the only recognizable glomerular antigens are synthesized by endothelial cells. It is possible that the rat does synthesize epithelial basement membrane in the renal glomerulus just as the human and mouse do, but that the molecular structure of the epithelial basement membrane is different enough in the rat so that the antigenic sites are not accessible in in uiuo or in vitro localization studies. Kefalides (1971) reported that the mammalian lens capsule, which is an epithelial basement membrane, contains at least three antigenic components: a glycoprotein of high molecular weight, a glycoprotein of small molecular weight, and collagen. He further postulated that the large glycoprotein molecules are associated with one another through disulfide bonds and hydrogen bonds. It is possible that there may be interaction among the disulfide bonds within the complexes of glycoprotein molecules and collagen molecules and within the molecules of collagen themselves. It is possible that slight variation in the environment at the time that the basement membrane is synthesized may make for discrepancies in the antigenicity of the basement membrane in the various basement membrane under consideration. The accessibility of antisera to a particular
VOLUME 42, 1975
tissue can be interfered with because of variations in the molecular structure of the basement membrane or there may be differences in the glycoprotein and collagenlike structure as it is being synthesized within the cell. It is unusual for the cytoplasm of the cells lining the glomerular basement membrane to localize antibodies from nephrotoxic antisera. Reichert’s membrane antiserum only localizes in RM and not in the cytoplasm of the PYS cells (Jensen and Brent, 1972; Jensen, 1973; Jensen and Brent, 1973). Antiserum against term whole rat VYS localizes in the cytoplasm of the VYS endodermal cells, RM, and the cytoplasm of the PYS cells, but fails to stain the basement membrane of the endodermal cell of the VYS (Brent et al., 1971) (Table 2). Since the VYS endoderma1 basement membrane is continuous with RM, the obvious explanation for the failure of in uiuo administered VYS antiserum to stain the visceral endodermal basement membrane is that the antibodies are either fixed or metabolized in the cytoplasm of the endodermal cell. The failure of RM antisera to localize in the cytoplasm of the visceral endoderm could be due to the fact that RM antiserum contains antibodies only to the basement membrane and not to the intracellular glycoproteins and collagen-like protein. Thus, VYS could have several species of antibodies against all components of the basement membrane from the smallest antigenic intracellular protein to the final extracellular basement membrane. It seems most likely from adsorbtion studies of term whole VYS antiserum that the teratogenic stimulating antigen is contained in the cytoplasm of the VYS endoderm and some kidney cells. It is also possible that these antigens could be other proteins that are unrelated to basement membrane. The answers to the questions raised to this paper will come from several areas.
JENSEN, KOSZALKA AND BRENT
Isolation and characterization of the antigen that stimulates teratogenic antibodies is essential as the first step in explaining the teratogenic processes.It is possible that an explanation of the events that follow the antigen-antibody reaction within the yolk sac may explain the limits of variation of some biochemical and physiological processesthat permit normal embryonic development. The authors wish to thank Mrs. Olga Zulak for the preparation of the histological material and Miss Barbara Hinkel and Mrs. Betty Ward for preparing the manuscript. This study was supported by NIH Grant No. HD 630. REFERENCES AL-ABBASS, A. H., and SCHULTZ, R. L. (1966) Phagocytic activity of rat placenta. J. Anat. 100,349-359. ALBRECHT, P. (1965). Detection of Group B arboviruses in chick embryo cell cultures by the fluorescent antibody method. Acta Viral. 9, 338-346. BARROW, M. V., and TAYLOR, W. J. (1971). The production of congenital defects in rats using antisera. J. Exp. Zool. 176: 41-60. BECK, F., LLOYD, J. B., and GRIFFITS, A. (1967). A histochemical and biochemical study of some aspects of placental function in the rat using maternal injection of horseradish peroxidase. J. Anat. 101, 461-478. BECK, F., and LLOYD, J. B. (1968). Methods for the study of embryotrophic nutrition. Lab. Anim. Care 2, 157-170. BRAGONIER, J. R., FRANK, M. M., and BRENT, R. L. (1970). Production of congenital malformations using tissue antisera. VIII. Effectiveness of reduced, alkylated and digested anti-kidney antibodies. J. Immunol. 105, 1175-1179. BRAMBELL, F. W. R. (1970). The transmission of passive immunity from mother to young. fn “Frontiers of Biology,” Vol. 18. North-Holland, Amsterdam. BRENT, R. L., AVERICH, E., and DRAPIEWSKI, V. (1961). Production of congenital malformations using tissue antisera. I. Kidney antisera. Proc. Sot. Exp. Biol. 106, 523-526. BRENT, R. L. (1964). The production of congenital malformations with tissue antibodies. II. The spec trum and incidence of malformations following the administration of kidney antiserum to the pregnant rat. Amer. J. Anat. 115, 525-541. BRENT, R. L. (1966a). Some biologic properties of teratogenic antisera. Official J. Cong. Anom. Res. Assoc. Japan 6, 12-14.
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Antiserum
11
BRENT, R. L. (1966b). The production of congenital malformations using tissue antisera. IV. Evaluation of the mechanism of teratogenesis by varying the route and time of administration of anti-rat-kidney antiserum. Amer. J. Anat. 119, 555-562. BRENT, R. L., HANCOCK, N., HEWON, J., and SHABER, G. S. (1967). Autoradiographic localization of teratogenic antiserum. American Pediatric Society. BRENT, R. L., and JOHNSON, A. (1967). The production of congenital malformations using tissue antisera. VII. Yolk sac. Fed. Proc. 26, 701. BRENT, R. L. (1968). The production of congenital malformations using tissue antisera. VI. Cross specificity between species. Fed. Proc. 27, 623. BRENT, R. L., BRAGONIER, J. R., and FRANK, M. M. (1970). Production of congenital malformations using tissue antisera. IX. Effectiveness of structurally modified anti-kidney antibodies. Teratology 3, 198. BRENT, R. L. (1970). The effect of immune reactions on fetal development. In “Advances in Biosciences 6. Schering Symposium on Intrinsic and Extrinsic Factors in Early Mammalian Development” (G. Raspe, ed.), pp. 421-455, Pergamon, Vieweg. BRENT, R. L. (1971a). Antibodies and malformation. In “Malformations Congenitales des Mammiferes” (H. Tuchmann-Duplessis, ed.), pp. 181-222, Masson, Paris. BRENT, R. L., JOHNSON,A. J., and JENSEN, M. (1971b). The production of congenital malformations using tissue antisera. VII. Yolk sac antiserum. Teratology 4, 255-276. DAVID, G., MERCIER-PAROT, L., and TUCHMANNDUPLESSIS, H. (1963). Action teratogene d’heteroanticorps tissulaires. I. Production de malformations chez le rat par action d’un serum anti-rein. C.R. Sot. Biol. 157, 939. DEMPSEY, E. W. (1953). Electron microscopy of the visceral yolk-sac epithelium of the guinea pig. Amer. J. Anat. 93, 331-363. DOBROWOLSKI, M. S. (1903). iiber cytotoxine der plazenta. Bull. Int.Acad. Sci. Cracouie, No. 5. 256. EVERETT, J. W. (1935). Morphological and physiological studies of the placenta in the albino rat. J. Exp. 2001. 70, 243-286. EYQUEM, A., BUTMAN, G., BISSON, J., MERCIER-PAROT, L., DAVID,. G., and TUCHMANN-DUPLESSIS, H. (1968). Etude en immunofluorescence et cytotoxicite des immunserum teratogenes. Ann. Inst. Pasteur 115, 841-854. GEBHARDT, D. 0. E., BAART, DE LA FAILLE-KUYPER, E. H., and NAGEL, J. (1970). The embryolethality and localization of anti-kidney serum in the pregnant mouse, Mus musculus. Teratology 3, 143-151. JENSEN, M., and BRENT, R. L. (1972). The production of congenital malformations using tissue antisera. XV. Reichert’s membrane antiserum. Teratology 5, 258.
12
DEVELOPMENTAL BIOLOGY
JENSEN,M., and BRENT, R. L. (1973). Comparative embryo pathology of Reichert’s membrane (RM) and visceral yolk sac antisera in the rat. Teratology 7, A-18. JENSEN,M. (1973). Histopathology and tissue cross specificity of embryotoxic rat Reichert’s membrane (RM) antiserum. Fed. Proc. 32, No. 3, Part 1. JENSH,R. P., JOHNSON,A. J., and BRENT,R. L. (1973). The effect of antisera on rat embryonic development after the period of organog&esis. Teratology 7, A-18. JOLLIE,W. P. (1968). Changes in the fine structure of the parietal yolk sac of the rat placenta with increasing gestational age. Amer. J. Anat. 122, 513-532. KEFALIDES,N. A. (1971). Chemical properties of basement membranes. Int. Rev. Erp. Pathol. 10,
VOLUME42,1975
FELDMAN,J. D. (1962). Parietal yolk sac carcinoma: Clue to the histogenesis of Reichert’s membrane of the mouse embryo. Amer. J. Pathol. 41, 549-557. PIERCE,G. B., MIDGLEY,A. R., and SRI RAM, J. (1963). The histogenesis of basement membranes. J. Exp. Med. 117, 339-348. PIERCE,G. B., JR., BEALS, T. F., SRI RAM, J., and MIDGLEY,A. R., JR. (1964). Basement membranes. IV. Epithelial origin and immunologic cross reactions. Amer. J. Pathol. 45, 929-961. PIERCE,G. B. (1966). The development of basement membranes of the mouse embryo. Develop. Biol. 13, 231-249. PIERCE,G. B., JR., and NAKANE,P. K. (1967). Antigens of epithelial basement membranes of mouse, rat and man. A study utilizing enzyme-labeled antibody. Lab. hue&. 1’7, 499-514. l-38. PIERCE,G. B., and NAKANE,P. K. (1969). Basement membranes. Synthesis and deposition in response KINGSBURY,F. B., CLARK, C. P., WILLIAMS,G., and to cellular injury. Lab. Znuest. 21, 27-41. POST, A. L. (1926). The rapid determination of albumin in urine. J. Lab. Clin. Med. 11, 981. PIERCE,G. B. (1970). Epithelial basement membrane: MERCIER-PAROT,L., DAVID, G., and TUCHMANN- Origin, development and role in disease. In “Chemistry and Molecular Biology of the Intercellular DUPLESSIS,H. (1963). Action teratogene d’heteroMatrix” (Endre A. Balazs, ed.), pp. 471-506, Acaanticorps tissulaires. II. Etude de l’action teratogene chez la souris de serums anti-rein. C.R. Sot. demic Press, London. Biol. 157, 974. PRESSMAN,D., and KORNGOLD,L. (1957). Localizing MICIGLEY,A., and PIERCE,G. (1963). Immunohistoproperties of antiplacenta serum. J. Zmmunol. 78, chemical analysis of basement membranes of the 75-78. mouse. Amer. J. Pathol. 43, 929-934. ROBERTSON, T. A., ARCHER,J. M., PAPADIMITRIOU, J. MIKHAILOV,V. M. (1967). Pathogenic action of neM., and WALTERS,M. N-I. (1971). Transport of phrocytotoxic serum on embryonic development of horseradish peroxidase in the murine placenta. J. albino rats. Biull. Eksp. Biol. Med. 63, 97-100. Patho2. 103, 141-147. MRENOVA,M., and ALBRECHT,P. (1966). Stabilization SEEGAL,B. C., and LOEB, E. N. (1940). Effect of of fluorescence in preparates treated by the fluoresanti-placenta serum on development of the fetus in cent antibody technique. Nature (London) 212, the pregnant rat. Proc. Sot. Exp. Biol. Med. 45, 1256-1257. 248-253. NEW D.A.T., and BRENT,R. L. (1972). Effect of yolk- SLOTNICK,V., and BRENT,R. L. (1966). The production of congenital malformations using tissue antisac antibody on rat embryos grown in culture. J. Emhryol. Exp. Morphol. 27,543-553. sera. V. Fluorescent localization of teratogenic antisera in the maternal and fetal tissue of the rat. PADYKULA,H., DEREN,J. J., and WILSON,T. H. (1966). J. Immunol. 95, 606-610. Development of structure and function in the mammalian yolk sac. I. Development morphology WISLOCKI,G. B., and DEMPSEY,E. W. (1955). Electron microscopy of the placenta of the rat. Anat. Rec. and vitamin B,, uptake of the rat yolk sac. Deuelop. 123, 33-63. Biol. 12, 311-348. PIERCE, G. B., MIDGLEY, A. R., SRI RAM, J., and
BIOLOGY 42, 1-12
Production
(19%)
of Congenital Malformations Antisera
XV. Reichert’s
Membrane
and Visceral
Using Tissue
Yolk Sac Antisera
MARCELA JENSEN, THOMAS R. KOSZALKA, AND ROBERT L. BRENT Stein Research Center, Thomas Jefferson
University,
Philadelphia,
Pennsylvania
19107
Accepted August 26, 1974 Rabbit anti-rat Reichert’s membrane (RM) serum, when injected into pregnant rats on the ninth day of gestation, produced embryolethality and stunting but a surprisingly low incidence of malformations. Immunofluorescent studies revealed that the maternally injected RM antiserum localizes in RM and in the basement membrane of some maternal kidney tubules but not in the maternal glomerulus or in the cytoplasm of the visceral yolk sac (VYS) cells as had teratogenic VYS and kidney antisera. When antiserum against rat RM was injected into mice, localization occurred in the glomerular basement membranes, thus confirming the findings of Pierce and co-workers. When teratogenic term VYS antiserum is adsorbed with RM, it still localizes in the VYS, does not localize in RM, and is still teratogenic. When this same antiserum is adsorbed with VYS, the antiserum is no longer teratogenic. It appears that the localization of antibodies in the VYS cells is the major contributor to the production of congenital malformations because localization of antisera in RM alone was associated with a very low incidence of teratogenesis. Since both structures participate in a multitude of biochemical processes, which include adsorption, transport, catabolism, and anabolism of nutrients, nonnutrients, and waste products, there are many plausible hypotheses to explain the nature of the parietal and visceral yolk sac dysfunction.
INTRODUCTION
Dobrowolski (1903) observed that specific anti-placental serum interrupted pregnancy in guinea pigs and rabbits. Seegal and Loeb (1940) reported that rabbit anti-rat placental serum was abortogenic and nephrotoxic. They did not report an increase in the incidence of congenital malformations, in spite of the fact that pregnant rats were injected on the ninth day of gestation. Pressman and Korngold (1957) found that cross-reacting antibodies were present in Seegal’s antiplacental and antikidney sera. Brent et al. (1961) first reported that heterologous kidney antiserum is teratogenic when injected into pregnant rats. This phenomenon was described in detail in a series of papers which included the spectrum and incidence of malformations following the administration of kidney antiserum to pregnant rats (Brent, 1964), some experiments testing Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
various hypotheses pertaining to the mechanism of teratogenesis (Brent, 1966b), the cross specificity of teratogenic antisera in mouse and rat (Brent, 1968), the biologic and biochemical properties of teratogenic antisera (Brent, 1966a; Bragonier et al., 1970) and several reviews of this subject (Brent, 1970; Brent, 1971a). Some of these findings were confirmed and extended by other investigators (David et al., 1966; Mercier-Parot et al., 1963; Mikhailov, 1967; Eyguem et al., 1968; Gebhardt et al., 1970; Barrow and Taylor, 1971). The teratogenic factor was demonstrated to be in the IgG fraction and reduction, and alkylation or preparation of the F(ab)” fragment of IgG from sheep anti-rat kidney teratogenic serum did not interfere with the potency of the teratogenic IgG (Bragonier et al., 1970; Brent, 1970). Thus, complement fixation was not an essential step in the teratogenic process. It was also
2
DEVELOPMENTALBIOLOGY
reported that lz51-labeled teratogenic gamma globulin and fluorescent-labeled teratogenic gamma globulin localized in the maternal glomerular basement membrane, in the Reichert’s membrane (RM), and in the cytoplasm of the visceral yolk sac (VYS) endodermal cells (Slotnick and Brent, 1966). However, no specific antibody localization was demonstrated in the embryo. Teratogenic kidney antiserum localized in the developing yolk sac structures and, therefore, it seemed logical to determine whether antigens in the visceral or parietal yolk sac were capable of stimulating teratogenic antibodies. Sheep antiterm whole-visceral rat yolk sac serum proved to be teratogenic (Brent et al., 1971b). Since localization of this antiserum also occurred in the maternal glomerular basement membrane, RM and in the cytoplasm of the VYS, it was not possible to determine whether localization in RM, VYS cells or both were primarily involved in the teratogenic process. If an antiserum which localized in one or the other of these structures could be prepared, then it might be possible to determine whether localizations in either the visceral or parietal yolk sac is associated with teratogenesis. Since RM is an epithelial basement membrane, it seemed logical as an initial step to study the localization, cross-reactivity, and embryotoxicity of antisera made against this epithelial basement membrane. MATERIALS
AND
VOLUME 42, 1975
structure. The trophoblastic layer can be removed from the PYS by simple stripping, leaving RM and the associated cells (Fig. 1). If the embryonic site is frozen and thawed before dissecting the uterine wall, the trophoblastic layer and the PYS cells can be removed by simply shaking the PYS in distilled water (Fig. 2). For each immunization six RM were homogenized in 2 ml of distilled water and emulsified with an equal volume of Freund’s incomplete adjuvant. Rabbits were injected at weekly intervals for 7 weeks before the first bleeding. The preparation of antisera with whole rat kidney and whole term rat VYS have been previously described (Brent et al., 1961; Brent et al., 1971b).
METHODS
Reichert’s membrane was prepared using the following protocol. Embryonic sites obtained from Wistar rats on the 15th day of pregnancy were placed in cold phosphate buffered saline at pH 7.2. The uterine musculature was carefully dissected from the surface of the extraembryonic membranes in order to prevent rupture of the parietal yolk sac (PYS). Recognition and dissection of the PYS is enhanced by the nonglossy appearance of this
FIG. 1. Fresh preparation of 15th day parietal yolk sacs immediately after dissection from the embryonic site. Reichert’s membrane (RM) was dissected free of the trophoblastic cells and then fixed in Bouin’s solution and embedded in paraffin. Sections cut at 5 pm and stained with hematoxylin and eosin. Note the RM and associated parietal yolk sac cells. x 125.
JENSEN, KOSZALKA AND BRENT
Reichert’s Membrane Antiserum
Adsorption
FIG. 2. Similar preparation to Fig. 1, except that the embryonic site was frozen at -60°C for at least 24 hr before the parietal yolk sac was dissected. Note the relatively pure preparation of RM. x 125.
Testing for Embryotoxicity Pregnant Wistar rats were obtained by mating rats from 4 PM to the following 9 AM. If sperm were detected in the vaginal smear, then the pregnancy was considered to be 0 hr and 0 days old at 9 AM, or the beginning of the first day of pregnancy. On the ninth day of pregnancy the rats were anesthetized with sodium pentobarbital (30 mg/kg) and a laparotomy was performed in order to determine the number and condition of the embryonic sites. After closing the incision, the various adsorbed and unadsorbed antisera were injected ip into the pregnant rat. On the 22nd day of pregnancy, the rats were sacrificed and the incidence of resorption and malformations was determined as well as the extent of fetal growth retardation (Tables 1 and 2).
3
Studies
Fresh splenic tissue was obtained from 12- to la-week-old female BALB/c mice weighing 18-25 g (Jackson Laboratory). The splenic tissue was homogenized in 0.85% saline to a final concentration of 10%. The cell suspension was disrupted by sonication for 15-30 min and the insoluble fraction collected by centrifugation at 1400 g for 30 min at 4°C. The precipitate was washed in distilled water and lyophilized. The insoluble splenic residue contained splenic reticulum and vascular basement membranes. Specific antisera were adsorbed by shaking a suspension of splenic residue in the conjugate at 4°C for 24 hr, centrifuging, and repeating the adsorption process twice (Pierce et al., 1963). Adsorption of teratogenic term VYS antiserum with 15th day RM and 15th day whole VYS was performed by combining 10 mg of lyophilized antigenic material per milliliter of antiserum at 4°C for 18 hr, centrifuging, and repeating the adsorption process three times. The adsorbed antisera were then utilized for localization and embryopathic studies as described in the sections dealing with immunofluorescent localization and embryotoxicity testing (Tables 1 and 2). Immunofluorescent
Localization
After the injection of rabbit anti-rat RM serum into pregnant rats on the ninth day of pregnancy, the rats were sacrificed 48 hr later. Frozen sections of adult kidney and embryonic tissue were cut at 4 pm. The direct fluorescent antibody technique was used for localization of the antiserum. The 7S fraction of goat anti-rabbit gamma globulin (Nutritional Biochemical Corp., Cleveland, Ohio) was conjugated with fluorescein isothiocyanate (FITC) in borate buffer at pH 9.0 for 3 hr at 4°C (Albrecht, 1965). Unreacted FITC was removed by adsorption with charcoal for 15 min (20 mg/ml of conjugate) and by adsorption of
4
DEVELOPMENTALBIOLOGY
VOLUME 42, 1975
TABLE
1
COMPARISON
OF THE EMBRYOPATHIC EFFECTS OF ANTISERUM PREPARED AGAINST (1) ~&DAY-OLD (15TH RM, (2) ADULT RAT KIDNEY, (3) ABSORBED AND UNAESORBEDTERM WHOLE-RAT VYS
DAY)
Tissue antiserum injected on the ninth day of pregnancy
Dose (mg/kg)
Number of pregnant rats
Total number of embryos on ninth day
Total number of embryos at term
Control sheep serum RM RM RM RM RM Whole kidney Term VYS Term VYS adsorbed with 15th day RM Term VYS adsorbed with 15th day RM Term VYS adsorbed with 15th day RM Term VYS adsorbed with 15th day yolk sac Term VYS adsorbed with 15th day yolk sac Term VYS adsorbed with 15th day yolk sac
350 50 70-100 110-150 160-400 240-600 175 100 70-100
9 2 6 14 9 2 4 5 3
94 22 65 169 105 24 50 65 34
84 22 49 91 20 0 20 47 30
0.0 24.6 46.1 81.0 100.0 60 27.6 12.3
4.68 4.91 4.26 4.19 3.12 3.89 3.90 4.02
1.2 0 0 0
125
5
58
20
41.2
3.35
100.0
140
2
30
7
70.0
2.16
100.0
100-120
3
34
29
14.7
4.89
0.0
140
2
23
18
21.7
4.37
0.0
160
2
26
8
69.2
4.12
0.0
(o/o) Resorbtions 10.6
Term fetal weight (4
(WI Malformations
100.0
TABLE 2 INTENSITY
OF TMMUNOFLUORESCENT
Antiseraprepared against
Intensity Maternal rat glomerular )asement membrane
Control: sheep serum 15th day embryonic rat Reichert’s membrane Whole adult rat kidney Term whole rat visceral yolk sac Term whole rat visceral yolk sac adsorbed with 15th day Reichert’s membrane Term whole rat visceral yolk sac absorbed with 15th day visceral yolk sac
LOCALIZATION AND EMBRYOTOXICITY EMBRYOTOXIC ANTISERA of immunofluorescent
Mousr maternal glomerular basement membrane
Maternal rat kidney tubular basement membrane
+++
Rat Reichert’s membrane
+++
+++
it+
++
tt+
+++
tt
++t+
++
+
ABSORBED AND UNABSORBED
localization
Cytoplasm of maternal rat kidney tubular cells
-
OF VARIOUS
+
Embryonic lethality
Embryonic growth retardation
+t++
++t+
*
+++
+ttt
+t+t
+++t
+tt
++tt
+tt+
++++
Cytoplasm of rat visceral wJJl;=
Embryonic malformations
t+
+++
+++
++++
i
t
*
*
JENSEN, KOSZALKA AND BRENT
the conjugate on acetone dried rat liver powder (100 mg/ml of conjugate). The conjugate was finally adsorbed with a 15% suspension of the particular tissue to be stained. Adsorption was performed for 1 hr at room temperature and overnight at 4°C. The adsorbed conjugate was then centrifuged and used for staining. Tissue sections that had been exposed to various teratogenic and nonteratogenic antisera prepared in the rabbit were fixed for 10 minutes in acetone and then stained for 30 min with goat anti-rabbit gamma globulin conjugated with FITC. After staining they were rinsed in three changes of phosphate-buffered saline. Sections were mounted in 1% Tris(hydroxymethyl)aminomethane in glycerine (Mrenova, and Albrecht, 1966). Examinations were made using a Leitz fluorescent microscope with an HBO 200 ultraviolet light source, using a dark-field condenser, BG 12 exciting filter, and OG 1 eyepiece filter. Photographs were made with ASA 3000 Polaroid roll film.
5
Reichert’s Membrane Antiserum
tions in surviving fetuses from a litter exposed to an LD,, dose of either whole rat kidney antiserum (Brent, 1966) or whole term rat VYS antiserum (Brent et al., 1971b) is greater than 50% (Table 3). In Vivo Localization
of Various Adsorbed
and Unadsorbed Antisera
In vivo localization of RM antiserum within the embryonic site revealed a linear staining along RM (Fig. 3, Table 2), but there was no localization in the cytoplasm of the VYS cells as has been observed with teratogenic antisera prepared against term whole VYS (Brent et al., 1971b) or whole rat kidney (Slotnick and Brent, 1966). Reichert’s membrane antiserum did not localize in the maternal glomerular basement membrane but did localize in the basement membrane of some kidney tubules adjacent to the glomeruli (Fig. 4). When rabbit anti-15th day rat embryonic RM serum is injected into the BALB/c goat anti-rabbit mouse, FITC-labeled gamma globulin does stain the glomerular basement membrane (Fig. 5). FurtherTesting for Nephrotoxicity more, if this antiserum is adsorbed with Male Wistar strain rats weighing insoluble splenic residue containing reticu230-270 g were injected intravenously with lum and vascular basement membrane and 200 mg/kg of pooled RM antiserum. Daily is then injected into the mouse, the glomerurine specimens were collected for 3 weeks ular masangium still localized the RM and tested for protein according to the antisera. Thus, rabbit anti-rat RM serum procedure of Kingsburg et al. (1926). After does not localize in the adult rat glomerurats were sacrificed, the kidneys were stud- lus but does localize in the mouse glomeruied for localization of antisera by the im- lus. munofluorescent technique described in TABLE 3 the previous section. RELATIONSHIP OF THE EMBRYONIC LD,, DOSE OF RESULTS
Embryotoxic
Effects of Various Antisera
Rabbit anti-rat RM serum when injected ip into pregnant rats on the ninth day of pregnancy produced embryolethality and fetal growth retardation (Table 1). The incidence of congenital malformations was very low (Tables l-3). This was unexpected since the incidence of malforma-
VARIOUS ANTISERA AND THE INCIDENCE OF MALFORMATIONS OBSERVED IN THE SURVIVING RAT FETUSES AT TERM LD,, dose injected on the ninth day of gestation Adult kidney antiserum Term chorioplacental antiserum Whole term yolk sac antiserum 15th day Reichert’s membrane and antiserum
Percent malformations in survivors 80 58 66 <8
6
DEVELOPMENTALBIOLOGY
VOLUME 42, 1975
bryonic rat RM are different from the embryotoxic effects of antisera prepared against whole rat kidney and whole term rat VYS. While the latter two antisera produce a high incidence of malformations in the survivors at term following an embryonic LD,, dose, the RM antiserum produced a very low incidence of malformations, in spite of the fact that embryonic lethality and severe growth retardation were observed (Table 3). None of these antisera, when administered t,o the mother, localized in the developing rat embryo, as demonstrated by the in uivo immunofluorescence studies during the embryonic and early fetal stages of development. Similarly, the antisera do not localize in the basement membrane of
FIG. 3. Frozen section of 11th day embryonic site from a rat injected with rabbit anti-rat RM serum on the ninth day of gestation. Stained with FITC-labeled goat anti-rabbit IgG as described in the Materials and Methods section. Note that the VYS is unstained and therefore not visible. Only RM fluoresces (arrow) having localized RM antisera. x 50.
Adsorption of teratogenic term VYS antiserum with 15th day RM did not remove the teratogenic properties of the antiserum, but did eliminate its localization in RM (Table 2, Fig. 6). Adsorbtion of teratogenic term VYS antiserum with 15th day whole VYS eliminated malformations, although some resorptions and growth retardation did occur (Table 1 and 2). When RM antiserum was injected into eight adult rats and 24-hr urines were tested for proteinuria over a period of 3 weeks, the daily urinary protein content ranged between 2 and 15 mg, which is the normal range of 24-hr protein for rats. DISCUSSION
The embryotoxic effects of heterologous antiserum prepared against 15th day em-
FIG. 4. Frozen section of kidney from a pregnant rat injected with rabbit anti-rat RM serum on the ninth day of pregnancy and sacrificed 48 hr later. Stained with FITC-labeled goat anti IgG serum as described in the Materials and Methods section. No staining of the glomeruli was observed, but the basement membrane of some cortical tubules located near glomeruli were stained. x 450.
JENSEN, KOSZALKA AND BRENT
Reichert’s
Membrane
Antiserum
7
body localization in the PYS or VYS can result in varying degrees of embryotoxicity, although localization in the VYS cytoplasm seems to have a greater embryotoxic effect. Since the yolk sac placenta is the major placental structure on the ninth, lOth, and 11th days of gestation, one could propose a number of mechanisms to explain the resulting embryopathology. Following fixation of antibody on RM, PYS dysfunction could result from the following: 1. Interference with transport of metabolic and catabolic products between mother and embryo following structural alterations in RM due to antibody localiza-
FIG. 5. Frozen section of mouse kidney obtained from a mouse injected with rat RM antiserum and sacrificed 6 days later. There is specific staining of glomerular basement membrane and renal tubular basement membrane. x 200.
the endodermal epithelium or mesothelial portion of the developing VYS within the first 48 hr of administration. Fixation of antibodies only in RM (antiserum against 15th day embryonic RM) results in embryonic death and fetal growth retardation in survivors (Table 2). In contrast, fixation of antibodies only in the cytoplasm of the VYS endodermal cells results in the triad of malformations, embryonic death, and fetal growth retardation (Table 2). It is of interest that the triad can be produced by antisera that localize in both the VYS endodermal cells and RM simultaneously (antisera against term whole rat VYS or adult rat kidney) or by antisera that localize only in the cytoplasm of the VYS endodermal cells (teratogenic antiserum against term whole rat VYS adsorbed with RM (Table 2)). Thus, anti-
FIG. 6. Four-micron frozen section of 11th day embryonic site from a pregnant rat injected with antiserum on the ninth day of pregnancy. The antiserum injected was teratogenic sheep anti-rat whole serum adsorbed with RM. The indirect fluorescent technique was utilized in this preparation (Brent et al., 1971b). Only the cytoplasm of the VYS cells stains with the VYS antiserum adsorbed with RM, yet this adsorbed antiserum is still teratogenic. x 210.
8
DEVELOPMENTALBIOLOGY
tion on RM or in the cytoplasm of the PYS cell. 2. Interference with the synthesis and growth of RM following localization of antibody on the developing RM or in the cytoplasm of the PYS cell. Jollie (1968) studied PYS permeability in the rat to ferritin and thorotrast by electron microscopy. He suggested that the overall function of the PYS is to regulate the passage of materials between maternal blood and yolk sac cavity. Reichert’s membrane and its associated cells apparently serve as the only barrier to this interchange through the 17th day of pregnancy. It was observed by Everett (1935) that trypan blue and toluidine blue readily penetrated RM but iron ammonium citrate did not. It has also been demonstrated that lithium carmine particles traversed the membrane, but lampblack did not (Al-Abbass and Schultz, 1966). Jollie’s observations suggested that, although ferritin is transferred readily across RM, thorotrast is not. Robertson et al. (1971) found that in term murine placenta, horseradish peroxidase (HRP) rapidly passes across RM. Padykula et al. (1966) showed that vitamin B,, labeled with 58Cois taken up by endoderma1 cells of the VYS but not by the PYS. It is difficult to explain why RM antiserum produced embryonic growth retardation and embryolethality but only results in a low incidence of malformations. Alterations of permeability and function of the PYS could conceivably interfere with growth and survival and not necessarily produce a high incidence of malformations. On the other hand, the mechanical restrictions produced by the retarded growth and development of RM could result in lethality and/or growth retardation without necessarily producing a high incidence of malformation in the surviving fetuses. The PYS consists of two cell layers, trophoblast and endodermal epithelium, which are separated by RM. Reichert’s membrane is an exceedingly thick base-
VOLUME 42, 1975
ment membrane which appears between the fifth and sixth days in the rat embryo (Pierce, 1970). Its fine structure reveals a uniform fine fibrillar or lamellar appearance throughout its width in the rat (Wislocki and Dempsey, 1955) and in the mouse (Pierce et al., 1962). Reichert’s membrane was thickened following exposure of the embryos on the ninth day of gestation to RM antiserum and teratogenic term VYS antiserum adsorbed with RM (Fig. 8). The number and distribution of the PYS cells was also abnormal (Fig. 8) compared to the control embryos at the same stage (Fig. 7). Reichert’s membrane antibody does not localize in the trophoblastic layer, the outer cellular layer on the maternal side of RM, and there appear to be some pathological changes in the appearance of these trophoblastic cells following exposure to RM antiserum. Antibody localization in the cytoplasm of the VYS endodermal cell may result in
FIG. 7. Histological section of a 15th day rat embryonic site. Note the normal thinness of RM. x 150.
JENSEN, KOSZALKA AND BRENT
Reichert’s Membrane Antiserum
9
processes occur in the VYS cell that do not occur in the PYS cell (Padykula et al., 1966). Studies of yolk sac function at term or in the latter stages of gestation may be quite inappropriate if one wishes to learn about yolk sac function during the period of organogenesis. There is some evidence to indicate that the antibody transport increases during the later stages of the yolk sac development (Brambell, 1970). Thus, it is important to perform those studies dealing with the teratogenic effects of altered yolk sac function during early organogenesis, since teratogenic kidney and visceral yolk sac antisera do not produce malformations later in gestation (Jensh et al., 1973). Transport studies, ultrastructural studies, and metabolic studies of the altered yolk sac placenta should be performed before the chorioplacenta becomes functional. The previous discussion has indicated that the visceral and parietal yolk sac FIG. 8. Fifteenth day rat embryonic site from the placenta are the primary sites of action of pregnant rats injected on the ninth day of pregnancy these embryotoxic antisera. None of these with rabbit anti-rat RM serum. This histological antisera localize in the embryo (Slotnick preparation exhibits significant thickening of RM when compared to control embryos the same age. x and Brent, 1966; Brent, 1970). Further150. more, there is no localization in the developing ectoplacental cone or in the chorio(1) interference with lysosomal function placenta following in uiuo administration and, therefore, embryotrophic function of of these embryotoxic antisera (Brent et al., the yolk sac as described by Beck and 1971b). Even when cultured rat embryos Lloyd (1968) following trypan blue adminare grown in antiserum against term whole istration, (2) interference with anabolic, rat yolk sac, the ectoplacental cone is catabolic, or transport functions of the unaffected while the VYS and embryo are VYS placenta secondary to antibody fixa- severely stunted (New and Brent, 1972). It tion by cytoplasmic constituents of the is well known that the chorioplacenta, VYS VYS endoderm, and (3) interference with placenta, and kidney contain some antigrowth and development of the VYS pla- gens in common (Pressman and Korngold, centa. 1957; Pierce et al., 1962; Pierce et al., 1964; It is not clear why specific antibody Slotnick and Brent, 1966; Brent and Johnlocalization in the VYS results in a much son, 1967; Gebhardt et al., 1970). Obvihigher incidence of malformations than ously, the antigenic sites of the chorioplaspecific antibody localization in RM. The centa are not accessible to the antisera structural complexity of the VYS would when it is administered in vivo. indicate that a larger number of processes Pierce and Nakane (1967) have reported are carried out by the cells, but they would the relationship among human, rat, mouse, only indicate that some functions and and other mammalian basement mem-
10
DEVELOPMENTALBIOLOGY
branes in an interesting and important series of experiments. This report confirms their findings that antiserum against rat RM localizes in RM but not in the maternal glomerular basement membrane of the rat. Furthermore, embryotoxic anti-rat RM serum does not produce proteinuria in the rat. Yet, rabbit anti-rat RM serum does localize in the adult mouse glomerulus and absorption with splenic endothelial basement materials does not remove all the antibodies which localize in the mouse glomerulus. These data, which confirm the findings of Pierce and co-workers, were the basis of their hypothesis that the mouse glomerular basement membrane is synthesized by both endothelial and epithelial cells, while in the rat the only recognizable glomerular antigens are synthesized by endothelial cells. It is possible that the rat does synthesize epithelial basement membrane in the renal glomerulus just as the human and mouse do, but that the molecular structure of the epithelial basement membrane is different enough in the rat so that the antigenic sites are not accessible in in uiuo or in vitro localization studies. Kefalides (1971) reported that the mammalian lens capsule, which is an epithelial basement membrane, contains at least three antigenic components: a glycoprotein of high molecular weight, a glycoprotein of small molecular weight, and collagen. He further postulated that the large glycoprotein molecules are associated with one another through disulfide bonds and hydrogen bonds. It is possible that there may be interaction among the disulfide bonds within the complexes of glycoprotein molecules and collagen molecules and within the molecules of collagen themselves. It is possible that slight variation in the environment at the time that the basement membrane is synthesized may make for discrepancies in the antigenicity of the basement membrane in the various basement membrane under consideration. The accessibility of antisera to a particular
VOLUME 42, 1975
tissue can be interfered with because of variations in the molecular structure of the basement membrane or there may be differences in the glycoprotein and collagenlike structure as it is being synthesized within the cell. It is unusual for the cytoplasm of the cells lining the glomerular basement membrane to localize antibodies from nephrotoxic antisera. Reichert’s membrane antiserum only localizes in RM and not in the cytoplasm of the PYS cells (Jensen and Brent, 1972; Jensen, 1973; Jensen and Brent, 1973). Antiserum against term whole rat VYS localizes in the cytoplasm of the VYS endodermal cells, RM, and the cytoplasm of the PYS cells, but fails to stain the basement membrane of the endodermal cell of the VYS (Brent et al., 1971) (Table 2). Since the VYS endoderma1 basement membrane is continuous with RM, the obvious explanation for the failure of in uiuo administered VYS antiserum to stain the visceral endodermal basement membrane is that the antibodies are either fixed or metabolized in the cytoplasm of the endodermal cell. The failure of RM antisera to localize in the cytoplasm of the visceral endoderm could be due to the fact that RM antiserum contains antibodies only to the basement membrane and not to the intracellular glycoproteins and collagen-like protein. Thus, VYS could have several species of antibodies against all components of the basement membrane from the smallest antigenic intracellular protein to the final extracellular basement membrane. It seems most likely from adsorbtion studies of term whole VYS antiserum that the teratogenic stimulating antigen is contained in the cytoplasm of the VYS endoderm and some kidney cells. It is also possible that these antigens could be other proteins that are unrelated to basement membrane. The answers to the questions raised to this paper will come from several areas.
JENSEN, KOSZALKA AND BRENT
Isolation and characterization of the antigen that stimulates teratogenic antibodies is essential as the first step in explaining the teratogenic processes.It is possible that an explanation of the events that follow the antigen-antibody reaction within the yolk sac may explain the limits of variation of some biochemical and physiological processesthat permit normal embryonic development. The authors wish to thank Mrs. Olga Zulak for the preparation of the histological material and Miss Barbara Hinkel and Mrs. Betty Ward for preparing the manuscript. This study was supported by NIH Grant No. HD 630. REFERENCES AL-ABBASS, A. H., and SCHULTZ, R. L. (1966) Phagocytic activity of rat placenta. J. Anat. 100,349-359. ALBRECHT, P. (1965). Detection of Group B arboviruses in chick embryo cell cultures by the fluorescent antibody method. Acta Viral. 9, 338-346. BARROW, M. V., and TAYLOR, W. J. (1971). The production of congenital defects in rats using antisera. J. Exp. Zool. 176: 41-60. BECK, F., LLOYD, J. B., and GRIFFITS, A. (1967). A histochemical and biochemical study of some aspects of placental function in the rat using maternal injection of horseradish peroxidase. J. Anat. 101, 461-478. BECK, F., and LLOYD, J. B. (1968). Methods for the study of embryotrophic nutrition. Lab. Anim. Care 2, 157-170. BRAGONIER, J. R., FRANK, M. M., and BRENT, R. L. (1970). Production of congenital malformations using tissue antisera. VIII. Effectiveness of reduced, alkylated and digested anti-kidney antibodies. J. Immunol. 105, 1175-1179. BRAMBELL, F. W. R. (1970). The transmission of passive immunity from mother to young. fn “Frontiers of Biology,” Vol. 18. North-Holland, Amsterdam. BRENT, R. L., AVERICH, E., and DRAPIEWSKI, V. (1961). Production of congenital malformations using tissue antisera. I. Kidney antisera. Proc. Sot. Exp. Biol. 106, 523-526. BRENT, R. L. (1964). The production of congenital malformations with tissue antibodies. II. The spec trum and incidence of malformations following the administration of kidney antiserum to the pregnant rat. Amer. J. Anat. 115, 525-541. BRENT, R. L. (1966a). Some biologic properties of teratogenic antisera. Official J. Cong. Anom. Res. Assoc. Japan 6, 12-14.
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BRENT, R. L. (1966b). The production of congenital malformations using tissue antisera. IV. Evaluation of the mechanism of teratogenesis by varying the route and time of administration of anti-rat-kidney antiserum. Amer. J. Anat. 119, 555-562. BRENT, R. L., HANCOCK, N., HEWON, J., and SHABER, G. S. (1967). Autoradiographic localization of teratogenic antiserum. American Pediatric Society. BRENT, R. L., and JOHNSON, A. (1967). The production of congenital malformations using tissue antisera. VII. Yolk sac. Fed. Proc. 26, 701. BRENT, R. L. (1968). The production of congenital malformations using tissue antisera. VI. Cross specificity between species. Fed. Proc. 27, 623. BRENT, R. L., BRAGONIER, J. R., and FRANK, M. M. (1970). Production of congenital malformations using tissue antisera. IX. Effectiveness of structurally modified anti-kidney antibodies. Teratology 3, 198. BRENT, R. L. (1970). The effect of immune reactions on fetal development. In “Advances in Biosciences 6. Schering Symposium on Intrinsic and Extrinsic Factors in Early Mammalian Development” (G. Raspe, ed.), pp. 421-455, Pergamon, Vieweg. BRENT, R. L. (1971a). Antibodies and malformation. In “Malformations Congenitales des Mammiferes” (H. Tuchmann-Duplessis, ed.), pp. 181-222, Masson, Paris. BRENT, R. L., JOHNSON,A. J., and JENSEN, M. (1971b). The production of congenital malformations using tissue antisera. VII. Yolk sac antiserum. Teratology 4, 255-276. DAVID, G., MERCIER-PAROT, L., and TUCHMANNDUPLESSIS, H. (1963). Action teratogene d’heteroanticorps tissulaires. I. Production de malformations chez le rat par action d’un serum anti-rein. C.R. Sot. Biol. 157, 939. DEMPSEY, E. W. (1953). Electron microscopy of the visceral yolk-sac epithelium of the guinea pig. Amer. J. Anat. 93, 331-363. DOBROWOLSKI, M. S. (1903). iiber cytotoxine der plazenta. Bull. Int.Acad. Sci. Cracouie, No. 5. 256. EVERETT, J. W. (1935). Morphological and physiological studies of the placenta in the albino rat. J. Exp. 2001. 70, 243-286. EYQUEM, A., BUTMAN, G., BISSON, J., MERCIER-PAROT, L., DAVID,. G., and TUCHMANN-DUPLESSIS, H. (1968). Etude en immunofluorescence et cytotoxicite des immunserum teratogenes. Ann. Inst. Pasteur 115, 841-854. GEBHARDT, D. 0. E., BAART, DE LA FAILLE-KUYPER, E. H., and NAGEL, J. (1970). The embryolethality and localization of anti-kidney serum in the pregnant mouse, Mus musculus. Teratology 3, 143-151. JENSEN, M., and BRENT, R. L. (1972). The production of congenital malformations using tissue antisera. XV. Reichert’s membrane antiserum. Teratology 5, 258.
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JENSEN,M., and BRENT, R. L. (1973). Comparative embryo pathology of Reichert’s membrane (RM) and visceral yolk sac antisera in the rat. Teratology 7, A-18. JENSEN,M. (1973). Histopathology and tissue cross specificity of embryotoxic rat Reichert’s membrane (RM) antiserum. Fed. Proc. 32, No. 3, Part 1. JENSH,R. P., JOHNSON,A. J., and BRENT,R. L. (1973). The effect of antisera on rat embryonic development after the period of organog&esis. Teratology 7, A-18. JOLLIE,W. P. (1968). Changes in the fine structure of the parietal yolk sac of the rat placenta with increasing gestational age. Amer. J. Anat. 122, 513-532. KEFALIDES,N. A. (1971). Chemical properties of basement membranes. Int. Rev. Erp. Pathol. 10,
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FELDMAN,J. D. (1962). Parietal yolk sac carcinoma: Clue to the histogenesis of Reichert’s membrane of the mouse embryo. Amer. J. Pathol. 41, 549-557. PIERCE,G. B., MIDGLEY,A. R., and SRI RAM, J. (1963). The histogenesis of basement membranes. J. Exp. Med. 117, 339-348. PIERCE,G. B., JR., BEALS, T. F., SRI RAM, J., and MIDGLEY,A. R., JR. (1964). Basement membranes. IV. Epithelial origin and immunologic cross reactions. Amer. J. Pathol. 45, 929-961. PIERCE,G. B. (1966). The development of basement membranes of the mouse embryo. Develop. Biol. 13, 231-249. PIERCE,G. B., JR., and NAKANE,P. K. (1967). Antigens of epithelial basement membranes of mouse, rat and man. A study utilizing enzyme-labeled antibody. Lab. hue&. 1’7, 499-514. l-38. PIERCE,G. B., and NAKANE,P. K. (1969). Basement membranes. Synthesis and deposition in response KINGSBURY,F. B., CLARK, C. P., WILLIAMS,G., and to cellular injury. Lab. Znuest. 21, 27-41. POST, A. L. (1926). The rapid determination of albumin in urine. J. Lab. Clin. Med. 11, 981. PIERCE,G. B. (1970). Epithelial basement membrane: MERCIER-PAROT,L., DAVID, G., and TUCHMANN- Origin, development and role in disease. In “Chemistry and Molecular Biology of the Intercellular DUPLESSIS,H. (1963). Action teratogene d’heteroMatrix” (Endre A. Balazs, ed.), pp. 471-506, Acaanticorps tissulaires. II. Etude de l’action teratogene chez la souris de serums anti-rein. C.R. Sot. demic Press, London. Biol. 157, 974. PRESSMAN,D., and KORNGOLD,L. (1957). Localizing MICIGLEY,A., and PIERCE,G. (1963). Immunohistoproperties of antiplacenta serum. J. Zmmunol. 78, chemical analysis of basement membranes of the 75-78. mouse. Amer. J. Pathol. 43, 929-934. ROBERTSON, T. A., ARCHER,J. M., PAPADIMITRIOU, J. MIKHAILOV,V. M. (1967). Pathogenic action of neM., and WALTERS,M. N-I. (1971). Transport of phrocytotoxic serum on embryonic development of horseradish peroxidase in the murine placenta. J. albino rats. Biull. Eksp. Biol. Med. 63, 97-100. Patho2. 103, 141-147. MRENOVA,M., and ALBRECHT,P. (1966). Stabilization SEEGAL,B. C., and LOEB, E. N. (1940). Effect of of fluorescence in preparates treated by the fluoresanti-placenta serum on development of the fetus in cent antibody technique. Nature (London) 212, the pregnant rat. Proc. Sot. Exp. Biol. Med. 45, 1256-1257. 248-253. NEW D.A.T., and BRENT,R. L. (1972). Effect of yolk- SLOTNICK,V., and BRENT,R. L. (1966). The production of congenital malformations using tissue antisac antibody on rat embryos grown in culture. J. Emhryol. Exp. Morphol. 27,543-553. sera. V. Fluorescent localization of teratogenic antisera in the maternal and fetal tissue of the rat. PADYKULA,H., DEREN,J. J., and WILSON,T. H. (1966). J. Immunol. 95, 606-610. Development of structure and function in the mammalian yolk sac. I. Development morphology WISLOCKI,G. B., and DEMPSEY,E. W. (1955). Electron microscopy of the placenta of the rat. Anat. Rec. and vitamin B,, uptake of the rat yolk sac. Deuelop. 123, 33-63. Biol. 12, 311-348. PIERCE, G. B., MIDGLEY, A. R., SRI RAM, J., and