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EFFECTS OF A TERATOGEN ON
MARTEN MURRAY KERNIS J
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
To my parents for their unparalleled encouragement and
concern and to my wife for her generous support, this dissertation
is lovingly dedicated.
The author takes this opportunity to express his genuine
appreciation to Dr. E. Marshall Johnson for his unfailing interest,
guidance and friendship
to Dr. Stanley Kaplan for his advice concerning
to the Faculty of the Department of Anatomical
Sciences for opening new horizons
S. and to the National Institutes of Health Training
Grant GM 00579 for financial support.
TABLE OF CONTENTS
ACK 0 O, D IT S. . ... iii
LIST OF TABLES . . . v
LIST OF FIGURES. . . .. vi
INTRODUCTION . . . 1
Morphology of the Rat's Yolk-Sac . .
Chemical Constitution of the Visceral Yolk-Sac 7
Function of the Visceral Yolk-Sac. . 9
Statement of the Problem . . 15
MATERIAL AND METHODS . . .. 18
Care and Breeding of Animals . . 18
Incidence of Gross Malformation at Term. . 18
Preparation of the Culture Medium for Ion Uptake in Vitro. 19
Preparation of Tissue for in Vitro Uptake of Radio-
isotopes . . . 21
Exposure of Tissues to Tagged Ions in Vitro. . 26
Ion Uptake in Vivo. . . 26
RESULTS. .. .. . 29
Teratogenic Action of Trypan Blue and of Niagara Blue 2B 29
Effects of Azo Dyes on the in Vitro Uptake of Labeled Ions 31
Effects of Trypan Blue on the in Vivo Uptake of -SO4--. 56
DISCUSSION . . . 65
Incidence of Gross Malformation. . 65
In Vitro Ion Uptak . . 69
In Vivo Uptake of5S4O--. . . 73
CONCLUSIONS. . . . 74
APPE.DICES . .. .. .. .. 76
APPE-2IX A Composition of Bouin's Fluid . 77
APPENDIX B Composition of Phosphate-Ringer Buffer .. 78
LITERATURE CITED . . 79
VITA . . . 4
LIST OF TABLES
1. INCID ECE OF MALFORMATION. . 30
2. DRY WEIGHT OF YOLK-SACS AND EMBRYOS. . 32
3. ORPTI OF 5Ca . . 38
4. ABSORPTION OF 35S0"-. . 44
5. ABSORPTION OF 22Na . ... 50
6. IN VIVO 35SO4-- INCORPORATION. . 57
7. INCIDENCE OF TRYPAN BLUE- AND OF NIAGARA BLUE 23-
INDUCED CONGENITAL MALFORMATION IN THE RAT 66
LIST OF FIGURES
1. Schematic drawing of a typical rat implantation site. 3
2. Schematic drawings demonstrating the microscopic
structure of the visceral yolk-sac and the
parietal yolk-sac . . 3
3. Embryo-in-yolk-sac preparations prior to the removal
of the chorio-allantoic placenta and uterine
muscle. . . 23
4. Embryo-in-yolk-sac preparations after the removal of the
chorio-allantoic placenta and uterine muscle. 25
5. Semi-logarithmic graph comparing mean tissue dry weights
with gestational age. . . 34
6. Bar graph depicting dye-treated tissue dry weights as
per cent of control. . . 36
7. Semi-logarithmic graph comparing mean tissue absorption
of Ca+ with gestational age. . 40
8. Semi-logarithmic graph comparing mean tissue 5Ca
specific activity with gestational age. 42
9. Semi-logarithmic graph comparing mean tissue absorption
of S04-- with gestational age . 46
10. Semi-logarithmic graph comparing mean tissue 35SO4
specific activity with gestational age. 49
11. Semi-logarithmic graph comparing mean tissue absorption
of -Na with gestational age . 52
12. Semi-logarithmic graph comparing mean tissue Na
specific activity with gestational age. 55
13. Autoradiographs of the functional zone of the chorio-
allantoic placenta. . . 60
14. Autoradiographs of villi from the visceral yolk-sac 62
15. Autoradiographs of embryonic mesenchyme in the
region of the notochord . 64
The rat embryo, unlike most mammals, undergoes the last two-
thirds of gestation and its true morphogenetic sequences enclosed
within the yolk-sac membrane (Fig. 1). The yolk-sac, which is of endo-
dermal origin, is composed of a proximal and a distal portion. The
proximal (visceral) membrane (Fig. 2A) is the more complex of the two
and separates the extraembryonic celom initially from the yolk-sac
cavity and, after rupture of the distal (parietal) component on day
14 of gestation, from the uterine lumen.
Although the function of the yolk-sac and its role in embryonic
and fetal differentiation are essentially unknown, studies of yolk-sac
physiology and morphology have indicated that the proximal yolk-sac
may function in the transfer of material to or from the developing
embryo and growing fetus. The present study was therefore designed to
first elucidate a possible absorptive function for the visceral yolk-sac
and second, to determine the effect of a potent teratogenic agent on
the concentrating ability of the yolk-sac.
Morphology of the Rat's Yolk-Sac
The parietal yolk-sac is not present as a complete surrounding
membrane throughout gestation (21 days), since it ruptures during day 141
1All days of gestation have been modified to correspond to that
described in Materials and Methods.
Fig. 1.-Schematic drawing
cap = chorio-allantoic placenta
cav = chorio-allantoic vessels
emb = embryo
ex = exocelom
ysc = yolk-sac cavity
vys = visceral yolk-sac
A = see legend for Figure 2A
of a typical rat implantation
see legend for Figure 2B
Fig. 2.-Schematic drawings demonstrating the microscopic
structure of the visceral yolk-sac and the parietal yolk-sac.
A.-The visceral yolk-sac. Abbreviations:
visceral basement membrane
sbm = serosal basement membrane
vv = vitelline vessel
fp = foot process
ac = apical canaliculi
ve = visceral endoderm
B.-The parietal yolk-sac. Abbreviations:
= parietal endoderm
= maternal labyrinth
rm = Reichert's membrane
gc = giant cell
of gestation resulting in loss of the antimesometrial portion. Remnants
of Reichert's membrane then recoil to the perimeter of the chorio-
allantoic placenta, thereby causing the yolk-sac cavity to become con-
fluent with the reforming uterine lumen (Wislocki and Padykula, 1953;
Padykula and Richardson, 1963).
Prior to its rupture, the distal yolk-sac consists of three
layers (Fig. 2B). The layer on the maternal side of the distal yolk-
sac is composed of the so-called giant cells or "central zone"
(Everett, 1935). These are large, spindle-shaped cells of unknown
derivation separated by intercommunicating spaces (the labyrinth),
through which flows maternal blood. This blood is completely replaced
every 20 minutes (Everett, 1935) contrary to previously published
reports by Sobotta (1903), Asai (1914) and Grosser (1927), who believed
it to be an immobile pool, gradually incorporated into the embryo. The
embryonic side of the distal yolk-sac consists of small, noncontiguous
parietal endoderm cells which seem capable of ameboid movement and
phagocytosis (Everett, 1935; Gerard, 1925). Between these two ill-
defined layers is an obvious basement (Reichert's) membrane which is
loosely connected to the innermost of the giant cells and has been
variously considered as an ectodermal cuticle (Duval, 1892), a proto-
plasmic membrane (Sobotta, 1903), a basement membrane (Grosser, 1927)
and a hyaline membrane (Mossman, 1937). More recent observations
(Wislocki and Padykula, 1953) have suggested that Reichert's membrane
is composed of fibers very similar to compact collagenous fibers. On
the basis of certain histochemical reactions, Reichert's membrane is
similar to the lens capsule and Descement's membrane in the cornea.
Ultrastructurally, Reichert's membrane is characterized by wavy
bundles of fibrils unlike other basement membranes. Wislocki and Dempsey
(1955) have suggested that the membrane is composed of a considerable
amount of ground substance which masks the usual visualization of col-
lagen with the electron microscope.
The visceral wall of the yolk-sac is composed of three cellular
layers separated by two basement membranes (Fig. 2A). The outer layer
is the visceral endoderm or vitelline epithelium consisting of columnar
cells over the mesometrial one-half and low cuboidal cells over the
antimesometrial hemisphere (Everett, 1935).
By day 12 of gestation, the free or apical surfaces of the
cells of the vitelline epithelium are evaginated into a microvillous
border. In the ensuing two days, the length and density of the micro-
villi increase and reach their maximum development at day 14. From
day 15 to term, they recede but do not disappear. Between the micro-
villi and projecting for variable distances into the apical cytoplasm
are a series of indentations (the apical canaliculi) which vary from
small pinocytotic invaginations to a series of interconnecting tubules.
These canaliculi are well-developed from day 12 until term, but, later
in gestation, they become increasingly dilated (Padykula, et al., 1966).
The microvilli are invested with a filamentous glycoprotein
coat (the glycocalyx; Bennett, 1963) which also seems to line all in-
vaginated canaliculi as well as the inner surfaces of some of the intra-
cellular vacuoles. Both colloidal gold (Luse, 1958) and ferritin
(Lambson, 1966) adhere to the glycocalyx on the surfaces of the cells
as well as within the canaliculi and vacuoles.
The lateral intercellular relationships also change during the
course of gestation. At days 12 and 13, the lateral cell membranes of
adjacent cells appear tightly sealed from the apical surface to about
two-thirds down the length of the cell where they are thrown into complex
folds and interdigitations. Near term, however, only the most apical
portions of the cells appear to be tightly bound together and below
this, large dilatations containing a finely granular material appear.
Only occasionally do the membranes of two adjacent cells meet to form
a desmosome. Excluding small foot processes which extend into the
underlying basement membrane, the basal cell membranes have no remarkable
specializations or sequential differentiation.
The middle cellular layer of the proximal yolk-sac, the mesoderm,
becomes vascularized by the irregular vitelline blood vessels beginning
on day 10. The inner cellular layer is a mesothelium composed of cells
unusually rich in endoplasmic reticulum (Wislocki and Dempsey, 1955).
Beneath the visceral endoderm is a narrow visceral basement
membrane which thickens as gestation continues. In contrast, the serosal
basement membrane, between the mesenchymal and mesothelial layers, is
stout and appears to be rich in collagen.
Villi begin to form in the visceral yolk-sac (Fig.1) during the
tenth day of gestation. The villi become taller and more branched as
gestation continues (Everett, 1935).
The proximal yolk-sac immediately surrounding the exit of the
chorio-allantoic blood vessels is covered by low cuboidal to squamous
cells. This area of the proximal yolk-sac is avascular.
The endodermal sinuses are invaginations of the yolk-sac cavity
into the chorio-allantoic placenta (Fig. 1). The placental surfaces
of the sinuses are lined by Reichert's membrane and distal endodermal
cells, both of which appear continuous with the parietal yolk-sac.
The opposite sides of the sinuses are lined by cuboidal cells which,
though continuous with the vitelline epithelium, differ in cytological
properties (Wislocki and Padykula, 1953). These cells are small and
cuboidal and contain no stores of glycogen. They demonstrate no brush
border, no well-defined basement membrane and rest on a stroma of
allantois rather than splanchnopleure.
Chemical Constitution of the Visceral Yolk-Sac
Wislocki and Padykula (1953) employed histochemical techniques to
demonstrate the presence of glycogen, glycoproteins, mucopolysaccharides
and lipids within the cytoplasm of the vitelline epithelium. These cells
exhibit alterations in chemical concentrations and composition with
increasing gestational age. For example, the visceral yolk-sac initiates
glycogen storage in both the vitelline epithelium and mesenchymal layer
by day 13. The stores increase through day 18, and then decrease to
Yolk-sacs explanted into a culture medium also have the ability
to store glycogen (Sorokin and Padykula, 1964), although in a different
temporal sequence. Under these conditions, glycogen accumulation begins
at day 13, reaches and maintains a maximum level at days 20 to 25 of
incubation and then declines. These results suggest two critical
aypotheses. First, the yolk-sac does not rely upon maternal or embryonic
influences to initiate or maintain glycogen storage. Second, the in
vivo decrease in glycogen concentration is apparently due to a stimulus
in the form of a decrease of substrate, change in the hormonal environ-
ment or other such environmental factor and is not the result of age or
senescence of the yolk-sac itself. Whether the glycogen is eventually
transferred to the embryo or is utilized as a substrate for yolk-sac
metabolism or is utilized elsewhere are questions which are, as yet,
Some of the enzyme constituents of the visceral yolk-sac also
have been characterized (Padykula, 1958). The enzymes studied were
succinic dehydrogenase, nonspecific esterases, acid phosphatase,
alkaline phosphatase and adenosine triphosphatase. In general, all
enzymatic activity was low at day 12, increased by day 14, reached a
peak on days 15 succinicc dehydrogenase), 16 (alkaline phosphatase) or
18 (adenosine triphosphatase) and thereafter declined. In addition,
enzymatic activity was localized over certain areas of the cell. For
example, alkaline phosphatase activity was most intense in the apical
cytoplasm and brush border, acid phosphatase and adenosine triphospha-
tase in the supranuclear cytoplasm and succinic dehydrogenase in the
General alterations in enzymatic activity during the ontogeny
of the yolk-sac recently have been demonstrated by Johnson and Spinuzzi
(1966). These investigators used electrophoresis to study the effects
of a teratogenic agent on the differentiation of various molecular
forms of enzymes in the yolk-sac. Other enzymes which have been
identified in the visceral yolk-sac of the rat are malate and lactate
dehydrogenases (Johnson and Spinuzzi, 1966), beta-glucuronidase (Bulmer,
1963; Beck et al., 1967) and ribonuclease and deoxyribonuclease (Beck
et al., 1967).
The visceral basement membrane of the proximal yolk-sac was
shown also to undergo a histochemical differentiation (Wislocki and
Padykula, 1953). At day 10 of gestation, the membrane is so thin that
it is barely perceptible with the light microscope but as gestation con-
tinues, it becomes increasingly thicker. At the height of its develop-
ment, it consists of reticular fibers, collagen and periodic acid-Schiff
(PAS)-positive ground substance. In these respects, the visceral base-
ment membrane is characteristic of other such membranes located through-
out the maternal and fetal tissues.
The serosal basement membrane, however, does not seem to demon-
strate the same characteristics as other basement membranes. Although
it becomes visible by day 14, it seems to degenerate by day 20, at which
time its outline becomes hazy. It is PAS-positive, though less intensely
so than the visceral basement membrane, and contains collagen, some
reticular fibers and a small amount of elastic tissue which is not histo-
chemically similar to elastic tissues located elsewhere (Wislocki and
Function of the Visceral Yolk-Sac
Prior to 1927, when Brunschwig proposed the idea that the
visceral yolk-sac is physiologically a placenta, it was customary to
consider the chorio-allantoic placenta as the primary organ for bringing
nourishment to and taking waste from the developing embryo and growing
fetus. Everett (1935) predicted that the proximal yolk-sac is at least
as functionally important as the chorio-allantoic placenta. Twelve
years later, Noer and Mossman (1947) suggested that due to its unique
morphology, the proximal yolk-sac functions in a substantially different
way than the chorio-allantoic placenta and is therefore, complementary,
rather than supplementary, to the chorio-allantoic placenta.
The possible functions of the visceral yolk-sac may be divided
into at least two broad categories. First, it may protect the embryo
from physical or chemical trauma and second, it may participate in the
transfer of material between mother and embryo.
The Visceral Yolk-Sac as an Organ
Brambell et al. (1951) published data indicating that homologous
gamma-globulin passed through the rabbit yolk-sac from the maternal side
to the fetal side, but that heterologous gamma-globulins did not. This
led to the question of whether or not the same phenomenon occurs in the
rat. Ferm et al. (1959) were able to study the distribution of homologous
and heterologous types of proteins in pregnant rats by combining them
with a diazotized dye. Both protein-dye complexes were found concentrated
in the vitelline epithelium at all tested stages of gestation, but neither
homologous nor heterologous proteins were found within the embryo itself.
There was, however, a distinct difference in the maternal distribution
such that the heterologous protein-dye complex was not distributed as
equally as the homologous protein-dye complex. This indicated that
there were indeed two different proteins, but that the yolk-sac seemed to
treat them as one. Although these investigators speculated that the
lack of color in the embryo and the apparent dense color in the yolk-
sac indicated that the yolk-sac was protecting the embryo from the
proteins, it is entirely possible that the yolk-sac contains the enzy-
matic machinery necessary to split the protein-dye complex. Under
these circumstances, a false representation of the situation could
easily have been achieved.
Two other studies have indicated that at least one of the
functions of the visceral wall of the yolk-sac is protection. Ferm
and Beaudoin (1960) demonstrated that yolk-sacs under in vitro con-
ditions accumulate and store both heterologous and homologous proteins
in the same manner as in vivo yolk-sacs. The conclusion was that the
yolk-sac has an intrinsic blockade mechanism useful to the embryo as a
Finally, using a known teratogenic agent, Wilson et al. (1959)
also concluded that the yolk-sac serves a protective function. Trypan
blue, an azo dye, is teratogenic in the rat before the end of the eighth
day of gestation. At the same time that the embryo becomes completely
enveloped by the visceral yolk-sac, the teratogenicity of trypan blue
is markedly reduced. When injected after day 8, the dye is absent from
the embryo proper and is absorbed and stored by the vitelline epithelium.
It was therefore suggested that the immobilization of trypan blue by
the yolk-sac protects the embryo from a teratogenic stimulus. The
efficiency of this proposed protective mechanism, however, is very low,
for even when the dye is administered to pregnant rats at day 9, the
incidences of both embryonic malformation and resorption are significantly
higher than the rate of spontaneous abnormality.
The Visceral Yolk-Sac as an Organ
On the basis of pure morphology and biochemistry, the most
likely and best documented function of the visceral yolk-sac is the
transfer of materials between mother and embryo. Anatomical studies
have indicated that the vitelline epithelium is composed of cells hav-
ing characteristics similar to the absorbing cells of the intestinal
villus and the active cells of the proximal convoluted tubule of the
kidney, e.g., dense microvillus border, apical canaliculi, glycocalyx.
In addition, the villous region of the visceral yolk-sac is remarkably
similar to the mucosa of the small intestine, having an absorbing
epithelium (the vitelline epithelium), a basement membrane (the visceral
basement membrane) and a vascularized lamina propria (the mesenchymal
layer containing vitelline blood vessels).
The closed vitelline circulatory pattern would also indicate the
possible importance of an absorptive function. The vitelline blood
vessels are conveyed between embryo and yolk-sac by the vitelline stalk
which is analogous to the umbilical cord carrying blood vessels to and
from the chorio-allantoic placenta. The vitelline artery is a branch
of the embryonic aorta while the vitelline vein empties into the umbilical
vein within the embryo. There is a dense capillary network in the
villous region of the yolk-sac, a less dense network in the antimeso-
metrial hemisphere and a totally avascular area at the hilus of the
The vitelline artery approaches the most mesometrial part of the
villous region, penetrates the mesothelial layer and serosal basement
membrane and divides into two main trunks, both of which are larger in
diameter than their parent vessel. These trunks then divide into many
branches which ramify throughout the mesenchymal layer. The probable
physiological significance of the larger branches from the main arterial
channel is to decrease the velocity of blood flow through the yolk-sac,
thereby increasing the time available for absorption (or secretion)
of materials (B3e, 1951).
One of the earliest attempts to characterize placental function
was in 1922, when Shimidzu injected 23 different dyes into pregnant
rats and mice for the purpose of ascertaining placental permeability.
Using the presence of color in fetal tissue as the criterion of perme-
ability, it was concluded that the permeability of the dyes paralleled
the colloidal state of their solution and their ability to diffuse
through a gel. Thus, the placenta would act as an unselective ultra-
filter, permitting the passage of small molecules while inhibiting the
passage of larger ones. This was followed by other experiments which
suggested that the proximal yolk-sac has the ability to absorb and
transfer iron (Brunschwig, 1927; Everett, 1935) and fats (Everett, 1935;
Koren and Shafrir, 1964).
In an attempt to correlate structure with function, Padykula
and Wilson (1960) suggested that with an apparent ultrastructural
degeneration of the yolk-sac at day 15, there was a steady decrease in
the ability of the visceral endoderm to absorb radioactive vitamin B 12-
intrinsic factor complex. Even though there was a fifty-fold increase
in yolk-sac weight between day 12 and term, there was only a five-fold
increase in absorptive capacity. In addition, Jollie (1964) noted that
the visceral endoderm was not labeled with tritiated thymidine between
days 16 and 20, and that this might indicate a process of aging.
Alternatively, however, the previously noted sharp rise in the
activity of certain enzymes at day 14 (Padykula, 1958) could indicate a
greater functional capacity when the yolk-sac becomes exposed to the
uterine lumen. Brambell and his colleagues have been able to show that
the yolk-sac of the rabbit is capable of absorbing and transferring
antibodies to the fetus at a late stage in gestation (Brambell et al.,
1951; Brambell, 1958). Halliday (1955) continued the work using rats
and found antibody absorption by the proximal yolk-sac at day 17.
Finally, Brambell and Halliday (1956), by lighting the vitelline vessels,
demonstrated that the vitelline epithelium and its underlying vascular
system were partly responsible for antibodies penetrating the embryo.
In addition, Deren et al. (1966a) noted that the rabbit yolk-
sac has the ability to concentrate vitamin B12 and further demonstrated
that the rabbit yolk-sac develops an active transport system for certain
amino acids at day 20 of the 32-day gestation period (Deren et al., 1966b).
Lambson (1966) was able to infer the transfer of ferritin across the
proximal yolk-sac of the rat from electron micrographs and Luse (1958),
using other particulate matter, suggested that the rabbit yolk-sac
absorbs those particles by pinocytosis.
It appears therefore that the visceral yolk-sac of the rat is a
dynamic organ having the ability to pursue certain functions which are
undoubtedly vital to normal embryonic development and fetal growth.
Statement of the Problem
From both morphological and biochemical evidence, it would seem
that the yolk-sac participates in an absorptive function such that
nutrients or other molecules enter the vitelline epithelium and pass
through the epithelial cytoplasm, the visceral basement membrane, the
basement membrane of the endothelium lining the vitelline capillaries,
the endothelium itself and, finally, enter the vitelline circulation to
be circulated throughout the embryo. It is unlikely, though as yet
unproven, that material would penetrate the total width of the yolk-sac
and pass to the embryo by simple diffusion across the exocelom, amnion
and amniotic fluid (Wislocki, 1921).
Proceeding on the hypothesis that the yolk-sac does indeed have
an absorptive function, it became of interest to test first, the
ability of the yolk-sac to absorb various radioactively-labeled ions
and second, the effect, if any, of a potent teratogenic agent on the
concentrating ability of the yolk-sac.
Trypan blue was chosen as the teratogenic agent because (1) it
is accumulated in the vitelline epithelium, but not in the chorionic
villi (Everett, 1935), and it has never been found to penetrate into
embryonic rat tissue per se; (2) its administration to pregnant rats
results in a high incidence of severely congenitally malformed fetuses
and (3) it may be used as a possible model system for analyzing the
complex interrelationships between the genome of the developing embryo
and its microenvironment.
Goldmann (1909) was one of the first to note that when a
pregnant rat was vitally stained with trypan blue, the dye was apparently
concentrated in the proximal endoderm of the yolk-sac (Everett, 1935).
He also suggested that with the initiation of pregnancy, the dye is
released by the maternal reticulo-endothelial system and liver. Follow-
ing this release it is free to circulate in the blood vascular system
(Wislocki, 1921). Zaretsky (1910), the first to use trypan blue in
avian embryos, also noted that the yolk-sac of a developing chicken has
the ability to absorb the dye and, furthermore, has the ability to pre-
vent the dye from penetrating the embryonic tissue proper (Hanan, 1927).
Apparently unaware that trypan blue concentrates in the yolk-sac
of the rat, Gillman et al. (1948) injected the dye into pregnant rats
under the hypothesis that particulate matter or abnormal proteins in
the maternal circulatory system (since trypan blue is adsorbed to serum
albumin, Rawson, 1943) could play a role in the production of congenital
malformations. Indeed, their results indicated that the dye was terato-
genic when administered during pregnancy and that the central nervous
system was usually the most severely affected system of the embryo.
Other investigators who have pursued the matter further have
confirmed the teratogenicity of trypan blue in the rat (Hogan, et al.,
1950), mouse (Hamburgh, 1952), rabbit (Harm, 1954) and chicken (Beaudoin
and Wilson, 1958). Wilson (1955) noted that the most susceptible period
for trypan blue-induced teratogenesis in the rat is on days 7, 8 and
9, the period during which the central nervous system is undergoing its
critical period of differentiation and development.
By correlating the distribution of trypan blue and the known
effect of the dye in producing abnormal young, Lloyd and Beck (1966)
and Beck et al. (1967) have suggested that trypan blue acts by inhib-
iting the passage of vital substances across the yolk-sac. The sup-
position that the yolk-sac is the site of action of trypan blue is a
reasonable one based on the generality that a teratogen will act on
only one or a combination of three possible locations: the embryo, the
mother, or the organ intervening between the two. The embryo seems to
have been eliminated as a possible site of action since no dye apparently
penetrates into its substance. The mother seems also to have been
eliminated since trypan blue is highly teratogenic when injected into
the yolk of a developing chicken embryo.
The present experiments were therefore initiated to determine
if trypan blue under both in vitro and in vivo circumstances does in-
deed have an effect on the absorptive ability of the yolk-sac. To
this end, three pertinent questions were asked:
1. Are the yolk-sacs from normally developing rat embryos
capable of absorbing ions?
2. Is there a difference between the amount of material that
can be taken up by normal control and trypan blue-treated yolk-sacs?
3. Does a nonteratogenic azo dye, Niagara blue 2B (Beaudoin
and Pickering, 1960), which also localizes in the visceral endoderm,
but is excreted more rapidly (Lloyd and Beck, 1966), have an effect on
MATERIALS AND METHODS
Care and Breeding of Animals
Virgin, black-hooded female rats of the Long-Evans strain,
weighing between 60 and 100 g, were obtained from Research Animals,
Inc.2 The animals were housed in wire-bottomed cages in a windowless,
well-ventilated room with an alternating 12-hour light-dark cycle. All
animals were fed a diet consisting of stock laboratory chow3 and tap
water ad libitum. The ration was supplemented twice weekly with lettuce
and once weekly with canned horse meat.
Late every afternoon, a smear of the vaginal contents of each
female weighing between 180 and 240 g and 80 to 120 days of age was
examined by light microscopy to detect those animals in proestrus (Long
and Evans, 1922). Each proestrus female was caged overnight with a
mature male of the same strain. The presence of sperm in a vaginal
smear at 10:00 AM the following morning was considered as day 0 of
Incidence of Gross Malformation at Term
Twenty-two pregnant rats were given a single, subcutaneous
injection of 1.8 per cent aqueous trypan blue4 at a dosage of 1 mg/6 g
3purina Rat Chow, The Ralston-Purina Co., St. Louis, Missouri.
4Specially purified and donated through the courtesy of Mr.
Floyd Greene of the Matheson, Coleman and Bell Division of the Matheson
Co., Inc., Norwood, Ohio.
maternal body weight (167 mg/kg) at 10:00 AM on day 8 of gestation.
Another group of 12 pregnant rats was similarly injected with Niagara
blue 2B5'6 and a third group of 12 females was untreated.
At day 20 of gestation (1 day before parturition) all females
were anesthetized with ether and killed by cervical dislocation. The
intact uterus was removed, opened along the antimesometrial border and
the fetuses were dissected free of their associated membranes. All
living fetuses were examined for gross external malformations and placed
in Bouin's fluid7 for later freehand sectioning (Wilson, 1965) and the
identification of any gross internal malformations.
Preparation of the Culture Medium for
Ion Uptake in Vitro
The culture medium was designed for use in manometric studies
of oxygen consumption by normally and abnormally developing rat embryos
(Netzloff et al., 1968). It consisted of bovine serum,8 chicken embryo
extract ultrafiltrate9 and a phosphate-Ringer buffer10 modified after
Kosan and Burton (1966) in a ratio of 3:1:1.
5Also named Benzo Blue 2B.
6Obtained from the Hartman-Leddon Co., Philadelphia, Pennsylvania.
7See Appendix A.
8Obtained from Microbiological Associates, Inc., Bethesda,
9Obtained from Microbiological Associates, Inc., Bethesda,
Maryland. Chicken embryo homogenized in an equal volume of Gey's bal-
anced salt solution with 100 units each of penicillin and streptomycin
added per ml before ultrafiltration.
10See Appendix B.
The serum and ultrafiltrate were both purchased as single lot
numbers in 100 ml bottles for the former and 20 ml bottles for the
latter and stored at -550 C prior to use. The buffer was mixed in
advance and stored at 50 C. On the day of an experiment, the culture
medium was freshly prepared and D-glucosell was added to a final con-
centration of 1 mg/ml. After the culture medium was warmed to a
temperature of 390 C, it was aerated for 2 minutes with air passed
through a water trap.
Twenty ul of a previously prepared stock solution of 45Ca++,12
35S04--3 or 22Na+14 were added to each milliliter of an aliquot of the
aerated culture medium. The final activities of ions were 0.210 uC/ml
for calcium, 0.072, uC/ml for sulfate and 0.019 uC/ml for sodium.
Enough labeled medium to completely submerse the tissue preparation was
placed into plastic, disposable 30 ml beakers and warmed to 380 400 C.
A second aliquot of the aerated culture medium was stored at 390 C in
small petri dishes.
110btained from the Fisher Scientific Co., Fair Lawn, New Jersey.
120btained from Nuclear-Chicago Corp., Des Plaines, Illinois.
Calcium-45 as calcium chloride in aqueous solution with a specific
activity of 8.73 uC/ug.
13Obtained from Nuclear-Chicago Corp., Des Plaines, Illinois.
Sulfur-35 as carrier-free sulfate in aqueous solution.
140btained from Nuclear-Chicago Corp., Des Plaines, Illinois.
Sodium-22 as sodium chloride in aqueous solution with a specific
activity of 9 uC/ug.
Preparation of Tissue for in Vitro Uptake
Thirty-four normal control, 34 trypan blue and 19 Niagara blue
2B injected pregnant females were stunned by a blow to the head at
10:00 AM of days 12, 13 and 14 of gestation. The animals were killed
by cervical dislocation, the uterus removed and placed in a petri dish
containing warmed, but unlabeled, medium. The number of implantation
sites and resorbing sites were noted and recorded.
One implantation site was separated from the remaining intact
uterus and transferred to another petri dish which contained warmed,
unlabeled medium. After opening the implantation site along the anti-
mesometrial wall, the decidua capsularis and parietal yolk-sac were
dissected free of the chorio-allantoic placenta and visceral yolk-sac.
A silk ligature was tied around the umbilical vessels at the point
where they enter the chorio-allantoic placenta (Fig. 3). The placenta
and uterus were then separated from the ligated proximal yolk-sac which
remained as a complete and vascularized membrane surrounding the embryo
The proximal yolk-sac, vitelline vessels and embryo were ex-
amined under a dissecting microscope to be certain that (1) there was
no puncture wound in the proximal yolk-sac, (2) no vitelline vessels
were ruptured and (3) the embryo had a beating heart which perfused
the vitelline vessels with blood. The latter parameter was used as
the criterion for diagnosing embryonic viability in all stages of the
experiment. No more than 6 embryos from each pregnant female were
Cd r-I '
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Exposure of Tissues to Tapged Ions in Vitro
The embryo-in-yolk-sac preparation was transferred to incubation
medium containing one of the radioactively labeled ions. All manipu-
lations of the preparations were done by using the free ends of the
ligature and care was taken to avoid brushing the preparation against
the walls of any of the vessels.
After 60 minutes of incubation, the preparations were removed
from the tracer-bearing medium, examined for the presence of a heart
beat and rinsed several times in 0.9 per cent saline. The proximal
yolk-sacs were separated from the embryos and each was rinsed 5 times
in saline. The tissues were transferred to individual 10 x 75 mm
test tubes containing 0.25 ml of concentrated nitric acid and dissolved
over a low flame. The resulting solution was placed on a tared, stain-
less steel, ringed planchet, dried for 24 hours at 700 C and for another
24 hours at 1300 C. The planchets were weighed and then counted for
radioactivity with a well-shielded, halogen-quenched Geiger-Muller tube.
Corrections were made for the decay rates of the isotopes and the results
were expressed as counts per minute per preparation (cpm/prep) and counts
per minute per mg dry weight (cpm/mg).
Ion Uptake in Vivo
To determine whether any differences in the uptake of radio-
actively-labeled ions between normally and abnormally developing embryos
could be detected under in vivo conditions, autoradiographic procedures
For this purpose, 8 pregnant rats were divided into 2 groups
of 4 each. One group was left untreated, while each rat in the other
group was given a single, subcutaneous injection of 1 mg trypan blue/
6 g maternal body weight on day 8 of gestation. At 10:00 AM of day 13,
each animal was injected intraperitoneally with 10 uC of carrier-free
35S04--/g body weight (Kochhar and Johnson, 1965). Two animals, one
from each group, were killed by cervical dislocation at 15 minutes, 30
minutes, 1 hour and 3 hours. The implantation sites were removed and
fixed in alcohol-formalin, dehydrated through increasing concentrations
of ethyl alcohol, cleared in terpineol and embedded in paraffin. The
tissue block was then serially sectioned at 5 u and placed on pre-
treated15 1" x 3" glass slides.
Autoradiography was done by a dipping method (Messier and
Leblond, 1957) in a completely light-proof darkroom under a No. 2
Wratten safelight. Eastman Kodak NTB 3 emulsion gel was placed in a
dipping container and warmed to a temperature of 400 450 C in a water
bath. After the emulsion had liquefied (1 hour), the slides, held by
the label end, were individually dipped, once each, for 1 2 seconds.
The slides were removed from the emulsion and their backs wiped dry.
They were then placed on a slide rack in a horizontal position and
allowed to dry at room temperature for 1 hour. The slides were inserted
into black, plastic slide boxes containing granular calcium chloride or
silica gel as drying agents and separated from one another by plain
15The glass slides were treated to provide for better adhesion
between the photographic emulsion and tissue sections. After being
soaked in dichromate solution for several hours, they were rinsed in
tap water, dipped in acetone and air dried. They were then irmersed
in a warm solution of 0.5 per cent gelatin and 0.05 per cent chrom-
alum in distilled water and dried at room temperature in a covered
staining dish (Boyd, 1955).
glass slides. The boxes were sealed with black tape and placed in a
vertical position in a dry atmosphere so that the emulsion sides
After 4 days of exposure, the slides were developed in the
darkroom in the following manner:
Kodak developer (type D 19) 5 min
Kodak stop bath (type SB 5a) 15 sec
Kodak acid fixer 10 min
Water rinse 15 min
They were immediately stained with hematoxylin (4 min) and eosin (12
sec), dehydrated through a graded series of ethyl alcohols, cleared in
xylene and permanently mounted in HSR16 mounting medium. The sections
were examined by light microscopy and comparisons of the number of
developed granules were made.
160btained from the Hartman-Leddon Co., Philadelphia,
Teratogenic Action of Trynan Blue
and of Niagara Blue 2B
The administration of a single, subcutaneous injection of trypan
blue at a dosage of 167 mg/kg maternal body weight on the eighth day of
pregnancy in the rat results in a high incidence of congenitally mal-
formed fetuses (Table 1). The same treatment with Niagara blue 2B in-
duces a much lower rate of malformation which is nonetheless, signifi-
cantly greater than the spontaneous incidence of malformation seen in
normal control animals. From 12 control animals sacrified 1 day before
parturition, 99 per cent of the fetuses were living and structurally
normal, while 1 per cent had been resorbed. The presence of trypan blue
resulted in a resorption rate of 46 per cent and a malformation rate of
62 per cent among the living fetuses. In many cases, the young bore
multiple congenital abnormalities of varying severity. The most common
site of malformation was the central nervous system and included defects
such as anencephaly, exencephaly, meningocele, meningomyelocele, anoph-
thalmia, microphthalmia and occasionally, hydrocephaly and spina bifida.
Micrognathia, microstomia, cleft palate, situs inversus and kinky tail
were only infrequently encountered.
The injection of Niagara blue 2B resulted in a ? per cent
resorption rate and a gross malformation rate of 2 per cent in the
living young. These defects were also of the central nervous system
and consisted of anophthalmia and hydrocephaly.
S-' 0o Z- 0 o
S- H- ,.0
0 *C 0
-= 0 0N 0
-li 4-' En
0- $L ; I
A4 ri C)(
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Effects of Azo Dyes on the in Vitro
Uptake of Labeled Ions
Dry Weights of Yolk-Sacs and Embryos
For the purpose of ascertaining the effects of trypan blue and
Niagara blue 2B on the general growth of yolk-sacs and embryos, the
dry weights of the tissues were compared (Table 2, Figs 5 and 6).
Table 2 shows the mean dry weights and standard errors in milligrans
for control and experimental yolk-sacs and embryos at days 12, 13 and
14 of gestation. On a semi-logarithmic graph (Fig. 5) of the dry weights,
the curves approximate the straight lines indicative of growth curves
in general. At days 12 and 13, yolk-sacs of the control preparations
weigh significantly more than the corresponding trypan blue-treated
group, while they do not statistically differ from those treated with
Niagara blue 2B. By day 14, there is an apparent recovery of yolk-sac
weights as there are no differences among the control and experimental
values. However, in the case of embryonic dry weights, day 13 control
embryos weighed more than the trypan blue-treated, while an increase
in embryonic weight at day 14 was noted in the Niagara blue 2B group.
Figure 6, which expresses experimental yolk-sac and embryonic
dry weights in terms of percentages of control values demonstrates the
apparent recovery of yolk-sac weight by day 14. Although both groups
of treated yolk-sacs approach control values, the trypan blue-treated
group demonstrates a greater rate of recovery. No such recovery
phenomenon can be described for embryonic dry weights.
v-I '-I --
0 CO 00
0 0 0
+1 +1 +1
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o 0 0
+1 +1 +1
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OD V"\r- \0
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o 0 0
+1 +1 +1
o *N O
v-I v-i r-I
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v-I 0 v-I
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c c- c
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<-4 vr-I i-I
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4 43-' C<
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6 CM~ w,
.-l C) 4->
r At ^ -i
**- -'a Cr
-I tt> $4
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Fig. 5.-Semi-logarithmic graph comparing mean tissue
dry weights with gestational age. Vertical bars represent
standard errors and are only present when a significant dif-
ference (p <0.05) exists. See Table 2 for numerical data.
0 O CONTROL
............... TRYPAN BLUE
0---0 NIAGARA BLUE 2B
DAY OF GESTATION
U0 *0 r
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The Untake of 45Ca
Table 3 indicates the uptake of 45Ca++ by control and trypan
blue-treated yolk-sacs and embryos on days 12, 13 and 14 of gestation.
In a graphic comparison between control and treated tissue (Fig. 7),
no significant differences in ion uptake on a preparation basis can be
detected. However, the manner in which these curves approach a
straight line and their slopes would tend to indicate that the rate
at which the label is taken up by the individual preparation is a
function of its dry weight.
A more realistic and possibly more accurate representation of
the amount of 45Ca++ absorbed is shown in Figure 8. The logarithm of
the specific activity of 4Ca (cpm/mg dry weight) is plotted against
the day of gestation. At day 13, control yolk-sacs demonstrate a de-
creased capacity to absorb 4Ca when compared to day 12, while the
specific activity is shown to undergo a rapid increase by day 14.
Trypan blue-treated yolk-sacs also show the same variation in specific
activity with gestational age, but the changes from day to day are con-
siderably less than control. More interesting, however, is that the
experimental yolk-sacs have a significantly greater specific activity
than the corresponding controls at day 13, while by day 14, the controls
are able to absorb more Ca than the treated group.
No such pronounced changes in the specific activities of the
control and experimental embryos were observed. Although the day 13
and 14 experimental embryos bear the same relationship to the controls
as in the cases of the corresponding yolk-sacs, no statistically sig-
nificant differences were noted. Furthermore, a comparison between the
/ 0 Eo 0
I > I
Fig. ?. -Semi-logarithmic graph comparing mean tissue
absorption of 5Ca+ with gestational age. See Table 3 for
YOLK SAC 0
S- o CONTROL
S.............* TRYPAN BLUE
DAY OF GESTATION
Fig. 8.-Semi-logarithmic graph comparing mean tissue
45Ca++ specific activity with gestational age. Vertical bars
represent standard errors and are only present when a signifi-
cant difference (p4 0.05) exists. See Table 3 for numerical
0- 0 CONTROL
S.............. TRYPAN BLUE
DAY OF GESTATION
i I i
uptakes of yolk-sacs and embryos in terms of both the preparation and
the dry weight would indicate that a very small proportion of the
45Ca+- that is available penetrates into the embryonic tissue proper.
The UDtake of 35so --
The means and their standard errors for the absorption of
35s04-- by control and dye-treated tissues are shown in Table 4. Both
yolk-sacs and embryos from the control and the two experimental groups
demonstrate a progressive increase in labeling with gestational age
and, therefore, with increasing tissue weight (Fig. 9). The slopes of
the lines indicate that the rate of increase by the yolk-sacs between
days 13 and 14 is less than the rate between days 12 and 13. Only
those embryos from Niagara blue 23-treated females show the correspond-
ing change in slope. Furthermore, the actual amounts, i.e., cpm, of
labeled 35S04-- measured in yolk-sac and embryonic tissues are nearly
A statistical comparison (Student's t-test) between the control
and experimental groups at each day of gestation indicates that the
day 12 Niagara blue 2B-treated yolk-sacs absorb a significantly
(p<0.05) greater amount of 35SO -- than the controls. Compared with
the control, trypan blue treatment results in a significantly greater
amount of the tagged ion being absorbed by the yolk-sacs at day 13.
Yolk-sacs laden with Niagara blue 2B show no difference in their ability
to accumulate 35S-- when compared with control or with trypan blue
treatment, while day 14 yolk-sacs absorb similar amounts of label regard-
less of treatment.
4 -- 0 0 'O __ -
C2t C). N Co C0 cd
-+-4 d c+
On Co-- C Q \04 '- ..O'-d
en '-' +1
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aI p .0
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-0 0 N "0 ON
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o c'- c-' oo -' o.
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0 C. cn
lq aa z
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4 0 -1 C C C CO .
Spq 0 C N P
S-a' -a- -C -Cd -Cd 4-
S+ +. + + + +
S' C N O
V1 (.% r-F r-4 4 C 0 0
C E-4 CM
C- 10 CM 0 ,EC*
o -d -8 ( E-:
+ +r +1 + + +N +i> '
S CO ON N C"-NJ -CN \ON 0C 0
V 0) 0 U)
l la l( -'
;-4 ,o- 4-. C)
N8 O> 0, r o
^ ^ ^ q ^ -a (
Fig. 9 -Semi-logarithmic graph comparing mean tissue
absorption of 3504-- with gestational age. Vertical bars repre-
sent standard errors and are only present when a significant dif-
ference (p 40.05) exists. See Table 4 for numerical data.
0- 0 -- CONTROL
* ............ 0 TRYPAN BLUE
*---9 NIAGARA BLUE 2B
O *' .
0- 0 CONTROL
* ............ 0 TRYPAN BLUE
S---o NIAGARA BLUE 2B
I I I
DAY OF GESTATION
Embryonic tissue also demonstrates no differences in its
ability to accumulate the label at day 14. However, at both days 12
and 13, Niagara blue 2B treatment results in a significantly greater
uptake than trypan blue treatment. The control values lie in between
the experimental values and are not significantly different from them.
By comparing the specific activities of 35S04-- (Fig. 10), it
can be seen that the trypan blue-treated yolk-sacs incorporate signifi-
cantly greater amounts of label than the corresponding controls. Yolk-
sacs laden with Niagara blue 2B accumulate more of the ion at day 12
only. By day 13, they apparently begin to recover and this recovery
is maintained to day 14. In all cases, yolk-sacs on day 12 exhibit a
relatively low specific activity of 35S04-- which increases greatly on
day 13 and levels off by day 14.
No significant differences in specific activities between con-
trol and experimental embryos at any stage of gestation were noted.
With the exception of the decrease in specific activity by Niagara blue
2B-treated embryos between days 13 and 14, all systems increased their
uptake with gestational age.
The Uptake of 22Na
Table 5 summarizes the actual amounts and specific activities
of 22Na absorbed by control and experimental yolk-sacs and embryos on
days 12, 13 and 14 of gestation. Figure 11 is a semi-logarithmic graph
showing the amount of 22Na+ absorbed per preparation as a function of
gestational age. The yolk-sacs of all groups appear to be capable of
absorbing more tagged ion with increased age and weight. At day 12, the
Fig. 10.-Semi-logarithmic graph comparing mean tissue
35S0 -- specific activity with gestational age. Vertical bars
represent standard errors and are only present when a significant
difference (p< 0.05) exists. See Table 4 for numerical data.
16 ......... ....................
141 -1 1
/ YOLK SAC
ee 0.. B
S....... TRYPAN BLUE
**---- NIAGARA BLUE 2B
12 13 14
DAY OF GESTATION
\O0 N CQOi
T-4 ^- .- -^ ^
+ NG N,
CMN NN M
O\-~ CM >-
Fig. 1. -Semi-logarithmic graph comparing mean tissue
absorption of a with gestational age. Vertical bars repre-
sent standard errors and are only present when a significant
difference (p4I0.05) exists. See Table 5 for numerical data.
S.........e.... TRYPAN BLUE
9---0 NIAGARA BLUE 2B
DAY OF GESTATION
YOLK SAC -
0- 0 CONTROL
S............. TRYPAN BLUE
S.--- ** NIAGARA BLUE 2B
i T -
yolk-sacs stained with Niagara blue 23 accumulate a significantly
greater amount of label than the controls, but then seem to recover
such that there are no differences at days 13 and 14. The presence of
trypan blue in the vitelline epithelium apparently results in a sig-
nificant increase in incorporation over control levels only at day
14. Prior to that time, trypan blue-treated and control values are
not different and no significant differences between yolk-sacs treated
with trypan blue or Niagara blue 2B are apparent.
The embryos also seem to increase their uptake of 22Na+ with
gestational age, though the rate of increase between days 12 and 13
is less than between days 13 and 14. Day 12 embryos from Niagara
blue 2B-treated females absorb a greater amount of label than the
controls, while control values at all other stages are not different
from either of the experimental groups. However, at day 14, Niagara
blue 2B-treated embryos absorb a significantly greater (p<0.05)
amount of label than those embryos exposed to trypan blue.
For each group tested, the specific activities of yolk-sacs
and embryos seem to parallel one another at each day of gestation
(Fig. 12). Both trypan blue- and Niagara blue 2B-treated yolk-sacs
have significantly greater 22Na+ specific activities than control
yolk-sacs at days 12 and 14, but are not different from one another.
At day 13, no differences between any of the groups are apparent.
The specific activities of the embryos are similar and the only statist-
ically significant differences which occur are on day 13, when the
Niagara blue 2B treatment results in a greater specific activity than
Fig. 12.-Semi-logarithmic graph comparing mean tissue
22Na+ specific activity with gestational age. Vertical bars
represent standard errors and are only present when a signifi-
cant difference (p< 0.05) exists. See Table 5 for numerical
0- 0 CONTROL
* *..........**..* TRYPAN BLUE
*---* NIAGARA BLUE 2B
DAY OF GESTATION
Effects of Frr'in Blue on the in Vivo
Uptake of 35S0O--
Autoradiographs were prepared to determine if the in vitro
changes in 35S04-- uptake reflect similar in vivo changes or if they
merely result from the artificial conditions employed. Table 6 is a
summary of the mean grain counts over the chorio-allantoic placenta,
visceral yolk-sac and embryo at 1 and 3 hours after injecting pregnant
females on day 13 of gestation with the isotope. The number of counts
over all tissues studied soon after injection (15 and 30 minutes) were
not significantly greater than background and are not reported.
All grain counts were taken in the following manner. A grid
containing 49 squares was placed in a 10X wide-field ocular. Under
oil immersion, the number of developed grains were counted over
several fields from several sections taken from 2 embryos from each
pregnant female. Only those grains in the 13 squares forming an X
in the center of the grid were counted. An exception was the yolk-sac
villi where the whole villus was counted. The means for the chorio-
allantoic placenta were calculated on the basis of the grain counts
over 3 fields each of the metrial gland, decidua basalis and junctional
zone. Background (the mean of 4 fields counted in the emulsion around
each section studied) was subtracted from each count and the sum of the
counts was divided by the number of fields. The mean grain count for
the visceral yolk-sac was calculated on the basis of fields over 8
villi and 2 fields over the nonvillous region, while the mean number
of counts over the embryo included fields counted over the neural tube,
limb bud, notochord and loose mesenchyme.
IN VIVO 35S04-- INCORPORATION
Control Trypan Blue Per Cent
Tissue Grain Count Grain Count of Control
Placenta 70 196 280
Visceral Yolk-Sac 30 51 170
Embryo 61 193 316
Placenta 433 361 88
Visceral Yolk-Sac 178 189 106
Embryo 202 273 135
aThe mean counts presented in
for background. See text for further
this table have
By 1 hour after injection of the label, the trypan blue-
treated tissue had absorbed 170 to 316 per cent more 35so04~ than the
control, with the largest percentage of increase being found in the
amount reaching the embryo. After 3 hours, the ratio of counts
between trypan blue-treated and control tissues had decreased so that
the number of grains over the control and experimental placentae
(Fig. 13) and yolk-sacs (Fig. 14) was essentially identical. The
abnormally developing embryos (Fig. 15), however, seem to absorb more
than the controls. Although the data indicate that trypan blue
treatment results in a greater uptake of 35S04--, the significance of
these increases is not assured until information from the progeny of
more than 1 pregnant female is included.
Fig. 13.-Autoradiographs of the junctional zone of the
chorio-allantoic placenta. Stained with hematoxylin and eosin.
A. Normal control, 1 hour after injection of label.
B. Trypan blue-treated, 1 hour after injection of label.
C. Normal control, 3 hours after injection of label.
D. Trypan blue-treated, 3 hours after injection of label.
Fig. 14.-Autoradiographs of villi from the visceral
yolk-sac. Stained with hematoxylin and eosin. 800X.
A. Normal control, 1 hour after injection of label.
B. Trypan blue-treated, 1 hour after injection of label.
C. Normal control, 3 hours after injection of label.
D. Trypan blue-treated, 3 hours after injection of label.
Fig. 15.-Autoradiographs of embryonic mesenchyme in
the region of the notochord. Stained with hematoxylin and
A. Normal control, 1 hour after injection of label.
B. Trypan blue-treated, 1 hour after injection of label.
C. Normal control, 3 hours after injection of label.
D. Trypan blue-treated, 3 hours after injection of label.
^<^7 ,^ ^
L^ '. ^ .'
Incidence of Gross Malformation
A review of the literature concerning experiments with trypan
blue and Niagara blue 2B-induced congenital malformations in rats
reveals much variation from investigation to investigation in the
incidence of malformation. In general, this variation depends upon
the strain of rats studied; the time, dose and route of administration
and, probably of even greater consequence, the purity of the dye.
Under the conditions of the experiments presented in this dissertation
(see Table 7 and Materials and Methods), a 49 64 per cent incidence
of malformation in surviving fetuses was noted. Each of these fetuses
exhibited at least one abnormality resulting from treatment with trypan
blue. The dye also caused a high rate of embryonic death as indicated
by the percentage of resorbing implantation sites.
On the other hand, Niagara blue 2B, an azo dye of quite similar
molecular structure and colloidal property (Wilson et al., 1959), appears
to be a relatively impotent teratogen; treatment with it resulting in a
low malformation rate and a resorption rate of less than one-half that
of trypan blue. Although Niagara blue 2B is absorbed by the vitelline
epithelium and maternal reticulo-endothelial system to much the same
extent as trypan blue (Wilson, et al., 1959), it does differ in other
physiological properties. For example, Niagara blue 2B is more toxic to
the mother even though it apparently has a shorter serum half-life (Lloyd
and Beck, 1966).
ONl -4- -Z- <1
-=t VN 1 N0
in 0 +
0 0 C
O v \ O -c
N- o (i .}
r-1 Y-l -
co co c
SON ON C'2
-a- 1 r-i C'
c) H O
1-4 SO M C
The results of the experiments reported in this dissertation
(Table 1) confirm the previously reported investigations with respect
to incidence of malformation and resorption. In addition, these data
indicate that at least 3 out of 5 of the trypan blue-treated preparations
incubated in vitro for the ion uptake studies were destined to have at
least one abnormality, while about 1 out of 50 Niagara blue 2B-treated
embryos would be malformed.
The observation that the primary site of malformation was the
central nervous system is not remarkable in the light of present
theories. Presumably, damage to a developing structure or system
occurs only at that time in differentiation which is critical to the
normal biochemical or morphological development of that system (Kalter
and Warkany, 1959). Consequently, the central nervous system, including
the special sense organs, which is undergoing its biochemical and morph-
ological differentiation at day 8, is affected most severely by treatment
at that day. The appearance of abnormalities of other organ systems may
be explained by postulating residual effects of the dye which result in
alterations of structures developing during a later stage in gestation.
The fact that both trypan blue and Niagara blue 2B are present in the
maternal tissues and visceral yolk-sac for an extended period of time
would tend to support the concept of residual effectiveness.
But what exactly are these effects? Gillman et al. (1948)
showed that trypan blue was absorbed in apparently high quantities by
the phagocytes of the maternal reticulo-endothelial system. The fact
that some nonteratogenic azo dyes were not phagocytized by this system
(Wilson, 1955) caused Wilson et al. (1959) to study whether or not only
teratogens were taken up by the phagocytes. Instead, they found both
Niagara blue 2B and India ink to be actively absorbed by the maternal
reticulo-endothelial system. It was therefore concluded that terato-
genesis is not influenced by a loading of this system.
Gillman et al. (1948) also demonstrated that trypan blue is
bound to the albumin fraction of maternal plasma protein, while Beau-
doin and Kahkonen (1963) showed a decrease in total fetal protein con-
centration as well as decreases in beta globulin, alpha-1-globulin and
albumin concentrations at day 20, after previous maternal injection with
trypan blue. No such information is available from studies with Niagara
blue 2B-treated pregnant rats. Whether these changes are directly con-
cerned with teratogenesis, either as causes or effects, remains to be
As a matter of fact, it is still not clear whether the embryo,
the maternal organism or the visceral yolk-sac is the direct site for
trypan blue action. Since the dye has never been seen to penetrate the
rat embryo and since it is a potent teratogen in chicks which would in-
dicate that no maternal influence is involved, Beck et al. (1967) have
concluded that the most reasonable site of action for trypan blue in
the rat is the visceral yolk-sac. Indeed, under in vitro circumstances,
they have demonstrated that increasing concentrations of trypan blue
are able to inhibit the activities of certain enzymes isolated from the
lysosomes of near-term visceral yolk-sacs. Accordingly, they suggest
that the inhibition of those lysosomal enzymes, i.e., beta-glucuronidase,
acid phosphatase, ribonuclease and deoxyribonuclease, results in the
inability on the part of the visceral endoderm to digest absorbed
material. Any possible barrier to these large undigested molecules
would result in a lack of transfer of nutritional elements to the embryo.
Although this theory is quite attractive, judgment should be reserved
until it is substantially shown that (1) trypan blue does indeed pene-
trate into the lysosomes, (2) the enzyme inhibition occurs in vivo and
(3) such an inhibition also occurs in yolk-sacs from earlier stages in
gestation, particularly at that critical time in development when
trypan blue is most effective in producing malformations.
In Vitro Ion Uptake
Since the available evidence indicated that trypan blue might
have an effect on yolk-sac function, it became desirable to test this
hypothesis in terms of the organ's ability to absorb ions under in vitro
and in vivo conditions.
The explanted embryo-in-yolk-sac preparations utilized for
these experiments appear to sustain themselves quite well in the culture
medium. This was indicated by heart beat and yolk-sac perfusion. In
addition, Netzloff et al. (1968) demonstrated that these preparations
could consume oxygen linearly with time for at least 13 hours. These
observations suggest that the preparations were indeed viable. There-
fore, the data presented in this dissertation were derived from robust,
living tissues rather than from moribund or necrotic preparations.
Unlike the uptakes of vitamin B12 or vitamin Bi2-intrinsic
factor complex (Padykula et al., 1966), the specific activities of
tagged ions do not seem to decrease with gestational age. Instead,
there is a general increase with time and weight such that there is no
reduction of yolk-sac ion absorption between days 12 and 14. No in-
formation concerning ion uptake by this tissue at later stages of
gestation is available. As a result, the suggestion that yolk-sac
function is reduced as gestation proceeds (Padykula et al., 1966;
Jollie, 1964) cannot be supported by these experiments.
With the exception of the 45Ca++ specific activities of day
14 yolk-sacs (Fig. 8), wherever there is a significant difference be-
tween the trypan blue-treated and control yolk-sacs or embryos, the
specific activity of the dye-treated tissue always is greater. Since
day 12 and 13 trypan blue-treated yolk-sacs weigh significantly less
than the corresponding controls, the increased specific activities
indicate that either a smaller amount of protein or a fewer number of
cells (or both) is capable of absorbing the same or greater amounts of
ion. Although Niagara blue 2B treatment does not result in reduced
tissue weights, where statistical differences do exist, the dye-treated
tissues have greater specific activities than controls. This phenomenon
would tend to suggest that the machinery used by the yolk-sac to absorb
ions is altered by some interaction with Niagara blue 2B. This inter-
action is as yet unidentified.
Although the absorption of ions by embryos generally parallels
the uptake by yolk-sacs as gestation proceeds, there appears to be no
consistency in the relative amounts of ions taken up when the yolk-sacs
are compared to similarly treated embryos at the same day of development.
For example, consider Figure 9 which depicts the absorption of 35S04--
on a preparation basis. On day 13, the trypan blue-treated yolk-sacs
absorb a significantly greater amount of label than the corresponding
controls, while the 13-day :Tiagara blue 2B-treated embryos incorporate
a significantly greater amount of label than the trypan blue-treated.
A more striking example of this situation is presented in Figure 12.
Statistical analysis demonstrates that at day 13, control and experi-
mental yolk-sacs show no differences in their capacity to absorb
Na The day 13 embryos after Niagara blue 2B treatment, however,
have a significantly greater 22Na+ specific activity than the controls,
but are not different from those treated with trypan blue.
These results suggest that the interrelationship between the
embryo and its yolk-sac is very complex. The presence of trypan blue
in the yolk-sac at day 13 increases the 35S04-- uptake, but the in-
crease is not reflected in the embryo. However, the presence of Niagara
blue 23 on day 13, has no effect on the yolk-sac's ability to absorb
sulfate, while it may cause an increase in the amount of ion passing
into the embryo. With regard to 22Na+, again no differences are seen
in day 13 yolk-sacs, while treatment with Niagara blue 2B causes an
increased amount of label to penetrate into the embryo. Since the
presence of trypan blue in the yolk-sac was never correlated with a
concomitant significant difference in the embryo, there is the pos-
sibility that the dye may prohibit the passage of these particular
ions at these particular stages in development. Whether the same holds
true for other stages of development and for other ions or organic
molecules has not been determined.
The relationship between treated and control tissues may also
change from day to day. For example, Niagara blue 2B-treated day 12
yolk-sacs (Fig. 9) absorb a significantly greater amount of sulfate
than the control, while at day 13 the presence of trypan blue causes
an increase in absorption when compared to controls. Figure 10 also
demonstrates these changing relationships. Trypan blue-treated yolk-
sacs have significantly greater specific activities than the controls
at each day of gestation. The day 12 Niagara blue 23-treated yolk-
sacs also absorb more sulfate than the controls. The change occurs
at day 13 when there is no difference between Niagara blue 2B and
controls, thus indicating a possible recovery. Conceivably, recovery
from Niagara blue 23 treatment could occur more rapidly than from treat-
ment with trypan blue, for the former is excreted from the maternal
tissues and the proximal yolk-sac at a greater rate than the latter.
The fact that there are changes in the specific activities or
amounts of different ions absorbed by control and experimental tissues
on varying days of gestation is not at all surprising. Embryonic and
yolk-sac tissues are undergoing a rapid and extensive biochemical and
morphological differentiation during this period of gestation. As a
result, it is quite likely that as the tissues differentiate, their
ionic and nutritional requirements are altered with their particular
needs at varying stages of development. More important, however, is
that the presence of azo dyes can affect the ability of yolk-sacs to
absorb and possibly transfer certain ions. For this reason, the com-
plex relationship between the embryo and its yolk-sac with particular
regard to the exchange of materials should be studied further. These
kinds of experiments might indeed show that the yolk-sac plays an im-
portant role in normal embryonic differentiation and growth, so that al-
terations in normal yolk-sac function could be a mechanism of teratogenesis.
In Vivo Uptake of 35S0,--
The increased in vitro uptake of 35S04-- by trypan blue-treated
tissues when compared to controls was confirmed by autoradiographic
procedures. Although the number of pregnant females utilized for this
purpose was not sufficiently great to warrant statistical analyses,
the results also confirm those seen by Kochhar et al. (1968). These
investigators noted a significant, dose-dependent increase in the
absorption of 35S04-- by trypan blue-treated mouse embryos at days 10,
11 and 12 of gestation.
Since the maternal administration of 35so 4- has been shown to
result in a high uptake by fetal mesenchyme or mesenchyme derivatives
(Bostrom and Odeblad, 1953) and since one of the end results of trypan
blue treatment is a paucity of embryonic mesenchyme (Chepenik, 1965),
it seems unlikely that less mesenchymal tissue is able to incorporate
more ion. Indeed, Kochhar.et al. (1968) found that the 35SO was not
incorporated into normal sulfated organic compounds, but,instead, was
present in greater amounts as the inorganic sulfate ion or in compounds
of low-molecular weight. The increase in sulfate ion shown in both
the current and previous studies could very well indicate that the
organ transferring material between mother and embryo is the affected
organ and thatsomehow, the presence of trypan blue results in an
increased placental permeability to sulfate. Whether the primary
effect is on the chorio-allantoic placenta or the proximal yolk-sac
is still unclear.
1. When injected into pregnant rats on day 8 of gestation,
trypan blue is a potent teratogen. It results in a high percentage of
congenital malformations, primarily of the central nervous system and
special sense organs. This effect is apparently due to the rapid
biochemical and morphological differentiation which is taking place in
these systems at the time of insult. Abnormalities of other systems
conceivably result from a residual effectiveness suggested by the slow
rate of excretion from the maternal organism.
2. Niagara blue 2B is considerably less effective as a
teratogen, but still results in a rate of congenital malformation
which is significantly greater than the spontaneous incidence of
malformation for the Long-Evans black-hooded strain of rats. Its
lower effectiveness could be the result of its more rapid rate of
3. On days 12, 13 and 14 of gestation, both dyes cause a sig-
nificant increase in the absorption of 4Ca++, 35S04-- or 22Na+ by
yolk-sacs and, in some cases, abnormally developing embryos. These
changes in the specific activities of yolk-sacs suggest that both dyes
aave an effect on normal yolk-sac function and indicate that alterations
Ln the function of the yolk-sac can conceivably bear a direct relation-
ship to the induction of congenital abnormalities. Future studies,
however, must utilize a teratogen which is effective at later days of
gestation, so that transfer phenomena may be studied before, during and
after the teratogenic insult.
4. Autoradiographic studies on day 13 control and trypan blue-
treated implantation sites confirmed the results of the in vitro
experiments. The preliminary data from teratogen-treated chorio-
allantoic placentae, yolk-sacs and embryos indicate that all three
tissue types absorb greater amounts of 35SO4-- than the corresponding
5. It is proposed that the proximal yolk-sac is a functioning
organ at this stage of gestation and that an alteration in the re-
lationship between the embryo and its yolk-sac could be significant
in the induction of congenital abnormalities.
Composition of Bouin's Fluid
Saturated aqueous picric acid
Formalin (40% formaldehyde)
Concentrated glacial acetic acid
75 parts by volume
25 parts by volume
5 parts by volume
Composition of Phosphate-Ringer u'uffer
Mix 8 parts A : 1 part B
Add to pH 7.4
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Marten Murray Kernis was born September 21, 1941, in Chicago,
Illinois. He received his primary and secondary education in the
public school system of Chicago and began his undergraduate training
at the University of Chicago in September, 1959. In 1961, he enrolled
at Roosevelt University in Chicago where he received his Bachelor of
Science degree with a major in Zoology in 1963. After one year as a
graduate student at the University of Illinois Department of Physi-
ology in Urbana, he began his graduate studies at the University of
Florida in September, 1964. During his studies toward the Doctor of
Philosophy degree at the University of Florida, he was supported by a
National Institutes of Health Predoctoral Traineeship.
He is a member of the American Association for the Advancement
of Science and an Associate member of Sigma Xi.
He has accepted the position of Assistant Professor of
Anatomy at the University of Illinois College of Medicine in Chicago.
He was married in August, 1966, to the former Michele Phyllis
Hinden of Sarasota, Florida.
This dissertation was prepared under the direction of the
chairman of the candidate's supervisory committee and has been
approved by all memberss of that committee. It was submitted to the
Dean of the College of Medicine and to the Graduate Council, and was
approved as partial fulfillment of the requirements for the degree
of Doctor of Philosophy.
De Coll g of Medicine
Dean, Graduate School
- -, -~ ~
UNIVERSITY OF FLORIDA
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