Effects of a teratogen on yolk-sac function

MISSING IMAGE

Material Information

Title:
Effects of a teratogen on yolk-sac function
Physical Description:
vi, 83 leaves : ill. ; 29 cm.
Language:
English
Creator:
Kernis, Marten Murray, 1941-
Publication Date:

Subjects

Subjects / Keywords:
Fetus   ( mesh )
Embryology   ( mesh )
Abnormalities   ( mesh )
Anatomical Sciences thesis Ph.D   ( mesh )
Dissertations, Academic -- Anatomical Sciences -- UF   ( mesh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1968.
Bibliography:
Bibliography: leaves 79-83.
Statement of Responsibility:
by Marten Murray Kernis.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000901169
oclc - 20059992
notis - AEK9994
System ID:
AA00009499:00001


This item is only available as the following downloads:


Full Text














EFFECTS OF A TERATOGEN ON
YOLK-SAC FUNCTION












By
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
1968

























DEDICATION


To my parents for their unparalleled encouragement and

concern and to my wife for her generous support, this dissertation

is lovingly dedicated.














ACKNOWLED3!-EI:7 S


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

statistical analysis

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

Page

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

Table Page

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


Figure Page

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
35 -
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
22 +
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












INTRODUCTION


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


Parietal 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
site. Abbreviations:

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


end
vi
vv
am
ul
pys
B


endodermal sinus
villi
vitelline vessels
amnion
uterine lumen
parietal yolk-sac
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:


mesothelium
mesenchyme
visceral basement membrane
pinocytotic vesicles
microvilli
yolk-sac cavity


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


mth
mes
vbm
pv
mv
ysc


























Fig. 1


A B
Fig. 2










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.








5

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.

Visceral Yolk-Sac

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.











TIXdodermal Sinuses

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

term.

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,

unanswered.

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

basal cytoplasm.

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

Padykula, 1953).


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
of Protection

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

protective device.

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
of Transfer

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

chorio-allantoic placenta.










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

ion uptake?














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

pregnancy.


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


2Pittsburgh, Pennsylvania.

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,
Maryland.

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
of Radioisotopes

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

(Fig. 4).

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

utilized.




































0)0
rHI


Sp4

C-
4H .-
05C



Cd r-I '
f--1 *d-
40 S-i



,-I 0 0
.e -4 VH
r-I 0 r-4
**- -to
*Sr-4 .co
i-H 0 0)



-t- O
***


CI-
0



0

P4




P4


o


o o
O >
Sr--
o 0
- 0
-4

Co






ed
r-4

oP







IC
0 0






04




* 0
10 -6
,Cd
U) d


03



o 1


$-4 P-4 n )0
P4 in 4- cd
CQ C to I
-i En w,


(=1 0 0
0) r-
It Cd


'* e--4 f-l0
*< pq r-4
(U 0) (
*H Cd
*r-4 A-4
0) 0;>


* *
T-N( C







23



















C4 CO
































0












-4

Cc

.o

00


F-4

4
o o

















0
IC

o r






J-4

Sr-
0

o

0


4-,


0

*-4
1--I




o CO
0o --






S**
C<-1-


* *


M ti o (D
pL, p. cSi-
ma)
c'N1 c D ,


>0 0~
CM C^ I W
r-I


cd r4
a,

>U
CO) 4-'


* *





25










U-
c 0o



!l











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

were employed.

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

faced down.

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,
Pennsylvania.













RESULTS


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.





















o
0 4-










c-'
5-4






























oI
S-' 0o Z- 0 o




S- ,




4 -
54-
























S- H- ,.0





S0
0 *C 0
















-= 0 0N 0
-li 4-' En
0- $L ; I

A4 ri C)(



Cdp pq a











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

ON La; CO















o 0 0
*








o oo











+1 +1 +1
0 -, I\ N
NM C N










o



OD V"\r- \0





0 9 0
N~ --N


co Xr\ CvN
C; c s
*
o 0 0











+1 +1 +1
o *N O
v-I v-i r-I














N N N
o4 0; 0
*
o 0 0

+1 +1 +1
'--I -=) ON
N ON 0
.
v-I 0 v-I


v-I v-i NO




c c- c


+1 +1 +1
ON O, 0
0N ONs V\
o 0 cI
v-I v-I -


J v-I -o

%lo)





+ + +
i', Co '






\0 Ui^ (
<-4 vr-I i-I










o, o 0









0 0
+1 +1 +

NO o
ON C\ C% -
* *












0\ 00
ON 0r- 0s

o 0 0
+1 +1 +1
-* ON -~t
0 CO v-I


SN


0N
r-4 OJ p


0 z


rIc
0 ? 0 0 $




00
C- 4 l a A
O2
o t c i c
| f g .&


a ti
+I
C)




Q
Id cs (n

4-,
CCO CI- 0
'0 0
4-
C> W



+j 0 0
a 0 -
4 43-' C<
C) C) ,0









-$i
U) 0 N
6 CM~ w,
$41 '-1
.-l C) 4->
r At ^ -i


**- -'a Cr


t0

-I tt> $4
S)
(=- r1 ,C

a! ,0 o


Cd
0












E-- 0


0

rI-

N


v-I




















v--I




























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.































EMBRYO










O .*












YOLK SAC


.'
*
. .L


0 O CONTROL

............... TRYPAN BLUE

0---0 NIAGARA BLUE 2B


DAY OF GESTATION


10 +


-/ ...-*





























U)

d d
U) U0

t) r-i
o-' a
,CO




U .'
U0 *0 r
*^*(


U) Cd
Q)





4-'CsO
W 40
>, ) C\)
rd tE

~C ) r-1


0
-t > W


u to ,





d0
+^ o p







a U
s:-' c3) 0








m0 ;d
I '1 i







u .4-'
hO eU )-





*do
u i










40)0
\0 0
*Cd





CI-4 ED
0
OC
cn
CS0 f-

(-10
OOl -
















-4


-1


Sz
I- (
z z 0
0 S
U z
Eli M


-4


z
4-, 0


I-
1,1,
0
U.
0
WE
0


F-


i


0lilNOD I0%











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
45 ++-
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
45 4++
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



















cll N
+i +i








+1 +1
.-3 cv~i
'-l
















cn- nC



-N %o
NO< '-4











+1 +1





0 0





+ +

V-4 -4
+1 +1
0 0
r-I
















N ct\
'N! N,


+ +




V-4
































+1 +1













0 T-l
+1 +1










o O


0 0
/ 0 Eo 0
I > I

->-4

-4 3


0 r








CQ

cQ cn
+- +!
T-4









+1 +1


n-4
o ON


1-4

+1 +1
-3- NO
C'- T-4
T-4








oR 0



ON -3-

+1 +1
N N
1L\ C~
0v- T-4


l11O
o
0

o !-


S-
0












cFi
*0



0 0


















Po
on














0




0
'0

















g-4
C-5






4-2
o- ,4




























Fig. ?. -Semi-logarithmic graph comparing mean tissue
absorption of 5Ca+ with gestational age. See Table 3 for
numerical data.


















YOLK SAC 0




00

S./0


EMBRYO


S- o CONTROL
S.............* TRYPAN BLUE


DAY OF GESTATION


600 -
50-


400+


300-


200 --

150 --


100 -

8-


z
0




C.

4-
(LI



t-
0
U
C,



a-
U


40

30-


10-+





























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
data.













-r
0


YOLK SAC..............




YOLK SAC


2-+


1l-+


EMBRYO


0- 0 CONTROL

S.............. TRYPAN BLUE


DAY OF GESTATION


101 -
8 -





4.- -
31--


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

equivalent.

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.











0C


4 -- 0 0 'O __ -



S O

FC4 *P





C2t C). N Co C0 cd
-+-4 d c+


On Co-- C Q \04 '- ..O'-d
en '-' +1
E-i
cd

134 0


+ + + + +- + +i
-4 C\ 2 C\N NCN OMM ,N ,\ V\ ,N c' .


aI p .0

o m cd F:
-\ c 0 :
0j a U
-0 0 N "0 ON
+C ++ + + + +r..
o c'- c-' oo -' o.
Scd 0V0- C,- C-..- O Z-. --- N rs

0 C. cn

lq aa z
004-
CH cd
o. F:4
1) 0 En P4
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


r-i N-r
C- 10 CM 0 ,EC*
o -d -8 ( E-:
+ +r +1 + + +N +i> '
S CO ON N C"-NJ -CN \ON 0C 0

8 *-H
Q)
4--20


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.

















YOLK SAC


0- 0 -- CONTROL

* ............ 0 TRYPAN BLUE

*---9 NIAGARA BLUE 2B


EMBRYO

O *' .


0- 0 CONTROL

* ............ 0 TRYPAN BLUE

S---o NIAGARA BLUE 2B


I I I


DAY OF GESTATION


400-
300-
200-

100-
60-


31-


11+


z
0
!R


UJ
0



I

0




U


10+










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.










I T
16 ......... ....................

141 -1 1
140



/ YOLK SAC








20"


ee 0.. B
0 /



-f


U '








S....... TRYPAN BLUE




**---- NIAGARA BLUE 2B


12 13 14
DAY OF GESTATION















co V


+ +
\O0 N CQOi
T-4 ^- .- -^ ^
(-I






c'- c


4+'
-a


fi






+c



C.)






















0
.4-















Cd
Cd
PL4














C)
'P-4







C.3


+N +f
00C^j %J-\
%,\- 00.


+ NG N,
+-N +N
CMN NN M
O\-~ CM >-


-4-




CO
T-1


N-I


0 -
-4


0,-
V-4


+.
0,-'-4
0-4
Cvt-'-


Cl-

+
o,, co
f T--l







a-.
0,
-c-


-4-


N-Co




-4* C-l












+N.
N CM
C\J
-1







C+-


xr^
0'.-C-


'4-
N
r-q CMl
00-








+
V. C,

CL .


CM
+CM
N

0



E-I
P-I
0


-I-

+
a) I-


0
T-i ~
CC%'
+^







0







0-,


+^

Q '-I


V-4
CM-"

+
C-
V-1


co
UD






1$4









ff1
0




Cd
441


dO

+-1



Cd

CdF





cdCH
U).






c)UD









0z
4-,' -

$4-
EO
0
.d



4-<
0 C"







Id 0
W0
4C3-4
E-1 -
PId C
4-,






t=>o
0
c e

Cdp
4-3
C)- c4f

OC)



C) -


f-i C)1


0 0H
C)* ^
i-I- U)
C)




u-4


0
S-4-

+<

\0 -
T-4


+1K
cv-'--


c-')



+4


+^
-4 C-


r-4


cv-
c'-


\0
-3t




























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.























N-

48-


7'

EMBRYO .*

0...
j.--^-0


o CONTROL
S.........e.... TRYPAN BLUE

9---0 NIAGARA BLUE 2B


13
DAY OF GESTATION


YOLK SAC -




0- 0 CONTROL

S............. TRYPAN BLUE

S.--- ** NIAGARA BLUE 2B


20

-r


z
0



LUI
ad

o
+
0
z
0-



U


U18-


20011+


116-
SI-

43-.


20

-"


f


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

the control.




























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
data.



















YOLK SAC


0


1L


EMBRYO


2+


0- 0 CONTROL

* *..........**..* TRYPAN BLUE

*---* NIAGARA BLUE 2B


DAY OF GESTATION


2N+


151+


a6+


I-











z

eC.
u~










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.











TABLE 6

IN VIVO 35S04-- INCORPORATION


Control Trypan Blue Per Cent
Tissue Grain Count Grain Count of Control

One Hour

Chorio-Allantoic
Placenta 70 196 280

Visceral Yolk-Sac 30 51 170

Embryo 61 193 316

Three Hours

Chorio-Allantoic
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
details.


been corrected










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.
00ox.

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.
/






















































v





























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.









































V
/





























Fig. 15.-Autoradiographs of embryonic mesenchyme in
the region of the notochord. 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.






64




OF
p1













e4e
/ -
. --<


^<^7 ,^ ^
L^ '. ^ .'













DISCUSSION


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


0 <0




4-Z

W )



PL0
0 CD






F o
-i









-0 0

+ C

4-,









o'C-
0 E-n






0d 0-d
in 0 +


0 0 C
n~


O v \ O -c
N- o (i .}
r-1 Y-l -


OrN




co co c


Sr
0C

o


rI
0
C 0


4 -

-4


0
0

Co



CO .
T- U


SON ON C'2
-a- 1 r-i C'


0









.
oM

d Q)
c) H O
plq *r-4


4-a
0







so
.4^ -I





N
5r-1 0



o a

5-1
-I- C)e


4-l
0








4-
, $-.t



1f.i ID
CD2
$U0
0)< ..-


M o
o 0



I 0
0 0
OJ a!
1'-I U


I
C 3

-D 0

iO


oa -
V-4 -


02
-3
0
o

Cd
hfG C)


6 ^
0
0 0
m U)
xr\


U=)





C-a
-0



NO n
1-a


NO 4
1-4 SO M C


I
CD) 4--

Cd z-
4-.; a)


4-: ci










u) CN

1-4ON
o


o
NOO


0 0
5-4 C)


02
w





0
1-1



02
*r-1 0
.3 *1*-<
4- 4--l
4-)c

,--1 0
-H )
~0 T
5-402


P-)



Spq


SN-
00 ^
Ci-'


02
CL)
P-4


f1)


4-

r-0
C)0
mno,


c'-
0
-t-




ON

o








j o
0j




0
0 .
cO \
,i-l U)


() 4-'


C 0
4-) (1)












0
02
.-
*1-






5-.





E-i










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

determined.

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
22 +
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.














COi3CLUSIOjS


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

excretion.

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

controls.

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.
































APPENDICES










APPENDIX A

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












APPENDIX B

Composition of Phosphate-Ringer u'uffer


Substance

Solution A

NaC1

KC1

Na2HPO4

H3PO 4
MgSO 4


Solution B

CaC12

Mix 8 parts A : 1 part B


millimoles/liter


grams/liter


145

5.6
4.2

Add to pH 7.4


2.16


8.50
0.42

0.60


0.145


0.24













LITERATURE CITED


Asai, T. 1914 Zur Entwicklung und Histophysiologie des Dottersackes
der :L~er mit Entypy des Keimfeldes. Anat. Hefte., 51:
467-641.

Beaudoin, A. R. 1962 Interference of Niagara blue 2B with the
teratogenic action of trypan blue. Proc. Soc. Exp. Biol.
Med., 109: 709-711.


, and D. Kahkonen 1963 The effect of trypan blue
serum proteins of the fetal rat. Anat. Rec., 147:


on the
387-395.


, and M. J. Pickering 1960 Teratogenic activity of several
teratogenic compounds structurally related to trypan blue.
Anat. Rec., 137: 297-305.

, and J. G. Wilson 1958 Teratogenic effects of trypan blue
on the developing chick. Proc. Soc. Exp. Biol. Med., 97:
85-90.


Beck, F., J. B. Lloyd and A. Griffiths 1967
hibition by trypan blue: A theory of
Science, 157: 1180-1182.


Lysozomal enzyme in-
teratogenesis.


Bennett, H. S. 1963 Morphological aspects of extracellular poly-
saccharides. J. Histochem. Cytochem., 11: 14-23.


Boe, F. 1951 Studies on placental circulation in rats. III.
Vascularization of the yolk sac. Acta Endocrinol., _7:


42-53.


Bostrom, H., and E. Odeblad 1953 Autoradiographic observations on
the incorporation of S3--labeled sodium sulfate in the rabbit
fetus. Anat. Rec., 115: 505-513.

Boyd, G. A. 1955 Autoradiography in Biology and Medicine.
Academic Press, New York.
Brambell, F. W. R. 1958 The passive immunity of the young animal.
Biol. Rev., 33: 488-531.

and R. Halliday 1956 The route by which passive immunity
is transmitted from mother to foetus in the rat. Proc. Roy.
Soc., B., 145: 179-185.











Bra-bell, F. W. R., W. A. R:emin-s and M. Henderson 1951 Antibodies
and Em.bryos. The Athlone Press, London.

Branschwig, A. E. 1927 Notes on experiments in placental permeability.
Anat. Rec., 3: 237-244.

3ulmer, D. 1963 The histochemical distribution of 3-glucuronidase
activity in the rat placenta. J. Anat., 97: 181-188.

Chepenik, K. P. 1965 Effects of trypan blue on embryonic and
placental development in the rat. Unpublished Master's
thesis, University of Florida, Gainesville, Florida.

Deren, J. J., H. A. Padykula and T. H. Wilson 1966a Development of
structure and function in the mammalian yolk sac. II.
Vitamin B12 uptake by rabbit yolk sacs. Develop. Biol., 13:
349-369.

1966b Development of structure and function in the mammalian
yolk sac. III. The development of amino acid transport by
rabbit yolk sac. Develop. Biol., 13: 370-384.

Duval, M. 1892 Le Placenta des Rongeurs. 2V. Alcan, Paris.

Everett, J. W. 1935 Morphological and physiological studies of
the placenta in the albino rat. J. Exp. Zool., 70: 243-284.

Ferm, V. H., and A. R. Beaudoin 1960 Absorptive phenomena in the
explanted yolk sac placenta of the rat. Anat. Rec., 137:
87-91.

H. J. Free and D. L. Shires 1959 Concentration of azo-
proteins in the yolk sac placenta of the rat. Proc. Soc.
Exp. Biol. Med., 100: 456-459.

GeTrard, P. 1925 Recherches morphologiques et experimentales sur
la vesicle ombilicale des rongeurs a feuillets inverses.
Arch. Biol., j5: 269-293.

Gillman, J., C. Gilbert, T. Gillman and I. Spence 1946 A preliminary
report on hydrocephalus, spina bifida and other congenital
anomalies in the rat produced by trypan blue: The significance
of these results in the interpretation of congenital mal-
formations following maternal rubella. S. Afr. J. Med. Sci.,
13: 47-90.

Goldmann, E. 1909 Die aussere und innere Sekretion des gesunden
und kranken Organismus im Lichte der "vitalen Farbung."
Teil I. Beitr. Z. Klin. Chir., 64: 192-265.










Grosser, 0. 1927 Fruhentwicklung, Eihautbildung und Placentation
des Menschen und der Saugetiere. Deutsche Frauenheilkunde,
5: 1-454.

Halliday, R. 1955 Prenatal and postnatal transmission of passive
immunity to young rats. Proc. Roy. Soc., B., 144: 427-430.

Hamburgh, M. 1952 Malformations in mouse embryos induced by trypan
blue. Nature, 169: 27.

Hanan, E. B. 1927 Absorption of vital dyes by the fetal membranes
of the chick. I. Vital staining of the chick embryo by
injections of trypan blue into the air chamber. Am. J.
Anat., 38: 423-450.

Harm, H. 1954 Der Einfluss von Trypanblau auf die Nachkommenschaft
trachtiger Kaninchen. Zeits. fur Naturforschung., 96:
536-540.

Hogan, A. G., B. L. O'Dell and J. R. Whitley 1950 Maternal nutrition
and hydrocephalus in newborn rats. Proc. Soc. Exp. Biol.
Med., 74: 293-296.

Johnson, E. M., and R. Spinuzzi 1966 Enzymic differentiation of
rat yolk-sac placenta as affected by a teratogenic agent.
J. Embryol. exp. Morphol., 16: 271-288.

Jollie, W. P. 1964 Radioautographic observations on variations in
deoxyribonucleic acid synthesis in rat placenta with increas-
ing gestational age. Amer. J. Anat., 114: 161-171.

Kalter, H., and J. Warkany 1959 Experimental production of con-
genital malformations in mammals by metabolic procedure.
Physiol. Rev., 39: 69-115.

Kochhar, D. M., and E. M. Johnson 1965 Morphological and autoradio-
graphic studies of cleft palate induced in rat embryos by
maternal hy-pervitaminosis A. J. Embryol. exp. Morphol.,
14: 223-238.

K. S. Larsson and H. Bostrom 1968 Embryonic uptake of
s35-sulfate: Change in level following treatment with some
teratogenic agents. Biol. Neonat., 12: 41-53.

Koren, Z., and E. Shafrir 1964 Placental transfer of free fatty
acids in the pregnant rat. Proc. Soc. Exp. Biol. Med.,
116: 411-414.

Kosan, R. L., and A. C. Burton 1966 Oxygen consumption of arterial
smooth muscle as a function of active tone and passive
stretch. Circulation Res., 18: 79-88.











Lambson, R. 0. 1966 An electron microscopic visualization of trans-
port across rat visceral yolk sac. Am. J. Anat., 118:
21-52.

Lloyd, J. B., and F. Beck 1966 The relationship of chemical
structure to teratogenic activity among bisazo dyes: A re-
evaluation. J. .-bryol. exp. Morphol., 16: 29-39.

Long, J. A., and H. M. Evans 1922 The Oestrus Cycle in the Rat and
its Associated Phenomena. Memoirs, V. 6. University of
California Press, Berkeley, California.

Luse, S. A. 1958 -he morphologic manifestations of uptake of
materials by the yolk sac of the pregnant rabbit. Fourth
Conference on Gestation. C. A. Villee, ed. The Macy
Foundation, pp. 115-141.

Messier, B., and C. P. Leblond 1957 Preparation of coated radio-
autographs by dipping section in fluid emulsion. Proc.
Soc. Exp. Biol. Med., 96: 7-10.

Mossman, H. W. 1937 Comparative morphogenesis of the fetal membranes
and accessory uterine structures. Carnegie Contributions to
Embryology, 26: 129-246.

Netzloff, M. L., K. P. Chepenik, E. M. Johnson and S. Kaplan 1968
Respiration of rat embryos in culture. Life Sci., In Press.

Noer, H. R., and H. W. Mossman 1947 Surgical investigation of the
function of the inverted yolk sac placenta of the rat.
Anat. Rec., 98: 31-37.

Padykula, H. A. 1958 A histochemical and quantitative study of
enzymes of the rat's placenta. J. Anat., 92: 118-129.

J. J. Deren and T. H. Wilson 1966 Developmento of structure
and function in the mammalian yolk sac. I. Developmental
morphology and vitamin Bl1 uptake of the rat yolk sac.
Develop. Biol., 13: 311-348.

and D. Richardson 1963 A correlated histochemical and
biochemical study of glycogen storage in the rat placenta.
Am. J. Anat., 112: 215-242.

and T. H. Wilson 1960 Differentiation of absorptive
capacity in the visceral yolk sac of the rat. Anat. Rec.,
136: 254 (Abstract).











Rawson, K. A. 1943 The binding of T-1824 and structurally related
diazo dyes by the plasma proteins. Am. J. Physiol., 138:
708-?17.

Shimidzu, Y. 1922 On the permeability to dyestuffs of the placenta
of the albino rat and the white mouse. Am. J. Physiol.,
62: 202-224.

Sobotta, J. 1903 Die Entwicklung des Eies der Maus vom Schlusse
der Furchungsperiode bis zum Auftreten der Amniosfalten.
Arch. f. Mikr. Anat., 61: 274-330.

Sorokin, S. P., and H. A. Padykula 1964 Differentiation of the
rat's yolk sac in organ culture. Am. J. Anat., 114: 457-477.

Wilson, J. G. 1955 Teratogenic activity of several azo dyes
chemically related to trypan blue. Anat. Rec., 123:
313-333.

1965 Methods for administering agents and detecting mal-
formations in experimental animals. In Teratology:
Principles and Techniques. J. G. Wilson and J. Warkany, eds.
The University of Chicago Press, Chicago, Illinois, pp.
262-277.

A. R. Beaudoin and H. J. Free 1959 Studies on the
mechanism of teratogenic action of trypan blue. Anat. Rec.,
133: 115-128.

Wislocki, G. B. 1921 Further experimental studies on fetal absorption.
III. The behavior of the fetal membranes and placenta of the
guinea pig toward trypan blue injected into the maternal blood
stream. IV. The behavior of the placenta and fetal membranes
of the rabbit toward trypan blue injected into the maternal
blood stream. In Carnegie Contributions to Embryology, 13:
89-101.

and E. W. Dempsey 1955 Electron microscopy of the placenta
of the rat. Anat. Rec., 123: 33-63.

and H. A. Padykula 1953 Reichert's membrane and the yolk
sac of the rat investigated by histochemical means. Am. J.
Anat., 92: 117-152.

Zaretsky, S. 1910 Versuche uber vitale Farbung Embryo. Virchow's
Arch. f. Path. Physiol., 201: 25-45.













VITA


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.


June, 1968




De Coll g of Medicine




Dean, Graduate School




Supervisory Committee:


- -, -~ ~










wy




































UNIVERSITY OF FLORIDA

3 1262 08554 5001




Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID ENL9P7W9C_0MU7F1 INGEST_TIME 2012-03-26T15:32:57Z PACKAGE AA00009499_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES