permeability of the rabbit blastocyst to trypan blue

PERMEABILITY O F THE RABBIT BLASTOCYST TO TRYPAN BLUE

VERGIL H. FERMa Department of Anatomy, University of Wisconsin, Madison

INTRODUCTION

Observations by many investigators (Goldmann, '09 ; Wis- locki, '20; Schmidzu, '22; Everett, '35; Gillman, '48; Ham- burgh, '54) that the placenta of the rodent is relatively im- permeable to moderate amounts of trypan blue injected subcutaneously into the pregnant animal, have been distracting when attempts have been made to explain the teratogenicity of this dye in mice and rats. In the absence of evidence that the dye traverses the placental membrane and affects the embryo directly, Gillman ('48) has suggested that some maternal metabolic defect caused by the presence of trypan blue in the maternal system produces the embryonic damage. Hamburgh ( '54) has postulated, as an alternative explanation for this teratogenic effect, that there is some alteration of placental permeability probably manifest in the yolk sac placenta because of its particular and peculiar affinity for this dye.

The author first investigated the possibility that the site of action of this teratogenic dye is related to the maternal adrenal, since certain corticoids are known to be teratogenic threats (Fraser, '54). There is no significant difference in adrenal weight stimulation among the teratogenic (trypan

lThis investigation was supported by a grant from the Wisconsin Alumni Research Foundation.

' A portion of a thesis submitted t o the Graduate Faculty of The University of Wisconsin, in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Present address : Department of Anatomy, Indiana University.

745

746 VERGIL H. FERM

blue) and non-teratogenic (Congo red, Bismarck brown) azo dyes when measured on male rats (Ferm, '55).

An examination of the possibility that trypan blue might exert some effect on placental permeability because of the affinity of the yolk sac endoderm for trypan blue aIso yielded no positive results when the placental transmission of the foreign proteins, bovine serum albumin and bovine gamma globulin was measured serologically.

The technique of collection of blastocyst fluid of the rabbit as described by Brambell ( '51), presents the opportunity for studying the composition of blastocyst fluid and the immediate embryonic environment under experimental condi- tions during critical stages of embryogenesis.

The following experiments were undertaken to determine : (1) if trypan blue can be detected in the yolk sac fluid of the rabbit after the intravenous injection of the pregnant doe, and (2) if trypan blue is a teratogen in rabbits when related to blastocyst permeability. Harm ( '54) has suggested that trypan blue causes certain eye defects when injected sometime between the first and eighteenth day of gestation in the rabbit. Congo red, an azo dye reported by Gillman ( '48) to be non- teratogenic in rats, was tested for blastocyst permeability, but not for teratogenicity, in rabbits.

Exper iment I - Permeabili ty o f the rabbit blastocyst t o t r y p a n blue and Congo red

MATERIALS AND METHODS

Accurately timed matings of rabbits were obtained and the does then isolated. One intravenous injection of a 1% solu- tion in saline of the azo dye (National Aniline) was made in a dosage of 25mgm/kgm of body weight at about 8 hour developmental age intervals beginning at 7 days - 18 hour post-coitus. Eight pregnant does were injected with trypan blue and five were injected with Congo red. Exactly two hours after the single injections of the dye, the animals were killed by 0.5 cc of 1/8 M Ca C1, intravenously and lOcc/kg of

PLACENTAL TRANSFER O F AZO DYES 747

1 M MgCl, subcutaneously. This method of killing tended to reduce the possibility of damage to the blastocysts by uterine contractions. All animals were dead and opened up within fifteen minutes. Maternal cardiac blood was drawn at the same time. The uterus was cut across the broad ligament and at the cervix. The entire uterus containing the blastocysts was then dropped into 95% alcohol previously chilled with dry ice. The tracts solidified almost instantly. The gestation sacs were cut free from the uterus and the thin uterine wall was thawed with the fingers. The yolk sac fluid was removed with ease as a solid piece of ice. Some animals, not used in these data, were killed by a blow on the head to determine if the method of killing influenced the appearance of the dye in the yolk sac fluid. No difference could be found. The yolk sac fluid was thawed with 0.1 cc of 1.6% potassium oxalate to prevent clotting, and the weight of each sample was determined. The samples were then diluted with 3.0cc of normal saline (buffered to pH 7.4), centrifuged, and then frozen until colorimetric determinations were made. Colorimetric analyses were made on 65 blastocysts of the trypan blue series and on 38 blastocysts of the Congo red series.

After light absorption curves were determined f o r trypan blue (600) and Congo red (490) from known concentrations of these dyes in saline, and it had been determined that these two dyes followed Beer’s Law, the concentrations of dye in all the experimental samples were then determined on the Beck- man spectrophotometer using for the control, normal yolk sac fluid after it had been treated in a manner similar to the experimental samples. Appropriate corrections for the dilutions were made so that the actual concentrations in mg/liter could be determined for each sample. Comparably treated maternal serum samples were used to determine the levels of the dye present in the serum of each doe at the time of sacrifice.

RESULTS

I n all animals injected with trypan blue o r Congo red during the 7th, 8th and 9th days of gestation, the dye material

83

1

..'

" '

131

P'91

93

1

1'E1 1.51

8'91 8'91

0'1Z2 O

'E9 CC'Z

90-6 L'1Z

63

2

L'61 6'91

Z'ZZ P'ZZ

2'1Z 6.18

E'9Z 9'22

0'99Z 9'29

9'2 00-6

EX9

L'69 P'9F

€'€G E'9E

P'O9

0'61%

0x

9

C9.Z 60-8

P.19 ~. . .

. . . ,

, ,

I'LF '.

.

''

_

L'IP

9'5'2 Z'8Z

6'62 8'FS

I'OP

9'IF

O'E91

O'L9

€7

€0-8

E'9E L'8P

6'1Z O'PE

9-92 1'EE

I'SE L'9Z

9'9F 8%

0'962

0'98 P'E

02-L

9K

NI

snnoD

?/OX

6 8

L 9

P P

E Z

1 rV

nnas 'OAT

'JB

X

NI m

a

3na ,!I0 3H

-&S

od

&

NnO

JYB

&

HO

IXhi

SllflOH

?

VJIO

&

(INV

snva n

%n

?/D

N

NI s&

snao&svm

NI 3a

a do N

OI,I,V

?I.LN

~~

NO

CI

'ON

03

Z 3T

CIV

L

z ffi w k

bi

G '

'

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 9

7

0'9 L'P

9'P 1.E

6'P 0'9

L'F Z'P

1%

Z'P

E'P '

' '

' 8'F

0'9 Z'P

€3

8%

8'9

E'E €

3

8'11 ' '

L'6 E'OI

8'6 2'01

L*II

6371 6'12

9'91 9'11

8'02 t'9Z

9'91 1'81

I'L1 E'9I

1.61 . .

. . . . .

. . .

67

2

"

"

"'

P'1Z

6'81 9'LZ

I'LZ I'OE

0'28 Z'LZ

1'91 '

' . .

6'91 9'91

O'PI

L'E1 E'91

6'81 L'91

L'91

.. ..

0 ffi

c/3 dr

t-

0'891 0'9P

l Z'P8 0'891 0'911 O

'PL1 0'621 0'6E1

9'E6

O'PO

1 9'28 0'89 S'L9 2'99 8'99 Z'99

9L'€ 9

13

OE'F SL'Z OL'Z 92'2 SZ'Z 92'2

20-21 00-01 10-6 00-6 02-8 01-8 €0-8 02-L

0 IV

sn

JI100

do J.Nn

OR

V

&H

91

3h

i S

nn

OH

?/OX

01

6 8

L 9

P P

E

Z 1

xnnm

NI ana

3H

-&Sod

TV

JIOL

a

NV

shva 'OAV

?IL&

IT/O

W

NI sm

mo

&sv

~a 61 m

a Y

O N

OI,I,V

?I&N

XC

INO

D

'3N

OD


appeared in every blastocyst. These data are summarized in tables 1 and 2. The permeability begins to decline after the ninth day of gestation so that animals injected with trypan blue on the 10th and 12th days of gestation revealed no detectable amounts of dye within the yolk sac fluid. Increase in blastocyst size was not sufficient to account f o r this observa- tion.

Experiment I I - Terntogenic effects of trypan, blue in rnbbits

With the evidence that trypan blue can be found in the yolk sac fluid of the rabbit during certain critical stages of embryogenesis, it was thought advisable to determine whether or not this dye is a teratogen in rabbits. The following experiment was devised as only a rough qualitative test of this possibility and no attempt was made to control the genetic background of the animals. Rabbits of all sizes and pigmentation were used.

MATERIALS AND METHODS

Timed matings of rabbit does were obtained with at least two males of different size and pigmentation. The does were isolated. Twelve pregnant does were then injected subcutaneously with various amounts of 1% trypan blue in saline at selected gestation ages (table 3). Most of the animals were killed on the 16th day of gestation and the embryos fixed in alcohol-formol-acetic acid. A total of 10 uninjected litters, killed between the 16th and 22nd day of gestation, served as controls as far as litter size and general external morphology were concerned. No gross morphological abnormalities were noted in 58 living embryos of this control group as compared to 12 abnormal embryos in a total of 56 living embryos of the experimental series. There was 28% resorption of embryos in the control group as compared to 41% in the experimental animals.

RESULTS

In the small number of animals used in this experiment, it is apparent that trypan blue has a teratogenic effect on rabbit

750 VERGIL 11. FERM

embryos (tablc: 3 and figs. 1-4). Some of the anomalies probabIy would have escaped detection if the animal had been sacrificed later than the 16th day of gestation, since after this time some of them would have undoubtedly resorbed. On the other hand, there were probably some embryos classified as grossly normal on the 16th clay which might have eventually become anomalous from some latent trypan blue-induced mechanism. Some anomalies were noted in the anterior thoracic region (figs. 3 and 4). These consisted of large dilated structures, apparently veins, containing fetal blood. The hearts in these animals were normal in size. The fixed material in some of these latter specimens was in poor con- dition, probably because they had died and begun to resorb.

DIXCUSSION

It is apparent that both trypan blue and Congo red will enter the blastocyst fluid of the rabbit from the maternal circulation up to the ninth day of gestation. Any explanation for the teratogenicity of trypan blue must now include the possibility that this compound has a direct toxic effect on the differentiating embryonic tissue. Differences in the teratogenicity of structurally related azo dyes may be due to slight differences in their cherriical structure, for Sugiura, e t al. ('54) have shown that the carcinogenicity of ethyl derivatives of p-aminoazobenzene can be radically changed by the substitution of a single hydroxy group.

The mechanism of transfer from the maternal circulation to the yolk sac fluid may be via molecules of maternal albumin which are known to have a high affinity for these compounds. There is the possibility, however, that this linkage may actually be broken during the transfer through the blastocyst wall. The variation in the concentrations of the dye in the maternal sera at the time of sacrifice is marked, although within a given litter, the amounts found within the blastocysts are fairly uniform. Approximately 50% of the injected trypan blue can be expected to disappear from the serum within two

PLACENTAL TRANSFER OF AZO DYES 751

hours after intravenous injection (Gregerson, '43). The rate of disappearance from the serum is apparently related to the strength of the bonds between the dye and the serum albumin molecule. This strength varies among the different dyes and probably accounts for the greater maternal sera and blastocyst levels in the Congo red-injected animals (tables 1 and 2).

Embryological differentiation of the major systems in the rabbit takes place between the 7th and 10th days of gestation (Waterman, '43). Thus trypan blue was present in the blastocyst fluid of the second group of experimental animals (table 3) during these critical stages of embryogenesis. This evidence tends to support the hypothesis that trypan blue has a direct toxic effect on the embryo proper.

The decline, after the ninth day of gestation, in the permeability of the rabbit blastocyst to maternal albumin and fibrinogen, homologous and heterologous gamma globulin (Brambell, '51), and to trypan blue and Congo red, is quite definite and consistent. Two possible explanations for this decrease in permeability should be considered.

First, there is the possibility that the developing yoll- sac splanchnopleure begins to take on its selective and absorptive functions at the ninth day, and removes foreign materials from the blastocyst fluid at increasing rates from then on. To examine this possibility, rabbits were killed at gestation days 10,12,14 and 16, approximately 12 hours after a subcutaneous injection of trypan blue. The earliest that the dye could be visually detected within the endoderm cells of the developing splanchnopleure was on day 16. It would be expected that, if this membrane were responsible for the removal of the dye from the blastocyst fluid after the ninth day of gestation, it would certainly concentrate the dye and appear to be deeply stained as early as the ninth o r tenth day. By the 16th day of gestation in the rabbit, the yolk sac is completely inverted and lies in immediate apposition to the uterine epithelium (Morris, '50).

TA

BL

E 3

Eff

ect o

f tr

ypan

blu

e on

rab

bit

embr

yos

-

DA

Y O

F

PR

EG

. IN

J.

WT

. K

U

CO

LO

R

NO

.

\

DA

Y

AM

T.

yz,f'

DY

E'

DY

E

KIL

LE

D

MG

INJ.

109

Whi

te

3.6

1 n

J 5 7

110

Bro

wn

4.85

104

Gra

y 5.

0 -7

1 7

106

Whi

te

2.6

6 7 8

119

Gra

y 2.

1 7 8 9

50

50

95

100

100

100

100

100

150

150

150 50

75

75

70

100

100

295

550

300

200

270

17

14

16

16

16

NO

.OR

N

O.O

F

LIV

ING

R

ESO

RP

, E

MB

RY

OS

SIT

ES

R

ES

UL

TS

1 5

1 -w

ell

pres

erve

d em

bryo

of

a ge

stat

ion

age

of

abou

t 12

da

ys - gr

ossl

y no

rmal

. 8 w 8 E W

of f

ourt

h ve

ntri

cle.

M

2 3

1 - d

efec

t of

hea

d - di

lata

tion

' 4

1 - gr

ossl

y no

rmal

.

11

0 1 - sm

all

ence

phal

ocoe

le.

10 - gr

ossl

y no

rmal

.

5 2

4 -c

ysti

c st

ruct

ure

on a

nter

ior

thor

acic

wal

l. 1 - ab

norm

al b

ody

twis

ting

. 1 - gr

ossl

y no

rmal

.

8 1

8 - al

l gr

ossl

y no

rmal

.


I I I I r l r l * m

I m

m m 0

0 W QJ

W rl W rl

0 0 cy

0 0 m

0 cy El

0 0 0 0 0 0

rlrlrl s m t - 0 0 0

t - m m a t - m

0 0.1 0.1 rl El rl

O M M In L. t-

m m o r l r l 0 . 1

2

h

a z

m 0 rl

1 M

0 0 0 m o t - rl

W t - m

m c\1 ri

4-

2 2

k.i 1

h m M

cy

m

0 3

W rl

0 e 0.1

0 0 0 o t - t - r(

w t - 0 0

x

m Y .3

E

W cy rl

- ii

EiJ

FI h m M

I M

*

M

W rl

0 0 m

0 0 0 0 0.1d

.I

t-t-

I

s

h

d m 0.1 rl

d Y

u 0 Y

FI

.3

.3 z 3 B %

m

CJ

m .(

754 VEBGIL H. FERM

The other explanation for the decrease in permeability of the blastocyst is based on more theoretical grounds arid would be difficult to prove experimentally. This hypothetical explanation is that basic changes in permeability occur when the definitive hemotrophic phase of embryonic nutrition is established. It is quite apparent that the free egg, morula, and the blastocyst are dependent upon the intrauterine environment for their nutritional demands. One indication of this early “histotrophic” phase of nutrition is the rapid expansion of the blastocyst cavity resulting from the considerable dif- fusion of intrauterine material into this cavity. The membrane making up most of the absorptive surface at this time is the bilaminar omphalopleure.

During the ninth day of gestation in the rabbit, there is the first direct contact of the embryonic trophoblast with the maternal blood (Mossman, ’26). This takes place in the region of the placental cotyledons or cctoplacenta and is ac- complished hy the formation of surface sinuses by rupture of the maternal surface capillaries. At this same time the sinus terminalis and many blood islands have formed on the yolk sac and, within a few hours, the allantoic niesentoic mesen- chynie containing fetal blood vessels also begins to spread over the inner surface of the trophoblast. Beginning at the ninth day then, there is a rapid transition from the histotrophic stage of nutrition to the more definitive hemotropliic type. From here on, the embryonic tissue can afford to be, and of necessity must be, more selective as to what it admits into its environment. The function of the developing definitive placenta becomes not unlike that of the adult renal epithelium which has the capacity to excrete waste materials and selec- tively absorb materials of value. There is a gradual reduc- tion in the functional role of the bilaminar omphalopleure and it begins to disappear, so that by the 15th day of gestation there remains only a small fringe of the original structure (Sansom, ’27).

It is paradoxical that the period of greatest permeability of the blastocyst should correspond with the period of greatest

PLACENTAL TRANSFER OF AZO DYES 755

susceptibility of the differentiating embryo to abnormal en- vironmental influences. Only after the definitive hemotrophic phase of embryonic nutrition has become established, does it appear that the extra-embryonic membranes begin to “pro- tect ” the embryonic tissues proper.

SUMMARY

1. Trypan blue and Congo red appear in the yolk sac fluid of the rabbit within two hours after the intravenous injection of the pregnant doe. This permeability declines rapidly after the ninth day of gestation.

Trypan blue is a teratogenic agent on rabbit embryos when injected into the mother prior to the ninth day of gestation.

The suggestion is made that the permeability of the rabbit blastocyst to azo dyes, and other foreign substances, during the first nine days of gestation, is related to the histotrophic phase of embryonic nutrition. Once the definitive hemotrophic phase of nutrition is established, the extra- embryonic tissues becomes more selective as to the type of materials admitted to the immediate embryonic environment.

It is concluded that the most likely explanation for the teratogenicity of trypan blue is that it has a direct toxic action on the differentiating embryonic tissue during critical stages of embryogenesis.

2.

3.

4.

ACKNOWLEDGMENT

I would like to express my sincere appreciation to Dr. H. W. Mossman f o r his suggestions and criticisms of this work. I would also like to acknowledge the generous help of Dr. W. H. McShan, of the Department of Zoology, in certain technical phases of the problem.

LITERATURE CITED

BRAMBELL, F. w. R., w. A. HENNINGS AND 11. HENDERSON

EVERETT, J. W.,

1951 Antibodies and Embryos. Univ. of London, The Athlone Press, 105 pp.

in the albino rat. J. Exp. Zool., 70: 243-285. 1935 Morphological and physiological studies of the placenta

756 VERGIL H. F E R M

FERM, V. H. 1955 Studies on the teratogenic activity of trypan blue. Ph. D. Thesis, University of Wisconsin.

GILLMAN, J., C. GILBERT, T. GILLMAN AND I. SPENCE 1948 A preliminary report on hydrocephalus, spina bifida and other congenital anomalies in the rat produced by trypan blue. So. Afr. J. Med. Sci., 13: 47-90.

GOLDMANN, E. E. 1909 Die aussere und innere Sekretion des gesunden und kranken Organismus im Lichte der ('vitalen Farbung". Beitr. z. klin. Chir., 6 4 : 192.

The disappearance of T-1824 and structurally related dyes from the blood stream. Am. J. Physiol., 138:

HAMBURGH, M. Malformations in mouse embryos induced by trypan blue. Nature, Lond., 269: 27.

HARM, H. Der Einfluss von Trypanblau and die Nachkommenschaft trach- tiger Kaninchen. Zeits. fur Naturforschung., 9b : 536-540.

MORRIS, B. 1950 The structure of the foetal yolk sac splanchnopleur of the rabbit. Quart. J. of Mic. Sci., 91: 237-249.

MOSSMANN, H. W. 1936 The rabbit placenta and the problem of placental transmission. Am. J. Anat., 37: 433-497.

SANSOM, G. S. 1927 The giant cells of the placenta of the rabbit. Proc. Roy. Soc. Lond. B. 102: 354-368.

SCHMIDZU, Y. 1922 On the permeability t o dyestuffs of the placenta of the albino rat and the white mouse. Amer. J. Physiol., 62: 202-224.

SUGIURA, K., M. L. CROSSLEY AND C. J. KENSLER 1945 The carcinogenicity of ethyl derivatives of p-aminoazobenzene. J. Nat. Can. Inst., 16:

WATERMAN, A. J. 1943 Studies of normal development of the New Zealand

WISLOCKI, G. B. 1920 Experimental studies on fetal absorption. Contrib. to

GREGERSEN, M. I., AND R. A. RAWSON 1943

698-707. 1952

1954

67-72.

white strain of rabbit. Amer. J. Anat., 72: 473-515.

Embry. Carnegie Inst. Wash., 11: 47-59.

PLATE 1

EXPLANATION OF FIGURES

1 Two 14 day old littermates from Rabbit # 110. Embryo a t the left is grossly norinal for this stage of development. Embryo a t the right has a large cystic cavity of the fourth ventricle. This cavity was markedly distended and filled with clear fluid prior to fixation. X 5.

2 Two 16 day old littermates from Rabbit # 104. Embryo at the left is grossly normal. Embryo a t the right has a small encephalocoele with sharp angulation of the head a t the cervical flexure. X 4.

3 Two 16 day old littermates from Rabbit #122. Embryo a t the left is grossly normal. The embryo a t the right has a marked curvature of the body with sharp angulation of the head. The loss of the normal contours of the brain and the large size of the head suggest that this might represent an early hydrocephalic stage.

A 16 day old embryo from Rabbit #106. The knob-like mass a t the upper pole of the photograph overlies the central axis of the body and presumably represents an early excencephalic stage. There is marked retardation of growth. x 9.

Three 16 day old littermates from Rabbit # 106. All of these had a moderately large cystic structure on the anterior thoracic wall. These structures were unilateral in one of the embryos and bilateral in the other two. Cross-see- tions through one of these revealed it to be a large dilated venous space which was filled with fetal blood. X 7.

A 16 day old embryo from Rabbit no. 122. This embryo has a large thin- walled cystic structure on the anterior thoracic wall. This sac ruptured easily and contained a darkish-brown material, presumably clotted fetal blood. There was no obvious gross connectioii to the thoracic or abdominal cavities. x 5.

X 4.

4

5

6

758

PLACENTAL TRANSFER OF A 2 0 DYES VERGIL H. FERM

PLATE 1

759

permeability of the rabbit blastocyst to trypan blue

Documents