/. Embryo/, exp. Morph. Vol. 30, 2, pp. 471-482, 1973
Printed in Great Britain
471
Passage of bovine serum albumin from the mother
to rabbit blastocysts
II. Passage from uterine lumen to blastocyst fluid
By ABRAHAM C. KULANGARA 1 AND
FLOY L. CRUTCHFIELD 1
From the Department of Anatomy, The Medical College of
Pennsylvania
SUMMARY
Passage of bovine serum albumin (BSA) into unimplanted rabbit blastocysts was studied
using intravenous or intrauterine injection of the mother and in vitro cultivation of 5-day
blastocysts. BSA concentration in blastocyst fluid was measured using a quantitative radial
immunodiffusion method and confirmed by double diffusion in agar.
Intravenous injection of up to 200 mg/kg body weight produced a mean concentration of
106 /<g/ml in uterine fluid and the blastocyst fluid was mostly negative. In vitro cultivation
with 1-20 mg BSA/ml resulted in appreciable passage which was confirmed by intra-uterine
injection. BSA appeared in blastocyst fluid after 2-3 h of exposure. After 24 h, 0-25-2-0/<g
BSA was accumulated. The highest concentration observed in blastocyst fluid was 338 /*g/ml
and this was about 1-7 % of the surrounding concentration. Combined with previous results
on passage from plasma into uterine fluid, it could be calculated that in 24 h the blastocyst
fluid acquired about 008 % of the maternal serum concentration of BSA.
The accumulation of BSA by the blastocyst is about half that of rabbit albumin in the
same period (5-6 days p.a), which suggests that the unimplanted blastocyst is capable of
discriminating between native and foreign serum albumin.
INTRODUCTION
Three days after mating, the rabbit blastocyst enters the uterus, where it
leads a free living existence bathed by uterine fluid, until about 6-5 days, when
it begins to implant on the uterine endometrium. During this period the blastocyst uses the uterine fluid for metabolic exchange and could be adversely affected
by substances in the fluid. Ions and small molecules injected into the mother
have been detected in uterine and blastocyst fluids (Lutwak-Mann, Boursnell
& Bennett, 1960; Lutwak-Mann, 1962). Larger molecules such as proteins,
introduced into the mother, have been found in mouse embryos in the oviduct
(Glass, 1963) and rabbit blastocysts after implantation (Brambell, Hemmings
& Rowlands, 1948; Smith & Schechtman, 1962). However, there are no quantitative data on passage of proteins into the unimplanted blastocyst.
1
Authors" address: Department of Anatomy, The Medical College of Pennsylvania, 3300
Henry Avenue, Philadelphia, Pa. 19129, U.S.A.
472
A. C. KULANGARA AND F. L. CRUTCHFIELD
Proteins from the maternal blood must enter the uterine fluid before they
can pass into free living blastocysts. Therefore, if studies of overall passage from
mother to embryo show no tracer in the blastocyst, it could be due to a limitation on passage from blood to uterine fluid rather than impermeability of the
blastocyst. Our studies indicate such a restriction. Albumins from the maternal
circulation are maintained in uterine fluid at about a fifth of the serum level
(Crutchfield & Kulangara, 1973). Also, if a difference is observed when overall
passage of two proteins is compared, it is impossible to decide whether selection
between the proteins occurred at the maternal or embryonic level (Smith &
Schechtman, 1962) or both. Our results suggest that there is no selection between rabbit and bovine albumin during passage from blood to uterine fluid
(Crutchfield & Kulangara, 1973).
The study of passage into the blastocyst is important from considerations such
as teratogenicity and contraception. There is considerable work on the embryotoxic effect of a variety of substances (Adams, Hay & Lutwak-Mann, 1961;
Lutwak-Mann, 1965), but the blastocysts were examined histologically as flat
mounts and concentration of the substances in blastocyst fluid was unknown.
Recently, Sieber & Fabro (1971) have measured the uptake of caffeine, barbital
and dextran by 6-day blastocysts in vitro.
We used bovine serum albumin in a quantitative study and the results on its
passage from blood to uterine fluid are reported in the preceding paper. Passage
into the blastocyst was studied by three methods, namely intravenous or intrauterine injection of the mother and in vitro cultivation of blastocysts with the
tracer protein added to the medium. These results are presented below.
MATERIALS AND METHODS
Dutch belted rabbits (1-8-3-0 kg body weight) were used for intravenous
injections and to obtain blastocysts for in vitro cultivation, whereas New Zealand
white rabbits (2-5-4-0 kg body weight) were used for intra-uterine injections.
All were obtained from commercial sources and housed in separate cages for
about two weeks before use.
Bovine serum albumin (BSA) from Mann Laboratories was made up as a
roughly 15 % solution in 0-9 % NaCl and filtered using a Millipore filter. Its
protein concentration was estimated by the biuret test (Gornall, Bardawill &
David, 1949) and the solution frozen in 5 ml lots and stored at - 2 0 °C.
In experiments using intravenous injection of BSA, Dutch belted does were
mated and at 5 days post coitum (p.c) they were given single injections of 100
or 200 mg/kg by ear vein. Five minutes later a sample of blood was taken from
the uninjected ear. At various times after injection the rabbits were anesthetized
and opened. Uterine fluid was obtained by direct aspiration through a capillary
tube inserted through the cervix. A sample of maternal blood was also collected
at this time. The uterine horns were then opened under a dissecting microscope
Passage from uterine lumen to blastocyst
fluid
473
and the blastocysts transferred to a dish of mineral oil, one at a time. The
diameter (both axes, if ellipsoid) of each blastocyst was measured with an ocular
micrometer. Next, it was rinsed thrice, with about 50 /i\ of saline each time, and
the last rinse was saved for testing. Finally, the fluid inside the blastocyst was
aspirated with a capillary tube drawn to a fine point.
Intra-uterine injection
In many cases, mated Dutch belted rabbits showed no blastocysts when
opened and therefore, intra-uterine injections were done in New Zealand white
rabbits, which showed a much better conception rate. Mated does at 5 days
p.c. were anesthetized and the left utero-tubal junction was exteriorized through
an incision 1 in. long in the lower left abdomen. A ligature was placed loosely
around the oviduct, carefully excluding blood vessels and a small incision made
in the oviduct, proximal to the ligature. The tip of a polyethylene tube (1-5 mm
O.D.) was passed through the incision into the anterior end of the uterine horn.
BSA solutions of different concentrations and volumes (Table 2) were injected
through the tube, after which the tube was withdrawn and the ligature tied
around the oviduct. The viscera were returned to the abdominal cavity, the body
wall was sutured and the animal allowed to recover. At different times after
injection, the rabbit was anesthetized and opened. Attempts were made to
obtain uterine fluid from both horns. Blastocysts were collected, measured,
rinsed and sampled as described above. Maternal blood was also collected at
this time.
In vitro cultivation
Blastocysts were obtained from Dutch belted rabbits at 5 days p.c, their
diameters measured and one or two were placed in a 5 ml glass vial containing
1 ml of Ham's F 10 medium. The vials were gassed with a mixture of 95 % O2
and 5 % CO2, capped and placed on a 3 rev/min rotator in a 37 °C incubator.
Blastocysts usually adhered to a spot on the inside of the vial and were submerged in medium only part of the time, since the rotator turned the vials end
over end. Different amounts of BSA were added to the medium (Table 3).
After 24 h the blastocysts were removed, their diameters measured and the
degree of development noted. They were then rinsed and their fluids withdrawn.
Judged by expansion and the progress of embryonic development, this method
of cultivation gave normal results for 48 h.
Estimation of BSA
The concentration of BSA in serum, uterine and blastocyst fluids and blastocyst rinses was estimated by a radial immunodififusion test using a rabbit antiserum against BSA. The test and its standardization were described previously
(Crutchfield & Kulangara, 1973). It was capable of measuring a minimum of
40 ng in samples containing at least 20 /<g BSA per ml, with 3-4 % error. Fluids
474
A. C. KULANGARA AND F. L. CRUTCHFIELD
Table 1. Concentration of bovine serum albumin (BSA) in uterine and blastocyst
fluids at various times after intravenous injection of pregnant rabbits at 5 days
post coitum
Sampled at
BSA in
(h after
BSA in uterine fluid blastocyst fluid
injection)
Og/ml)
C"g/ml)
Rabbits given 100 mg BSA/kg body weight
12
neg (1/3)
neg (1)
24
neg (1/4)
neg (7)
24
30
neg (3)
48
< 20; 59*
neg; < 20f (2)
48
neg; < 20
neg (2); < 20f (4)
Rabbits given 200 mg BSA/kg body weight
12
< 20
neg (7)
24
69; 87
neg (3)
24
109; 160
neg; < 20 (2)
36
92; 92
< 20 (2)
* Empty horn of a unilaterally pregnant rabbit.
f These blastocysts were sampled in situ without rinsing, since they had begun to implant,
neg = negative; neg (1/3) = a 1/3 dilution of the sample was tested and was negative.
Numbers in parentheses in the last column are numbers of blastocysts sampled.
with less than 20 /<g/ml also gave precipitin rings, but since the error was larger,
these results are recorded below as < 20 /£g/ml. When no rings were observed,
they are referred to as negative.
RESULTS
Rabbits given intravenous injection
Dutch rabbits at 5 days p.c. were given 100 or 200 mg/kg doses of BSA intravenously. At various times after this, blood and uterine and blastocyst fluids
were obtained. BSA concentrations in serum and uterine fluid were reported
earlier (Crutchfield & Kulangara, 1973) and concentrations in blastocyst fluid
are given in Table 1. Thirty-three samples of blastocyst fluid were tested19 from rabbits given 100 mg/kg and 14 from rabbits given 200 mg/kg doses.
All blastocyst rinses were negative, except two which showed a trace of BSA and
in both cases the blastocyst fluid was negative. Therefore, the rinsing in these
two cases must have been inadequate.
In the 100 mg/kg series, 14 out of 19 blastocysts showed no BSA in their
fluids. The remaining five which showed a trace were sampled in situ without
rinsing because they had begun to implant, at 48 h after injection. In the
200 mg/kg series, 11 out of 14 samples were negative. Three blastocysts showed
a trace of BSA, but their trophoblasts had withdrawn from the lemmas and
were perhaps damaged. Thus passage into blastocyst fluid did not occur, even
475
Passage from uterine lumen to blastocyst fluid
Table 2. Concentration of bovine serum albumin (BSA) in blastocyst
fluid after infra-uterine injection of rabbits at 5 days post coitum
Blastocysts from
un injected horn
Injected solution
Volume
Rabbit
number
(ml)
Sampled
at(h
BSA
cone,
after
(mg/ml) inject.)
Volume
range
(/*!)
BSA in
fluid
Og/ml)
94
95
96
0033
0033
0033
150
150
150
3
6
16
1-21-1-43
1-00-1-66
1415-2019
neg (2)*
neg (7)*
neg (4)
107
0033
150
24
4-70-18-51
neg (3)
119
10
5
24
20-92-27-12
neg (4)
life
0-25
20
24
27-22
neg (1)
106
01
50
24
1113-1308
neg (5)
50
100
150
22
22
2
12-56-16-44
10-98-19-63
1-05-1-09
neg (4)
neg (2)
neg (2)*
44
45
66
005
01
0067
Blastocysts from
injected horn
Volume
109-1-65
1-12-1-78
11-92
15-65
16-24
17-47
4-52
4-52
602
11-44
2101
26-64
21-25
24-73
10-23
14-70
15-85
16-44
13-26
1-23-2-69
0-68-1-37
BSA in
fluid
(/ig/ml)
176 (4)*
188 (3)*
148
140
137
85
289
119
46
127
73
69
53
48
107
71
85
123
19
242 (3)*
244 (6)*
The BSA concentrations in maternal serum at sampling in rabbits 94, 95, 96, 107, 119,
118, 106, 44, 45 and 66 were 27, < 20, < 20, 22, < 20, < 20, < 20, neg, neg, and 21 /tg/ml
respectively.
Values in parentheses are numbers of blastocysts tested; fluids were pooled from those
marked with *.
with a maternal dose of 200 mg/kg. The mean BSA concentration in uterine
fluid, at the 24 h peak in these animals, was 106/^g/ml. Higher ambient concentrations seem to be required for significant passage to occur into blastocysts.
Passage after intra-uterine injection
High concentrations of BSA in the vicinity of blastocysts were achieved by
this method. Ten New Zealand white rabbits at 5 days p.c. were given injections
of various doses of BSA into their left uterine lumens. The right horn in each
case served as control. Concentrations observed in maternal serum and blastocyst fluid are given in Table 2. BSA passed from the uterine lumen into the
31
EM B 30
476
A. C. KULANGARA AND F. L. CRUTCHFIELD
maternal circulation, since < 20 to 27 jng/m\ were observed in the serum after
intra-uterine injection. Two of these sera were obtained 2 and 3 h after injection.
Assuming a plasma volume of 45 ml/kg body weight, roughly 3 mg BSA had
passed into plasma, which is one third to over half the amount injected into the
uterine lumen.
Blastocysts in the injected horns always showed BSA in their fluids, ranging
from 19 to 289/tg/ml, but all samples from uninjected horns were negative.
The rinses were all negative, except 3 which showed a trace of BSA and one about
28 /tg/ml. The blastocyst fluid in these four cases had 71-176 /<g/ml of BSA.
Passage of BSA into blastocyst fluid occurs fairly rapidly, since 2 and 3 h
after injection 244 and 176 /tg/ml were recorded in this fluid (rabbits 66 and 94,
Table 2). This means an accumulation of 0-235-0-258 fig BSA in 2-3 h. The
concentration of BSA to which these blastocysts were exposed is uncertain. The
injected solutions were 150mg/ml, but attempts to obtain uterine fluid at
sampling gave highly variable results. Assuming a uterine fluid volume of 16 fi\
at 5 days p.c. (Kulangara, 1972), they were exposed to 100-120 mg/ml at the
start.
Some experiments were designed to test the effects of amount and concentration of BSA injected and of the time after injection. However, blastocysts varied
tremendously in volume, even when they were from the same uterine horn. Also,
the erratic expansion of blastocysts during the experiment made the results
difficult to interpret. Nevertheless, some conclusions may be drawn if, instead
of BSA concentrations which are volume-dependent, accumulated amounts of
BSA in blastocyst fluid are compared.
Rabbits 94, 95, 96 and 107 received the same dose, but their blastocysts were
sampled 3, 6, 16 and 24 h after injection and the average contents were 0-257,
0-261,1-916 and 0-707 fig BSA respectively. It looks as if blastocysts from rabbit
96 had accumulated more BSA in a shorter time than those from rabbit 107,
but a comparison of their volumes (Table 2) with the mean and range for 6 day
blastocysts (11-5/4; 3-06-31-0/tl; Daniel, 1964) shows that the former had expanded normally or better during the experiment, whereas the latter had not.
Thus, these results indicate greater accumulation of BSA with time.
Rabbits 119, 118, 106 and 107 were given appropriate volumes of 5, 20, 50
and 150 mg/ml solutions to constitute a 5 mg dose to each and their blastocysts
were sampled 24 h later. The amount and concentration of BSA observed in
blastocyst fluid do not seem to be related to the concentration of injected solution. Since volumes ranging from 0-033 to 1-0 ml were injected, it may be
expected that blastocysts in different rabbits were not equally well exposed to
BSA, especially when the injected volume was small. Thus, the maximum difference between concentrations is among blastocysts of rabbit 107, which
received 0-033 ml. When observed blastocyst concentrations are converted to
percentage of BSA concentrations they were exposed to at the start, the mean
values are 1-79, 0-27, 0-22 and 0-15 % in rabbits 119, 118, 106 and 107 given
Passage from uterine lumen to blastocyst
fluid
411
Table 3. Concentration of bovine serum albumin (BSA) in
blastocyst fluid after in vitro cultivation for 24 h
BSA in
medium
(mg/ml)
1*
5
5
10
15
15
15
20
20*
Blastocyst volumes (/tl)
Initial
Terminal
6-47
8-91
0-91
1 66
0-92
0-87
0-74
110
0-69
2-50
316
2-30
22-35
43-34
34-32
3-12
3-33
717
303
2-82
4-53
1-24
10-48
1108
2-79
50-26
BSA in
blastocyst fluid
Og/ml)
neg
neg
20f
< 20
32|
106
74
44f
338
53
Each line of entry above refers to one blastocyst.
* These blastocysts were 6 days and the others were 5 days old at the beginning of
cultivation.
t Fluid from the two blastocysts pooled for assay.
1-0, 0-25, 0-1 and 0-033 ml respectively. Apparently, BSA passes better into
blastocysts exposed to larger volumes.
Individual blastocyst concentrations in this series of ten rabbits varies from
0-05 to 0-3 % of the injected solution, except in rabbit 119 where it is 1-4-2-5 %.
The maximum concentration observed in blastocyst fluid is 289 /*g/ml. Comparison among blastocysts of different sizes from the same uterine horn shows that
the larger ones have a lower BSA concentration and vice versa (rabbits 96,
107, 118 and 119), with the exception of two in rabbit 106.
Passage in vitro
Thirteen blastocysts were cultivated as described earlier for about 24 h with
different amounts of BSA. Three were 6 days and 10 were 5 days p.c. at the
beginning of cultivation. In contrast to the intra-uterine experiments, there
seems to be a direct relationship between the concentration of BSA in the
culture medium and in the blastocyst fluid (Table 3). Two 6-day-old blastocysts
exposed to 1 mg/ml showed no BSA in their fluid. But with higher concentration
in the medium, BSA was detectable in blastocysts (20-338/tg/ml). It may be
pointed out that blastocysts exposed in vivo to 5 mg/ml (Table 2) achieved
4-6 times the concentration in those exposed to the same dose in vitro (Table 3).
31-2
478
A. C. KULANGARA AND F. L. CRUTCHFIELD
Fig. 1. Agar diffusion pattern of normal 6-day blastocyst fluid (right well) and fluid
collected after 24 h exposure of a 5-day blastocyst to 10 mg/ml BSA in vitro (left
well). Sheep antiserum against normal rabbit serum (top well), 200/^g/ml BSA
(central well) and rabbit antiserum against BSA (bottom well). The most dense band,
due to BSA, is absent in normal blastocyst fluid and present in fluid from blastocyst
exposed to BSA. At least four other precipitin bands due to normal rabbit serum
proteins are visible, one confluent with a protein in normal blastocyst fluid.
Breed differences may partly account for this, since blastocysts from Dutch
belted rabbits were used in vitro, but New Zealand white rabbits were used for
intra-uterine injections. Also, the conditions in vitro may not have been optimal.
The inverse relationship between blastocyst volume and BSA concentration,
observed after intra-uterine injections, is confirmed by results obtained in vitro
(Table 3). A blastocyst, exposed to 20 mg/ml BSA, which expanded least in
24 h, showed the highest concentration in this group. More generally, BSA in
blastocyst fluid was 0-27-0-67 % of the surrounding concentration.
Confirmation of BSA in blastocyst fluid
Representative samples of blastocyst fluid were tested by Ouchterlony type
double diffusion in agar (Fig. 1). Antiserum against BSA showed no lines in
fluid from normal blastocysts, but produced a precipitin line, which showed
reaction of identity with BSA, in fluid from blastocysts exposed to BSA. This
line was clearly different from lines due to rabbit proteins, detected by a sheep
antiserum against normal rabbit serum. Attempts were made to confirm the
identity by immunoelectrophoresis as well, but the level of BSA and available
volumes of samples were inadequate.
Passage from uterine lumen to blastocyst
fluid
479
DISCUSSION
It was Brambell & Mills (1946) who first showed the presence of a maternal
protein in fluid from implanted rabbit blastocysts. Subsequently, heterologous
antibodies and serum proteins injected into the mother have been shown to
appear in this fluid (Brambell et ah 1948; Smith & Schechtman, 1962). Glass
(1963) found immunofluorescence due to proteins injected into the mother in
oviducal stages of the mouse embryo. Recently, uteroglobin and/or blastokinin,
a protein secreted into the uterine lumen during 3-9 days gestation, has been
identified in 6-day blastocyst fluid by immunoelectrophoresis (Petry, Kuhnel
& Beier, 1970) and its Sephadex filtration profile (Krishnan & Daniel, 1967).
Other protein fractions such as prealbumin, albumin, uterine /?-globulin and
immunoglobulin G appear to be common between uterine and blastocyst fluids
(Schwick, 1965; Beier, 1970). The present studies clearly show that molecules
similar to BSA appear in fluid from unimplanted blastocysts, after the protein
was introduced into the mother.
These experiments have also obtained quantitative data, which enable characterization of such passage for the first time. BSA is hardly detectable in fluids of
blastocysts exposed to 1 mg/ml or less (Tables 1, 3), but fair amounts of it occur
in those exposed to 5 mg/ml or more (Tables 2, 3). It passes rapidly into blastocysts, since about 0-25 jug accumulates in 2-3 h, producing concentrations of
176-244//g/ml. Our efforts to study the kinetics and dose-dependence of passage
were frustrated by the enormous variability in size and the erratic expansion of
blastocysts. The mean volume of 5- and 6-day blastocysts in this series was
1-37 /4 (range, 0-48-3-16/d) and 11-6/d (range, 0-58-31-54 /d). These ranges
overlap and are somewhat more than those reported by others (Daniel, 1964).
Also, while some blastocysts expanded rapidly during the experiment, tripling
or quadrupling their volume, others hardly expanded at all (Table 3).
Nevertheless, the following estimate may be made from the results of intrauterine injection and in vitro cultivation. In cases where maximum passage was
recorded, BSA in blastocyst fluid was 1-7-1-8 % of the surrounding concentration. It was shown earlier that, 24 h after intravenous injection of the mother,
BSA in uterine fluid reaches 4-5 % of the serum level (Crutchfield & Kulangara,
1973). Thus, passage from uterine to blastocyst fluid is restricted even more
than passage from blood to uterine fluid. Combining these results to estimate
overall passage shows that after 24 h exposure, BSA in 6-day blastocyst fluid
reaches 0-08 % of the maternal serum level.
Entry of BSA into blastocyst fluid may be compared to the appearance of
rabbit albumin in this fluid. From the data of Hafez & Sugawara (1968), the
protein content of 5- and 6-day blastocyst fluid may be calculated as 1-92 and
20-4 //g respectively. All the protein at 5 days p.c. seems to be albumin (Hafez,
1971), but at 6 days p.c, albumin, uteroglobin and a broad globulin band are
the major components in this fluid (Beier, 1970; Hafez, 1971). Quantitative
480
A. C. KULANGARA AND F. L. CRUTCHFIELD
estimates of the amount of albumin at 6 days p.c. are not available, but it seems
to be roughly a fourth to a third of the total. Therefore, approximately 4 //g
of rabbit albumin accumulates during this 24 h (5-6 days p.c). Blastocysts
exposed to BSA in vivo accumulated 0-25-2-0 [ig in the same period (Table 2).
Apparently, rabbit albumin preferentially enters the blastocyst in greater quantities than BSA. It may be pointed out that rabbit albumin forms only 13 % of
uterine fluid proteins at 6 days p.c. (Beier, 1970), but constitutes about 25-33 %
of the proteins in blastocyst fluid. Thus, there seems to be a selection among
available homologous and between homologous and heterologous proteins by
the blastocyst wall for admission into the fluid.
Smith & Schechtman (1962) injected human or guinea-pig serum into rabbits
at 6 days p.c. and obtained titers of these proteins in yolk sac fluid of implanted
(9 days) blastocysts. They found about 2-4 times more human proteins than
guinea-pig proteins, except guinea-pig alpha globulin which was 11 times more
than the human. This selection among proteins may have occurred before or
after implantation or both. Also, as the authors cautioned, there may have been
selection at the maternal level during passage from blood to uterine fluid. Our
studies indicate no selection at the maternal level in non-pregnant rabbits
(Crutchfield & Kulangara, 1973). The present experimental design also narrows
selectivity to the bilaminar wall of the unimplanted blastocyst.
The mammalian embryo was once thought to be isolated from maternal
metabolism, except for exchange of gases and small molecules. Discovery of the
mechanism of erythroblastosis fetalis (Levine, Burnham, Katzin & Vogel, 1941)
and the demonstration that bacteriophages can pass from mother to fetus (Kulangara & Sellers, 1959) have shown that the placental barrier is permeable to
proteins and larger particles. The recent human tragedy of thalidomide-induced
malformations emphasizes the perils of existence in utero. With different substances and at different stages of development, the embryo appears to be in
unique states, which are combinations of some measure of protection and vulnerability. Only quantitative and kinetic studies can characterize these states in
each case. Present experiments indicate that the 6-day blastocyst acquires no
more than 0-08 % of the serum level of BSA in 24 h. However, some substances
that enter the blastocyst fluid remain there long after they become undetectable
in maternal serum (Lutwak-Mann, 1962, 1965; Lutwak-Mann & Hay, 1965), so
that even small concentrations could prove deleterious. After implantation, proteins in blastocyst fluid increase abruptly (Hafez & Sugawara, 1968). Sugawara
& Hafez (1967) also showed that appearance of protein in fluid of 7- and
7-5-day blastocysts was prevented by ovariectomy 1 day earlier and that progesterone corrected this effect to some extent.
Several major roles have been suggested for proteins normally passed from
the mother to the fetus. (1) Maternally derived proteins may serve as the
source of not only amino acids, but larger fragments of protein. Francis &
Winnick (1953) showed that embryonic tissue in vitro utilized such large frag-
Passage from uterine lumen to blastocyst
fluid
481
ments. (2) Schechtman (1956) persuasively argued that the mammalian embryo
is dependent upon presynthesized macromolecules supplied by the mother, for
enzymes, templates and molecules necessary for normal embryonic development. Blastokinin (Krishnan & Daniel, 1967) is secreted by the mother just
before implantation and promotes blastocyst development. Perhaps other protein fractions are also involved in developmental processes (Beier, 1970). (3) Hormones, which have obvious functions, may be passed into the fetus (Knobil &
Josimovich, 1959). (4) Maternal antibodies may be beneficial or injurious.
Immunological protection of the newborn by antibodies from the mother
has been well documented since the classical experiments of Ehrlich (1892).
Brambell, Hemmings & Henderson (1951) suggested that antibodies help in
maintaining asepsis in the uterine lumen. Antibodies can also cause damage
and death due to hemolytic disease of the newborn in human and other species
(Brambell, 1970).
Heterologous protein passed into early developmental stages may play a
fundamental immunological role. Induction of tolerance by proteins introduced
from the mother into the fetus has been shown (Hanan & Oyama, 1954;
Humphrey & Turk, 1961), but it is not known whether tolerance can result from
proteins introduced into the blastocyst. Recognition of a foreign protein,
sufficient to discriminate against it, seems to be already present at the blastocyst
stage, although hemopoiesis has not yet begun. Further experiments, now under
way, are necessary to establish the mechanism and cellular basis of recognition
in the 5- to 6-day blastocyst. It would be highly significant to study the effect
of maternally introduced proteins on the future development of immunologic
function in the organism.
This work was supported by a grant from the Lalor Foundation. One of us (F.L. C.) was
the recipient of a predoctoral fellowship from the National Institutes of Health.
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{Received 23 January 1973, revised 21 February 1973)