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 μg/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 ·0μg BSA was accumulated. The highest concentration observed in blastocyst fluid was 338 μg/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 0· 08 % 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.c.), which suggests that the unimplanted blastocyst is capable of discriminating between native and foreign serum albumin.

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 blasto-cyst 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.

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.

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 −20 °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 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 μl 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.

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

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
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
Table 2.

Concentration of bovine serum albumin (BSA) in blastocyst fluid after intra-uterine injection of rabbits at 5 days post coitum

Concentration of bovine serum albumin (BSA) in blastocyst fluid after intra-uterine injection of rabbits at 5 days post coitum
Concentration of bovine serum albumin (BSA) in blastocyst fluid after intra-uterine injection of rabbits at 5 days post coitum

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 °/0 CO2, capped and placed on a rev/min rotator in a 37 °C incubator. Blastocysts usually adhered to a spot on the inside of the vial and were sub-merged 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.

Table 3.

Concentration of bovine serum albumin (BSA) in blastocyst fluid after in vitro cultivation for 24 h

Concentration of bovine serum albumin (BSA) in blastocyst fluid after in vitro cultivation for 24 h
Concentration of bovine serum albumin (BSA) in blastocyst fluid after in vitro cultivation for 24 h

Estimation of BSA

The concentration of BSA in serum, uterine and blastocyst fluids and blastocyst rinses was estimated by a radial immunodiffusion test using a rabbit anti-serum 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 μg BSA per ml, with 3–4 % error. Fluids with less than 20 μg/ml also gave precipitin rings, but since the error was larger, these results are recorded below as < 20 μg/ml. When no rings were observed, they are referred to as negative.

Rabbits given intravenous injection

Dutch rabbits at 5 days p.c. were given 100 or 200 mg/kg doses of BSA in-travenously. 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 tested - 19 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 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 con-centrations 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 maternal circulation, since < 20 to 27 μg/ml 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 μg/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 μg/ml. The blastocyst fluid in these four cases had 71–176 μg/ml of BSA.

Passage of BSA into blastocyst fluid occurs fairly rapidly, since 2 and 3 h after injection 244 and 176 μg/ml were recorded in this fluid (rabbits 66 and 94, Table 2). This means an accumulation of 0·235–0·258 μg 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 μ1 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 μg 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 μ1; 3·06–31·0μ1; 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 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 μg/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 (20–338μg/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). 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.

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), 200μ 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.

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), 200μ 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.

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 al. 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, Kühnel & Beier, 1970) and its Sephadex filtration profile (Krishnan & Daniel, 1967). Other protein fractions such as prealbumin, albumin, uterine β -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 μ g accumulates in 2–3 h, producing concentrations of 176–244μ 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 1·37μ 1 (range, 0·48–3·16μ 1) and 11·6μ 1 (range, 0·58–31·54μ 1). 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 μg 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 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 μg of rabbit albumin accumulates during this 24 h (5–6 days p.c.). Blastocysts exposed to BSA in vivo accumulated 0·25–2·0μg 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 sub-stances 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 7μ5-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 fragments. (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.

Adams
,
C. E.
,
Hay
,
M. F.
&
Lutwak-Mann
,
C.
(
1961
).
The action of various agents upon the rabbit embryo
.
J. Embryol. exp. Morph
.
9
,
468
491
.
Beier
,
H. M.
(
1970
).
Protein patterns of endometrial secretion in the rabbit
.
In Ovo-Implantation, Human Gonadotropins and Prolactin
, pp.
157
163
.
Basel, Munich, New York
:
Karger
.
Brambell
,
F. W. R.
(
1970
).
The Transmission of Passive Immunity From Mother to Young
.
Amsterdam
:
North-Holland Publ. Co
.
Brambell
,
F. W. R.
,
Hemmings
,
W. A.
&
Henderson
,
M.
(
1951
).
Antibodies and Embryos
.
London
:
Athlone Press
.
Brambell
,
F. W. R.
,
Hemmings
,
W. A.
&
Rowlands
,
W. T.
(
1948
).
The passage of antibodies from the maternal circulation into the embryo in rabbits
.
Proc. R. Soc. Lond. B
135
,
390
403
.
Brambell
,
F. W. R.
&
Mills
,
I. H.
(
1946
).
Presence of fibrinogen in the yolk sac content of rabbits
.
Nature, Lond
.
158
,
24
.
Crutchfield
,
F. L.
&
Kulangara
,
A. C.
(
1973
).
Passage of bovine serum albumin from the mother to rabbit blastocysts. I. Passage from the circulation to uterine lumen
.
J. Embryol. exp. Morph
.
30
,
459
469
.
Daniel
,
J. C.
Jr.
(
1964
).
Early growth of rabbit trophoblast
.
Am. Nat
.
48
,
85
98
.
Ehrlich
,
P.
(
1892
).
Über Immunität durch Vererbung und Säugung
.
Zeit. Hyg. InfektKrankh
.
12
,
183
203
.
Francis
,
M. D.
&
Winnick
,
T.
(
1953
).
Studies on the pathway of protein synthesis in tissue culture
.
J. biol. Chem
.
202
,
273
289
.
Glass
,
L. E.
(
1963
).
Transfer of native and foreign serum antigens to oviducal mouse eggs
.
Am. Zool
.
3
,
135
156
.
Gornall
,
A. G.
,
Bardawill
,
C. J.
&
David
,
M. M.
(
1949
).
Determination of serum proteins by means of the biuret reaction
.
J. biol. Chem
.
177
,
751
766
.
Hafez
,
E. S. E.
(
1971
).
Some maternal factors affecting physico-chemical properties of blastocysts
.
In Biology of the Blastocyst
, pp.
139
191
(ed.
R. J.
Blandau
).
Chicago, London
:
University of Chicago Press
.
Hafez
,
E. S. E.
&
Sugawara
,
S.
(
1968
).
Maternal effects on some biochemical characteristics of the blastocyst in the domestic rabbit
.
J. Morph
.
124
,
133
142
.
Hanan
,
R.
&
Oyama
,
J.
(
1954
).
Inhibition of antibody formation in mature rabbits by contact with the antigen at an early age
.
J. Immun
.
73
,
49
53
.
Humphrey
,
J. H.
&
Turk
,
J. L.
(
1961
).
Immunological unresponsiveness in guinea pigs. I. Immunological unresponsiveness to heterologous serum proteins
.
Immunology
4
,
301
309
.
Knobil
,
E.
&
Josimovich
,
J. B.
(
1959
).
Placental transfer of thyrotropic hormone, thyroxine, triiodothyronine and insulin in the rat
.
Ann. N. Y. Acad. Sci
.
75
,
895
904
.
Krishnan
,
R. S.
& DANIEL,
J. C.
Jr
. (
1967
).
‘Blastokinin’: inducer and regulator of blasto-cyst development in the rabbit uterus
.
Science, N. Y
.
158
,
490
492
.
Kulangara
,
A. C.
(
1972
).
Volume and protein concentration of rabbit uterine fluid
.
J. Reprod. Fert
.
28
,
419
425
.
Kulangara
,
A. C.
&
Sellers
,
M. I.
(
1959
).
Passage of bacteriophages from mother to foetus in the rat
.
Proc. Soc. exp. Biol. Med
.
101
,
207
211
.
Levine
,
P.
,
Burnham
,
L.
,
Katzin
,
E. M.
&
Vogel
,
P.
(
1941
).
The role of isoimmunization in the pathogenesis of erythroblastosis fetalis
.
Amer. J. Obstet. Gynec
.
42
,
925
937
.
Lutwak-Mann
,
C.
(
1962
).
Glucose, lactic acid and bicarbonate in rabbit blastocyst fluid
.
Nature, Lond
.
193
,
653
654
.
Lutwak-Mann
,
C.
(
1965
).
Experimental embryopathy as a tool in research on animal reproduction
.
In Embryopathie Activity of Drugs
(ed.
J. M.
Robson
,
F.
Sullivan
&
R. L.
Smith
), pp.
138
151
.
London
:
J. and A. Churchill
.
Lutwak-Mann
,
C.
,
Boursnell
,
J. C.
&
Bennett
,
J. P.
(
1960
).
Blastocyst-uterine relationships: uptake of radioactive ions by the early rabbit embryo and its environment
.
J. Reprod. Fert
.
1
,
169
185
.
Lutwak-Mann
,
C.
&
Hay
,
M. F.
(
1965
).
Maternally transmitted embryotropic agents
.
In Agents Affecting Fertility
(ed.
C. R.
Austin
&
J. S.
Perry
), pp.
261
274
.
London
:
J. and A. Churchill
.
Petry
,
G.
,
Kühnel
,
W.
&
Beier
,
H. M.
(
1970
).
Studies on hormonal regulation of pre-implantation stages in pregnancy. I. Histological, topohistochemical and biochemical studies in the normal rabbit uterus
.
Cytobiologie
.
2
,
1
32
.
Schechtman
,
A. M.
(
1956
).
Uptake and transfer of macromolecules by cells with special reference to growth and development
.
Int. Rev. Cytol
.
5
,
303
322
.
Schwick
,
H. G.
(
1965
).
Chemisch-etwicklungsphysiologische Beziehungen von Uterus zu Blastocyste des Kaninchens Oryctolagus cuniculus. Wilhelm Roux Arch. EntwMech. Org
.
156
,
283
343
.
Sieber
,
S. M.
&
Fabro
,
S.
(
1971
).
Identification of drugs in the preimplantation blastocyst and in the plasma, uterine secretion and urine of the pregnant rabbit
.
J. Pharmac. exp. Ther
.
176
,
65
75
.
Smith
,
A. E. S.
&
Schechtman
,
A. M.
(
1962
).
Significance of the rabbit yolk sac. A study of the passage of heterologous proteins from mother to embryo
.
Devi Biol
.
4
,
339
360
.
Sugawara
,
S.
&
Hafez
,
E. S. E.
(
1967
).
Electrophoretic patterns of proteins in the blasto-coelic fluid of the rabbit following ovariectomy
.
Anat. Rec
.
158
,
115
119
.