1. A study of certain chemical components of the rabbit blastocyst was carried out using material available at 6, 7, 8, and up to 12 days after copulation. For chemical determinations unimplanted blastocysts were used whole; from the implanted blastocysts the fluid filling the interior was withdrawn by aspiration. The content of nitrogen, chlorides, Na, K, Mg, and Ca was determined. Glucose was identified in yolk-sac fluid and its concentration before and after implantation was determined.

  2. The passage from maternal blood into the blastocyst fluid was investigated, during a 30-60-minute interval following parenteral administration, of glucose, C. LUTWAK-MANN—PROPERTIES OF RABBIT BLASTOCYST 13 fructose, sucrose; sulphapyridine, thiocyanate. Under the experimental conditions of this study all these substances, except thiocyanate, passed freely into the fluid of the fully implanted blastocyst 8 days after mating. None, however, was able to penetrate into the unimplanted blastocysts. During the initial phase of implantation glucose entered the fluid more readily than either fructose or sucrose.

  3. In hyperglycaemic alloxan-diabetic rabbits the glucose concentration in the blastocyst fluid represented only a fraction of that found in the maternal blood.

  4. Data are presented for the distribution of glycogen and phosphorus in the rabbit uterus, –8 days after copulation, at the level of implantation, and in the interimplantation areas.

A Considerable, though far from complete, collection of data is available on the chemical composition of the mammalian embryo and foetal fluids in the more advanced stages of pregnancy. But the information on pre-implantation foetal physiology and biochemistry, or the stage immediately succeeding implantation, is still meagre. In the past research on the mammalian blastocyst has depended to a large extent, though not exclusively, upon microscopic methods of study. A notable departure in this respect was created by the investigations initiated by Brambell and his co-workers (Brambell, Hemmings, & Henderson, 1951); based on a combination of immunological and chemical methods, these experiments have provided a new and original approach to problems in this field. The sensitivity of the immunological reactions, coupled with elegant surgical technique, has made it possible to obtain results which have materially widened our understanding, especially of the protein transfer processes between the maternal body and the early embryo.

The present study to which this paper represents an introduction was undertaken in an endeavour (1) to increase our knowledge concerning the chemical composition of the inner milieu of the mammalian blastocyst, and (2) to examine the ability of certain relatively simple chemical substances introduced into the maternal blood-stream to reach the fluid filling the blastocyst cavity, before and immediately after implantation.

Experimental animals

Sixty-five adult does were used for the experiments; as a routine measure, all were allowed to pass through their first pregnancy and were mated for the actual experiment within a few days after parturition when showing signs of oestrous behaviour. The majority of the experiments were carried out 6, 7, and 8 days after copulation, but on a few occasions foetal fluids were also examined on the 10th and 12th day. A reference hereafter to ‘6-days-old’, ‘7-days-old’, &c., is meant to indicate the interval in days between copulation and the actual experiment.

In some experiments the does were superovulated by treatment with a gonadotrophin preparation, ‘Gonadophysin’, presented to the author by Dr. M. C. Chang, whose superovulation procedure (1948) was followed. To obtain the experimental material the animals were first injected intravenously with 05 ml./kg. of Nembutal, then thoroughly bled; blood and urine samples were collected in most experiments. The uterus was removed together with the ovarian tubes and ovaries; the number of the corpora lutea was recorded and compared with that of the embryos. On the whole, few discrepancies were noted, except in superovulated does in which the corpora lutea exceeded by 2−3 times the embryos found on the 7th or 8 th day. When ovaries were intended for chemical analysis, the fresh luteal tissue was excised and the ovarian tissue containing the follicles was used.

Blastocysts

Six days after mating all blastocysts in our rabbits were found lying free in the uterine cavity, their diameter varying from 1 to 3 mm. Generally, at this stage they were fairly evenly spaced, except in superovulated animals, but quite often their size varied markedly within the same uterine horn.

At 7 days the blastocysts in most of the does appeared implanted, but in a considerable proportion of animals (38 per cent, in this series) the implantation areas were relatively small and the blastocysts were still found lying free, ranging from 3 to 4 mm. in diameter. Actually, at this stage, external inspection usually permitted an approximate differentiation between implanting and non-implanted blastocysts: in the latter case, the implantation sites were not prominent and the blastocysts could be shifted from their positions by gentle pressure upon the uterine wall. The unimplanted blastocysts obtained at 6 and 7 days appeared as transparent colourless spheres; whereas the younger blastocysts were resilient and appeared perfectly round, many of the larger ones removed 1 day later flattened out distinctly when deprived of the support of the uterine wall. The 6-day blastocysts could be handled with some degree of impunity, but the later ones were very delicate and much care had to be devoted to their collection from the uterus preparatory to transfer into chemical glassware.

The surface of the blastocysts appeared to be covered with some material of considerable adhesive power, because when they were placed against the inside of the vertical wall of a glass test-tube they remained firmly in position and did not roll down even if the tube was tilted. It was found that the blastocyst membrane at this stage was remarkably resistant to the action of zinc hydroxide at 100° : when, for the purpose of glucose determination, the blastocysts were placed in a solution of Zn(0H)2 and boiled for 3 minutes, the membrane did not rupture and it required considerable mechanical pressure to pierce it with a pointed glass rod.

The unimplanted blastocysts were lifted from the longitudinally dissected uterus with a small sharp-edged stainless-steel spoon and used whole for chemical determinations, without rinsing, but after careful wiping of their surface with strips of filter-paper. On the other hand, the fluid filling the blastocyst cavity in implanted blastocysts at 7 and 8 days was collected by puncturing the uterine wall and aspirating with a glass syringe. From the blastocysts at 7 days 0·02–0·05 ml. could usually be collected in this manner per single blastocyst; 1 day later 0·4–0·6 ml. could easily be withdrawn. The fluid which filled the blastocyst cavity at 7 days was colourless, transparent, and did not clot on standing; its pH immediately upon withdrawal was roughly equal to that of maternal plasma, but within a few minutes the pH increased and reached a value of about 8 8. The fluid removed from blastocysts 8 days after mating was faintly yellowish and opalescent, and showed a distinct blue fluorescence in ultra-violet light; it clotted almost at once, owing to the presence at this stage of fibrin (Brambell, 1948). The latter property necessitated prompt pipetting, or the clot was allowed to form and was then broken up mechanically; after it retracted, the fluid portion was used for the experiments. The pH value of the 8-days-old fluid was 7·6−7·8, and did not change appreciably on standing. The difference in pH value and clotting ability between the 7- and 8-days-old blastocyst fluid was so striking that it alone provided a means of distinguishing these two stages of development. Furthermore, whereas upon the addition of trichloroacetic acid or ethanol, no precipitate formed in the 7-day fluid, a heavy precipitate invariably followed the addition of these reagents to the fluid collected one day later from the implanted blastocysts.

On the whole, it was convenient at 7 days to pool the fluid recovered from the blastocysts of any given doe; but on several occasions, especially in superovulated does with numerous embryos, or in others at that stage in which the blastocysts were well developed, sufficient material could be obtained from single blastocysts to permit comparative analyses, e.g. of the glucose content. The amount of fluid available from a single blastocyst a full 8 days after mating usually sufficed for most chemical determinations; otherwise pooled fluid was used. When intact, unimplanted blastocysts were used, they were collected in the bottom part of a conical graduated tube and the volume which they occupied was noted.

Dissection of uterine implantation and interimplantation sites

Several experiments were carried out to determine the glycogen content of the uterus (1) at the level of implantation, (2) in adjacent interimplantation portions, and (3) in the non-pregnant rabbit uterus. For this purpose the fluid was first drained from the blastocysts and next the entire uterine portion containing the implantation site was dissected; similarly, a length of pregnant uterus between two implantation sites, of roughly the same fresh weight, was cut and cleared from adhering fatty tissue; fresh and dry weights were determined. In a limited series of experiments the phosphorus (P) content in such material was also examined.

Alloxan diabetes

To examine the effect of diabetic hyperglycaemia on the glucose level in the blastocyst fluid, diabetes was induced in rabbits by intravenous injection of alloxan, 80 mg./kg. This amount was well tolerated and with the majority of our rabbits resulted in an elevated blood-sugar level and glucosuria within 3−4 days. Some does were first rendered diabetic and then mated for the experiment, but others were mated first and then treated with alloxan on the 3rd or 4th day of pregnancy. Also several superovulated does were given alloxan, which did not appear to interfere with superovulation; as a matter of fact, in one mildly diabetic superovulated doe 30 blastocysts were counted 7 days after mating. In general, however, except where diabetes was slight, the uterus in the alloxan-treated animals appeared thin and poorly vascularized, the embryos were small for their age and seemed to be degenerating.

Administration of sugars and other substances

In these experiments a 50 per cent, solution of sugar (glucose, fructose, sucrose) was injected intravenously, 1·7 g. /kg. body weight, into pregnant does 6,7, and 8 days after copulation; after an interval of 30−60 minutes the animal was nem-butalized; blood, blastocysts, urine, and occasionally also ovaries, were used for chemical determinations. In other experiments a solution of Na-sulphapyridine was injected intravenously, 100 mg./kg. body weight. Similarly, the effect of sodium thiocyanate (NaSCN) was examined; a solution of the salt was administered intravenously, or more often subcutaneously, 300 mg. / kg. body weight.

Chemical methods

Glucose was determined by the method of Hagedorn & Jensen (1923); the content of ‘true’ glucose, as distinct from the total reducing value obtained by the Hagedorn & Jensen method, was determined with glucose oxidase (Mann, 1946). Glucose in blastocyst fluid was also identified by paper chromatography, using the method of Trevelyan, Procter, & Harrison (1950). Fructose was determined by the method of Roe (1934); in experiments which involved fructose administration, in addition to fructose glucose was also estimated, both in blood and in the fluid collected from blastocysts. The presence of glucose in urine was detected with the Benedict reagent. Sucrose was determined by carrying out the estimation of reducing sugar in two identical samples, of which one was first hydrolysed for 25 minutes at 100° in the presence of 0·02 N-HC1; the sucrose content was calculated from the difference between these two values. Glycogen estimations were carried out according to Good, Kramer, & Somogyi (1933) using the anthrone reagent (Morris, 1948). Phosphorus was estimated by the method of Fiske & Subbarow (1925). Chloride content was determined according to Whitehorn (1921); nitrogen, potassium, sodium, calcium, and magnesium according to methods described by King (1946) and Pincussen (1928). Sulphapyridine was determined by the method of Bratton & Marshall (1939). Thiocyanate deter minations were carried out by the method of Goldstein (1950); 15−20 μg. NaSCN can be accurately estimated by this method.

Dry weight, nitrogen, and phosphorus content of blastocyst fluid

The appearance of the blastocysts and a few general properties of the blastocyst fluid have been remarked upon earlier (p. 3); the dry weight was determined in 2 samples of fluid withdrawn at 7 and days after mating and in 3 samples at 10 days; the values were 2·5 and 2·2 per cent, and 5·0,5·3, and 5·8 per cent, respectively, against 7·2 and 7·5 per cent, in rabbit serum. The total N content in fluid aspirated from 7-days-old blastocysts was 30·2−38·5 mg./100 ml. A sample of fluid from the yolk-sac cavity of 10-days-old embryos contained 282·9 mg./100 ml. total N, of which 24·5 mg./100 ml. was non-protein-N; maternal serum contained 845 mg./100 ml. total N and 24·5 mg./100 ml. non-protein-N. The content of phosphate in the pooled fluid from 6 blastocysts collected 7 days after mating was too small to be detected by the chemical method used. But a sample of yolk-sac fluid at 10 days was found to contain 5·6 mg. /100 ml. total P, as compared with 10·4 mg. /100 ml. in maternal serum.

Chloride, sodium, potassium, magnesium, and calcium content

A determination of chloride was carried out in 7-days-old unimplanted blastocysts, and 390 mg. /100 ml. was found. In 2 samples of fluid withdrawn at 10 days 595 and 600 mg./100 ml. were found; at 12 days an estimation gave 640 mg./ 100 ml.; maternal serum contained 560−595 mg. /100 ml. So far the analyses of Na, K, Mg, and Ca have all been carried out in pooled fluid from 10-days-old embryos; the values in mg./100 ml. were: Na 300, K 26·5, Mg. 1·3, Ca 11·2; equivalent values in rabbit serum were: Na 329, K 21·5, Mg. 2·0, Ca 10·2.

Glycogen content of the uterus

The following animals were used for these experiments: 1 pregnant nonsuperovulated doe, 4 pregnant superovulated does, all 5 animals at 8 days after copulation, and 1 non-pregnant adult doe. The implantation sites in the superovulated animals varied markedly in size, but, in general, those towards the tubal end of the uterus were smaller than the rest; in most non-superovulated animals a similar tendency was evident. The determinations were carried out separately with the small and large implantation sites. Dry-weight determinations on 100 mg. fresh-weight pieces of (1) pregnant uterus, (2) uterus at the level of implantation, and (3) non-pregnant uterus were 17·9, 18·3, and 16·9 mg. respectively.

The findings on glycogen distribution are summarized in Table 1. It can be seen that the size of the individual implantation sites in a given doe varied a great deal. As to the uterine glycogen content, it is likely that the variations found between the animals were to some extent due to breed and age differences. Concerning the glycogen content of the implantation sites as compared with the interimplantation areas, in some instances, e.g. in does A and C, not much difference was found; but in B there appeared to be a significantly higher concentration in the well-developed implantation sites, as compared both with the poorly developed ones and with the interimplantation areas of the uterus. A relatively high glycogen level in the uterus was found in the two mildly diabetic animals, D and F, of which the latter was non-pregnant.

TABLE 1

Glycogen distribution in the rabbit uterus

Glycogen distribution in the rabbit uterus
Glycogen distribution in the rabbit uterus

The content of phosphorus in the uterus

So far three experiments have been carried out in which the content of acidsoluble and total P was determined, at the level of implantation and in interimplantation areas of the uterus, at days after mating. The mean value in mg. 1100 g. fresh weight was: acid-soluble P in implantation sites 50, interimplantation sites 83, total P in the former 225, in the latter 329. Much further work is needed to determine the significance of these findings at this early stage of pregnancy.

The content of glucose in blastocyst fluid

In intact unimplanted 6-days-old blastocysts the glucose content as determined by the ferricyanide reduction method of Hagedorn & Jensen was 3−10 mg. /100 ml. Determinations carried out with unimplanted blastocysts removed from the uterus 7 days after mating gave values ranging from 18 to 25 mg./100 ml.; but the fluid withdrawn at that stage from what appeared to be already partly implanted blastocysts contained 46−68 mg. /100 ml. At 8 days values ranged from 68 to 77 mg./100 ml. In two different samples of 10-days-old yolk-sac fluid the content of total anthrone-reactive carbohydrate was 93·0 and 104 mg. /100 ml.; in the same fluids ‘true’ glucose was determined by incubation with glucose oxidase and a content of 90·0 and 99·2 mg./100 ml., respectively, was found. Another sample of this fluid was examined by paper chromatography; the only sugar detected under these conditions was glucose; fructose and inositol were absent. The glucose content was also examined in the fluid of 8-days-old poorly developed blastocysts from 2 diabetic does which had a blood-sugar level of 247 and 178 mg./100 ml. respectively; however, not more than 82 and 79 mg./100 ml. respectively was found in the blastocyst fluids.

The effect of intravenous administration of glucose, fructose, and sucrose on the sugar content of the blastocyst.

(a) Glucose

Table 2 gives the results of experiments carried out with does injected with glucose; two of the animals were 6 days and four were 7 days after concentration of maternal blood glucose and the concomitant glucosuria, no glucose was found in the fluid present in the 6-days-old blastocysts. Furthermore, insignificant amounts were found in unimplanted blastocysts at 7 days (Expt. Nos. 7 and 8); in these experiments the blastocysts were large enough for every two to provide sufficient material for a single glucose analysis; but results obtained in this manner failed to indicate any appreciable differences in glucose content. The animals in Expt. Nos. 3,5, and 6 possessed well-developed implantation sites 7 days after mating; 30−60 minutes after the administration of glucose its presence could be unequivocally shown in the fluid withdrawn from the blastocysts. In Expt. No. 4 it was difficult to decide whether or not the blastocysts were already implanted; the concentration of glucose in the aspirated fluid was within the range of values found in implanted blastocysts from untreated animals.

TABLE 2

Glucose concentration in blood, blastocysts, and urine following intravenous injection of glucose,1·7 g-/kg. body weight

Glucose concentration in blood, blastocysts, and urine following intravenous injection of glucose,1·7 g-/kg. body weight
Glucose concentration in blood, blastocysts, and urine following intravenous injection of glucose,1·7 g-/kg. body weight

(b) Fructose

Following intravenous injection of fructose, not only the fructose concentration but also that of glucose was determined as a routine in the maternal blood and, if sufficient material was available, also in the blastocyst fluid. The experiments in Table 3, numbered 1-7, were carried out with animals which had been mated 7 days before; some had unimplanted and others what appeared to be already implanting blastocysts. However, except for a trace in Expt. No. 4, no fructose was detected in the blastocyst fluid at that stage. It was, however, present in the ovarian tissue and in urine.

TABLE 3

Fructose and glucose concentration in blood, blastocysts, ovaries, and urine, following intravenous injection of fructose, 1·7 g/kg. body weight

Fructose and glucose concentration in blood, blastocysts, ovaries, and urine, following intravenous injection of fructose, 1·7 g/kg. body weight
Fructose and glucose concentration in blood, blastocysts, ovaries, and urine, following intravenous injection of fructose, 1·7 g/kg. body weight

On the other hand, already by half a day later, and better still at 8 full days after mating, the presence of fructose could be demonstrated clearly in the blastocyst fluid 30 minutes after fructose administration (Expt. Nos. 8−11). As a matter of fact, the farther advanced the pregnancy, the more the fructose concentration in the blastocyst fluid tended to approach that found in the ovaries.

As is well known, in the rabbit fructose is readily converted into glucose. When the blood of the above animals was analysed, it was found that there was not only considerable fructosaemia but in addition an elevated concentration of glucose; moreover, there was an indication, as in Expt. Nos. 1,4, and 7, that some of that glucose found its way into the blastocyst fluid, which nevertheless was fructose-free.

(c) Sucrose

Four experiments have been carried out with intravenous sucrose; the results are presented in Table 4. The amounts of sucrose found in the blastocyst fluid 7 days after mating were so slight as to be insignificant, though possibly in Expt. No. 3 a small amount was present. But 8 days after copulation an appreciable amount of sucrose was found in the blastocyst fluid, as indicated by results based on the determination of the reducing value as well as a positive Seliwanoff reaction.

TABLE 4

Sucrose and glucose concentration in blood and blastocyst fluid, following intravenous injection of sucrose, 1·7 g. / kg. body weight

Sucrose and glucose concentration in blood and blastocyst fluid, following intravenous injection of sucrose, 1·7 g. / kg. body weight
Sucrose and glucose concentration in blood and blastocyst fluid, following intravenous injection of sucrose, 1·7 g. / kg. body weight

Effect of sulphapyridine

The results obtained with this substance are shown in Table 5. Following the injection of Na-sulphapyridine, the concentration in blood reached a high level after 30 minutes, but even after an hour a considerable amount still remained in the blood. Unimplanted -days-old blastocysts removed after 30 minutes from an injected doe were analysed without previous rinsing; a small amount of the sulphonamide drug was detected, but it seems questionable as to whether this was due to its penetration into the blastocysts, or was the result of external surface contamination with sulphapyridine-containing uterine secretion (Expt. No. 1). Ovarian tissue showed a concentration approaching that of blood. Very little sulphapyridine was present in the fluid aspirated from apparently implanted 7-days-old blastocysts (Expt. No. 2). On the other hand, significant amounts were found in fully implanted 8-days-old blastocysts 30 to 60 minutes after the administration of the drug (Expt. Nos. 3 and 4).

TABLE 5

Sulphapyridine concentration in blood, blastocysts, and ovaries, following intravenous injection of Na-sulphapyridine, 100 mg./kg. body weight

Sulphapyridine concentration in blood, blastocysts, and ovaries, following intravenous injection of Na-sulphapyridine, 100 mg./kg. body weight
Sulphapyridine concentration in blood, blastocysts, and ovaries, following intravenous injection of Na-sulphapyridine, 100 mg./kg. body weight

Effect of sodium thiocyanate

As can be seen from Table 6, when 300 mg. /kg. of this salt was injected intravenously or subcutaneously into rabbits, 30-60 minutes later its presence could be shown in considerable concentration in the maternal blood and in urine. But the fluid collected by aspiration from blastocysts at 7, or even at 8, days after mating contained barely distinguishable amounts of thiocyanate (Expt. Nos. 2, 3, and 4). The fact that some thiocyanate appeared to be present in unimplanted 6|-days-old blastocysts was presumably due to traces of thiocyanate in the uterine secretion adhering to the blastocyst surface (Expt. No. 1).

TABLE 6

Thiocyanate concentration in blood, blastocysts, and urine, following injection of NaCNS, 300 mg. I kg. body weight

Thiocyanate concentration in blood, blastocysts, and urine, following injection of NaCNS, 300 mg. I kg. body weight
Thiocyanate concentration in blood, blastocysts, and urine, following injection of NaCNS, 300 mg. I kg. body weight

The main obstacle to an extensive study of the biochemical properties of the early mammalian blastocyst is that, unless unlimited numbers of experimental animals are available, the material for analytical purposes is forthcoming slowly and in rather inadequate quantity. In addition, as in all such work, much individual variation must be anticipated. With that in mind, it was decided to study in the first place the behaviour of substances which could be estimated accurately in small amounts, were well tolerated by the animals, were capable, after parenteral administration, of reaching the maternal blood-stream promptly, and were likely to remain there for a reasonable length of time at a fairly high level of con-centration. As far as it was practicable, an effort was made to repeat each type of experiment a sufficient number of times to obtain evidence of reproducibility.

The choice of rabbit as experimental animal was guided by the fact that in an oestrous doe ovulation follows copulation with regularity within 10 hours (Walton & Hammond, 1928); this ensured the time of conception within the limits of individual variations of approximately 1 hour. The existence of appreciable individual differences in the progress of early pregnancy was evident, amongst other observations, in the degree of development of blastocysts 7 days after copulation. In some 38 per cent, of the animals in this series the blastocysts at that stage were found lying free in the uterine lumen; but the rest of the animals exhibited well-developed, prominent implantation sites and the blastocysts were presumed to be in the process of incipient implantation. While of course the exact extent of implantation could only be judged accurately by histological methods, it was found that certain simple features of the blastocyst fluid were sufficiently characteristic to allow an approximate estimate of the condition of the blastocysts. Thus, whereas the fluid withdrawn from blastocysts a full 8 days after mating invariably clotted rapidly, contained material precipitable with trichloro-acetic acid or ethanol, and had a pH value of not more than 7 8, the fluid obtained from blastocysts 1 day younger did not clot, became distinctly alkaline on standing, and did not form a precipitate in the presence of proteinprecipitating reagents. This behaviour is not surprising in view of the low content of total nitrogen in the fluid at that stage. From experiments still in progress there is evidence of a significant difference in the content of bicarbonate in the blastocyst fluid at 7 and 8 days respectively after copulation.

It is believed that the results of experiments which involve the introduction of simple chemical substances into the maternal blood-stream, and a study of their ability to enter the embryonic fluid within a certain period of time, are likely to form a useful contribution to our knowledge of the extent of contact which is being built up between the maternal organism and the embryo during the early stages of pregnancy. It was interesting to note that, in the rabbit at any rate, simple molecules such as glucose and fructose did not readily penetrate into the unimplanted blastocyst, even when their concentration has reached a high level in the maternal blood and in tissues which form part of the female reproductive tract, e.g. the ovaries. But with incipient implantation, as soon as material links, however tenuous, between the uterine wall and the blastocyst began to form, glucose, though not fructose or sucrose, appeared to be able to pass into the blastocyst fluid. The evidence for this preference for glucose was strengthened by the experiments with fructose administration, in which an increased amount of glucose, but not fructose itself, was found in the blastocyst fluid. Once the process of implantation has been completed, a full 8 days after copulation, all three sugars could be detected within the blastocysts a short time after injection.

The study of the behaviour of these sugars was made easier by the fact that the fluid of the rabbit blastocyst at 6, and even at 7, days after mating contains relatively little reducing material of its own; there is good evidence that nearly all of it is glucose; fructose and inositol, two carbohydrates known to be present in certain foetal fluids of other species, being absent. It was interesting to observe that the glucose concentration in the blastocyst fluid appeared to be unaffected by maternal hyperglycaemia due to alloxan diabetes. It remains for future experiments to demonstrate to what extent the glucose level in the yolk-sac fluid can be influenced by the action of insulin.

As to the behaviour of parenteral sulphapyridine, while it could be readily detected in the fluid aspirated from fully implanted blastocysts, it is difficult to decide whether the small amount found when whole unimplanted blastocysts were used for analysis indicated its presence inside the blastocyst or, as seems likely, was merely due to contamination of the blastocyst surface with sulpha-pyridine-containing secretion of the uterine glands. A similar impression was gained in experiments with thiocyanate, so far as early unimplanted blastocysts are concerned; but, unlike the sulphonamide, thiocyanate failed to penetrate in detectable amounts into the fluid of obviously implanted blastocysts, at any rate within the restricted experimental time-interval.

Studies on the distribution of glycogen in the uterus have been hitherto largely concerned with the more advanced stages of pregnancy; as regards the very early stage of pregnancy in the rabbit, which was the object of the present investigation, there was as yet no clear indication of a differential distribution as between the implantation and interimplantation sites of the uterus, although in one experiment at least, distinctly more glycogen was accumulated in the well-developed implantation areas than in the rest of the uterus. Perhaps a clearer picture might emerge if it were possible to limit the investigation to animals of identical age and breed. The suggestion of a significant difference in the pattern of phosphorus distribution in the early pregnant uterus at the level of implantation, as compared with adjacent uterine portions, merits further investigation. Similarly, as more experimental material becomes available it will be interesting to obtain more detailed information of the chemical nature of the fluid which fills the cavity of the 6- and 7-days-old blastocyst, of its inner cell mass, and of the membrane which surrounds it.

This study was carried out on behalf of the Agricultural Research Council. The author is greatly indebted to Dr. P. Tate for an opportunity to carry out much of the chemical work at the Molteno Institute, University of Cambridge.

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