The in vitro uptake and incorporation of [3H] uridine by blastocysts of the tammar wallaby showed a 16- and 30-fold increase from day 0 to day 10 after removal of pouch young, respectively. Two of the six non-expanded blastocysts recovered on day 5 showed a tenfold increase in incorporation. During the first ten days after removal of pouch young the diameter of the blastocyst increased threefold. Endometrial exudate from gravid uteri had a higher protein concentration than exudate from nongravid uteri (39·5 ±0·9 and 320 ± 2·0 mg/ml (mean + s.E.M.), respectively). Endometrial exudates from uteri where the blastocyst was actively growing were found to contain six uterine-specific proteins. These were separated by gradient polyacrylamide gel electrophoresis. Two of the proteins were prealbumins and the others were larger molecules (M.W. 153000-670000). Two proteins were only present at particular stages of pregnancy: the other four were present at all stages from diapause to birth, in exudate from gravid and nongravid uteri. The specific binding of progesterone and androstenedione to proteins in endometrial exudates or uterine flushings from pregnant wallabies was less than one per cent of the value obtained from day-5 pregnant rabbits. The ability of mouse blastocysts to take up and incorporate [3H] uridine into acidinsoluble material increased threefold in the presence of day-10 endometrial exudates from wallabies. However, this was less than ten percent of the values obtained in the presence of bovine serum albumin.

The concentration of calcium in endometrial exudates increased from 23·6 to 45·2μg/ml during pregnancy; in endometrium it remained at 88·7μg/g (wet weight) throughout pregnancy, and in plasma it was 53·3 μg/ml. The concentration of zinc in endometrial exudates was 4·5 μg/ml; in endometrium it decreased from 21·8 to 13-3μg/g (wet weight) during pregnancy and in plasma it was 0·6 μg/ml.

The role of the uterine environment in fetal development has received increasing attention in recent years. In particular, uterine-specific proteins have been detected in a range of species, and have been found to vary with the hormonal state of the animal (Roberts & Parker, 1974a; Roberts, Parker & Symonds, 1976; Aitken, 1977; Dixon & Gibbons, 1979; Zavy, Bazer, Sharp & Wilcox, 1979). Some of these proteins have enzymic activity (Roberts & Parker, 1974b; Roberts, Parker & Henderson, 1976) but their precise functions are not understood. Blastokinin (uteroglobin) was thought to be uterine-specific and have a role there related to its ability to bind progesterone, but was later found to occur in a number of tissues (Krishnan & Daniel, 1967; Fridlansky & Milgrom, 1976; Beato & Beier, 1978). Uterine-specific prealbumins have been found in several species, including marsupials and primates, and it has been suggested that they may be able to pass into the blastocyst and exert some effect on its growth (Peplow, Breed & Eckstein, 1974; Renfree, 1973, 1975; Hearn & Renfree, 1975).

Changes in the concentration and metabolism of various ions in uterine tissues and fluids throughout pregnancy have indicated that these ions might be important in embryogenesis. Aitken (1974) found increased levels of calcium in uterine endometrium of roe deer at the time of rapid embryonic elongation. Dhar, Roy and Kar (1976) measured in vivo uptake of 65Zn in the female rat and found a marked increase at the time of implantation, throughout the whole genital tract. The role of zinc in uterine endometrium is not fully understood, though there is some evidence of its involvement in the binding of steroid hormones to receptors in the endometrium (Emanuel & Oakey, 1969) and as a component of several enzymes such as carbonic anhydrase.

The tammar wallaby provides an ideal opportunity to examine the influence of the uterine environment on embryogenesis. For much of the year the mother carries a blastocyst in diapause (Berger, 1966). If the pouch young, born from the previous pregnancy, is removed before the end of May (Western Australia), the blastocyst in the uterus is activated, grows and birth occurs 26–27 days later. When the pouch young is retained, or is lost after the end of May, the blastocyst remains in diapause until late December. Activation of the blastocyst then occurs and twenty-six days later the young is born. The mother then mates and the cycle recommences (Renfree & Tyndale-Biscoe, 1973). As the blastocyst in diapause is not attached to the uterus, the maternal signal for activation must pass via the uterine fluids.

The reproductive tract of the female tammar wallaby has two distinct and separate uteri, each opening via its own cervix into the median vaginal sinus. Since ovulation occurs alternately from right and left sides, normally only one uterus is occupied by an embryo at any one time. This is designated the ‘gravid uterus’. The unoccupied, contralateral uterus is designated the ‘nongravid uterus’. Thus, gravid and nongravid uteri of pregnant animals can act as physiological controls for each other.

Two series of experiments were conducted on tammar wallabies (1) to examine biochemically the response of the blastocyst at activation, and (2) to characterize the changes in the uterine environment at activation. The latter were assessed by determination of protein concentration, molecular weights of uterine-specific proteins, and specific steroid binding; and the metabolic response of mouse blastocysts to endometrial exudates.

Collection of blastocysts

Tammar wallabies, originating from Kangaroo Island, South Australia, were maintained in grassed yards in the Native Fauna Research Unit at Murdoch University, Western Australia. Oats, lucerne hay and vegetables were provided as additional food. Tammar wallabies at various stages of pregnancy were obtained by removal of pouch young during the period of lactational-diapause (January-May): this procedure activates the blastocyst and causes it to resume development (Renfree & Tyndale-Biscoe, 1973). The animals were killed by cervical dislocation and the uterine horns were dissected out. Blastocysts were recovered by flushing the uteri with ice-cold buffered saline.

Virgin 10-week-old albino mice were induced to super-ovulate by an intraperitoneal injection of 5–10 i.u. human Chorionic Gonadotrophin (hCG) (Intervet) after priming 48 h previously with 5–10 i.u. pregnant mare serum gonadotrophin (Intervet) (Brinster, 1967). The mice were mated and then killed by cervical dislocation 93–96 h after administration of hCG. Embryos at the morula or early blastocyst stage were collected by flushing the reproductive tract with ice-cold buffered saline.

Incubation of blastocysts from tammar wallabies and mice

Tammar blastocysts collected at days 0, 5 and 10 after removal of pouch young were washed twice in ice-cold buffered saline and then transferred individually to a 50 μl drop of incubation medium under paraffin oil in a tissue culture dish (Biggers, Whitten & Whittingham, 1971). The incubation medium was Krebs-Ringer phosphate supplemented with bovine serum albumin (1 mg/ml), sodium pyruvate (35 μg/ml) and [3H]uridine (54 μ Ci/ml, 25·2 Ci/m mol). The blastocysts were incubated for 5 h at 37°C. Each blastocyst was then washed twice in 2 ml ice-cold Dulbecco’s phosphate-buffered saline containing non-radioactive uridine (12 μ g/ml) and bovine serum albumin (1 mg/ml), and deposited between two pieces of Whatman fibre-glass filter (Bitton-Casimiri, Brun & Psychoyos, 1976). These filters were placed in 1 ml cold 5% trichlor-acetic acid (TCA) for 20 min, then treated twice with 1 ml cold 95% ethanol for 10 min, and finally with 1 ml cold ether for 5 min. The filters were allowed to dry in air. The dried filters and 0·5 ml TCA supernatant were counted with 5 ml scintillation fluid (toluene: Triton X100, in the ratio 2:1, 0·5% 2,5-di-phenoxazole (PPO)) in a liquid scintillation spectrometer (Packard Tri-carb 3255). The radioactivity in the filters gave an estimate of [3H] uridine incorporated into TCA-insoluble material containing the RNA. The radioactivity in the supernatant gave an estimate of [3H] uridine taken up by the blastocysts but not incorporated into RNA. Back-ground radioactivity was determined in an equal amount of the wash solution.

Mouse blastocysts were treated in a similar manner except that they were incubated in groups of 20–40 in each 25 μl drop of incubation medium. The incubation medium for mouse blastocysts was Dulbecco’s phosphate-buffered saline (Dulbecco & Vogt, 1954) supplemented with either bovine serum albumin or tammar endometrial exudate (1 mg protein/ml). Sodium pyruvate (35 μg/ml) and (3H]uridine (54 μCi/ml, 25·2Ci/mmol) were added in both situations. After incubation, the blastocysts were washed and extracted.

Intrauterine injections

Endometrial exudate from uteri where the blastocyst had recently resumed development (blastocyst diameter 300–1000 μm, estimated age equivalent to 8–11 days after removal of pouch young) was introduced into the uteri of recipient wallabies which were predicted to have a blastocyst in diapause. This was done either via an indwelling cannula fixed in the neck of the cervix, or directly by injection through the wall of the uterus. Controls received foetal calf serum or wallaby serum instead of exudate. Four groups of wallabies were given either 1, 2, 4 or 8 injections of 100, 50, 30 or 20 μl respectively. After various intervals of time (the longest interval being 20 days after the first injection) the uteri were flushed to recover the blastocysts.

Collection and storage of endometrial exudates

Exudates were obtained from the endometrium of the tammar wallaby. For some samples the stage of pregnancy was estimated from the diameter of the blastocyst (Table 2) which was recovered from the uterus.

The uterus was opened longitudinally and laid flat. Endometrial tissue was stripped from the underlying myometrium using fine curved forceps, and frozen at − 20°C in a small vial. On thawing, a clear fluid exuded from the tissue. Between 5 and 320 μl fluid was obtained from each uterus, depending on stage of pregnancy (Renfree, 1973). The fluid was stored in sealed microcapillary tubes at − 20°C. Protein concentrations were measured by Coomassie blue binding (Bradford, 1976).

Gradient gel electrophoresis

Measurements of molecular weight of proteins in endometrial exudates were carried out using Pharmacia polyacrylamide gradient (4–30%) gels and the Pharmacia high molecular weight calibration kit. The electrode vessel buffer was 0·09 M tris, 0·08 M boric acid, Na2EDTA 0·93 g/1, pH 8-4. Electrophoresis was carried out for 16–20 hours at 150 V. Gels were stained for 2 h in a mixture of 10 g naphthalene black and 4 g Coomassie blue per litre 7% acetic acid, and destained electrophoretically in 7% acetic acid.

Uterine endometrial exudates and fresh uterine flushings from various stages of pregnancy were tested for specific binding of progesterone, and androstenedione. These results were compared with those obtained using uterine flushings from pregnant rabbits (day 5). Rabbit uterine flushings were diluted to 22μg protein/ml with Dulbecco’s phosphate-buffered saline containing 30-8 mg dithiothreitol/ml. For each steroid tested, incubations were carried out in the presence and absence of 1000-fold excess of non-radioactive steroid over radioactive steroid. Incubations were carried out in a volume of 200 μl containing radioactive steroid at 50 nM, dithiothreitol 30·8 mg/ml, protein (22μg/ml for rabbit, 600/óg/ml for tammar) and Dulbecco’s phosphate-buffered saline. Half of the vials also contained non-radioactive steroid at 50 μM. All vials were incubated at 30°C for 1 hour. They were then chilled in ice prior to chromatography on 15 × 54 mm columns of Sephadex G-25 to separate protein-bound steroid from free steroid. Elution was carried out with phosphate-buffer 0·02 M pH 7·5 containing 0·1 M-NaCl. The first 2·2 ml of eluate was.discarded. The next 3·5 ml was mixed, and. 0·5 ml samples taken for scintillation counting (Ginsberg, et al., 1974). The concentration of protein in this fraction was assayed by the method of Bradford (1976).

Specific binding was calculated as:
formula

Determination of calcium and zinc concentrations

Atomic absorption spectrophotometry was used to measure the concentration of calcium and zinc in sample solutions after dilution with lanthanum chloride solution (5000μg La/ml) to the required concentration range. Standards covering the range 0–1·0/μg Zn/ml and 0-1-5 μg Ca/ml were prepared. Both endometrial exudate and plasma were assayed directly after appropriate dilution. Tissue was digested before assay. A weighed amount of uterine endo-metrium was digested in nitric/perchloric acid (9/1) and taken to dryness. The digest was reconstituted with one or two millilitres of water and analysed after appropriate dilution. Each sample was assayed in duplicate. Samples from the first few days of pregnancy were dated from the beginning of oestrus rather than the removal of pouch young.

Chemicals

Bovine serum albumin and sodium pyruvate were obtained from Sigma Chemical Co. and [3H] uridine (25·2 Ci/mmol, 1 μCIμ1), [3H]progesterone (pregn-4-ene-3,20-dione) (101 Ci/mmol, 1 mCi/ml) and [3H]androstenedione (4-androstene-3,17-dione) (60mCi/mmol, 1μCí/58μA1) were supplied by the Radiochemical Centre, Amersham. Special-grade perchloric acid (BDH) was used in tissue digestion prior to atomic absorption spectrophotometry. All other chemicals were of analytical grade.

Statistics

The data were analysed using nonparametric statistics (Colquhoun, 1971).

Uptake and incorporation of [3H]uridine by tammar blastocysts during activation

All the blastocysts recovered on the day of removal of pouch young (day 0) had diameters in the normal range for blastocysts in diapause (263 ± 19 μm S.D.) (Renfree & Tyndale-Biscoe, 1973). Likewise, none of the blastocysts recovered on day 5 had expanded. However, two of these blastocysts incorporated uridine at a much increased rate (Table 1). By day 10 after removal of pouch young the blastocysts had all expanded, though their diameters varied considerably. This increase in size was reflected in a 13- to 23-fold increase in the uptake of [3H] uridine and a 23- to 50-fold increase in the incorporation of [3H] uridine into the TCA-insoluble fraction.

Table 1

Uptake and incorporation of [3H]uridine by tammar blastocysts

Uptake and incorporation of [3H]uridine by tammar blastocysts
Uptake and incorporation of [3H]uridine by tammar blastocysts
Table 2

Concentration of protein in endometrial exudate during pregnancy in the tammar wallaby

Concentration of protein in endometrial exudate during pregnancy in the tammar wallaby
Concentration of protein in endometrial exudate during pregnancy in the tammar wallaby

Protein concentration in uterine exudates

Protein concentrations were measured in exudates from both the gravid and nongravid uteri of six wallabies 14–17 days after removal of pouch young, and in a single uterus of 14 wallabies at other stages of pregnancy. Of the 14–17 day samples, the protein concentration in the gravid uteri was 20% higher than in the nongravid uteri, though the difference was not statistically significant in this small sample. A highly significant difference (P < 0·01) was observed when all the data from 8·23 days after removal of pouch young were compared (Table 2); the exudate from gravid and nongravid uteri contained 39·5 ±0-9 and 32·0 ± 2·0 mg protein/ml (mean ± s.e.m.) respectively.

Identification of uterine-specific proteins

Uterine endometrial exudates from tammar wallabies at various stages of pregnancy were subjected to gradient gel electrophoresis and the protein composition compared with that obtained from serum. The samples of exudate were grouped according to the size of the blastocyst. Thus, pregnancy was divided into eight stages; from one to three samples were examined at each stage. Exudate from nongravid uteri was also examined. There were no qualitative differences in protein composition between samples from individual animals at the same stage of pregnancy, though there appeared to be some quantitative variation. After electrophoresis exudates were found to contain six proteins which were not present in serum. Many major and minor bands were present in both. Some of the uterine-specific proteins were present in all exudates and some were limited to a restricted stage of pregnancy.

The uterine-specific proteins present in exudates from gravid and nongravid uteri at all stages of pregnancy had Rf values of 0·265 (M.W. 670000), 0-397 (M.W. 400000) and 0·947 (prealbumin(s) beyond the calibration range of the gel). There were two protein bands which, besides being uterine-specific, were also specific to the stage of pregnancy. A protein with an Rf value of 0·385 (M.W. 487000) first appeared when the blastocyst had expanded to at least 400 /on diameter (equivalent to about 9 days after removal of pouch young) and disappeared when the (now) vesicle reached 9000 μm diameter (equivalent to day 16 after removal of pouch young, i.e. 1-2 days before implantation). The other protein had an Rf value of 0·692 (M.W. 153000) and appeared when the blastocyst began to expand (diameter 300 μm) and remained present until the vesicle reached 15000 μm diameter (the day before implantation).

After 16–20 h electrophoresis the prealbumin section of the gel was overcrowded, so some samples were electrophoresed for only 2 h to enable closer examination of this section. Proteins of low molecular weight were run in conjunction with the exudate samples to provide a calibration. Under these conditions three prealbumin bands were resolved in exudate samples, the band with the highest mobility comigrating with the pre-albumin band in serum. Valid molecular weight estimations can only be made when the migration rate of the protein is essentially zero, or when all proteins may be assumed to have the same charge density and shape due to treatment with sodium dodecylsulphate. Although neither of these criteria were met in the 2 h electrophoresis, it was nevertheless decided to calculate the molecular weights of the two uterine-specific prealbumins, in the absence of any more definitive data. The approximate molecular weights were 43000 and 47000.

There was one other protein which was present in exudates and in serum, but only in trace amounts in the latter. It appeared in exudates from gravid and nongravid uteri at all stages of pregnancy, and had an Rf value of 0·724 (M.W. 132000).

Steroid binding to uterine proteins

Uterine flushings from day-5 pregnant rabbits, which had been stored at −20°C, showed highly specific binding of progesterone (Table 3). This was probably due to the presence of blastokinin which constitutes a large proportion of the total protein in rabbit uterine flushings at day 5 of pregnancy. As uterine-specific proteins in tammar wallabies were only present in trace amounts, the binding incubation was carried out at a 27-fold higher protein concentration than for rabbit. However, the binding of progesterone and androstenedione was negligible. The binding incubations were repeated using fresh uterine flushings, in case the binding protein was unstable to freezing and thawing. Tammar wallabies 10 and 15 days after removal of pouch young were sampled, but again no specific binding of progesterone or androstenedione was observed.

Table 3

Specific steroid binding to uterine proteins in exudate and flushings from gravid uteri

Specific steroid binding to uterine proteins in exudate and flushings from gravid uteri
Specific steroid binding to uterine proteins in exudate and flushings from gravid uteri

Response of mouse blastocysts to endometrial exudates from wallabies

This experiment was designed to test whether or not mouse blastocysts were capable of detecting differences in composition of uterine endometrial exudates taken from the tammar wallaby at various stages of pregnancy. Bovine serum albumin was included as a control (Table 4). All of the test solutions contained the same concentration of protein, but the uptake and incorporation of [3H] uridine by blastocysts in the presence of exudate were never greater than 6·5 and 7·1% respectively, of the values obtained in the presence of bovine serum albumin. Similar results were obtained when day 0 and day 5 exudates were used. However, there was a threefold increase in uptake and incorporation in the presence of exudates obtained 10 days after removal of pouch young. No further increase was observed when day 15 exudates were used.

Table 4

Uptake and incorporation of [3H]uridine by mouse blastocysts in presence of uterine endometrial exudates from the tammar wallaby

Uptake and incorporation of [3H]uridine by mouse blastocysts in presence of uterine endometrial exudates from the tammar wallaby
Uptake and incorporation of [3H]uridine by mouse blastocysts in presence of uterine endometrial exudates from the tammar wallaby

Calcium and zinc concentrations in endometrium and endometrial exudates

The concentration of calcium in endometrial exudates of gravid uteri obtained from wallabies between day 0 and 2 post coitus was 23·6 ± 3·3μg/ml (n = 8). Animals sampled later in pregnancy had significantly higher (P < 0·01) levels of calcium, 45·2 ± 1·8μg/ml (n = 27, mean ± s.e.m.). The concentration of calcium in endometrium was 88·7 ± 3·6 μg/g (wet weight) throughout pregnancy and in plasma it was 53·3 ±4·0 μg/ml.

The concentration of zinc in endometrium was quite variable throughout pregnancy. Up to the time of implantation (day 17) the mean level was 21·8 ± 2·0 μg/g (wet weight) (n = 29). The values for days 21–23 after removal of pouch young were more uniform, and significantly lower (P < 0·01) at 13·3 ± 0·8 /zg/g (n = 7). The concentration of zinc in endometrial exudates was 4-5 ± 0·4/zg/ml, and did not vary significantly throughout pregnancy. The plasma level was 0-6 ±0·1 μg/ml.

Intrauterine injections

Twelve wallabies received endometrial exudate or serum via an intrauterine cannula, and a blastocyst was recovered from one of them.

This one (0008) had received 6 daily doses of 30 pX exudate. Its pouch young was removed on day 5 and its uterus flushed on day 20 (day 0 was the day of the first injection). The blastocyst had dimensions of 340 × 290 /zm. In an additional control animal simple dilation of the cervix did not lead to loss of the blastocyst, but two further controls fitted with sealed cannulae did not contain blastocysts after 16 days.

From 22 wallabies receiving direct intrauterine injections, three blastocysts were recovered as follows:

No. 0053 received 2 × 100 μ I injections of exudate, 2 days apart. The uterus was flushed immediately and the blastocyst had a diameter of 300 μm.

No. 0277 was treated as No. 0053 except that the uterus was flushed 2 days after the second injection. The diameter of the blastocyst was 278 μm.

No. 0197 received 100/4 of foetal calf serum in a single injection and the uterus was examined 2 days later. An unfertilized ovum (diameter 220 μm) was recovered.

Blastocyst reactivation

For several days after the removal of pouch young, the tammar blastocyst shows no increase in diameter. Indeed it is not until day 8 that there is a change of reasonable significance (Renfree & Tyndale-Biscoe, 1973). Thereafter the blastocyst expands rapidly, its diameter more than doubling in the next two days. The metabolic steps necessary for such expansion probably entail synthesis of new proteins, hence the choice here to measure uptake and incorporation of [3H]uridine. Although none of the day-5 blastocysts had expanded, two of them showed a tenfold increase in incorporation of [3H]uridine. Thus their metabolism had increased before any marked expansion in size. Moore (1978) incubated tammar blastocysts in the presence of [3H]UMP, and counted the silver grains after autoradiography. He called this ‘RNA polymerase activity’, and found that the fifth day after removal of pouch young was the earliest time at which he could detect an increase in such activity. The TCA-insoluble radioactivity denoted ‘[3H]uridine incorporation’ in the present report may to some extent have been a measure of the same parameter. The two ‘activated’ day-5 blastocysts incorporated [3H]uridine five times faster than the other four blastocysts of the same age. Between-animal variation is to be expected and becomes more apparent at times of rapid development. This is also illustrated by the range of diameters, uptake and incorporation of the three day-10 blastocysts (Table 1). The experiments on uptake and incorporation of [3H]uridine did not demonstrate any net uptake or incorporation of uridine. However, there is no evidence that blastocysts differentiate between []H]uridine and [3H]uridine.

As the diapause blastocyst is not attached to the uterus the maternal signal for activation must be mediated via the uterine fluids. Tyndale-Biscoe (1970) found that quiescent tammar blastocysts, when transferred to the uteri of day-8 recipient tammars, resumed development. An attempt was made by the present authors to do the converse experiment, and activate blastocysts in diapause by manipulation of the uterine environment. However, the recovery rate was low: three blastocysts were obtained from 20 wallabies treated with endometrial exudate, and one unfertilized egg from 14 wallabies treated with serum. The blastocysts appeared normal, but only one (No. 0008) was large enough to be classified ‘reactivated’, and it was much smaller than expected for a blastocyst 15 days after removal of pouch young. This recovery rate was too low to enable any conclusions to be drawn about the influence of changes in the intrauterine environment on the blastocyst in diapause. Whilst the cause of this low recovery rate is not understood, these experiments have clearly shown that it is difficult to manipulate the uterine environment in this way and maintain viable blasto-cysts.

Composition of uterine exudate

The wallaby blastocyst expands rapidly after activation and, as it is not attached to the uterine wall until shortly before birth, it must rely on the uterine fluids to supply nutrients for this rapid growth. Exudates from uteri containing a blastocyst contained 23% more protein than those where no blastocyst was present. It is not known how early in pregnancy this difference becomes apparent, but such an increase in protein concentration might be of nutritional significance to the growing blastocyst. However, blastocysts transferred into nongravid uteri after day 8 will develop normally (Tyndale-Biscoe, 1970).

Mouse blastocysts have been used before as indicators of a change in composition of uterine fluid from other species. Aitken & Matthius (1978) incubated mouse blastocysts in the presence of human uterine flushings collected at various stages of the menstrual cycle. Blastocyst hatching and attachment were not impaired by flushings collected before or after ovulation: they were impaired by a flushing collected on the last day of a menstrual period. In the experiments reported here, mouse blastocysts were three times more active metabolically when in the presence of exudate from day 10 or day 15 than day 0 or day 5. Two new uterine specific proteins appeared in exudates between days 5 and 10. These might be biologically significant to the activation of the blastocyst. In the rabbit, one such protein has been shown to have the very specific property of binding progesterone (Fridlansky & Milgrom, 1976). No such binding could be measured in endometrial extracts from the tammar wallaby. This might be partly attributable to the fact that in the wallaby the uterine specific proteins constitute an extremely small ( < 1%) fraction of the total endometrial proteins. Even if they had high affinity for binding a hormone, this is not likely to affect the total intrauterine milieu.

Another component of the intrauterine environment which changed within 10 days of removal of pouch young was the concentration of calcium in exudate. It is not known whether the twofold increase occurred before or after day 5, but in either case it might be an important factor in the activation and expansion of the blastocyst. Calcium is known to have a role in the regulation of ion transport and to be necessary for the activation of certain enzymes. The former is likely to be of prime significance at the time of rapid expansion of the blastocyst. In species which display delay of implantation there is a marked increase in response to macromolecules and divalent cations from the 2-cell to the blastocyst stage and this might control the metabolic activity of blastocysts (Surani, 1980). Certainly, the presence of adequate concentrations of calcium ions is known to be essential for blastocyst growth (Wales, 1970).

The 40% decrease in zinc concentration in uterine endometrium at the time of implantation was substantially larger than that observed in roe deer by Aitken (1974), which he attributed to oedema of the endometrium (Aitken et al. 1973). Such an explanation seems unlikely to account for the large decrease in zinc concentration observed in the wallaby. Zinc is a component of carbonic anhydrase, peptidases and several dehydrogenases, and a decrease in activity of one of these might facilitate implantation.

E. J.T. was a Research Fellow of the Lalor Foundation, which also provided funds for the project. Support from the National Institutes of Health, U.S.A. HD09387, and the Common-wealth Scientific and Industrial Research Organization, Australia, is also gratefully acknowledged. Assistance and advice from Dr C. H. Tyndale-Biscoe, CSIRO Division of Wildlife Research, Canberra, was most welcome. Thanks are due to Dr I. Pike and Dr M. Cake, both of Murdoch University, for advice on technical matters and to Dr D. Lincoln, University of Bristol, for advice on the manuscript.

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