/. Embryol. exp. Morph. Vol. 62, pp. 325-338, 1981
Printed in Great Britain © Company of Biologists Limited 1981
325
Biochemical studies of intrauterine
components of the tammar wallaby Macropus
eugenii during pregnancy
By ELIZABETH J. THORNBER,1 MARILYN B. RENFREE
AND GREGORY I.WALLACE
From the School of Environmental and Life Sciences,
Murdoch University, Western Australia
SUMMARY
The in vitro uptake and incorporation of [3H]ui idine 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 45-2/*g/ml
during pregnancy; in endometrium it remained at 88-7/*g/g (wet weight) throughout
pregnancy, and in plasma it was 53-3/Mg/ml. The concentration of zinc in endometrial
exudates was 4-5/fg/ml; in endometrium it decreased from 21-8 to 13-3/^g/g (wet weight)
during pregnancy and in plasma it was 0-6 /*g/ml.
INTRODUCTION
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 &
1
Author's address: Department of Community Health Science, Western Australian
Institute of Technology, Bentley, Western Australia 6102.
326
E. J. THORNBER, M. B. RENFREE AND G. I. WALLACE
Symonds, 1976; Aitken, 1977; Dixon & Gibbons, 1979; Zavy, Bazer, Sharp &
Wilcox, 1979). Some of these proteins have enzymic activity (Roberts & Parker,
19746; 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
Intrauterine
components of the tammar wallaby
327
uterine-specific proteins, and specific steroid binding; and the metabolic response
of mouse blastocysts to endometrial exudates.
MATERIALS AND METHODS
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 (A 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 /tg/ml) and [3H]uridine (54 JLL 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 nonradioactive uridine (12 /*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% trichloracetic 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-diphenoxazole (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
328
E. J. THORNBER, M. B. RENFREE AND G. I. WALLACE
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 /i\ 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 ^g/ml)
and [3H]uridine (54 JLL Ci/ml, 25-2 Ci/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 ^m, 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/*1, 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 /tl 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, Na 2 EDTA 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.
Intrauterine components of the tammar wallaby
329
Steroid binding
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 22 jtig 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 fi\
containing radioactive steroid at 50 nM, dithiothreitol 30-8 mg/ml, protein
(22/tg/ml for rabbit, 600/tg/ml for tammar) and Dulbecco's phosphatebuffered saline. Half of the vials also contained non-radioactive steroid at
50 /IM. All vials were incubated at 30 °C for 1 hour. They were then chilled in
ice prior to chromatography on 15 x 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:
[(counts with radioactive steroid only) - (counts with radioactive and
nonradioactive steroid present)]/protein concentration.
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 (5000/*g La/ml) to the required concentration range. Standards
covering the range 0-1-0/tg Zn/ml and 0-1-5 /ig 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 endometrium 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 jid//i\), [3H]progesterone
(pregn-4-ene-3,20-dione) (101 Ci/mmol, 1 mCi/ml) and [3H]androstenedione
(4-androstene-3,17-dione) (60 mCi/mmol, l/*Ci/58/*l) were supplied by the
330
E. J. THORNBER, M. B. RENFREE AND G. I. WALLACE
Table 1. Uptake and incorporation of [zH]uridine by tammar blastocysts
Days after
removal of
pouch young
Diameter of
blastocyst
Om)
—
—
—
—
244
230
300
258
258
Uptake of [3H]uridine
TCA-soluble fraction
(dpm/ blastocyst/
5 h + 100)
68
70
111
127
x±s.e.m. 94±14
56
59
132
44
63
Incorporation of [3H]uridine
TCA-insoluble fraction
(dpm/blastocyst/5 h + 100)
01
1-5
1-8
41
20 ±0-7
1-6
4-5
4-5
50
18-5*
244
10
172
211*
*±s.e.m. 88 ±21
9-2 ±3-4
800
1482
46-5
900
1178
700
2063
1500
100-3
*±s.e.m. 1574 ±260
72-3 ±15-6
* Note increased incorporation by two day-5 blastocysts.
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).
RESULTS
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/^m 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.
Intrauterine
components of the tammar
wallaby
331
Table 2. Concentration of protein in endometrial exudate during
pregnancy in the tammar wallaby
Diameter of
blastocyst
0*m)
Estimated
age*
(days)
Gravid or
nongravid
uterus
< 300
Quiescent
300-400
400-500
500-1000
1000-2200
2200-5000
8
9
11
12
14
G
NG
NG
G
G
5000-9000
16
9000-15000
17
Embryo, crownrump length
(mm)
9-11
11-15
NG
G
NG
G
NG
G
NG
21
23
G
G
Protein content
of exudate
(mg/ml)
32-4
31-2
25-2
41-2
39-2
37-2
38-511
590/
40-81
32-4»
41-21
270/
42-5
41-2
28-8
29-81
360/ 4 6 0
360 V 4001
38-8/ 22-8/
31-5
29-5
39-4
35-2
* Age estimated from blastocyst size, as determined by the removal of pouch young
(Renfree & Tyndale-Biscoe, 1973).
t Brackets indicate data from both uteri of the same animal.
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
332
E. J. THORNBER, M. B. RENFREE AND G. I. WALLACE
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 /tm diameter (equivalent to about 9 days after removal of pouch young) and
disappeared when the (now) vesicle reached 9000 /im 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 /im) and remained present until the
vesicle reached 15000/*m 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 uterinespecific 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
- 2 0 °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 uterinespecific proteins in tammar wallabies were only present in trace amounts, the
binding incubation was carried out at a 27-fold higher protein concentration
Intrauterine components of the tammar wallaby
333
Table 3. Specific steroid binding to uterine proteins in exudate and
flushings from gravid uteri
Binding
cpm//*g protein
Frozen endometrial exudate
(tammar wallaby)
Progesterone
Androstenedione
Fresh uterine flushings
(tammar wallaby
Progesterone
Androstenedione
Frozen uterine flushings
(rabbit)
Pooled
Samples
Days after removal
of pouch young
55
22
10
15
10
15
77
126
Pregnant
day 5
< 20
< 20
30000
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.
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.
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 ± 3-3 /tg/ml (n = 8).
Animals sampled later in pregnancy had significantly higher (P < 0-01) levels
of calcium, 45-2 ± 1-8/*g/ml (n = 27, mean ± s.e.m.). The concentration of
334
E. J. THORNBER, M. B. RENFREE AND G. I. WALLACE
Table 4. Uptake and incorporation of [*YL]uridine by mouse blastocysts
in presence of uterine endometrial exudates from the tammar wallaby
Days after
removal of
pouch young
No. of
assays
Uptake of [3H]uridine
(TCA-soluble fraction)
dpm/blastocyst/5 h
(±s.e.m.)
0
5
5
4
105 ±22
97 ±26
Incorporation of [3H]uridine
(TCA insoluble fraction)
dpm/blastocyst/5 h
(±s.e.m.)
< 55
< 55
P < 005
P < 005
10
3
318±1O
159±19
15
4
287±38
142±13
bovine serum
5
4487 ±912
2436 ±85
albumin
Concentration of protein in the incubation medium was 1 mg/ml.
calcium in endometrium was 88-7 ± 3-6 fig/% (wet weight) throughout pregnancy
and in plasma it was 53-3 ±4-0 /tg/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/*g/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 /^g/g (n = 7). The concentration of zinc in endometrial exudates was 4-5 ±
0-4 /Ag/ml, and did not vary significantly throughout pregnancy. The plasma
level was 0-6 ±0-1 fig/m\.
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 fil 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 x 290 /*m. 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 2x 100/*1 injections of exudate, 2 days apart. The uterus was flushed
immediately and the blastocyst had a diameter of 300 /im.
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 fim.
No. 0197 received 100/tl of foetal calf serum in a single injection and the uterus was
examined 2 days later. An unfertilized ovum (diameter 220 /^m) was recovered.
Intrauterine components of the tammar wallaby
335
DISCUSSION
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 blastocysts.
336
E. J. THORNBER, M. B. RENFREE AND G. I. WALLACE
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 ah
1973). Such an explanation seems unlikely to account for the large decrease in
Intrauterine components of the tammar wallaby
2>2>1
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 Commonwealth 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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{Received 30 June 1980, revised 24 October 1980)