/. Embryol. exp. Morph. Vol. 41, pp. 295-300, 1977
Printed in Great Britain © Company of Biologists Limited 1977
295
The culture of mouse blastocysts in the presence
of uterine flushings collected during normal
pregnancy, delayed implantation and pro-oestrus
By R. J. AITKEN
From the Department of Genetics, University of Edinburgh
SUMMARY
When day-3 mouse embryos were cultured in a simple medium supplemented with uterine
fluids of mice autopsied on day 4 of pregnancy, 48 h after administration of oestradiol, or
during pro-oestrus, the percentage of blastocysts hatching from the zona pellucida was
significantly greater than in unsupplemented medium. In the presence of uterine fluids
recovered during delayed implantation this stimulation of blastocyst hatching was not
observed. When the culture medium was supplemented with dialysed uterineflushingscontaining 20 or 30fig protein/ml, both 'day 4' and 'delay' uterine proteins were equally
effective in enhancing hatching frequency {P < 005). The results suggested that 'delay'
uterine fluids may contain a dialysable inhibitor of blastocyst activity. The putative inhibitor
was not effective in the presence of serum, since uterine fluids recovered both on day 4 of
pregnancy and during delayed implantation significantly increased the size attained by
blastocyst outgrowths in the presence of foetal calf serum (P < 0001). The percentage of
blastocysts exhibiting giant cell transformation and outgrowth was also increased (P < 002)
by these uterine fluids when the concentration of FCS in the medium was minimal (0-25 %).
INTRODUCTION
The mouse blastocyst may exist in one of two alternative metabolic states.
On day 4 of normal pregnancy, the day of implantation, the blastocysts are
metabolically active, exhibiting high rates of nucleic acid and protein synthesis,
cell division and carbon dioxide production (Weitlauf & Greenwald, 1965,1968;
Bowman & McLaren, 1970a; McLaren & Menke, 1971; McLaren, 1973). All
these parameters are diminished during lactational or experimentally induced
delay of implantation, when the blastocysts enter a state of dormancy or diapause. Some circumstantial evidence has been obtained to suggest that these
metabolic transitions are the result of hormonally induced changes in uterine
secretions (Surani, 1975; Aitken, 1977). It has been suggested that embryonic
diapause is brought about by the presence of inhibitory substances (Psychoyos &
Bitton-Casimiri, 1969; Psychoyos, 1973; Finn, 1974; Weitlauf, 1976) or the
absence of embryotrophic factors (Surani, 1975; Aitken, 1977) in the uterine
Address for reprints: M.R.C. Unit of Reproductive Biology, 2 Forrest Road, Edinburgh,
EH1 2QW, U.K.
296
R. J. AITKEN
lumen. An experiment was therefore designed to examine the influence of
uterine fluid recovered from 'active' or 'dormant' uteri on the growth of mouse
blastocysts in vitro.
MATERIALS AND METHODS
Mice of the random-bred Q strain were used in this study. Delayed implantation was induced by ovariectomizing females on day 2 of pregnancy (day of
plug = day 0) and administering 1 mg of a long acting progestin, medroxyprogesterone acetate (Depo-provera, Upjohn). In certain instances diapause was
terminated by the injection of 50 ng oestradiol-17/? (Sigma).
Embryos were harvested on day 3 of pregnancy and cultured for up to 5 days
in a modified Brinster's medium containing 3 mg/ml Bovine Serum Albumin
(BSA) (Bowman & McLaren, 1910b) under paraffin oil at a temperature of
37 °C and a gas phase of 10 % carbon dioxide in air. This medium is suboptimal
for blastocyst attachment and does not normally support outgrowth. In certain
experiments the medium was supplemented with Foetal Calf Serum (FCS)
and/or mouse uterine fluids. The cultures were examined daily and counts made
of the blastocysts that had hatched from the zona pellucida. An attachment and
outgrowth score was also obtained for each culture using a procedure similar
to that devised by Spindle & Pedersen (1973). The scoring system was attachment 1 point, giant cell transformation 1-5 points and extensive outgrowth
2 points; the result was expressed as a percentage of the highest possible score
(2 points per blastocyst). An assessment of the size of each outgrowth was also
obtained by taking the mean of two diameters measured at right angles on day 5
of culture.
RESULTS
The first experiment was designed to determine the effect of supplementing
the culture medium with uterine fluids obtained during (a) day 4 of pregnancy,
(b) delayed implantation, (c) 48 h after the administration of oestradiol during
diapause, and (d) pro-oestrus. In each case the uterine fluids from three mice
were used to supplement each ml of medium. The hatching frequency observed
in media containing the luminal fluids of animals in diapause was not significantly different from that recorded for unsupplemented medium. In contrast,
a highly significant increase (P < 0-001) in the number of hatched blastocysts
was observed in the presence of uterine fluids recovered on day 4 of pregnancy,
48 h after the administration of oestradiol and during pro-oestrus (Table 1).
Only pro-oestrous uterine fluids induced a degree of attachment significantly
greater than that observed in the unsupplemented control medium (P < 0-005).
Attachment was normally followed, within 2 or 3 days, by a gradual loss of
adhesion and dissociation of the blastomeres. In the presence of pro-oestrous
uterine fluid, however, 38 % of the blastocysts exhibited giant cell transformation and limited outgrowth.
Delayed implantation
297
Table 1. Hatching and attachment of blastocysts on day 4 of culture in
media supplemented with uterine fluid
Supplement
No supplement
Delayed implantation
Day 4
48 h Post oestradiol
Pro-oestrus
% Hatching
% Attachment
and outgrowth
No.
No.
±S.E.
±S.E.
blastocysts
trials
510±4-9
46-3 + 7-9
l6-8±4-7
5-0 ±3-2
164
116
8
6
**82-8±4-7
**95-2±0-6
**91-6±l-6
**P < 0001
10-6 ±4-4
28-4 ±3-5
*55-l±3-8
*P < 0005
184
115
33
7
4
2
In each group the uterine fluids from three animals were used to supplement each ml of
medium. A 2ml reservoir of cold medium was flushed through the uterine horns of six mice
in rapid succession (0-5 ml/horn), extensive precautions being taken to avoid contaminating
the samples with blood. The flushings were then centrifuged, filter-sterilized and dispersed
in 005 ml droplets under paraffin oil. pH determinations on the culture medium following
equilibration indicated that the presence of uterine fluids did not influence this factor.
The second experiment was designed to determine the effect of supplementing
the culture medium with a fixed quantity of macromolecular luminal protein
recovered on either day 4 of pregnancy or during diapause. At both the 20 and
30 /tg dose levels the hatching frequencies observed in the presence of 'day 4'
and ' delay' proteins were not significantly different from each other, but were
significantly above that observed in unsupplemented control medium (P < 0-05).
Blastocyst attachment was not significantly influenced by the addition of uterine
proteins in this experiment and trophoblastic outgrowth was not observed
(Table 2).
A third experiment was designed to determine whether the presence of uterine
fluids recovered during diapause or at the time of implantation would enhance
or suppress serum-induced giant cell transformation and outgrowth of the
trophoblast. The number of blastocysts hatching from their zonae was not
significantly influenced by the quantity of FCS in the medium or the presence
of uterine fluids. The degree of blastocyst attachment and outgrowth was influenced by the concentration of FCS, however, the score observed in the presence of 1 % FCS being significantly greater than that recorded for 0-5 % or
0-25 % (P < 0-05). The subsequent addition of either 'day 4' or 'delay' uterine
fluids significantly enhanced blastocyst attachment and outgrowth when the
concentration of FCS was minimal (0-25 %) (P < 0-02). The size attained by the
outgrown blastocysts showed a highly significant correlation with the concentration of FCS in the medium (P < 0-001; r = 0-99). The addition of 'day 4' and
'delay' uterine fluids induced a further significant increase (P < 0001) in the
size attained by the outgrowths in a majority of experimental groups (Table 3).
298
R. J. AITKEN
Table 2. Hatching and attachment of blastocysts on day 4 of culture in
media supplemented with known quantities of uterine protein
% Hatching
Supplement
20 fig delay protein
20 fig day-4 protein
30 fig delay protein
30 fig day-4 protein
No supplement
±S.E.
% Attachment
and outgrowth
iS.E.
blastocysts
No.
trials
33-8 ±2-2
38-2±7-8
45-2 ±2-6
361 ±5-6
34-6 ±3-1
51
48
49
53
78
2
2
*92-liO-l
*980±20
*94-4i5-6
*96-6i3-5
70-115-4
*P < 005
No.
2
2
4
Uterine flushings were collected from six to eight mice, using 0-5 ml phosphate buffered
saline per horn, then dialysed, lyophilized and finally reconstituted with doubledistilled water.
The protein content of each preparation was determined by the Lowry technique (Lowry,
Rosebrough, Farr & Randall, 1951) and 20 fig or 30 fig of protein added to each ml of medium
in a volume of 30 fi\. Each ml of the control medium was supplemented with 30 fi\ of double
distilled water.
Table 3. Hatching and attachment of blastocysts on day 4 of culture and mean
outgrowth diameter on day 5 of culture in media supplemented with foetal calf
serum and uterine fluids
°/
/o
Foetal
calf
serum
0-25
0-50
1 -00
% Attachment
and
No.
/o
Hatching outgrowth blasto±S.E.
±S.E.
cysts
0/
Supplement
No supplement
Delayed implantation
Day 4
No supplement
Delayed implantation
Day 4
No supplement
Delayed implantation
Day 4
820 ±7-9 59-6 ± 6 0
87-7±2-3 *831 ±3-8
90015-1 *82-9±4-9
83-816-3 77-315-3
73-9110-2 67-219-9
93-413-6 90-013-6
87-112-8 82-513-9
90-315-7 88-214-6
93-114-3 90-713-6
*P < 002
109
110
88
143
72
69
267
97
96
Outgrowth
diameter
±S.E.
(mm)
0-23810-007
*0-31710-007
*0-29210-007
0-294 ± 0008
*0-341 10-014
0-289 + 0-008
0-404 + 0008
*O-447iOO12
*0-494± 0-022
*P < 0001
No.
outgrowths
83
77
81
94
43
39
119
43
36
The three concentrations of FCS used in this experiment (1 %, 0-5% and 0-25%) were
equivalent to a supplement of 660 fig, 330 fig and 165 fig protein per ml culture medium.
The uterine fluids of three mice were used to inoculate each ml of medium.
DISCUSSION
The hatching frequencies observed in the first experiment suggest that uterine
fluids recovered from mice on day 4 of pregnancy or following the administration of oestradiol-17/? contain embryotrophic factors which are absent during
diapause. The high concentration of protein present in the uterine lumen at the
time of implantation (Table 4) may account for this embryotrophic stimulus
Delayed implantation
299
Table 4. Relationship between the hatching frequencies observed in the first
experiment (Table 1) and the estimated concentration of uterine protein in the
culture media
Supplement
No supplement
Delayed implantation
Day 4
48 h Post oestradiol
Pro-oestrus
t Aitken (1977).
% Hatching ±S.E.
Estimated concentration of uterine protein in culture media
(/tg/ml)
510 + 4-9
20-7f
46-3 ±7-9
59-7f
82-8 ±4-7
95-2 ±0-6
39-9t
48-OJ
91-6±l-6
% R. J. Aitken, unpublished observations.
(Aitken, 1977). A high luminal concentration of embryotrophic protein cannot
be the only uterine requirement for the initiation of implantation however.
Pro-oestrous uterine fluid was found to be highly embryotrophic in this study,
and yet blastocysts transferred to pro-oestrous uteri do not implant. Evidently
the endometrium must differentiate into a receptive state under the combined
influence of progesterone and oestrogen (Psychoyos, 1973) before the stimulation
of blastocyst activity can be translated into implantation.
Although the macromolecular fractions of both 'day 4' and 'delay' uterine
flushings were found to be equally stimulatory in this study (Table 2), some
evidence was obtained for the presence of a dialysable inhibitor of blastocyst
activity in the uterine lumen during diapause. This is suggested by the hatching
rates observed in the presence of unfractionated (46-4 %; Tables 1 and 4) and
dialysed 'delay' uterine fluids (92 1 %; Table 2). In both instances an estimated
20 /tg of uterine protein was added to each ml of medium, but only after dialysis
was the hatching frequency elevated over the control (P < 0-05). The possibility
t h a t ' delay' uterine fluids inhibit blastocyst hatching in a non-specific manner
(e.g. suboptimal salt concentration) must not be overlooked. However, evidence
for the existence of a small molecular weight inhibitor of blastocyst activity
has also been obtained by Psychoyos (1973) in the rat and Weitlauf (1976) in
the mouse.
The results obtained in the third experiment indicate that if such an inhibitor
does exist, its influence can be neutralized by the presence of serum. The ability
of serum to negate the inhibitory influence exerted by 'delay' uterine fluids
may be relevant to the mechanism of blastocyst activation in the mouse. The
termination of diapause in this species is associated with an influx of high
molecular weight proteins into the uterine lumen (Aitken, 1977) and some of the
most prominent proteins are of serum origin.
I am grateful to Dr Anne McLaren for her encouragement during this study and to the
M.R.C. for financial support.
300
R. J. AITKEN
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(Received 1 February 1977, revised 19 April 1977)