/. Embryol exp. Morph. Vol. 71, pp. 75-82, 1982
75
Printed in Great Britain © Company of Biologists Limited 1982
Correlation between blastocyst oxygen
consumption and trophoblast cytochrome oxidase
reaction at initiation of implantation of delayed
mouse blastocysts
ByB. OVE NILSSON1, CLAES MAGNUSSON2,
SIBYLLE WIDfiHN1 AND TORBJORN HILLENSJO2
From the Reproduction Research Unit, Biomedical Centre, Uppsala
and the Department of Physiology, University of Gothenburg
SUMMARY
Delayed blastocysts had an oxygen consumption of 0-24 nl/h, while only 4 h after an
oestrogen injection the respiration had increased nearly two fold, remaining at this level both
8 and 18 h after activation for implantation. The mitochondria of delayed blastocysts exhibited
no positive cytochrome oxidase reaction, neither in the trophoblast nor in the embryoblasts.
A few mitochondria at 8 h and most of those of blastocysts activated for 18 h were positive.
It is suggested that the activation of blastocysts for implantation is initiated by a surge of
substrates for glycolysis into the uterine secretion causing an increased energy production
by glycolysis which in turn makes possible an increase of the cytochrome oxidase activity of
the mitochondria thus getting oxidative phosphorylation into action.
INTRODUCTION
When a pregnant mouse is spayed within three days after mating and subsequently given progesterone, its blastocysts will slowly attain a low metabolic
activity and then remain in a state of experimental delay. From this state of
delay, the blastocysts can be activated once again and induced to implant within
24 h by giving the animal an injection of oestrogen.
It is known that both in the mouse (Mills & Brinster, 1967) and in the rabbit
(Fridhandler, Hafez & Pincus, 1957) the morula has low respiratory activity,
while the blastocyst has a high oxygen consumption. The oxygen requirement
of the blastocyst during delayed implantation is, however, not known. It can be
inferred from the marked increase in carbon dioxide production (Menke &
McLaren, 1970; Menke, 1972; Weitlauf, 1974) by delayed mouse blastocysts
1
Authors* address: Reproduction Research Unit, Biomedical Centre, Box 571, S-751 23
Uppsala, Sweden.
2
Authors' address: Department of Physiology, University of Gothenburg, S-400 33
Goteborg, Sweden.
76 B. O. NILSSON, C. MAGNUSSON, S. WIDEHN AND T. HILLENSJO
0-6 -
~
0-5
-
0-4 —
a.
I 0-3
0-2
-
01-
12
18
4
20
16-18
Time (h)
Fig. 1. Oxygen consumption by blastocysts recovered from mice in an experimental
delay of implantation at various hours after an injection of oestrogen. The figure
shows data from two experiments (mean±s.E.). The number of samples (each
including 1-2 blastocysts) is indicated within the bars. Thirty-four mice were used.
** = P < 001 v.Oh.
activated for implantation that these blastocysts also increase their oxygen need
at implantation. One purpose of the present study was therefore to measure the
oxygen consumption of mouse blastocysts at different times after activation
for implantation by oestrogen. A highly sensitive microspectrophotometric
technique, using haemoglobin as an indicator of oxygen tension, was applied
(Hultborn, 1974; Magnusson et al 1977).
One site where metabolic control may be exerted in the cell is the cytochrome
system, which is the last link in the electron transport chain which governs the
oxidative component of metabolism. One way of judging the capacity of uterine
blastocysts to utilize oxygen is to evaluate the cytochrome oxidase activity by
the 3,3'-diaminobenzidine tetrahydrochloride (DAB) reaction and electron
microscopy. This technique measures the mitochondrial respiratory potency
and was therefore applied in the present study to compare delayed and activated
mouse blastocysts to find out whether a difference in the activity of the cytochrome system might explain possible differences in their capacity for aerobic
metabolism. In this part of the study also, blastocysts were examined at different
times after activation for implantation.
Blastocyst oxygen consumption and cytochrome oxidase
11
Fig. 2. Trophoblast mitochondria in a blastocyst recovered from a mouse in experimental delay of implantation 18 h after an injection of oestrogen. The DAB reaction
is positive in most of the mitochondria, as demonstrated by the darkly stained
mitochondrial cristae. x 40000.
MATERIALS AND METHODS
Mated mice (NMRI) were sprayed three days after mating and kept in a state
of delayed implantation by giving a depot dose (1 mg) of progesterone (DepoProveraR, Upjohn AB) each 5th day. After at least eight days of delay, their
blastocysts were activated by a subcutaneous injection of 0-1 /ig of oestradiol.
After various lengths of time, the activated blastocysts were flushed out of the
uterus, delayed control blastocysts being obtained in the same way from noninjected animals.
Oxygen consumption was determined in four groups of blastocysts, namely
blastocysts obtained 0, 4, 8 and 16-18 h after the injection of oestrogen.
In these experiments the uteri were flushed with a phosphate-buffered
culture medium without glucose, arginine and leucin (Naeslund, 1979).
One to two blastocysts were incubated in gas-tight microchambers
(12-3 nl) filled with oxyhaemoglobin-containing (30-35 mM) culture medium
and the shift in absorbance of monochromatic light (435 nm) was recorded as
the oxygen tension decreased (Magnusson et al. 1977). Between one and three
batches of blastocysts were examined from each mouse. All measurements from
78 B. O. NILSSON, C. MAGNUSSON, S. WIDEHN AND T. HILLENSJO
*"*.»<
t*
'r
« , '
f--^»' : - • ? / • • ' . ^ ^ r i *.«
Fig. 3. A trophoblast mitochondrion from a mouse blastocyst in experimental
delay of implantation. The DAB reaction is negative, x 80000.
Fig. 4. A trophoblast mitochondrion from a mouse blastocyst 18 h after oestrogen
injection following experimental delay of implantation. The DAB reaction is
positive, x 80000.
one animal were completed within 30 min after obtaining the blastocysts. There
was no systematic difference in oxygen consumption between the first and last
sample of blastocysts examined from each animal. Controls, that is measurements of samples without any blastocysts, did not show any oxygen consumption. Oxygen consumption is expressed as nl 0 2 /h/blastocyst and given as mean
±S.E. for each treatment group. Statistical differences were calculated by
analysis of variance followed by the Student-Newman-Keul multiple range test
(Woolf, 1968). A P value of <0-05 was considered significant.
For examination of the cytochrome oxidase activity, blastocysts taken 0, 8
and 16-18 h after the injection of oestrogen were used. At each stage six-eight
histochemical experiments were performed, each experiment including four-five
blastocysts.
The blastocysts were flushed out of the uterine cavity with a freshly prepared
fixative of 2-5% pure glutaraldehyde (Serva, Heidelberg, W-Germany) in
0-1 M Na-cacodylate buffer, pH 7-3. The total fixation time was 2-5 min.
Following fixation, which took place at room temperature (+18°C), the
Blastocyst oxygen consumption and cytochrome oxidase
79
blastocysts were rinsed for at least 2 h at + 4 ° C , beginning with 0-2 M
Na-cacodylate buffer, pH 7-3, but with a change to 0-05 M Tris-HCl buffer,
pH 7-3.
The DAB technique used was that described by Andersson & Perotti (1975).
Before incubation with DAB, the specimens were again brought to room
temperature. The incubation was carried out for 90 min at + 30 °C, immediately
after the addition of H2O2, in freshly prepared medium 0-1% 3,3'-diaminobenzidine tetrahydrochloride, DAB (Serva, Heidelberg, W-Germany) in
0-05 M Tris-HCl buffer, pH 7-8, with 0-002% H2O2 (Perhydrol, Merck, Darmstadt, W-Germany, final pH 7-3). The control medium used was the incubation
medium without DAB. After incubation, the blastocysts were washed several
times, first in Tris-HCl buffer, then in the Na-cacodylate buffer, and post-fixed
in 1% osmium tetroxide in the Na-cacodylate buffer for 60 min at room
temperature.
The dehydration in ethanol and part of the Epon embedding were carried out
at + 4 °C, which renders handling of the blastocysts easier. The embedded
blastocysts were sectioned on an LKB ultrotome, stained with uranyl acetate
and lead citrate, and examined in a JEOL 100B electron microscope.
RESULTS
Oxygen consumption
The delayed blastocysts had an oxygen consumption of 0-24 ± 0-01 nl/h
(Fig. 1). Already 4 h after the oestrogen injection it had increased two fold and
did not increase further up to 18 h after injection.
Cytochrome oxidase activity
The trophoblast and embryoblast cells were usually found to be well preserved.
Mitochondria, endoplasmic reticulum, ribosomes, microfibrils, and other cell
constituents were recognized. The mitochondria of blastocysts in delay
did not show any positive cytochrome enzyme reaction (Fig. 3), while in those
of blastocysts activated for implantation for 18 h, activity was demonstrated
(Figs 2 and 4). Most mitochondria were stained, but the staining within a
mitochondrion was not always homogeneous. At the 8 h stage, the mitochondrial
reaction varied more, some blastocysts showing a positive reaction while
others were negative.
DISCUSSION
The oxygen consumption of mouse blastocysts during implantation delay
was about 0-2 nl/h, but 18 h after the oestrogen injection, which corresponds
to a stage when the blastocysts are about to implant, it had increased by more
than 100%, to reach about 0-5 nl/h. This value is similar to that found in
normal implanting mouse blastocysts by Mills & Brinster (1967) using a
80
O. NILSSON, C. MAGNUSSON, S. WIDEHN AND T. HILLENSJO
micromanometric technique. Thus, implanting blastocysts, whether they are
implanting by the normal process or following a delay, have the same rate of
respiration.
The cytochrome oxidase reaction of trophoblast mitochondria was negative
during delay but positive at implantation. Thus, the low oxygen consumption
during delay might be explainable by an incompletely functioning respiratory
chain in the mitochondria of the trophoblast cells.
The lack of visible cytochrome oxidase in mitochondria of delayed blastocysts would seem to imply that they are totally unable to carry out oxidative
phosphorylation. However, as seen from the measurements of oxygen consumption, this is not true, since delayed blastocysts were respiring, though at a lower
level. Even if a certain amount of oxygen is consumed by the endoplasmic
reticulum (Gonzales-Cadavid & Saez de Cardova, 1974; Robbi, Berthet &
Beaufay, 1978), the mitochondria are probably responsible for the major part
of the oxygen consumption. The discrepancy between the results of the two
techniques can be explained by a lower sensitivity of the histochemical as
compared with the respirometric method. It is also possible that mitochondrial
activity, as measured by the DAB technique, may not be rate limiting for
respiration at this stage of development. Respiration might also be controlled
by other factors, such as substrate availability.
The oxygen consumption of implanting blastocysts increased rapidly and
already 4 h after injection of oestrogen, the same level was reached as in
blastocysts recovered 8 and 18 h after the initiation of implantation. This rapid
increase suggests that the underlying mechanism for augmenting the amount
of enzymes might be either an activation of enzyme precursors or a translation
of ready-made RNA templates. Thus, the delayed blastocyst is prepared for a
rapid increase of its metabolic activity. This is also evident from experiments on
the reversibility of the activation for implantation (Naeslund, 1979). The
results of these experiments demonstrated that a delayed blastocyst, being
activated for more than 3 h but less than 6 h in vitro by culture in a glucosecontaining complete medium, could no longer be brought back to its delayed,
inactive state by being transferred to a growth-arrest medium. By this time the
capacity for oxidative phosphorylation will keep the growth of the blastocyst
unimpeded.
The energy required by the blastocyst during the first hours of growth after a
breaking of delay should derive from glycolysis. This view is strengthened by
the finding that culturing blastocysts in a medium without glucose but with free
access of oxygen will markedly impede the outgrowth of the blastocysts (Naeslund, 1979; van Blerkom, Chaez & Bell, 1979). Since an injection of oestrogen,
which will end the period of blastocyst delay in an animal, causes a rapid
production of a uterine secretion (Nilsson & Lundkvist, 1979), we assume that
the newly-produced secretion contains substrates for glycolysis. In fact, analyses
Blastocyst oxygen consumption and cytochrome oxidase
81
of the early uterine secretion have shown that it contains glucose (Nilsson,
Ostensson, Eide & Hellerstrom, 1980).
It can be imagined that the uterine mucosa can restrict the secretion of
substrate for glycolysis but not easily of oxygen for oxidative phosphorylation.
Therefore, it could be that an insufficient capacity for oxidative phosphorylation
in the mitochondria together with a lack of substrate for glycolysis in the
secretion are factors of importance for keeping the blastocysts in delay. Since
depleting blastocyst culture medium of arginine and leucin in addition to glucose
results in a growth arrest of a longer duration than that caused by a lack of
glucose only (Naeslund, 1979), it is probable that other substrates in the
uterine secretion are crucial for the activation of a delayed blastocyst. Of
course, another factor contributing to blastocyst delay can be the postulated
inhibitor of blastocyst activity suggested to be present in the uterine secretion
during delay (Psychoyos & Bitton-Casimiri, 1969; Weitlauf, 1978).
Financial support was received from the Swedish Medical Research Council (Project No.
B80-12X-00070), the Magnus Bergvall Foundation and the 'Expressen' Prenatal Research
Foundation. Mrs Barbro Einarsson provided much able technical assistance. Upjohn AB,
Partille, Sweden, kindly offered the Depo-ProveraR.
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{Received 29 September 1981, revised 1 March 1982)