Delayed blastocysts had an oxygen consumption of 0 ·24 nl/h, while only 4h 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.

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 (Fridhändler, 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 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.

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 (Depo-ProveraR, Upjohn AB) each 5th day. After at least eight days of delay, their blastocysts were activated by a subcutaneous injection of 0·1 μg 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 non-injected 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 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 02/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 blastocysts were rinsed for at least 2h 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.

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.

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 (meantS.E.). The number of samples (each including 1 –2 blastocysts) is indicated within the bars. Thirty-four mice were used. ** = P < 0 ·01 v.Oh.

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 (meantS.E.). The number of samples (each including 1 –2 blastocysts) is indicated within the bars. Thirty-four mice were used. ** = P < 0 ·01 v.Oh.

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.

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. × 40000.

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. × 40000.

Fig. 3.

A trophoblast mitochondrion from a mouse blastocyst in experimental delay of implantation. The DAB reaction is negative, × 80000.

Fig. 3.

A trophoblast mitochondrion from a mouse blastocyst in experimental delay of implantation. The DAB reaction is negative, × 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, × 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, × 80000.

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 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 blasto-cysts 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 4h 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 glucose-containing 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 (Naes-lund, 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 of the early uterine secretion have shown that it contains glucose (Nilsson, Östensson, Eide & Hellerström, 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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