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The duration of the preimplantation period is not rigidly fixed in mice and rats. Although attachment of embryos to the uterine epithelium normally occurs late on the 5th day after fertilization, it may be delayed for days or weeks by concurrent lactation (naturally delayed implantation) or maternal ovariectomy (experimentally delayed implantation). It has long been known that the development of such 'delayed implanting' embryos is arrested at the blastocyst stage, and it has recently been shown that their overall embryonic metabolism is reduced. This depressed metabolic rate may be related to the ability of embryos to survive the prolonged free-living phase associated with delayed implantation.

How delayed implanting embryos are rendered metabolically dormant and then reactivated to implant at a later time is unknown. However, a comparison of changes in the rates of

Summary. Fluid flushed from the uteri of 'delayed implanting' and 'implanting' mice was fractionated by gel-filtration. The fractions were freeze-dried, individually resuspended in culture medium containing [³H]uridine, and used for incubating 'implanting' blastocysts. Several factors were found that reduced incorporation of [³H]uridine by the blastocysts. The amount of inhibitory activity in corresponding factors was generally similar in flushings from both types of uteri, but there was significantly more inhibitory activity in the void volume fractions of flushings from 'delayed implanting' animals and it is suggested that this factor may be responsible for the metabolic dormancy of embryos during the diapause associated with delayed implantation.

Summary. A dual-label ratio method was used in conjunction with two-dimensional polyacrylamide gel electrophoresis to measure the relative changes in rates of production of individual secreted proteins by mouse uteri at the start of the process of decidualization. A characteristic pattern of differential changes in the rates of synthesis and secretion of the proteins was found to be associated with development of a positive Pontamine Blue reaction at the site of embryo implantation. These changes were compared with those associated with development of experimentally induced deciduomata and although the patterns were similar, presumably reflecting common processes in transformation of the endometrium, there was preferential enhancement of a subset of small (M _r 14 000–20 000) acidic proteins in the authentic implantation sites. It is suggested that this embryo-dependent modification of constitutive changes associated with decidualization reflects a form of embryo–maternal signal–response mechanism that may be important for the process of implantation in mice.

Keywords: implantation; uterine proteins; embryonic signalling; decidual reaction; mouse

Changes in the expression of antigens on mouse uterine or embryonic tissues were examined by enzyme immunocytochemistry. Cryostat sections of uteri from days 1, 8 and 15 of pregnancy were probed with the monoclonal antibodies M3/38 and M3/84, originally defined by their reactivity with macrophage surface antigens (Mac-2 and Mac-3, respectively). In the uterus of pregnant mice, reaction of these antibodies was not limited to leucocytes. M3/38 did not react at detectable levels with cells in the uterus on day 1 but did react with decidual cells immediately surrounding the embryo on day 8. By day 15, the placenta, decidua basalis and metrial gland were intensely positive but the embryo was negative. M3/84 reacted with the luminal side of the endometrium on day 1, the entire decidual mass on day 8, and with all maternal and fetal tissues on day 15. M3/38 was detected in saline-soluble preparations of uterine tissue and had a molecular mass of approximately 32–35 kDa as determined by SDS–PAGE and western blot analysis. The pattern of expression of these molecules suggests a functional relationship to developmental changes that occur at the maternal–fetal interface.

The ontogeny of immunospecific cell surface determinants on preimplantation mouse embryos was determined by means of an antibody-dependent complement-mediated cell lysis assay. The determinants conferring sensitivity to lysis in that assay were first observed at the late blastocyst stage on embryos recovered from normal pregnancy on day 5 or grown to an equivalent stage in vitro. Although blastocysts recovered during the dormant phase associated with delayed implantation were not lysed, those recovered following reactivation with an injection of oestrogen to the mother were sensitive. Furthermore, it was found that treating the dormant embryos with neuraminidase rendered them sensitive to lysis. These results demonstrate that the appearance of specific cell surface determinants on mouse embryos is temporally associated with the process of attachment to the uterus in both normal pregnancy and at termination of the dormant phase associated with delayed implantation. They also indicate that those determinants may be 'masked' with sialic acid during embryonic diapause. It is suggested that such cell surface determinants could be important for embryo attachment and that the mechanism responsible for their expression may explain some aspects of the synchronization between the preimplantation conceptus and its mother at the time of implantation.

Summary.

`Normal' and `delayed' implanting' mouse blastocysts were incubated in non-radioactive culture medium for various lengths of time (preincubation), and then placed in culture medium with [¹⁴C]-glucose for 2 hr. The rate of embryonic CO₂ production was estimated from ¹⁴CO₂ given off by the blastocysts. Normal embryos were unaffected by the length of preincubation. By contrast, `delayed implanting' embryos had a low level of CO<sub>2</sub> production with short preincubations (i.e. less than 2 hr), but approached the normal range with longer preincubations (i.e. more than 8 hr) and thus, were `activated' in vitro. Incubation of the `delayed implanting' embryos with actinomycin D prevented the expected increase in CO<sub>2</sub> production, indicating that synthesis of new RNA is necessary for their activation. Preincubation of normal and `delayed implanting' blastocysts with oestradiol-17β and/or progesterone had no effect on the level of CO₂ production, suggesting that the hormones do not directly stimulate or inhibit carbohydrate metabolism in the embryos.

Menke & McLaren (1970) reported that CO₂ production by `delayed implanting' mouse embryos is significantly lower than that of blastocysts implanting at the normal time. They suggested that the metabolic quiescence associated with delayed implantation could result either from an inhibitory effect of progesterone, or from a lack of stimulation by oestrogen. The present experiments were undertaken to distinguish between these possibilities; maternal levels of oestradiol and progesterone were manipulated and the effect on CO₂ production by preimplantation embryos was determined.

Sexually mature, virgin, white Swiss mice at random stages of the oestrous cycle were induced to ovulate with injections of gonadotrophins (Fowler & Edwards, 1957). The females were placed with fertile males and mating was confirmed by the presence of a vaginal plug on the next

Summary. The possibility that the embryonic diapause associated with delayed implantation in mice is maintained by limitation of an essential amino acid, energy substrate or concentration of ions was examined by comparing the rates of DNA synthesis in delayed implanting embryos that were 'reactivated' by incubation in 'complete' medium or in one of several specially formulated 'deficient' media. It was found, in agreement with earlier observations, that an increase in the rate of DNA synthesis could be detected within 12 h and continued through 72 h in complete medium. An identical pattern was found when embryos were incubated in medium deficient in amino acids and vitamins. Similar patterns of activation were observed in the absence of all metabolizable substrates, a drastically reduced concentration of Na⁺, and even in a medium consisting only of 25 mm-bicarbonate buffer, NaCl and KCl. The embryos incubated in the more drastically deficient media appeared to be damaged after 18–24 h. Nevertheless, the observation that the rate of DNA synthesis did not remain depressed suggests that such deficiencies are not the means by which embryonic dormancy is maintained in utero.

Summary. Mouse embryos collected before implantation were incubated in vitro for 24 h with fluid rinsed from the uteri of ovariectomized female mice injected with progesterone, oestradiol-17β + progesterone, or oestradiol-17β alone. Although none of the zonae was completely dissolved, those incubated in fluid from animals treated with oestradiol + progesterone were subsequently more soluble in sodium thiocyanate (NaSCN) than those incubated similarly in control buffer, indicating a sublytic change during the incubation with uterine washings. Zonae incubated in fluid from animals injected with either hormone alone did not undergo such a change.

Summary.

Mature mice were ovariectomized on the 4th day post coitum (Day 1 = vaginal plug) and given no further treatment. Unimplanted (delayed) blastocysts were recovered from these females between 5 and 40 days post coitum. They were tested for viability by determining the proportion that developed into normal foetuses following transfer to Day-4 pseudopregnant recipients.

It was found that the proportion of transferred blastocysts developing into foetuses was similar after delays of from 0 to 10 days. After delays of more than 10 days, the proportion of blastocysts that were capable of developing into normal foetuses decreased; 30 to 32% developed following transfer on Days 5 to 15, 18% with transfer on Day 20, and 3% with transfer on Day 30. The finding that blastocysts can be delayed in the absence of ovarian hormones and yet remain capable of developing into normal foetuses demonstrates that progesterone is not essential for blastocyst survival in mice.