Delayed Implanting Embryos Research Articles

Synthesis and secretion of stage-specific proteins by peri-implantation mouse embryos.

Blastocysts were recovered from intact mice at various times on the fourth and fifth days of pregnancy and incubated in vitro with 35S-methionine. Labeled proteins synthesized by the embryos and secreted into the medium were separated in two-dimensions on polyacrylamide gels by electrophoresis and localized by fluorography. The array of proteins synthesized and secreted by late stage blastocysts was found to be qualitatively and quantitatively different from those released by embryos at earlier stages of development. Similar changes were also observed in secreted proteins when delayed-implanting embryos were reactivated after an injection of estrogen. Furthermore, there was a temporal correlation between the appearance of certain proteins secreted by the embryos and changes in specific proteins synthesized and released by the uterus. It is suggested that these various secreted proteins constitute a signal-response mechanism that is important for the process of embryo implantation in mice.

Effects of metabolic substrates and ionic environment on in-vitro activation of delayed implanting mouse blastocysts.

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.

Regulation of glycolysis in the mouse blastocyst during delayed implantation.

The rate of oxidation of glucose is reduced in mouse embryos in the prolonged free living phase associated with delayed implantation and increases when the embryos are reactivated by estrogen. To determine how these changes in metabolism are regulated, several aspects of glucose metabolism were evaluated in dormant and reactivated blastocysts: 1) Embryos were exposed to 14C-pyruvate in vitro and evolved 14CO2 was measured. It was found that the rate of production of CO2 was equal in the two types of blastocysts, suggesting that aerobic pathways are fully functional during delayed implantation. 2) Production of lactate in the presence of O2 was measured and a decrease of 30% was found in delayed implanting embryos, suggesting that the overall regulatory mechanism for glucose metabolism resides in the glycolytic portion of the pathway. 3) Capacity for uptake and phosphorylation of glucose was evaluated using 3H-2-deoxyglucose and was found to be equal in the two types of embryos. 4) Total amounts of the rate-controlling enzymes for glycolysis (i.e., hexokinase and phosphofructokinase) in lysates of delayed and reactivated embryos were found to be equal, indicating that amounts of these enzymes are not limiting in delayed implantation. 5) Lactate production, measured under anaerobic conditions, was found to be equal, demonstrating that it is not the capacity for glycolysis but a difference in the degree of allosteric inhibition that is responsible for reduced glucose oxidation in delayed implantation. 6) Levels of ATP, ADP, and hexose-6-phosphates were found to be consistent with allosteric inhibition of the glycolytic pathway at phosphofructokinase during delay and a release of this inhibition with reactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of the rates of accumulation of nonpolyadenylated and polyadenylated RNA in normal and delayed implanting mouse embryos

Two experiments were done to examine changes in the rates of accumulation of RNA by mouse blastocysts entering and leaving the metabolically quiescent phase associated with delayed implantation. In both cases, normal embryos were labeled with [ 14C]uridine while delayed implanting embryos were labeled with [ 3H]uridine. The differentially labeled blastocysts were mixed together and their RNA was extracted. The overall rates of accumulation of both rRNA and mRNA were determined in delayed implanting embryos and expressed as percentages of normal. In the first experiment, delay of implantation was caused by removing the maternal ovaries at noon on the fourth day of pregnancy. The rates of accumulation of both rRNA and mRNA were found to decrease steadily over the subsequent 3–12 hr as the blastocysts entered the metabolically quiescent phase. In the second experiment, dormant embryos were reactivated by injecting ovariectomized females with estradiol. A slow but steady increase was observed in the rate of accumulation of rRNA in the following 18–21 hr; by contrast, a sharp increase was found in the accumulation of mRNA within the first 3 hr. Interestingly, this early burst was transient and subsequently the rate of accumulation of mRNA increased only slowly along with rRNA. Although the significance of the rapid increase in accumulation of mRNA during reactivation was not determined, it seems likely that it is involved with the regulation of metabolism and is part of the mechanism responsible for controlling embryonic diapause.

Resumption of DNA synthesis during activation of delayed implanting mouse blastocysts.

Cell division ceases in mouse blastocysts during the extended dormant period associated with delayed implantation but resumes following activation of the embryos by administration of 17 beta-estradiol to the mother. To determine the temporal and spatial aspects of the resumption of DNA synthesis during activation, blastocysts were recovered from delayed implanting females at various intervals after an injection of 17 beta-estradiol, incubated with 3H-thymidine in vitro, and prepared for light microscopic autoradiography. Although less than 4% of the cells were labeled in delayed implanting embryos, the proportion of labeled cells increased soon after the administration of 17 beta-estradiol and reached a maximum of over 50% by 24 hours. This increase in labeling was not uniform in all regions of the embryo, i.e., the labeling index of the inner cell mass began a steady increase immediately after the injection of 17 beta-estradiol while labeling of the polar and proximal mural trophoblast remained depressed for 6 and 12 hours, respectively, and only then began to increase. No labeling was present over the distal mural trophoblast in delayed implanting or activated blastocysts although cytological changes characteristic of primary giant cell transformation were present in activated embryos. These results indicate that the resumption of DNA synthesis is part of the overall increase in metabolic activity associated with activation. Furthermore, the sequential pattern of resumption of synthesis suggests that the ICM may influence the initiation of DNA synthesis in the surrounding trophoblast.

Comparison of overall rates of RNA synthesis in implanting and delayed implanting mouse blastocysts in vitro

Implanting and delayed implanting mouse embryos were incubated in vitro with [ 3H]uridine for 2–24 hr. The size and specific activity of the [ 3H]UTP pools were determined by means of a double isotope technique using copolymer synthesis with the [ 3H]UTP in the embryos, exogenous [ 14C]ATP, and E. coli RNA polymerase. Using the rate of incorporation of [ 3H]uridine into acid-insoluble material and the specific activity of the [ 3H]UTP pools, it was possible to calculate the overall rate of incorporation of uridine into RNA by the embryos. In implanting embryos it was constant for 24 hr. In contrast, the initial rate of uridine incorporation by the delayed implanting embryos was only 31% of that in implanting embryos (i.e., per cell); this increased steadily during the incubation period, reaching 81% of the rate in implanting embryos after 24 hr. This activation of RNA synthesis by delayed implanting embryos in vitro occurred in the absence of any uterine stimulatory factors. Further, it was shown that although 10% mouse serum would support trophoblastic outgrowth in vitro, it did not influence uptake, distribution of label into nucleotides, or rate of uridine incorporation into RNA in either implanting or delayed implanting embryos. Therefore, it is suggested that if depression and activation of metabolic activity in blastocysts are part of the mechanims of delayed implantation, and if trophoblast outgrowth in vitro is analogous to the process of implantation in vivo, then these two aspects of embryo activation are under different controls.

Effect of uterine flushings on RNA synthesis by 'implanting' and 'delayed implanting' mouse blastocysts in vitro.

Blastocysts were collected from the uteri of ‘normal’ and ‘delayed implanting’ mice and incubated with either Brinster’s medium containing H uridine, or the same medium mixed with material flushed from the uteri of ‘implanting’ or ‘delayed implanting’ animals. The rate of embryonic RNA synthesis was estimated from the incorporation of H uridine by the embryos in vitro. RNA synthesis by normal embryos incubated in Brinster’s medium was linear for at least 6 h. RNA synthesis by delayed implanting embryos in Brinster’s medium was initially low and increased to the level of normal embryos by the fourth hour in vitro. In contrast, RNA synthesis was low in both types of embryos incubated in medium containing uterine flushings; material from the uteri of ‘implanting’ animals was as effective an inhibitor of RNA synthesis as that from ‘delayed implanting’ animals. The apparent inhibitory activity is dialyzable and heat resistant. It is not known whether or not the inhibitory effect of uterine flushings on embryonic RNA synthesis found in vitro has any significance in vivo.

Effect of actinomycin D on protein synthesis by delayed implanting mouse embryos in vitro.

AbstractBlastocysts were collected from the uteri of intact or ovariectomized mice and incubated with or without actinomycin D in a medium containing [14C] amino acids. In the absence of actinomycin D the rate of amino acid incorporation by blastocysts from intact mice (i.e., normal embryos) was constant; the rate of amino acid incorporation by blastocysts from ovariectomized mice (i.e., delayed implanting embryos) was initially low and increased to the normal value after four hours in vitro. In the presence of actinomycin D the rate of amino acid incorporation by normal embryos was only slightly reduced but the expected increase in the rate of amino acid incorporation by delayed implanting embryos was completely blocked. It is suggested that metabolic “activation” of delayed implanting mouse embryos requires the synthesis of new RNA.

Changes in the protein content of blastocysts from normal and delayed implanting mice.

AbstractTotal protein content of preimplantation mouse embryos was measured at 06.00 and 16.00 hours on day 5 of pregnancy (day 1, the day of vaginal plug formation). During this ten hour period, just prior to implantation, the amount of protein increased from 23 to 35 ng per blastocyst; a net increase of 52%. In contrast, protein content was unchanged in delayed implanting embryos recovered from ovariectomized animals during a comparable period on days 5 or 10 (day 5, 24 ng at 06.00 hours and 24 ng at 16.00 hours; day 10, 29 ng at 06.00 hours and 30 ng at 16.00 hours).These results demonstrate that the different uptake and incorporation of amino acids between normal and delayed implanting embryos (Weitlauf and Greenwald, '65, '68; Weitlauf, '73) reflect a difference in the actual net synthesis of protein by the two types of blastocysts.