The effect of oxygen on the development of preimplantation mouse embryos in vitro

The optimal oxygen tension for development of preimplantation mouse embryos to the blastocyst stage in vitro was found to be between 2.5% and 5%. One- and two-cell embryos had a more sharply defined range of oxygen tension capable of supporting development than 8-cell and morula stages. At all stages of development, more embryos developed to the blastocyst stage under 5% O2 compared to the numbers of developing under higher oxygen tensions (20% and 40% O2). The blastocysts developing under 20% O2 had fewer blastomeres than those which developed under 5% O2. As the time required for development to the blastocyst stage in vitro increased, there were fewer blastomeres present at the blastocyst stage. These results indicate that the cleaving mouse embryo has an optimal oxygen requirement in vitro of about 5%. At higher oxygen tensions, fewer embryos develop to the blastocyst stage and in those which do develop, there are fewer cell divisions. If a gradient of oxygen tension exists across the blastomeres from the outside of the embryo to its centre, the blastomeres might be using this gradient to obtain imformation about their location within the embryo and respond accordingly. Thus blastomeres on the outside at a higher oxygen tension would divide at a slower rate and form trophectoderm whereas those on the inside at a lower oxygen tension would divide more rapidly and contribute to the inner cell mass.     

The effect of biogenic monoamine antagonists on the development of preimplantation mouse embryos cultured in vitro

The effect of serotonin and adrenaline antagonists was tested on the early embryos of mice of three lines. All the substances tested produced an arrest or inhibition of cleavage division and the appearance of anomalies. Serotonin introduced in the incubation medium was effective against some serotoninolytics. We were unable to test the protective effect of adrenaline, as in the concentrations used it has its own effect on the development. From the data obtained, a conclusion is made of the existence in early mouse embryos of the structures sensitive to serotonin and adrenaline antagonists. The assumptions is made from the previously obtained data on the presence of biogenic monoamines in early mouse embryos, of functional activity of prospective mediators of the nervous system at the earliest stages of embryonic development of mammals.     

Effects of human diabetic serum on the in vitro development of mouse preimplantation embryos

The effects of sera from different types of human diabetes (type I with and without ketoacidosis; type II treated with insulin or Daonil or untreated) on the in vitro development of early preimplantation mouse embryos were studied. In controls, 20% of blastocysts failed to develop successfully when grown for 72 h in RPMI medium supplemented with 10% fetal bovine serum and 50% nondiabetic human serum. In experiments using 50% diabetic serum, the highest embryotoxic effect was found in type-I diabetes with and without ketoacidosis: The percents of undeveloped embryos were 66 and 58, respectively. In type-II diabetes, embryotoxic effects were found among all studied types: The percent of undeveloped blastocysts varied from 36% in insulin-treated type-II diabetes to 44% in untreated type-II diabetes. A high correlation was found between the number of undeveloped embryos and the blood concentrations of metabolic diabetic factors: glucose (r = .53-.64 in type-I diabetes), B-HOB (r = .7-.77 in type-II diabetes untreated or treated with Daonil), acetoacetate (r = .66 in insulin-treated type-II diabetes), and HbA1c (r = .89 in insulin-treated type-II diabetes or .99 in Daonil-treated type-II diabetes). A concentration of 80% serum was embryo-toxic when obtained from nondiabetic or from diabetic human. The possible role of diabetic metabolic factors in causing increased risk of spontaneous abortions and infertility among diabetic women is discussed.     

Effect of serum on cell-to-cell associations during in vitro development of preimplantation mouse embryos

We have identified an activity which alters the morphology and developmental timing of post-compaction mouse embryos. A 15-min exposure of 4- and 8-cell mouse embryos to sera containing this activity induced monolayer formation, changing the normal positions of blastomeres at the 16- to 64-cell stages. Recovered embryos form normal blastocysts, based on morphology and in vitro production of trophectoderm and inner cell mass derivatives. These results suggest that under certain circumstances blastomeres remain developmentally labile as late as the sixth or seventh cleavage cycle.     

Inhibitory effect of IGF-I on induced apoptosis in mouse preimplantation embryos cultured in vitro

Insulin-like growth factor I (IGF-I) has been shown to promote mammalian early embryo development. Increased cell division or decreased cell death have been proposed as two main possible mechanisms in its effect. Here we examine the nature of this promoting effect in a model situation. Camptothecin (0.01 microg/ml) and actimomycin D (0.005 microg/ml) were used to induce apoptosis. Four-cell mouse embryos were cultured in vitro to blastocyst stage in the temporary (15 h) presence or absence of apoptotic inductors and in the permanent presence or absence of IGF-I (100 ng/ml). Embryos were assessed by morphological triple staining (Hoechst 33342, propidium iodide, Calcein AM) and comet assay on Day 5, 120 h after administration of hCG. The number of nuclei, the blastocyst formation, the proportion of embryos containing fragmented DNA and the percentage of apoptotic and secondary necrotic nuclei were assessed. Both inductors of apoptosis significantly increased the percentage of apoptotic and secondary necrotic cells and reduced total cell counts (camptothecin, P>0.001; actinomycin D, P>0.001). When IGF-I was added to the culture medium in the presence of an apoptosis inductor, apoptosis incidence was significantly decreased (P<0.001). The addition of IGF-I into control samples also decreased the percentage of apoptotic and secondary necrotic cells. In contrast, IGF-I addition had no significant influence on embryo development (P>0.05). Our data suggest a primary role for IGF-I as an apoptotic survival factor in mouse preimplantation embryos in specific conditions.     

Effect of Shigella toxin on preimplantation mouse embryos in vitro

Preimplantation mouse embryos at the 4-cell to 8-cell stage were exposed to Shigella dysenteriae toxin at concentrations of 0.001-100 pg/ml in vitro. The effect of the toxin was studied by morphological observation of the embryos to the blastocyst stage, by assessing protein synthesis with 14C-leucine incorporation, and by measuring embryonic adenosine triphosphate (ATP) content. Preimplantation mouse embryos were highly sensitive to the toxin. All variables investigated were adversely influenced by the toxin. After a lag period of 24 hr, 0.01 pg/ml toxin inhibited development to the blastocyst stage and protein synthesis. Toxin concentrations of 1.0 pg/ml resulted in a significant decrease in ATP content.     

The role of calcium in DNA synthesis and development of mouse preimplantation embryos in vitro.

The effect of calcium upon embryonic growth was studied using cultured mouse preimplantation embryos. Both morphological development of the embryos and embryo DNA synthesis were shown to be dependent on the Ca2+ concentration in the medium in which the embryos were grown. Reduction of the calcium concentration below 10(-5) M completely blocked cell division and blastocyst formation in the cultured embryos, but only moderately inhibited embryo DNA synthesis. Trifluoperazine, a calmodulin antagonist, strongly inhibited the Ca(2+)-dependent DNA synthesis in the embryos. On the other hand, the drug only slightly affected the morphological development of the embryos. These results demonstrate that calcium independently affects two different aspects of the embryo development, i.e. DNA synthesis and cell division. It is suggested that the former effect is calmodulin-dependent, while the latter involves the calcium-dependence of metabolite transport through the cell membranes.     

Effect of polyamine limitation on DNA synthesis and development of mouse preimplantation embryos in vitro

In-vitro treatment of preimplantation mouse embryos with spermine and spermidine biosynthesis inhibitor, methylglyoxal-bis-(guanylhydrazone) (MGBG), arrested embryo development at the 8-cell or morula stage. In addition, the embryo DNA synthetic rate, as measured by [3H]thymidine incorporation, was strongly inhibited. The inhibition of blastocyst formation and DNA synthesis by MGBG was readily reversible by an exogenous supply of spermine and/or spermidine to the culture medium. DL-alpha-Methylornithine or DL-alpha-difluoromethylornithine (alpha-DFMO), inhibitors of putrescine biosynthesis, had no effect on embryos cultured for 1 or 2 days, but on the 3rd day embryo DNA synthesis was significantly depressed in the presence of alpha-DFMO. These observations suggest that, during early development of the preimplantation mouse embryo, spermine and spermidine are involved in regulation of embryo growth and DNA synthesis. They may also indicate a role of putrescine at a later stage of mouse embryo development.     

Influence of short-term maternal zinc deficiency on the in vitro development of preimplantation mouse embryos

In this study, we evaluated the use of mouse preimplantation embryos as a model to study zinc deficiency-induced abnormal development. In Experiment 1, the effect of culture medium Zn concentrations on blastocyst development was studied. Preimplantation embryos (2 and 4 cells) obtained from superovulated females developed normally in media containing 0.7-30 microM Zn for up to 72 hr; higher levels of medium Zn resulted in abnormal development. In Experiment 2A, females were fed diets containing 50 (+Zn) or 0.4 (-Zn) micrograms Zn/g (760 vs 6 nmol/g, respectively) from 1 day before to 1 day after mating (3 days total). Preimplantation embryos were removed from the dams and cultured for 72 hr in 0.7 microM Zn medium. Embryos from the -Zn dams were morphologically normal at time zero; however, over the 72-hr period, these embryos tended to develop at a slower rate than controls, although compaction and cavitation frequency were similar. By the end of the 72-hr culture period, embryos from -Zn dams had significantly fewer cells than did embryos from control dams. In Experiment 2B, an extended period of maternal Zn deprivation (6 days) was used to investigate the potential for further impairment of in vitro preimplantation embryo development observed in Experiment 2A. Results from this experiment were consistent with those from Experiment 2A, in addition to providing evidence that the developmental progress of embryos obtained from mice fed Zn-deficient diets for 6 days was significantly impaired. In Experiment 3, the potential for supplemental Zn in culture medium to overcome the impairment in development due to maternal Zn deficiency was investigated. Embryos from female mice subjected to the same dietary regimen described in Experiment 2A were cultured to the blastocyst stage in medium containing Zn at a concentration of either 0.7 or 7.7 microM. Medium Zn supplementation did not improve development of embryos from dams fed Zn-deficient diets. In summary, embryos from mice fed -Zn diets for a 3- or 6-day period encompassing oocyte maturation and fertilization exhibited impaired development in vitro. This impairment was not overcome by medium Zn supplementation.     

In vitro exposure of preimplantation mouse embryos to cocaine and benzoylecgonine inhibits subsequent development.

Cocaine is used by over 20% of women of reproductive age. Although there have been numerous studies focusing on its effects on reproductive processes, none has evaluated its direct effect on preimplantation development. We have investigated the effect of cocaine and its major metabolite, benzoylecgonine, on in vitro preimplantation mouse embryogenesis. One-cell embryos were exposed at the one-, two-, four-, or eight-cell stage for 24 hr to medium containing 0-400 micrograms/ml cocaine or benzoylecgonine and then cultured to the blastocyst stage. Cocaine had its strongest inhibitory effect at the earliest stages of development. At the one- and two-cell stages, there was a significant inhibition of blastocyst formation following exposure to cocaine concentrations of 25-400 micrograms/ml, and at the four-cell stage there was an inhibitory effect at 100 and 400 micrograms/ml cocaine. Benzoylecgonine inhibited the development of embryos to blastocyst only at the one- and two-cell stages, at concentrations of 100-400 micrograms/ml. These findings suggest that cocaine is capable of blocking preimplantation embryogenesis, particularly following exposure at the earliest stages, and that this toxicity may abate as cocaine is biotransformed to benzoylecgonine.     

Biopsy of preimplantation mouse embryos: development of micromanipulated embryos and proliferation of single blastomeres in vitro

We have developed a technique to sample the preimplantation embryo, which may, in the future, be applied to prenatal diagnosis of genetic disease. Using micromanipulation, we aspirated a single blastomere from 4-cell mouse embryos. This procedure had no effect on in vitro development; 98% of control and 94% of biopsied embryos reached the blastocyst stage after 48 h in culture. Furthermore, after transfer to pseudopregnant recipient mice, the rate of fetal development of biopsied embryos was not significantly different from control embryos, although implantation rate was significantly reduced (mean +/- SD: biopsied 53.1 +/- 4.0, control 81.8 +/- 8.4, p less than 0.001). For the first time we have produced monolayer cell cultures derived from single preimplantation blastomeres. Individual biopsied blastomeres were cultured in vitro on different extracellular matrix components. Significantly greater cell proliferation was obtained in wells coated with fibronectin (FN), laminin (LN), and a complex of laminin and nidogen (LNC) than in a less specific matrix of swine skin gelatin (SSG). Mean (+/- SE) cell nuclei number per well after 6 days in culture was 6.4 +/- 2.1, 11.9 +/- 1.5, 19.8 +/- 2.9, and 20.9 +/- 2.6 in wells coated with SSG, LN, FN, and LNC respectively.     

Compensatory development in preimplantation mouse embryos derived from delayed mating.

Rodent embryos resulting from delayed mating grow relatively faster than those resulting from normal mating. To evaluate this phenomenon quantitatively, in the present study we compared the number of cells at the preimplantation stage of mouse embryos derived from normal mating and those derived from delayed mating (3 and 6 h after ovulation). The mean cell numbers (45.4 and 43.0 for delayed mating at 3 and 6 h postovulation, respectively) of embryos at 77 h postcoitus (hpc) in the delayed mating groups were greater than that (38.4) of the normal mating group. Further, when the mean cell numbers (38.8 and 38.5) in the delayed mating groups were counted at 74 hpc, they were almost equal to that of the normal mating group at 77 hpc. The study demonstrated that preimplantation mouse embryos derived from delayed mating progress more rapidly than their normally mated counterparts. However, a 3-h advance in development seems to be the limit of this increased rate of growth, even when the time interval from ovulation to mating is longer than 3 h. The mechanism(s) of this interesting compensatory phenomenon should be investigated.     

Effects of opioids on the development of preimplantation mouse embryos

Opioid peptide DAGO, agonist of opiate mu-receptors and naloxone antagonist of mu-, delta- and kappa-receptors in concentration 3 x 10 M inhibit embryonic development of CBA mice. Inhibition was stage-specific with maximal effect after addition of opioids to zygotes: in the presence of Naloxone no more than 6.7% of embryos reached morula and blastula stages and in the presence of DAGO--36.8%. The other embryos were arrested at two-, four- or, sometimes even, at eight-cell stages without any signs of fragmentation. Four and eight-cell embryos were less sensitive to drug action. Inhibitory effects of these opioids were reduced when they were added simultaneously to zygotes. Agonist of opiate delta-receptors, opioid peptide DADLe, failed to affect embryonic development.