The effect of ethanol upon early development in mice and rats. XXIV. The effect of acute ethanol intoxication upon implantation in mice.

The effect of acute ethanol intoxication (ethanol 33%, i.v.) on day 4 of pregnancy (at 9 o'clock a.m.) upon the initial stages of implantation in mice was investigated. The pregnant animals (treated and controls) were killed in the afternoon and in the evening of day 5 of pregnancy (at 30 and 36 hours after ethanol administration). After histological proceeding, the pregnant uteri were microscopically examined, using the following criteria: the position of embryos, the stage of embryos differentiation, the aspect of uterine epithelium, the decidual reaction and the presence of oedema. The results obtained are analysed and compared with some literature data and with our previous results. Some apparent contradictions between the data presented and those previously reported, require new investigations.

There are no in vivo pulsations of mouse blastocysts.

An important event at the onset of the implantation process in mammals is the hatching of the blastocyst from the zona pellucida. Microcinematographic studies of in vitro preimplantation development in mice revealed the pulsatile activity of blastocysts before and during the hatching period. It is generally accepted--up to now--that the in vitro hatching is at least partially the result of repeated contractions and reexpansions of the blastocyst. The presence of pulsatile activity in vivo may confirm this hypothesis. Mouse blastocysts were obtained by a rapid flushing (5-10') from uterine horns on day 4 of pregnancy and the presence or absence of contracted blastocysts was noted. From 410 examined blastocysts only 3% were contracted as compared with the very frequent in vitro pulsations. This result suggests that in mice the in vivo pulsatile activity of blastocysts, practically, does not exist. The in vivo contractions are probably determined by the suboptimal culture conditions.

The effect of ethanol upon early development in mice and rats. XXIII. The effect of indomethacin on beer-induced disturbances of preimplantation development in rats.

The effect of Indomethacin on beer-induced modifications of preimplantation development was investigated in rats (control on day 5 of pregnancy), using the following criteria: the mean number of embryos/animal, topographical distribution of embryos, the developmental stage attained, the appearance of pathological forms, the mean cell number/embryo. It resulted that previous administration of Indomethacin antagonized the main deleterious effect of repeated acute administration of beer in the preimplantation period, supporting the hypothesis of prostaglandins (PG) being involved in the pathogenetic action of this alcoholic beverage.

The effect of ethanol upon early development in mice and rats. XXI. The effect of acute maternal ethanol intoxication upon in vitro hatching of mouse embryos.

The effect of acute ethanol intoxication (ethanol 33% in a.d.) in mice on day 4 of pregnancy (at 9 o'clock a.m.) upon in vitro hatching has been investigated. The embryos were cultured in modified Brinster medium (1963), at 37 degrees C and 5% CO2 in air beginning at 2 o'clock p.m. on day 4 of pregnancy. The control of the developmental stage of embryos from experimental and control groups on day 4 of pregnancy (at 9 o'clock a.m.) showed some numerical differences between the embryos found in early and mid blastocyst stages but no difference between advanced blastocysts. The control after 24 hours of culture showed in both experimental and control groups the presence of expanded blastocysts. No hatched or degenerated embryos were observed. The control at 48 hours revealed the following aspects: the inhibition of hatching revealed by a significantly lower percentage of in vitro hatching (16.3%) in the experimental as compared with the control group (36.6%); the percentage of degenerated embryos is significantly increased in the ethanol group (13%) as compared with the untreated group (6.10%); the percentage of embryos with the zona pellucida fractured does not differ significantly between experimental and control groups. Some problems related to the mechanism of in vitro and in vivo, hatching are discussed.

The effect of early hypoxy upon the development of rats and mice. II. Foetal effects.

The late, foetal effect of temporary hypoxy--performed by uterine vascular clamping--during the preimplantation period (day 2, 3 and 4 of pregnancy) was controlled in rats. The main detected effects were: lowering of the number of implantation sites and of the mean number of living foetuses; lowering of the mean foetal weight: increased percentage of resorptions and partial retardation of skeletal development. No structural anomalies were registered.

The effect of ethanol upon early development in mice and rats. XVII. In vivo effect of chronic biparental alcoholization upon preimplantation development in rats.

The effect of chronic biparental alcoholization upon preimplantation rat embryos was investigated. On day 5 of pregnancy the following were controlled: developmental stage attained; mean number of embryos/animal; mean number of cells/embryo; mean number of cells/blastocyst; mean number of cells/early blastocyst; oviductal-uterine migration rate and the number of pathological embryos. Chronic biparental alcoholization induced a delay of the onset of cavitation and a significantly increased number of pathological embryos. Chronic biparental alcoholization has a more marked effect than chronic maternal alcoholization. In mice (RAP strain), the same experimental schedule induced more marked effects than in rats.

The effect of early hypoxy upon the development of rats and mice. 1. Preimplantation effects.

The effect of temporary hypoxy obtained by uterine vascular clamping during the preimplantation developmental period, was controlled on rats and mice (on the last day of preimplantation development). The following main effects were detected: retarded preimplantation development in rats; increased number of pathologically modified embryos and retarded oviduct-uterus migration in both species. The changes of the uterine wall due to vascular clamping disappear two days after the experimental intervention. The existence of an experimental hypoxic blastopathy is attested (at least its early, preimplantation aspect).

On the prenatal noxious effects of trypan blue and of a related azo dye.

Since 1948 trypan blue has been a well-known and extensively used experimental teratogen, belonging to the group of azo dyes. Chemically, trypan blue consists of a biphenyl molecule (0-tolidine or benzidine) combined by means of azo linkages with two molecules of a substituted naphthalene. Between 1987-89 the effect of the replacement of the biphenyl molecule by a molecule of p,p'-diaminobenzanilide upon the prenatal noxious action of trypan blue has been controlled. Investigations were carried out on three species: chick embryos, albino rats and albino mice. In the species used, the replacement annihilates the teratogenic properties of the dye, with the persistence of some embryotoxic effects. On the other hand, the control of o-tolidine and of p,p'-diaminobenzanilide revealed that no one had teratogenic properties (only some embryotoxic effect, more marked in the case of o-tolidine). It results that the teratogenic action of trypan blue cannot be attributed to the o-tolidine molecule proper but to an effect which results (in a for the moment unknown manner) from its combination with the other parts of the dye molecule.

In vitro studies on normal and pathological preimplantation development. I. Events of normal mouse preimplantation development as revealed by microcinematography.

After briefly presenting the main historical data of in vitro culture of preimplantation mouse embryos and their filming, the first own observations on normal preimplantation development made by using microcinematography are presented: development from two-cell to eight-cell embryos; compaction and cavitation. The timing and the duration of various developmental events were recorded. Own observations were compared with previous cinematographic data reported by other authors. Some processes needing further investigations are evidenced: rotation within the zona pellucida, penetration of cytoplasmic emissions through the zona, contraction and reexpansion.

The effect of ethanol upon early development in mice and rats. VIII. The effect of chronic consumption of some beverages upon preimplantation development in rats.

The effects of chronic consumption of some beverages (plum-brandy 24% and cognac 20%) upon preimplantation development in rats were studied. The control of possible effects was performed on day 5 by usual flushing, examination and photographying of oviductal and uterine embryos. In order to evaluate the effect of the beverage applied, the following criteria were used: mean litter size, migration of the embryos from the oviduct to the uterus, the developmental stage attained by the pre-implantation embryos and the appearance of pathological embryos. The main results were the following: both beverages applied influenced the preimplantation development; with respect to the developmental rate and to the induction of pathological changes, the effect of both beverages was similar (retardation and an increased, number of pathological morulae and blastocysts); a different action could be detected as to the mean litter size and to the migration of preimplantation embryos: plum-brandy reduced more substantially the mean litter size, whereas cognac had a more marked retarding effect upon the migration of embryos from the oviduct to the uterus: all the changes detected show a more or less marked "litter-effect". The present data were compared with the corresponding effects of chronic ethanol administration observed previously in our laboratory. No obvious potentiating effect of beverage congeners could be established. The findings are discussed in connection with other experimental models of alcohol embryo and fetopathy.

The effect of ethanol upon early development in mice and rats. VIII. Cytogenetic analysis of preimplantation stages of alcoholized mice.

Cytogenetic analysis was performed on preimplantation embryos (morulae and blastocysts) from control (untreated) and experimental groups of RAP strain female mice. The experimental groups underwent chronic alcoholization, acute ethanol intoxication of a combined treatment. The results suggest that the treatments applied did not induce, in our experimental conditions, chromosomal aberrations.

The effect of ethanol upon early development in mice and rats. VI. In vivo effect of acetaldehyde upon preimplantation stages in rats.

In completion of the previously outlined "experimental alcohol blastopathy", the role of acetaldehyde in the induction of preimplantation pathological changes in rat embryos has been controlled. Two experimental models were used: the direct administration of acetaldehyde by gavage and the blockage of acetaldehyde metabolization by ANTALCOL (an aldehyde-dehydrogenase blocking compound). The main results were as follows: The exogenous acetaldehyde in the blood of pregnant animals has an obvious effect upon the developmental rate during the late preimplantation period (retarding segmentation, blastulation), and in one of the experimental models upon the oviductal-uterine migration rate. The increase of the blood acetaldehyde level by blockage of its further metabolization has a more marked effect as compared with the direct intravenous administration of the substance. According to our previous observations the intravenous application of ethanol on the same day (day 4) has no such effect. The direct noxious influence upon the developing preimplantation embryos (fragmentation) of the increased level of acetaldehyde obtained by ANTALCOL treatment is similar but more marked than this effect obtained previously by ethanol administration. The same effect observed after the direct administration of the substance is less marked than the effect of ANTALCOL treatment but more marked than the effect of intravenous ethanol administration. These results attest that acetaldehyde may contribute (alone or together with the effect of ethanol) to the induction of "experimental alcohol blastopathy". The less marked action of the substance proper introduced into the blood stream may be due--in our opinion--to its possible alteration during the period between distillation and application.

The shaping of the mesometrial proamniotic cavity in the mouse. Are there various mechanisms?

The existence of an alternative pathway of the shaping of the mesometrial proamniotic cavity (based upon the examination of several hundreds 6-day mouse embryos), which occurs more frequently than mentioned by other authors, is described. The observed pathway begins with the invagination of the mesometrial pole of the implanted blastocyst, the early proamniotic cavity thus communicating with the uterine lumen. Subsequently, it is occluded by the ectoplacental cone. The phylogenetic aspects of these observations are discussed.

The effect of ethanol upon early development in mice and rats. V. In vivo effect of acute preimplantation intoxication with or without previous chronic alcoholization.

Albino rats (Wistar) and albino mice (RAP) were either injected intravenously with ethanol during the preimplantation period (day 4 and 3, respectively) or injected in the same way after a previous chronic alcoholization (peroral consumption of 20% ethanol for 50-60 and 32-35 days, respectively before mating, adding the days until killing). The control of possible effects was performed on day 5 (rats) and 4 (mice) by usual flushing, examination and photographing of oviductal and uterine embryos. A group of albino rats, with chronic alcoholization, was controlled for late, fetal effects (resorption rate, skeletal control, possible ocular anomalies). The main results obtained were as follows: Acute ethanol intoxication. Rats: significant increase of pathological, fragmented preimplantation embryos with a marked "litter effect". Mice: no deleterious effect upon preimplantation development. Chronic alcoholization + acute ethanol intoxication. Rats: significant retardation of the preimplantation development rate and a significant increase of the number of pathological, fragmented embryos with a marked "litter effect". Mice: demonstrable advance of preimplantation development and migration rate. Chronic alcoholization--late fetal control in rats: the increase of resorption rate; the more frequent absence of sacral vertebrae; very rare rib anomalies and the absence of ocular malformations.

The effect of ethanol upon early development in mice and rats. IV. The effect of acute ethanol intoxication of day 4 of pregnancy upon implantation and early postimplantation development in mice.

Acute ethanol intoxication in albino mice (RAP) induced by intravenous administration of ethanol on day 4 of pregnancy delayed or inhibited implantation in about 25 per cent of the cases. The noxious action upon the implantation process showed a clear-cut "litter effect" and the mean litter was not affected by the experimental intervention. In very early postimplantation stage (day 6 of pregnancy) a statistically significant advance of some main morphogenetic indices was detected in treated specimens. As a possible explanation of this finding, a "selection" of more resistant and viable embryos by the acute ethanol intoxication is presumed. The data discussed in the present paper, together with authors' previous findings suggest a possible noxious action of acute ethanol intoxication during preimplantation stages upon implantation.