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At least 19 recordsLinked to original sources
PubMed1978

In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts. II. Inner cell masses from 3.5- to 4.0-day p.c. blastocysts.

This paper describes the development in culture of inner cell masses isolated immunosurgically from C3H/He mouse blastocysts immediately after collection between 3.5 and 4.0 days p.c. By 24--48 h most of the inner cell masses isolated from half-expanded blastocysts, and about 50% of those from expanded blastocysts, regenerate an outer layer of trophectoderm-like cells and so resemble mini-blastocysts. With further in vitro culture these structures attach to the substratum and give rise to trophoblast-like giant cells, together with clusters of parietal endoderm cells or inner cell masses surrounded by visceral endoderm. Many of the inner cell masses from the remaining expanded blastocysts develop into floating structures with an outer layer of endoderm cells, and by 7 days consist of a large fluid filled cyst surrounding a collapsed vesicle of epithelial cells. Mesodermal cells line the cysts and form numerous blood islands. When mechanically disrupted, and grown as attached sheets of cells, these cystic structures give rise to patches of trophoblast-like giant cells similar to those described in the previous paper. These results suggest that the inner cell mass of normal mouse blastocysts contains cells which are capable of giving rise to trophoblast in culture.

Animals↗
PubMed1978

In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts. I. Inner cell masses from 3.5-day p.c. blastocysts incubated for 24 h before immunosurgery.

This paper describes the in vitro development of inner cell masses isolated immunosurgically from mouse blastocysts which had been collected on 3.5 days p.c. and then incubated for 24 h. The inner cell masses continue to grow in culture and develop through a series of stages with increasing complexity of internal organization. By day 1 all of the cultured ICMs have an outer layer of endoderm, and by day 3 some of them have two distinct kinds of inside cells; a columnar epithelial layer and a thin hemisphere of elongated cells. Later, mesodermal cells appear to delaminate from a limited region of the columnar layer, close to where it forms a junction with the thinner cells. By day 5, about 25% of the cultured ICMs have a striking resemblance to normal 7.5-day p.c. C3H embryos, with embryonic ectoderm, extra-embryonic ectoderm and chorion, embryonic and extra-embryonic mesoderm, and visceral endoderm. When mechanically disrupted and grown as attached clumps of cells in a tissue dish, these embryo-like structures give rise to trophoblast-like giant cells. These results suggest that the inner cell mass of 4.5-day p.c. blastocysts contains cells which can give rise to trophoblast derivates in culture.

Animals↗
PubMed1979

Scanning electron microscopy of the surface of normal and implantation-delayed mouse blastocysts during development in vitro.

Mouse blastocysts undergo developmental steps in culture analogous to those occurring during implantation in utero. We examined cultured blastocysts by scanning electron microscopy (SEM) as they passed through these stages. From the time of hatching to the acquisition of adhesiveness, most blastocysts were exhanded, with flattened cells possessing relatively small numbers of microvilli, centrally raised areas (presumably reflecting the location of the nuclei) and intercellular ridges often possessing microvilli. At, or shortly before, the trophoblast outgrowth stage, blastocysts appeared to contract; the cells bulged noticeably, microvilli covered the entire surface of most cells and intercellular ridges were no longer observable. Blastocysts removed from uteri on the seventh day of ovariectomy delay possessed a variety of morphologies and shapes. The blastocoel was frequently collapsed and cell outlines were difficult to discern. These blastocysts were initially adhesive in vitro, but subsequently disengaged from the substratum before becoming permanently adherent several hours later. During the initial phase of adhesiveness, blastocysts were elongated and had prominent intercellular ridges, particularly in the equatorial region. Detached blastocysts contained bulging cells with contours which obscured the intercellular ridges. Surface ultrastructure during subsequent phases resembled non-delayed blastocysts during attachment and outgrowth. On the basis of our studies, we propose that intercellular ridges play some role in blastocyst adhesiveness. However, we must conclude that there are other factors involved in the acquisition of adhesiveness by the blastocyst which are at least equally important but of a nature too subtle to be identified by our SEM analyses. Insofar as delayed blastocysts are concerned, we find that, within limits, the surface alterations that take place when blastocysts are activated in culture mirror those observed following reversal of delay in vivo by administration of hormones. Since delayed blastocysts placed in saline also undergo morphological changes resembling those seen at the onset of activation in utero, we suggest that reversal of implantation delay requires initially neither direct contact with steroid or macromolecular inducers nor an exogenous supply of metabolites.

Adhesiveness↗
PubMed1978

The in vivo transport of 14C-alpha aminoisobuturic acid into mouse blastocysts during activation for implantation.

The in vivo transport of 14C-AIB (14C-alpha-aminoisobuturic acid) into mouse blastocysts was studied during activation for implantation. Mice, kept in experimentally delayed implantation, were given estrogen to induce implantation and then injections of 14C-AIB i.v. at 0, 4 and 8 h after the estrogen. After in vivo incubation times for 1/3 or 4 h with the labeled amino acid the blastocysts were flushed out of the uterus and collected. A distinct uptake of 14C-AIB occurred in the blastocysts 8 h (the highest uptake) and 12 h after the induction, provided that the in vivo incubation time was 4 h. At these times the blastocysts are lying free in the uterine lumen and consequently there is a transport of 14C-AIB from the epithelium via the uterine secretion into the blastocysts. This uptake indicates that amino acids transported by the system A are important nutrients during early activation. The uptake and retention of 14C-AIB in the uterus was tested at 4 and 8 h after the induction of implantation. The highest uptake was observed when the labeled amino acid was given at 8 h while the longest retention time occurred when 14C-AIB was given at 4 h. Since the transport ratio between the blastocysts and the uterine tissue is not maintained constant it is concluded that the metabolic rates for 14C-AIB transport are different for the uterus and the blastocysts. The AIB transport into uterine tissue preceeds that into the blastocysts. The AIB transport into the blastocysts is maintained as long as they have a negative surface charge.

Aminoisobutyric Acids↗
PubMed1979

Steroidogenesis and prostaglandin synthesis by cultured bovine blastocysts.

Bovine blastocysts were collected at Days 13, 15 and 16 and placed in TCM-199 supplemented with 5% fetal calf serum; some blastocysts were immediately frozen while the others were cultured for 48 h and then frozen. Samples (tissue + medium, 5--12/group) were thawed, homogenized and analysed by radioimmunoassays. Measurable amounts of progesterone were found in all blastocysts but values were higher (P less than 0.01) after culture. Testosterone was not found in the cultured or uncultured blastocysts at Day 13, but was detectable on Days 15 and 16 and in greater amounts (P less than 0.05) in the cultured blastocysts. PGF and PGE-2 were increased (P less than 0.05) in the cultured blastocysts in all 3 days. Oestradiol was measurable in some but not all blastocysts. It is suggested that PG synthetase and enzymes capable of synthesizing progesterone, testosterone and, possibly, oestradiol are present in these early bovine blastocysts.

Animals↗
PubMed1979

The effect of glucose-, arginine- and leucine-deprivation on mouse blastocyst outgrowth in vitro.

To estimate the degree of trophoblast outgrowth in vitro, mouse blastocysts obtained after delay of implantation were cultured either in a modified Brinster medium or in the same medium with exclusion of various combinations of glucose, arginine and leucine. Trophoblast outgrowth was prevented only in a medium from which all three substances were excluded. In this medium the blastocysts remained expanded for 5 days without signs of trophoblast outgrowth--a growth arrest in vitro. After transfer of blastocysts growth arrested in vitro for 5 days to a complete medium including both glucose and the two amino acids, normal outgrowths occurred within two or three days. The growth-arrested blastocysts also developed normally for at least one week when transplanted into salpingectomized foster mothers. It is concluded that blastocysts activation in vitro can be controlled by a few nutrients in a way reminiscent of the activation prior to implantation in utero. Blastocysts activated in utero by systemic administration of oestrogen for various lengths of time before the start of culture, or in vitro by preincubation in a medium containing glucose and all amino acids, also grew out in the growth arrest medium if they had been activated for a sufficiently long time, 18 h in utero and 1 h in vitro, thus indicating that when a blastocyst has reached a certain degree of activation its growth can not be arrested by exclusion of glucose, arginine and leucine.

Animals↗
PubMed1976

Cyclic adenosine monophosphate-induced changes in the surface morphology of diapausing blastocysts and the effects on implantation.

Estrogen induces blastocyst implantation in diapausing female mice, increases uterine levels of adenyl cyclase and cyclic adenosine monphosphate (cylic AMP), and stimulates synthesis of ribonucleic acid and protein in both the uterus and blastocyst. Furthermore, the surface ultrastructure of trophoblast cells seen with scanning electron microscopy (SEM) changes with activation of the diapausing blastocyst by estrogen. Cyclic AMP-activated blastocysts were investigated with the use of SEM and compared to blastocysts activated by estrogen. Intraperitoneal administration of cyclic AMP was generally ineffective, whereas administration of cyclic AMP intraluminally in the uterus effectively mimicked the early estrogen activation. These findings are discussed in relation to other known activation changes in preimplantation blastocysts and with regard to similar findings after administration of various antiestrogens.

Animals↗
PubMed1978

Effects of indomethacin on uterine capillary permeability and blastocyst development in rabbits.

Increased capillary permeability at implantation sites was demonstrated in rabbits by extravasation of intravascular blue dye on day 7 of pregnancy. Subcutaneous administration of indomethacin (Id, 8 mg/kg twice daily) on days 4-6 of pregnancy inhibited this uterine blueing response and appeared to reduce the size of implantation swellings. To test the latter observation blastocyst diameter and development of the embryonic disk were assessed at 144 hr post coitum. In females receiving indomethacin at the dose level which inhibited uterine blueing, blastocysts were significantly smaller than those from control females. Developmental staging of embryonic disks revealed only slight differences between the smaller (Id-treated) blastocysts and control blastocysts. No effect of Id was seen on ovarian function as judged by luteal weights and plasma progesterone and estradiol levels. Since the major biological effects of indomethacin are due to its inhibition of prostaglandin synthesis, it appears that prostaglandins may play a role in the uterine response to blastocyst stimulation and in the expansion of blastocysts in the rabbit.

Animals↗
PubMed1979

Time-dependent effects of alpha-amanitin on blastocyst formation in the mouse.

Early compacting mouse morulae were placed in alpha-amanitin at various times after HCG and the effect of this transcriptional inhibitor during formation of the blastocyst was noted. No cavitation was observed in those embryos transferred into alpha-amanitin before 77 h after HCG but the percentage of cavitation increased dramatically in those embryos transferred after 80 h post HCG. The large increase in amino acid incorporation which normally occurs during fomration of the blastocyst was blocked in those embryos placed in the inhibitor before 80 h post HCG. Two-dimensional polyacrylamide gel electrophoresis of radiolabelled embryos showed that the changes in synthesis of certain polypeptides normally associated with blastocyst formation did not occur in those embryos placed in alpha-amanitin before 80 h after HCG. Only one cleavage division appears to occur in the presence of alpha-amanitin. However, groups of embryos which had completed their fifth division before exposure to the drug subsequently were able to form blastocysts and showed the quantitative and qualitative changes associated with blastocyst formation despite the continued presence of the inhibitor. These results suggest that a critical transcriptional event concerned with blastocyst formation occurs around 80 h after HCG and may be associated with the fifth cleavage division.

Amanitins↗
PubMed1978

Changes in the surface of the mouse blastocyst at implantation.

Implantation is a critical event, and perhaps the earliest one, in the maternal recognition of pregnancy. Information transfer from conceptus to mother might occur during, and subsequent to, implantation at the level of cell surface interaction. Therefore, attempts have been made both to identify the phases of implantation during which changes in the blastocyst surface occur and to characterized such changes. In vitro, blastocysts have been found to go through a series of discrete steps which are analogous to implantation in utero, and these steps can be retarded or prevented by the use of either suboptimal culture media or an inappropriate substratum. Morphological surface changes are not apparent when the blastocyst becomes adherent to the substratum; however, marked differences in blastocyst surface structure are revealed by scanning electron microscopy at the onset of trophoblast outgrowth. Studies at the molecular level implicate collagen as having a role in blastocyst adhesiveness, but other cell surface components are also likely to be involved.

Animals↗
PubMed1978

Effect of inorganic lead on the mouse blastocyst in vitro.

In vitro culture of mouse blastocysts over the implantation period was used (1) to find out whether embryos recovered from uteri of mice given an injection of lead, which has been shown in a previous study to inhibit implantation in vivo, were capable of attachment and outgrowth when transferred to a lead free culture medium; (2) to study the effect of different amounts of inorganic lead in the culture medium on blastocyst viability and ability to attach and grow out. The levels of lead used in the medium were comparable to those found in uterine tissue of animals given an implantation inhibiting dose of lead. It was found that the preimplantation blastocyst was unaffected by the administration of lead to the mother as regards the ability to attach and grow out. It was also shown that normal blastocysts were adversely affected by the presence of lead in the culture medium. Addition of increasing amounts of inorganic lead (5, 10 and 20 mumol/l) caused a corresponding decrease in the ability of the blastocysts to attach and grow out, the highest concentration also causing abnormal appearance of the embryos within 48 hours of culture.

Animals↗
PubMed1979

Tumorigenicity of embryonal carcinoma as an assay to study control of malignancy by the murine blastocyst.

A bioassay, based on tumorigenicity, has been developed to determine the mechanism whereby the blastocyst of the mouse controls malignant expression of embryonal carcinoma. The assay is based upon the incidence of tumors obtained when known numbers of cells of the 402AX strain of embryonal carcinoma are injected into strain 129 mice, compared to the incidence obtained when the same number of embryonal carcinoma cells are incorporated into Swiss-Webster blastocysts that are then injected in strain 129 animals. The results indicate that the blastocyst can regulate one embryonal carcinoma cell consistently; it may have a slight effect on three, but it cannot regulate four or five of them. The position of the embryonal carcinoma cell in the blastocyst is important. Regulation occurs if the embryonal carcinoma cell is placed in the blastocoele cavity, but enhancement of tumorigenicity is obtained if it is placed between the zona pellucida and the trophectoderm. By contrast, the blastocyst is unable to regulate a single B-16 melanoma cell placed in the blastocoele cavity, indicating a degree of specificity for the regulatory process.

Animals↗
PubMed1979

The effect of alpha-aminoisobuturic acid and 2,4-diaminobuturic acid on mouse blastocyst outgrowth in vitro.

Blastocysts recovered from mice in a state of delay of implantation were incubated for 10 h with either alpha-aminoisobuturic acid (AIB) or 2,4-diaminobuturic acid (DAB), two non-metabolizable amino acids. The incubation medium was composed so as to maintain growth arrest of the inactive, delayed blastocysts in vitro. The blastocysts were then transferred to a complete outgrowth medium without the two non-metabolizable amino acids to test the capacity for trophoblast outgrowth. AIB, which displays saturation kinetics, was harmless to the blastocysts even at a high concentration, while DAB at a low concentration irreversibly damaged the trophoblast cells and prevented outgrowth, probably due to nonsaturation kinetics resulting in a high intracellular accumulation. The harmful effect of DAB could be abolished by concomitant incubation with L-alanine and L-methionine, which compete with DAB for the same transport system, while the D-forms of the same amino acids had little or no effect. The results suggest the presence of transport System A in mouse blastocysts growth arrested in vitro, indicating an operative carrier mechanism already during delay of implantation.

Alanine↗
PubMed1978

Effect of removal of the zona pellucida on subsequent development of mouse blastocysts in vitro.

Removal of the zona pellucida allowed mouse blastocysts incapable of hatching in vitro to develop in culture. Blastocysts denuded by pronase always developed further than those of identical age that had hatched spontaneously. More blastocysts mechanically denuded and treated with pronase developed egg cylinder-like structures than did blastocysts mechanically denuded and not treated with pronase. Plastic support gave better development of blastocysts than did glass.

Animals↗
PubMed1978

A stimulatory effect of the fluid from preimplantation rabbit blastocysts upon luteinization of monkey granulosa cell cultures.

Blastocyst fluid was aspirated from Day 6 1/2--7 rabbit blastocysts and was added to cultures of granulosa cells obtained from preovulatory follicles of untreated rhesus monkeys or from follicles of monkeys or from follicles of monkeys treated with PMSG. The stimulation of progesterone secretion was measured and equated with that produced by hCG. The hCG-like activity was also measured in a radioreceptor assay using 125I-labelled hCG and porcine granulosa cells. In 8 out of 10 experiments with cultured cells from untreated monkeys, addition of 20% blastocyst fluid from Days 6--9 of culture stimulated progesterone secretion by 2- to 6-fold. Similar findings were obtained in 5 experiments with cultures from PMSG-treated monkeys except that the blastocyst fluid was added from Days 0 to 6 of culture. The granulosa cells in such cultures underwent morphological luteinization. Compared to a standard of purified hCG the blastocyst fluid contained about 0.76--2.5 ng hCG-like activity/ml which was non-dialysable. The radioreceptor assay indicated the presence of 0.5--2.5 ng hCG-like material/ml.

Animals↗
PubMed1978

Effect of the endocrine state of blastocyst donors on the time required for initiation of trophoblast outgrowth.

Blastocysts from mice in a state of delayed implantation after ovariectomy were recovered on day 5, 7 or 9 (day 1 was the day a vaginal plug was found). Blastocysts were also recovered on day 7 from animals that had received an injection of oestradiol-17beta 8, 16 or 24 hours earlier. The blastocysts were incubated in a modified Brinster's medium to which serum had been added and the time of initiation of blastocyst outgrowth was recorded. Blastcysts from early delay grew out before those from late delay. A steady state of outgrowth time was achieved on day 7. It is therefore suggested that blastocysts for culture experiments in which the metabolic activity level is crucial should not be recovered before the steady state is attained. Oestrogen injections caused earlier outgrowths, at least after 16 h, which indicates that the earlier outgrowth soon after ovariectomy might also be caused by a residual effect of the oestrogen.

Animals↗
PubMed1977

The culture of mouse blastocysts in the presence of uterine flushings collected during normal pregnancy, delayed implantation and pro-oestrus.

When day-3 mouse embryos were cultured in a simple medium supplemented with uterine fluids of mice autopsied on day 4 of pregnancy, 48 h after administration of oestradiol, or during pro-oestrus, the percentage of blastocysts hatching from the zona pellucida was significantly greater than in unsupplemented medium. In the presence of uterine fluids recovered during delayed implantation this stimulation of blastocyst hatching was not observed. When the culture medium was supplemented with dialysed uterine flushings containing 20 or 30 microgram protein/ml, both 'day 4' and 'delay' uterine proteins were equally effective in enhancing hatching frequency (P less than 0.05). The results suggested that 'delay' uterine fluids may contain a dialysable inhibitor of blastocyst activity. The putative inhibitor was not effective in the presence of serum, since uterine fluids recovered both on day 4 of pregnancy and during delayed implantation significantly increased the size attained by blastocyst outgrowths in the presence of foetal calf serum (P less than 0.01). The percentage of blastocysts exhibitng giant cell transformation and outgrowth was also increased (P less than 0.02) by these uterine fluids when the concentration of FCS in the medium was minimal (0.25%).

Animals↗