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Biomedical subjects

M I Sherman

Publications and source records attributed to M I Sherman.

At least 55 records · Page 3Linked to original sources
PubMed1981

Studies on the mechanism of induction of embryonal carcinoma cell differentiation by retinoic acid.

On the basis of our studies, we conclude that RA is a potent promoter of differentiation of EC cells. We have demonstrated that the tumorigenicity of PCC4-azalR EC cultures can be effectively reduced following exposure of the cells to RA. This is presumably a result of differentiation of the EC cells to nontumorigenic derivatives. However, even after several weeks of exposure to RA, there remains in the culture a subpopulation of unresponsive EC cells. The reason why these cells do not differentiate in the presence of RA is currently under investigation. We have also derived by mutagen treatment and clonal selection EC cells that fail to respond to RA. Preliminary indications are that these cells have lost the capacity to differentiate when subjected to other manipulations which stimulate differentiation of the parental EC cells. This is an important observation since the mutants were selected only by their lack of response to RA. Unlike the parental cells, which have relatively large amounts of RABP, dif- cells appear either to lack RABP or to possess an altered binding protein which has a greatly reduced affinity for RA. These observations are consistent with the view that some function of the RA-RABP complex is critical in the sequence of events leading to differentiation of EC cells. However, further studies are required before we can establish the generality of this proposal. We are presently investigating whether the level and/or function of RA-RABP complexes in cells from the EC lines listed in TABLE 1 can explain their varying tendencies to differentiate.

Animals↗
PubMed1981

Isolation and characterization of mouse mutant embryonal carcinoma cells which fail to differentiate in response to retinoic acid.

Murine embryonal carcinoma cells from line PCC4.aza1R differentiate readily in response to retinoic acid. By treating PCC4.aza1R cells with the mutagen N-methyl-N' -nitro-N-nitrosoguanidine, we derived two embryonal carcinoma lines in which the cells failed to differentiate during exposure to retinoic acid. Although these dif(RA)- cells maintained the tumorigenic potential of the parental cells, they differentiated poorly in tumor form. Similarly, the tendency of dif(RA)- cells to differentiate when aggregated in vitro was diminished relative to that of PCC4.zaz1R cells. The rate of retinoic acid uptake in cells from the two mutant lines did not appear to be reduced compared with the rate in cells from the parental line; however, specific cytoplasmic retinoic acid-binding protein activity was virtually absent in both mutants. These results strengthen the view that differentiation of embryonal carcinoma cells in response to retinoic acid requires formation of retinoic acid-cytoplasmic retinoic acid-binding protein complexes.

Animals↗
PubMed1981

Protein and free amino acid content in preimplantation mouse embryos and in blastocysts under various culture conditions.

By using a rapid and sensitive method for determination of total protein and free amino acid contents we have shown that mouse preimplantation embryos developing in vivo and in vitro contain decreasing amounts of protein until the late blastocyst stage. Free amino acid contents in embryos developing in vivo drop following fertilization and then rise gradually until the late blastocyst stage. Embryos developing in vitro from the 2-cell stage have much lower levels of free amino acids than those in vivo. Embryos placed in culture at the blastocyst stage show a decrease in protein content within 24 h, but protein levels rise rapidly thereafter in medium containing serum and amino acids. In the same medium, total amino acid pools increase from the time the blastocysts are placed in culture. Blastocysts cultured in the absence of amino acids show a very slight net increase in protein content and free amino acid pools are eventually depleted. In the presence of amino acids but the absence of serum, there is only a modest increase in the protein content of cultured blastocysts, suggesting that, in addition to an exogenous supply of amino acids, other factors are required for optimal growth.

Amino Acids↗
PubMed1981

In vitro studies of mouse embryos bearing mutations in the T complex: effects of culture in suboptimal medium upon t6/t6 and normal embryos.

We have compared the behaviour of normal and t6/t6 embryos in PCMF, a suboptimal 'delay' medium which arrests normal development. Morphologically, the two types of embryos are indistinguishable in this medium. Although beta-glucuronidase activity in embryos does not rise as quickly in delay medium as in cNCTC, a nutritive medium, the rate of increase is similar in t6/t6 and normal embryos. We conclude from these observations that the lethality of t6/t6 embryos is not a consequence of their reaching a given absolute age. Together with previous studies, our data suggest that embryo lethality correlates more closely with metabolic state than with morphological stage. Blastocysts maintained in PCMF are unable to give rise to trophoblast outgrowths but do so upon transfer into cNCTC medium. When a mixture of fourth-day t6/t6 and normal embryos are transferred to cNCTC medium after lengthy pre-incubation periods in PCMF, trophoblast outgrowth is initiated from both types of embryos at approximately the same time. However, when embryos are removed from the genital tract on the second or third day of gestation, t6/t6 embryos are slower to produce trophoblast outgrowths than are normal embryos upon transfer from PCMF to cNCTC medium. Although the reason for this differential behaviour is not yet clear, it is hypothesized that some product(s) required for the outgrowth process is (are) more unstable in third-day t6/t6 embryos than in normal third-day embryos or fourth-day t6/t6 embryos. Our ability to separate t6/t6 from normal embryos by their delayed initiation of trophoblast outgrowth provides us with a convenient way to identify, and to isolate for analysis, enriched populations of homozygous mutant embryos prior to the time at which they show gross morphological abnormalities.

Animals↗
PubMed1980

H-2 analyses of blastocyst-derived cell lines from mice bearing T and tw18 mutations.

T/T and tw18/tw18 blastocysts appear to survive in culture for periods of 3 weeks at frequencies equal to that of normal embryos. We have, therefore, undertaken to generate cell lines with a homozygous mutant genotype. We attempted to identify these cells by H-2 haplotype analysis. The results indicate that barring recombination, which would be expected to occur at a frequency of about 15%, 1 of 10 blastocyst-derived cell lines from a (+/tw18 x +/tw18) cross had a tw18/tw18 genotype and 1 of 4 lines from a (T/+ x T/+) cross had a T/T genotype. Unexpectedly, all of the remaining lines were heterozygous; no +/+ lines were observed. The putative homozygous mutant lines might be useful in attempts to characterize tw18-specific antigens.

Absorption↗
PubMed1980

Effects of culture conditions on the developmental programme of mouse blastocysts.

We have investigated the developmental potential of mouse blastocysts cultured under a variety of conditions. A number of parameters were used as criteria for development and differentiation, namely hatching of blastocysts from the zona pellucida and their adhesion to the substratum, outgrowth and polyploidization of trophoblast cells, increase in cell number, protein content, beta-glucuronidase activity, appearance of lactate dehydrogenase A subunits, plasminogen activator production, and delta 5,3 beta hydroxysteroid dehydrogenase activity. Under optimal culture conditions, embryos grew relatively rapidly and expressed all the differentiative markers for which they were tested. Under less supportive conditions, the production of the markers was usually reduced quantitatively; the expression of some markers could also be considerably delayed or even totally prevented. In fact, embryos cultured in the least nutritive medium (one designed to support development only through pre-implantation stages) appeared to be in a state of metabolic quiescence closely resembling that of blastocysts in ovariectomy-induced delay. Overall, the results of our investigations lead us to propose that the expression of each of the aforementioned markers is probably under independent control and subject to intrinsic programming. Finally, the observation that some markers are produced by embryos in suboptimal media whereas others are not, suggests that the minimum metabolic level necessary for expression varies from one marker to another.

3-Hydroxysteroid Dehydrogenases↗
PubMed1980

Studies on the temporal correlation between secretion of plasminogen activator and stages of early mouse embryogenesis.

Fibrinolytic activity is associated with mouse embryos during preimplantation stages, but thorough analyses indicate that activity is restricted to the zona pellucida and is not likely to be of embryonic origin. Investigations on embryos developing further both in utero and in vitro suggest that plasminogen activator is unlikely to be involved in either hatching from the zona pellucida or the acquisition of adhesiveness by trophoblast cells. However, the enzyme is invariably secreted by blastocysts that have reached the stage of trophoblast invasiveness; fibrinolytic activity is even produced by most embryos which have failed to hatch from their zona but remain viable at the time of acquisition of invasive properties by trophoblast cells from other, normally developing, embryos. These studies are, therefore, consistent with the view that plasminogen activator is involved in trophoblast invasiveness. Possible functions of the enzyme during migration of trophoblast cells through tissue layers are considered.

Animals↗
PubMed1979

In vitro development of core cells of the inner cell mass of the mouse blastocyst: effects of conditioned medium.

Blastocysts submitted to two rounds of immunosurgery give rise to cores of presumptive ectoderm cells, many of which do not survive for more than 48 hours when cultured individually. Precoating of the culture plates with conditioned medium (CM) from PYS-2 cells increases the incidence with which cores regenerate an outer layer. This procedure also improves the survival frequency of the cores, but only for a limited period of time. The small number of cores which survive for two weeks or more, either in uncoated or CM-coated plates, give rise to any array of cell types, including giant cells resembling trophoblast.

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↗
PubMed1979

Enzyme analysis of mouse extra-embryonic tissues.

We have separated for enzyme analysis the following layers that surround the conceptus at midgestation: decidua, trophoblast, parietal endoderm (including Reichert's membrane), visceral endoderm, yolk-sac mesoderm and amnion. Measurement of several catabolic enzyme activities (N-acetyl-beta, D-hexosaminidase, beta-glucuronidase, alkaline and acid phosphatases and non-specific esterases) in these tissues indicates that they are biochemically distinct, perhaps reflecting the different functions that they perform in providing the embryo proper with a desirable environment for differentiation and development. Our studies also provide an example of how visceral endoderm cells can effectively block passage of maternal macromolecules (in this case a serum esterase) in the fetal circulation. Finally, since there is often difficulty in distinguishing among early embryonic and extra-embryonic cell types produced in teratocarcinoma cultures, we have considered how our observations might be of use in the respect, particularly in discriminating between visceral and parietal endoderm.

Acid Phosphatase↗