Steroid metabolism by mouse preimplantation embryos in vitro

Summary Mouse preimplantation embryos were incubated with radioactive pregnenolone, progesterone or dehydroepiandrosterone for various periods of time. These substrates were not converted to metabolites even after incubation of 120 h. We suggest that preimplantation mouse embryo does not possess enzyme activities for steroid metabolism.Acknowledgments. The authors would like to thank Prof. L. Saxén for giving the animals and the facilities of his tissue culture laboratory.     

Close association of erythrocytes with surface of late mouse blastocysts: SEM analysis

SEM analysis of preimplantation mouse blastocysts reveals closely associated red blood cells on involuted surfaces of trophoblasts, and indication of the capability for initial phases of phagocytosis at an early developmental stage.     

Close association of erythrocytes with surface of late mouse blastocysts: SEM analysis

Summary SEM analysis of preimplantation mouse blastocysts reveals closely associated red blood cells on involuted surfaces of trophoblasts, an indication of the capability for initial phases of phagocytosis at an early developmental stage.Acknowledgment. This study was supported by NICHD grant HD-06234 and NIH grant RR-05384. Author for reprint requests.     

Die degenerationsrate bei präimplantierten ratteneiern

Summary The material studied consisted of 941 rat ova in various stages of cleavage. The preimplantation period has been subdivided into 5 stages, and for each period the amount of degenerated eggs has been recorded. There was only one degenerated egg found between the first and the second cleavage divisions but a considerably higher frequency afterwards during the second and the third segmentation step.     

Effect of low dose of 70 kVp X-rays on the intrauterine development of mice

Pregnant Swiss albino mice were exposed to low doses of X-rays (approximately 9 mGy) in the range used for diagnostic exposure, on day 3.5 of gestation (preimplantation period), day 6.5 (early organogenesis period) or day 11.5 (late organogenesis period). The fetuses were examined on the 18th day of gestation. Exposure at 3.5 days post coitus (d.p.c.) resulted in a significant increase in prenatal mortality, and an increased incidence of retarded fetuses was observed after exposure at 3.5 and 6.5 d.p.c. The major effect of exposure at 11.5 d.p.c. was a significant decrease in the fetal head size and brain weight.     

Allophenic mice produced from embryos aggregated with antibody

One difficulty in the production of allophenic mice by aggregation of preimplantation embryos is that they frequently roll apart before the bonds between the blastomeres have had time to form. One solution to the problem, described here, is to pretreat one of the embryos with rabbit anti-mouse serum just prior to pushing them together. Blastocyst formation is unhampered by antibody treatment, and numerous allophenic mice have already been produced with this new procedure.     

Effect of low dose of 70 kVp X-rays on the intrauterine development of mice

Summary Pregnant Swiss albino mice were exposed to low doses of X-rays (9 mGy) in the range used for diagnostic exposure, on day 3.5 of gestation (preimplantation period), day 6.5 (early organogenesis period) or day 11.5 (late organogenesis period). The fetuses were examined on the 18th day of gestation. Exposure at 3.5 days post coitus (d.p.c.) resulted in a significant increase in prenatal mortality, and an increased incidence of retarded fetuses was observed after exposure at 3.5 and 6.5 d.p.c. The major effect of exposure at 11.5 d.p.c. was a significant decrease in the fetal head size and brain weight.     

The importance of HLA-G expression in embryos,trophoblast cells,and embryonic stem cells

The nonclassical HLA-G molecule is a trophoblast-specific molecule present in almost every pregnancy. It differs from classical HLA class I molecules by the low degree of allelic variants and the high diversity of protein structures. HLA-G is reported to be a tolerogenic molecule that acts on cells of both innate and adaptive immunity. At the maternal–fetal interface HLA-G seems to be responsible largely for the reprogramming of local maternal immune response. This review will focus on the HLA-G gene expression profile in pregnancy, in preimplantation embryos, and in human embryonic stem cells with emphasis on the structural diversity of the HLA-G protein and its potential functional and diagnostic implications.     

Current applications of single-cell PCR

The advent of the polymerase chain reaction (PCR) has revolutionised the way in which molecular biologists view their task at hand, for it is now possible to amplify and examine minute quantities of rare genetic material: the limit of this exploration being the single cell. It is especially in the field of prenatal diagnostics that this ability has been readily seized upon, as it has opened up the prospect of preimplantation genetic analysis and the use of fetal cells enriched from the blood of pregnant women for the assessment of single-gene Mendelian disorders. However, apart from diagnostic applications, single-cell PCR has proven to be of enormous use to basic scientists, addressing diverse immunological, neurological and developmental questions, where both the genome but also messenger RNA expression patterns were examined. Furthermore, recent advances, such as optimised whole genome amplification (WGA) procedures, single-cell complementary DNA arrays and perhaps even single-cell comparative genomic hybridisation will ensure that the genetic analysis of single cells will become common practice, thereby opening up new possibilities for diagnosis and research.     

A simple method for counting nuclei in the preimplantation mouse embryo

An easy and rapid method of counting the number of cells in the preimplantation mouse embryo is described. The procedure increases the speed with which large numbers of embryos can be processed using a simple squash technique. Cell numbers are determined by exposing the embryos to the fluorescent DNA-binding dye, Hoechst 33258, removing the zona pellucida and simply squashing the embryo and counting the number of fluorescent nuclei. An increase in fluorescent intensity and maintenance of nuclear conformation of the squashed preparations are greatly improved by the use of the non-ionic detergent Triton X-100. Viability of dye-treated fertilized one-cell and blastocyst stage embryos is maintained at least up to day 13 of pregnancy following transfer of the embryos to the uteri of pseudopregnant recipients. Additional uses for this staining technique are discussed.     

A simple method for counting nuclei in the preimplantation mouse embryo

Summary An easy and rapid method of counting the number of cells in the preimplantation mouse embryo is described. The procedure increases the speed with which large numbers of embryos can be processed using a simple squash technique. Cell numbers are determined by exposing the embryos to the fluorescent DNA-binding dye, Hoechst 33258, removing the zona pellucida and simply squashing the embryo and counting the number of fluorescent nuclei. An increase in fluorescent intensity and maintenance of nuclear conformation of the squashed preparations are greatly improved by the use of the non-ionic detergent Triton X-100. Viability of dye-treated fertilized one-cell and blastocyst stage embryos is maintained at least up to day 13 of pregnancy following transfer of the embryos to the uteri of pseudopregnant recipients. Additional uses for this staining technique are discussed.Acknowledgment. Financial support was provided by NIH Grant HDD-06210 (KME) and by Basil O'Connor Starter Research Grant No. 5-379 from the March of Dimes Birth Defects Foundation (VEP). We thank Steven Halpern for help with the photography and Jon Flax for expert technical assistance.     

Epigenetic differences between naïve and primed pluripotent stem cells

It has been 8 years since the concept of naïve and primed pluripotent stem cell states was first proposed. Both are states of pluripotency, but exhibit slightly different properties. The naïve state represents the cellular state of the preimplantation mouse blastocyst inner cell mass, while the primed state is representative of the post-implantation epiblast cells. These two cell types exhibit clearly distinct developmental potential, as evidenced by the fact that naïve cells are able to contribute to blastocyst chimeras, while primed cells cannot. However, the epigenetic differences that underlie the distinct developmental potential of these cell types remain unclear, which is rather surprising given the large amount of active investigation over the years. Elucidating such epigenetic differences should lead to a better understanding of the fundamental properties of these states of pluripotency and the means by which the naïve-to-primed transition occurs, which may provide insights into the essence of stem cell commitment.     

Blastomere biopsy influences epigenetic reprogramming during early embryo development,which impacts neural development and function in resulting mice

Blastomere biopsy is used in preimplantation genetic diagnosis; however, the long-term implications on the offspring are poorly characterized. We previously reported a high risk of memory defects in adult biopsied mice. Here, we assessed nervous function of aged biopsied mice and further investigated the mechanism of neural impairment after biopsy. We found that aged biopsied mice had poorer spatial learning ability, increased neuron degeneration, and altered expression of proteins involved in neural degeneration or dysfunction in the brain compared to aged control mice. Furthermore, the MeDIP assay indicated a genome-wide low methylation in the brains of adult biopsied mice when compared to the controls, and most of the genes containing differentially methylated loci in promoter regions were associated with neural disorders. When we further compared the genomic DNA methylation profiles of 7.5-days postconception (dpc) embryos between the biopsy and control group, we found the whole genome low methylation in the biopsied group, suggesting that blastomere biopsy was an obstacle to de novo methylation during early embryo development. Further analysis on mRNA profiles of 4.5-dpc embryos indicated that reduced expression of de novo methylation genes in biopsied embryos may impact de novo methylation. In conclusion, we demonstrate an abnormal neural development and function in mice generated after blastomere biopsy. The impaired epigenetic reprogramming during early embryo development may be the latent mechanism contributing to the impairment of the nervous system in the biopsied mice, which results in a hypomethylation status in their brains.     

Ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase and arginine decarboxylase from Mycobacterium bovis (BCG)

Ornithine decarboxylase (ODC), S-adenosyl-L-methionine decarboxylase (AMDC) and arginine decarboxylase (ADC) activities were detected for the first time in extracts of Mycobacterium bovis (BCG). All the decarboxylases differed from corresponding known bacterial decarboxylases in that: a) ODC did not require GTP for activity; b) ODC was not inhibited by any known inhibitor of bacterial ODCs; c) AMDC and ADC did not require Mg2+-ion for activity and were not markedly inhibited by any known inhibitor of the decarboxylases of other bacteria.     

Ornithine decarboxylase,S-adenosyl-L-methionine decarboxylase and arginine decarboxylase fromMycobacterium bovis (BCG)

Summary Ornithine decarboxylase (ODC), S-adenosyl-L-methionine decarboxylase (AMDC) and arginine decarboxylase (ADC) activities were detected for the first time in extracts ofMycobacterium bovis (BCG). All the decarboxylases differed from corresponding known bacterial decarboxylases in that: a) ODC did not require GTP for activity; b) ODC was not inhibited by any known inhibitor of bacterial ODCs; c) AMDC and ADC did not require Mg²⁺-ion for activity and were not markedly inhibited by any known inhibitor of the decarboxylases of other bacteria.     

Dextranase-producing organisms in dental plaque from caries-free and caries-active naval recruits

Summary Dental plaque samples from caries-free and caries-active naval recruits were assayed for the prevalence of dextranase-producing organisms. These organisms were found in the plaque of all of the subjects. Mean percentages of dextranase-producing organisms with respect to total colony count for the 2 groups of subjects were not significantly different.The authors thank DTl Samuel Shelton and Mr Eric Mandel for technical assistance. The research was supported by Naval Medical Research and Development Command Project ZF58.524.012-0026, Bethesda, Maryland. The opinions expressed herein are those of the authors and are not to be construed as reflecting the views of the Navy Department or the Naval Service at large. The use of commercially available products does not imply endorsement of these products or preference to other similar products on the market.     

Influence of diet on plasma tryptophan and brain serotonin levels in mice

Groups of mice were maintained for up to 78 weeks on tryptophan restricted, protein restricted and control diets. Plasma tryptophan levels were significantly reduced by both forms of dietary restriction. Brain serotonin levels were significantly reduced only in mice on the tryptophan restricted diet, but not for mice on the protein restricted diet. The protein-restricted diet contains less of the large neutral amino acids which compete with tryptophan to enter the brain. It is known that protein restriction and tryptophan restriction extend lifespan. The results presented here suggest that extension of lifespan and lowering of brain serotonin are not related.     

Influence of diet on plasma tryptophan and brain serotonin levels in mice

Summary Groups of mice were maintained for up to 78 weeks on tryptophan restricted, protein restricted and control diets. Plasma tryptophan levels were significantly reduced by both forms of dietary restriction. Brain serotonin levels were significantly reduced only in mice on the tryptophan restricted diet, but not for mice on the protein restricted diet. The protein-restricted diet contains less of the large neutral amino acids which compete with tryptophan to enter the brain. It is known that protein restriction and tryptophan restriction extend lifespan. The results presented here suggest that extension of lifespan and lowering of brain serotonin are not related.     

Radioimmunoassay of cortisol in blood of buffaloes (Bubalus bubalis) during the oestrous cycle

Summary Changes in the concentration of cortisol were measured by radioimmunoassay in the blood plasma of buffaloes (Bubalus bubalis). There were minor fluctuations in the level during the oestrous cycle, but the differences between the days were not significant. The study revealed that cortisol under normal conditions does not appear to be involved in the regulation of the cycle.The authors are indebted to Dr D. Sundaresan for his keen interest in the study and to Dr A.E. Leek, Technia Diagnostics, for providing cortisol antiserum.