One-cell CF-1 x B6SJLF1/J embryos, which usually exhibit a 2-cell block to development in vitro, have been cultured to the blastocyst stage using CZB medium and a glucose washing procedure. CZB medium is a further modification of modified BMOC-2 containing an increased lactate/pyruvate ratio of 116, 1 mM-glutamine and 0.1 mM-EDTA but lacking glucose. Continuous culture of one-cell embryos in CZB medium allowed 83% of embryos to develop beyond the 2-cell stage of which 63% were morulae at 72 h of culture, but blastocysts did not develop. However, washing embryos into CZB medium containing glucose after 48 h of culture (3-4-cell stage) was sufficient to allow development to proceed, with 48% of embryos reaching the blastocyst stage by 96 h of culture. Exposure of embryos to glucose was only necessary from the 3-4-cell stage through the early morula stage since washing back into medium CZB without glucose at 72 h of culture still promoted the development of 50% of embryos to the blastocyst stage. The presence of glucose in this medium for the first 48 h of culture (1-cell to 4-cell stage) was detrimental to embryo development. Glutamine, however, exerted a beneficial effect on embryo development from the 1-cell to the 4-cell stage although its presence was not required for development to proceed during the final 48 h of culture. Blastocysts which developed under optimum conditions contained an average of 33.7 total cells. The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.

/pdf/an-improved-culture-medium-supports-development-of-random-4ommimicna.pdf

An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro

We have developed an in vitro method for amplifying a large fraction of the DNA sequences present in a single haploid cell by repeated primer extensions using a mixture of 15-base random oligonucleotides. We studied 12 genetic loci and estimate that the probability of amplifying any sequence in the genome to a minimum of 30 copies is not less than 0.78 (95% confidence). Whole genome amplification beginning with a single cell, or other samples with very small amounts of DNA, has significant implications for multipoint mapping by sperm or oocyte typing and possibly for genetic disease diagnosis, forensics, and the analysis of ancient DNA samples.

https://www.pnas.org/content/pnas/89/13/5847.full.pdf

Whole genome amplification from a single cell: implications for genetic analysis

La presente revue considere les donnees recentes concernant les produits impliques dans l'activation du genome de l'embryon chez plusieurs especes

https://onlinelibrary.wiley.com/doi/epdf/10.1002/mrd.1080260113

Transition from maternal to embryonic control in early mammalian development: a comparison of several species.

Cellular senescence is a response to nonlethal stress that results in persistent cytostasis with a distinct morphological and biochemical phenotype The senescence phenotype, detected in tumors through the expression of mRNA and protein markers, can be generated in cancer cells lacking functional p53 and retinoblastoma protein Current research suggests that therapy-induced senescence (TIS) represents a novel functional target that may improve cancer therapy TIS can be induced in immortal and transformed cancer cells by selected anticancer compounds or radiation, and accumulating data indicate that TIS may produce reduced toxicity-related side effects and increased tumor-specific immune activity This review examines the current status of TIS-regulated mechanisms, agents, and senescence biomarkers with the goal of encouraging further development of this approach to cancer therapy Remaining hurdles include the lack of efficient senescence-inducing agents and incomplete biological data on tumor response The identification of additional compounds and other targeted approaches to senescence induction will further the development of TIS in the clinical treatment of cancer

/pdf/therapy-induced-senescence-in-cancer-olnwdrmevr.pdf

Therapy-Induced Senescence in Cancer

Culture of preimplantation mammalian embryos and cells can influence their subsequent growth and differentiation. Previously, we reported that culture of mouse embryonic stem cells is associated with deregulation of genomic imprinting and affects the potential for these cells to develop into normal fetuses. The purpose of our current study was to determine whether culture of preimplantation mouse embryos in a chemically defined medium (M16) with or without fetal calf serum (FCS) can affect their subsequent development and imprinted gene expression. Only one third of the blastocysts that had been cultured from two-cell embryos in M16 medium complemented with FCS developed into viable Day 14 fetuses after transfer into recipients. These M16 1 FCS fetuses were reduced in weight as compared with controls and M16 fetuses and had decreased expression of the imprinted H19 and insulin-like growth factor 2 genes associated with a gain of DNA methylation at an imprinting control region upstream of H19. They also displayed increased expression of the imprinted gene Grb10. The growth factor receptor binding gene Grb7, in contrast, was strongly reduced in its expression in most of the M16 1 FCS fetuses. No alterations were detected for the imprinted gene Mest. Preimplantation culture in the presence of serum can influence the regulation of multiple growth-related imprinted genes, thus leading to aberrant fetal growth and development. developmental biology, gene regulation, IGF receptor, implantation/early development

https://bioone.org/journals/Biology-of-Reproduction/volume-64/issue-3/biolreprod64.3.918/Culture-of-Preimplantation-Mouse-Embryos-Affects-Fetal-Development-and-the/10.1095/biolreprod64.3.918.pdf

Culture of Preimplantation Mouse Embryos Affects Fetal Development and the Expression of Imprinted Genes

Although the embryos of certain mammals, including man, can be cultured in vitro through several cleavage divisions1,2, it is only in the mouse that complete development through the preimplantation period (one-cell to the blastocyst stage3) occurs without affecting subsequent embryonic viability after transfer1,4. But even in the mouse, development in vitro from the one-cell to the blastocyst stage is restricted to certain inbred strains and F1 hybrids1,3,5,6. In most randomly bred strains the one-cell embryo becomes blocked at the two-cell stage in vitro and it will only continue development if transferred back into the oviduct7. Here we present evidence that this so-called ‘in vitro two-cell block’ can be obviated by injecting small amounts of cytoplasm from embryos which do not exhibit this block.

Cytoplasmic control of preimplantation development in vitro in the mouse

Hybrid cells derived from whole-cell fusions of replicating phase-II normal fibroblast cells (WI-38s) with SV40 transformed WI-38 fibroblast cells (CL-1s) demonstrated that the majority of the hybrid experimental cells still maintained a finite life-span. Approximately 2% demonstrated sustained and possibly indefinite replication. Experimental binucleate cells and subsequent hybrid synkaryons were also formed by fusing CL-1 karyoplasts into phase-II WI-38 replicating normal fibroblasts. In addition, viable cells were constructed from WI-38 fibroblast cytoplasts with CL-1 karyoplasts. Sustained replication was not observed in these crosses.

Replicative potentials of various fusion products between WI-38 and SV40 transformed WI-38 cells and their components.

As a model for the detection of human genetic disease in preimplantation embryos, we describe a method in which trophectoderm biopsy samples from viable mouse blastocysts are simultaneously analyzed for the presence of a normal or mutant allele of the myelin basic protein gene by the polymerase chain reaction. The biopsied embryos are kept in culture during analysis of biopsied material and later reintroduced to a foster mother. Prenatal diagnosis can be completed in less than 7 hr. The identity of either amplification product was proved conclusively by direct sequence analysis of amplified products. Ninety-six percent of recovered blastocysts survived biopsy, as judged by re-formation of a blastocyst cavity in culture. Fifty-nine percent of the biopsied embryos established pregnancy by day 6.5, compared to 88% of unmanipulated controls. This approach can be applied to preimplantation diagnosis of human genetic diseases by using extraembryonic cells from blastocysts obtained after in vitro fertilization or uterine lavage. It will make possible the elimination of a mutant allele from a family in a single generation.

Rapid preimplantation detection of mutant (shiverer) and normal alleles of the mouse myelin basic protein gene allowing selective implantation and birth of live young. implantation and birth of live young

Methods for nuclear transfer and fusion of individual viable nuclei into cells is described. Experimental binucleates have been formed by fusing nuclei from normal fibroblasts, SV40 virus-transformed fibroblasts, and HeLa carcinoma cells into individual diploid fibroblasts which have undergone their total “life span” (population doublings) and have been maintained in culture for a period of 10–12 weeks (late phase III) prior to the nuclear transfer. Many of the cells receiving additional nuclei replicate and form clones of cells with a limited life span, and the morphology of the cells remains fibroblast-like.

Nucleocytoplasmic interactions in experimental binucleates formed from normal and transformed components

Neonatal, adult, and aged donor human skin fibroblast cell cultures have been characterized for the population doubling potential and in vitro-in vivo relationship. Hybrid cells derived from individual whole-cell fusions of replicating GRC 387 (aged) and CSC 303 (neonatal) cells demonstrated that an intermediate mode of replication between that of the two parental cell lines occurred; therefore the longevity of the aged fibroblast cells was enhanced. The GRC 387 cells have an extended post-replicative phase-out period in comparison with the CSC 303 cells, and the experimental hybrids demonstrated a 20–25 % increase of this period over that of the GRC 387 cells.

Audrey L. Muggleton-Harris

Papers

Cytoplasmic control of preimplantation development in vitro in the mouse

Replicative potentials of various fusion products between WI-38 and SV40 transformed WI-38 cells and their components.

Rapid preimplantation detection of mutant (shiverer) and normal alleles of the mouse myelin basic protein gene allowing selective implantation and birth of live young. implantation and birth of live young

Nucleocytoplasmic interactions in experimental binucleates formed from normal and transformed components

Replicative potential of individual cell hybrids derived from young and old donor human skin fibroblasts