Chromosomal Abnormalities in Embryonic and Somatic Stem Cells.

The potential use of stem cells (SCs) for tissue engineering, regenerative medicine, disease modeling, toxicological studies, drug delivery, and as in vitro model for the study of basic developmental processes implies large-scale in vitro culture. Here, after a brief description of the main techniques used for karyotype analysis, we will give a detailed overview of the chromosome abnormalities described in pluripotent (embryonic and induced pluripotent SCs) and somatic SCs, and the possible causes of their origin during culture.

Spermatogenesis is seasonal in the large hairy armadillo, Chaetophractus villosus (Dasypodidae, Xenarthra, Mammalia).

Very little is known about the distinct reproductive biology of armadillos. Very few studies have investigated armadillo spermatogenesis, with data available only for Euphractus sexcinctus and Dasypus novemcinctus. In the present study, we analysed male germ cell differentiation in the large hairy armadillo Chaetophractus villosus throughout the year, describing a cycle of the seminiferous epithelium made of eight different stages. Evaluation of the testis/body mass ratio, analysis of the architecture of the seminiferous epithelium and the frequency of defective seminiferous tubules allowed identification of a temporal interruption of spermatogenesis during the period between mid-May to July (mid-end autumn) in correlation with very low testosterone levels. Overall, these results suggest that spermatogenesis is seasonal in C. villosus.

Gatekeeper of pluripotency: a common Oct4 transcriptional network operates in mouse eggs and embryonic stem cells.

BACKGROUND: Oct4 is a key factor of an expanded transcriptional network (Oct4-TN) that governs pluripotency and self-renewal in embryonic stem cells (ESCs) and in the inner cell mass from which ESCs are derived. A pending question is whether the establishment of the Oct4-TN initiates during oogenesis or after fertilisation. To this regard, recent evidence has shown that Oct4 controls a poorly known Oct4-TN central to the acquisition of the mouse egg developmental competence. The aim of this study was to investigate the identity and extension of this maternal Oct4-TN, as much as whether its presence is circumscribed to the egg or maintained beyond fertilisation. RESULTS: By comparing the genome-wide transcriptional profile of developmentally competent eggs that express the OCT4 protein to that of developmentally incompetent eggs in which OCT4 is down-regulated, we unveiled a maternal Oct4-TN of 182 genes. Eighty of these transcripts escape post-fertilisation degradation and represent the maternal Oct4-TN inheritance that is passed on to the 2-cell embryo. Most of these 80 genes are expressed in cancer cells and 37 are notable companions of the Oct4 transcriptome in ESCs. CONCLUSIONS: These results provide, for the first time, a developmental link between eggs, early preimplantation embryos and ESCs, indicating that the molecular signature that characterises the ESCs identity is rooted in oogenesis. Also, they contribute a useful resource to further study the mechanisms of Oct4 function and regulation during the maternal-to-embryo transition and to explore the link between the regulation of pluripotency and the acquisition of de-differentiation in cancer cells.

The NOBOX protein becomes undetectable in developmentally competent antral and ovulated oocytes.

The oocyte-specific NOBOX protein is an important player during oocyte growth. Its absence in Nobox-/- mice arrests the transition from primordial to growing follicles and down-regulates the expression of a number of genes, including Oct4, a transcription factor crucial in the acquisition of oocyte developmental competence. Despite its role during folliculogenesis, a clear description of the expression of NOBOX throughout oocyte growth is lacking. Here, we have analysed the pattern of expression of both the Nobox gene (qRT-PCR) and its protein (immunofluorescence) during folliculogenesis, classifying the oocytes based on their size (six classes: 10-30, 31-40, 41-50, 51-60, 61-70, 71-80 microm) and chromatin organisation (NSN, Non Surrounded Nucleolus or SN, Surrounded Nucleolus). Significant differences were observed in Nobox transcription in the group of 41-50 microm (NSN > SN), 71-80 microm (NSN > SN) and in developmentally incompetent metaphase II-derived NSN (MII(NSN)) or competent metaphase II-derived SN (MII(SN)) oocytes (MII(NSN) > MII(SN)). The NOBOX protein is expressed throughout oocyte growth in the nucleus of ovarian NSN and in MII(NSN) oocytes; in contrast, beginning with SN oocytes of 61-70 microm, it becomes almost undetectable. Our data, while being in line with the hypothesis of a regulative role of NOBOX on Oct4 gene expression at the primordial/primary stage, when both transcription factors are coincidentally expressed, also indicate that this role might not be maintained in the subsequent growing stages. Furthermore, the sharp difference of NOBOX expression in developmentally incompetent or competent oocytes makes this protein a putative marker of their quality.

The biopolitics of frozen embryos.

The unresolved debate about frozen embryos has left open the discussion on "what to do with them". There are only three ways to deal with frozen embryos: 1) to leave them frozen indefinitely; 2) to defrost and discard them and 3) to use them for research. In this paper, we suggest that the application of current scientific knowledge, instead of inappropriately referring to ethical principles or to the concept of person, could help with the decision about what to do with hundreds of thousands of frozen embryos, thus bringing the sensitive debate on bioethical issues to shared practical solutions. We face a new individual only when a new functional copy of his genome is formed. In both natural and artificial animal and plant reproduction, this principle applies. This status occurs in humans at the 4-8 cell stage. Acknowledgement of this factual datum would allow advocates of all religious and ideological beliefs to defend their principles and to realign their positions to a setting within the boundaries of current scientific knowledge.

The 2-cell block occurring during development of outbred mouse embryos is rescued by cytoplasmic factors present in inbred metaphase II oocytes.

In mice, completion of preimplantation development in vitro is restricted to certain crosses between inbred strains. Most of the outbred and inbred strains cease development at the 2-cell stage, a phenomenon known as the "2-cell block". Reciprocal mating between blocking and non-blocking strains has shown that the 2-cell block is dependent upon female, but not male, developmental information. One question that still remains unanswered is whether the genome of the metaphase II (MII) oocyte is genetically programmed to express, during the very early stages of development, some factor(s) required to determine developmental competence beyond the 2-cell stage. In the present study, we have addressed this question by performing reciprocal MII-chromosome plate transfer between MII oocytes of a non-blocking inbred strain and MII oocytes of a blocking outbred strain. Here, we report that development beyond the 2-cell stage does not depend on the MII genome, but instead it relies on a cytoplasmic factor(s) already present in ovulated non-blocking oocytes, but absent, inactive or quantitatively insufficient in blocking oocytes. Further evidence of the ooplasmic origin of this component(s) was obtained by transferring a small quantity of ooplasm from non-blocking MII oocytes to blocking MII oocytes or 2-cell embryos. Following the transfer, a high percentage of blocking oocytes/embryos acquired developmental competence beyond the 2-cell stage and reached the blastocyst stage. This study shows that development beyond the 2-cell stage relies also on a factor(s) already present in the ovulated oocyte.

Variable reprogramming of the pluripotent stem cell marker Oct4 in mouse clones: distinct developmental potentials in different culture environments.

A prevailing view of cloning by somatic-cell nuclear transfer is that reprogramming of gene expression occurs during the first few hours after injection of the nucleus into an oocyte, that the process is stochastic, and that the type of reprogramming needed for cloning success is foreign and unlikely to be readily achieved in the ooplasm. Here, we present evidence that the release of reprogramming capacity is contingent on the culture environment of the clone while the contribution of aneuploidy to altered gene expression is marginal. In particular, the rate of blastocyst formation in clones and the regional distribution of mRNA for the pluripotent stem cell marker Oct4 in clonal blastocysts was highly dependent on the culture environment after cumulus cell nuclear transfer, unlike that in genetically equivalent zygotes. Epigenetic modifications of genetically identical somatic nuclei continue after the first cell division of the clones and are amenable to a degree of experimental control, and their development to the blastocyst stage and appropriate expression of Oct4 predict further outcome, such as derivation of embryonic stem (ES) cells, but not fetal development. This observation indicates that development to the blastocyst stage is not equivalent to full reprogramming and lends support to the novel concept that ES cells are not the equivalent of the inner cell mass, hence the discrepancy between ES cell derivability and fetal development of clones.