Michele Boiani

5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming

The epigenomes of early mammalian embryos are extensively reprogrammed to acquire a totipotent developmental potential. A major initial event in this reprogramming is the active loss/demethylation of 5-methylcytosine (5mC) in the zygote. Here, we report on findings that link this active demethylation to molecular mechanisms. We detect 5-hydroxymethylcytosine (5hmC) as a novel modification in mouse, bovine and rabbit zygotes. On zygotic development 5hmC accumulates in the paternal pronucleus along with a reduction of 5mC. A knockdown of the 5hmC generating dioxygenase Tet3 simultaneously affects the patterns of 5hmC and 5mC in the paternal pronucleus. This finding links the loss of 5mC to its conversion into 5hmC. The maternal pronucleus seems to be largely protected against this mechanism by PGC7/Dppa3/Stella, as in PGC7 knockout zygotes 5mC also becomes accessible to oxidation into 5hmC. In summary, our data suggest an important role of 5hmC and Tet3 for DNA methylation reprogramming processes in the mammalian zygote.

Regulatory networks in embryo-derived pluripotent stem cells.

Mammalian development requires the specification of over 200 cell types from a single totipotent cell. Investigation of the regulatory networks that are responsible for pluripotency in embryo-derived stem cells is fundamental to understanding mammalian development and realizing therapeutic potential. Extracellular signals and second messengers modulate cell-autonomous regulators such as OCT4, SOX2 and Nanog in a combinatorial complexity. Knowledge of this circuitry might reveal how to achieve phenotypic changes without the genetic manipulation of Oct4, Nanog and other toti/pluripotency-associated genes.

Oct4 is required for primordial germ cell survival

Previous studies have shown that Oct4 has an essential role in maintaining pluripotency of cells of the inner cell mass (ICM) and embryonic stem cells. However, Oct4 null homozygous embryos die around the time of implantation, thus precluding further analysis of gene function during development. We have used the conditional Cre/loxP gene targeting strategy to assess Oct4 function in primordial germ cells (PGCs). Loss of Oct4 function leads to apoptosis of PGCs rather than to differentiation into a trophectodermal lineage, as has been described for Oct4‐deficient ICM cells. These new results suggest a previously unknown function of Oct4 in maintaining viability of mammalian germline.

Sperm from neonatal mammalian testes grafted in mice

Spermatogenesis is a productive and highly organized process that generates virtually unlimited numbers of sperm during adulthood. Continuous proliferation and differentiation of germ cells occur in a delicate balance with other testicular compartments, especially the supporting Sertoli cells. Many complex aspects of testis function in humans and large animals have remained elusive because of a lack of suitable in vitro or in vivo models. Germ cell transplantation has produced complete donor-derived spermatogenesis in rodents but not in other mammalian species. Production of sperm in grafted tissue from immature mammalian testes and across species has not yet been accomplished. Here we report the establishment of complete spermatogenesis by grafting testis tissue from newborn mice, pigs or goats into mouse hosts. This approach maintains structural integrity and provides the accessibility that is essential for studying and manipulating the function of testes and for preserving the male germ line. Our results indicate that this approach is applicable to diverse mammalian species.

Developmental cell biology: Regulatory networks in embryo-derived pluripotent stem cells

Mammalian development requires the specification of over 200 cell types from a single totipotent cell. Investigation of the regulatory networks that are responsible for pluripotency in embryo-derived stem cells is fundamental to understanding mammalian development and realizing therapeutic potential. Extracellular signals and second messengers modulate cell-autonomous regulators such as OCT4, SOX2 and Nanog in a combinatorial complexity. Knowledge of this circuitry might reveal how to achieve phenotypic changes without the genetic manipulation of Oct4, Nanog and other toti/pluripotency-associated genes.

Dynamic link of DNA demethylation, DNA strand breaks and repair in mouse zygotes

In mammalian zygotes, the 5‐methyl‐cytosine (5mC) content of paternal chromosomes is rapidly changed by a yet unknown but presumably active enzymatic mechanism. Here, we describe the developmental dynamics and parental asymmetries of DNA methylation in relation to the presence of DNA strand breaks, DNA repair markers and a precise timing of zygotic DNA replication. The analysis shows that distinct pre‐replicative (active) and replicative (active and passive) phases of DNA demethylation can be observed. These phases of DNA demethylation are concomitant with the appearance of DNA strand breaks and DNA repair markers such as γH2A.X and PARP‐1, respectively. The same correlations are found in cloned embryos obtained after somatic cell nuclear transfer. Together, the data suggest that (1) DNA‐methylation reprogramming is more complex and extended as anticipated earlier and (2) the DNA demethylation, particularly the rapid loss of 5mC in paternal DNA, is likely to be linked to DNA repair mechanisms.

Progeny from Sperm Obtained after Ectopic Grafting of Neonatal Mouse Testes

Abstract Ectopic grafting of testicular tissue is a promising new approach that can be used to preserve testicular function. This technique has been used recently to differentiate the neonatal testes of different species, up to the level of complete spermatogenesis. This approach can be applied successfully to generate live progeny using sperm extracted from grafts originating from testes of newborn donors. The sperm are capable of supporting normal development and producing fertile male and female offspring after intracytoplasmic injection into mouse oocytes and embryo transfer into surrogate mothers. The grafted tissue was also capable of significantly normalizing reproductive hormone levels in the castrated recipients. This technique presents new avenues for experimentation. The recipient mouse can be regarded as a living incubator and a culture system of testicular tissue, allowing the experimental manipulation of several aspects of testis development and spermatogenesis. The successful generation of pups indicates that this technqiue can be used to study the testicular phenotype and to breed mutant or transgenic mouse strains with lethal postnatal phenotypes. The ability to generate sperm from the germ line ex vivo also paves the way for the development of new strategies for preserving fertility in boys undergoing cancer therapy.

Pluripotency deficit in clones overcome by clone–clone aggregation: epigenetic complementation?

Abnormal gene expression patterns in somatic cell clones and their attrition in utero are commonly considered a consequence of errors in nuclear reprogramming. We observe that mouse clone blastocysts have less than half the normal cell number, and that higher cell number correlates with correct expression of Oct4, a gene essential for peri‐implantation development and embryonic pluripotency. To increase the cell number, we aggregated genetically identical clones at the 4‐cell stage. Clone–clone aggregates did not form more blastocysts, but the majority expressed Oct4 normally and had higher rates of fetal and postnatal development. Fertilized blastocysts with low cell numbers, induced by removal of two blastomeres at the 4‐cell stage, did not exhibit abnormal Oct4 expression, indicating that improved gene expression and post‐implantation development of clone–clone aggregates is not a consequence of increased cell number. Rather, we propose that complementation of non‐cell‐autonomous defects of genetically identical, but epigenetically different, embryos results in improved gene expression in clone–clone aggregates.

The analysis of chromatin organisation allows selection of mouse antral oocytes competent for development to blastocyst.

Mouse antral oocytes can be classified in two different types termed SN or NSN oocytes, depending on the presence or absence, respectively, of a ring of Hoechst 33342-positive chromatin surrounding the nucleolus. The aim of the present study was to test the developmental competence to blastocyst of the two types of oocytes. Here we show that following isolation, classification and culture of cumulus-free antral oocytes, 14.7% and 74.5% of NSN and SN oocytes, respectively, reached the metaphase II stage. When fertilised and further cultured none of the metaphase II NSN oocytes developed beyond the 2-cell stage whilst 47.4% of the metaphase II SN oocytes reached the 4-cell stage and 18.4% developed to blastocyst. The findings reported in this paper may contribute to improved procedures of female gamete selection for in vitro fertilisation of humans and farm animals. Furthermore, the selection of oocytes with better developmental potential may be of interest for studies on nuclear/cytoplasm interaction, particularly in nuclear-transfer experiments.

Production of cloned mice by somatic cellnuclear transfer

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), the success rate for producing live offspring by cloning remains <5%. Nevertheless, the techniques have potential as important tools for future research in basic biology. We have been able to develop a stable NT method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although manipulation of the piezo unit is complex, once mastered it is of great help not only in NT experiments but also in almost all other forms of micromanipulation. In addition to this technique, embryonic stem (ES) cell lines established from somatic cell nuclei by NT can be generated relatively easily from a variety of mouse genotypes and cell types. Such NT-ES cells can be used not only for experimental models of human therapeutic cloning but also as a backup of the donor cells genome. Our most recent protocols for mouse cloning, as described here, will allow the production of cloned mice in ≥3 months.