[show abstract][hide abstract] ABSTRACT: Oct4A is a core component of the regulatory network of pluripotent cells, and by itself can reprogram neural stem cells into pluripotent cells in mice and humans. However, its role in defining totipotency and inducing pluripotency during embryonic development is still unclear. We genetically eliminated maternal Oct4A using a Cre/loxP approach in mouse and found that the establishment of totipotency was not affected, as shown by the generation of live pups. After complete inactivation of both maternal and zygotic Oct4A expression, the embryos still formed Oct4-GFP- and Nanog-expressing inner cell masses, albeit non-pluripotent, indicating that Oct4A is not a determinant for the pluripotent cell lineage separation. Interestingly, Oct4A-deficient oocytes were able to reprogram fibroblasts into pluripotent cells. Our results clearly demonstrate that, in contrast to its role in the maintenance of pluripotency, maternal Oct4A is not crucial for either the establishment of totipotency in embryos, or the induction of pluripotency in somatic cells using oocytes.
[show abstract][hide abstract] ABSTRACT: Tolerance induction toward allogeneic organ grafts represents one of the major aims of transplantation medicine. Stem cells are promising candidates for promoting donor-specific tolerance. In this study, we investigated the immunomodulatory properties of murine embryonic stem cells (ESCs), obtained either by in vitro fertilization (IVF-ESCs) or by nuclear transfer (NT-ESCs), in heart transplant mouse models. IVF-ESCs did not prolong the survival of fully allogeneic cardiac transplants but significantly prolonged the survival of semiallogeneic hearts from the same ESC donor strain for >100 d in 44% of the animals. However, 28% of transplanted animals infused with IVF-ESCs experienced development of a teratoma. NT-ESCs similarly prolonged semiallogeneic heart graft survival (>100 d in 40% of the animals) but were less teratogenic. By in vitro studies, IVF-ESC and NT-ESC immunoregulation was mediated both by cell contact-dependent mechanisms and by the release of soluble factors. By adding specific inhibitors, we identified PGE(2) as a soluble mediator of ESC immunoregulation. Expansion of regulatory T cells was found in lymphoid organs and in the grafts of IVF-ESC- and NT-ESC-tolerized mice. Our study demonstrates that both IVF-ESCs and NT-ESCs modulate recipient immune response toward tolerance to solid organ transplantation, and that NT-ESCs exhibit a lower tendency for teratoma formation. Because NT-ESCs are obtained by NT of a somatic cell from living individuals into an enucleated oocyte, they could represent a source of donor-derived stem cells to induce tolerance to solid organ allograft.
The Journal of Immunology 03/2011; 186(7):4164-74. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Germ cells are a unique population of cells responsible for transmitting genetic information from one generation to the next. Our understanding of the key mechanisms underlying germ cell development in vivo remains scarce because of insufficient amounts of cell materials available for conducting biological studies. The establishment of in vitro differentiation models that support the generation of germ cells from mouse pluripotent stem cells provides an alternative means for studying reproductive development. The detection and analysis of stem cell-derived germ cells, however, present technical challenges. Methods for determining the developmental stage of germ cells ex vivo, such as gene expression and/or immunochemical analyses are inadequate, frequently necessitating the use of alternative, elaborate methods to prove germ cell identity. We have generated putative oocytes and granulosa cells in vitro from mouse embryonic stem cells and utilized electron microscopy to characterize these cells. Here, we report on the striking ultrastructural similarity of in vitro-generated oocytes and granulosa cells to in vivo oocytes developing within follicles.
Stem cells and development 01/2011; 20(12):2205-15. · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Oct1 (Pou2f1) is a transcription factor of the POU-homeodomain family that is unique in being ubiquitously expressed in both embryonic and adult mouse tissues. Although its expression profile suggests a crucial role in multiple regions of the developing organism, the only essential function demonstrated so far has been the regulation of cellular response to oxidative and metabolic stress. Here, we describe a loss-of-function mouse model for Oct1 that causes early embryonic lethality, with Oct1-null embryos failing to develop beyond the early streak stage. Molecular and morphological analyses of Oct1 mutant embryos revealed a failure in the establishment of a normal maternal-embryonic interface due to reduced extra-embryonic ectoderm formation and lack of the ectoplacental cone. Oct1(-/-) blastocysts display proper segregation of trophectoderm and inner cell mass lineages. However, Oct1 loss is not compatible with trophoblast stem cell derivation. Importantly, the early gastrulation defect caused by Oct1 disruption can be rescued in a tetraploid complementation assay. Oct1 is therefore primarily required for the maintenance and differentiation of the trophoblast stem cell compartment during early post-implantation development. We present evidence that Cdx2, which is expressed at high levels in trophoblast stem cells, is a direct transcriptional target of Oct1. Our data also suggest that Oct1 is required in the embryo proper from late gastrulation stages onwards.
Development 09/2010; 137(21):3551-60. · 6.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 gammaH2A.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.
The EMBO Journal 06/2010; 29(11):1877-88. · 9.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mouse and human stem cells with features similar to those of embryonic stem cells have been derived from testicular cells. Although pluripotent stem cells have been obtained from defined germline stem cells (GSCs) of mouse neonatal testis, only multipotent stem cells have been obtained so far from defined cells of mouse adult testis. In this study we describe a robust and reproducible protocol for obtaining germline-derived pluripotent stem (gPS) cells from adult unipotent GSCs. Pluripotency of gPS cells was confirmed by in vitro and in vivo differentiation, including germ cell contribution and transmission. As determined by clonal analyses, gPS cells indeed originate from unipotent GSCs. We propose that the conversion process requires a GSC culture microenvironment that depends on the initial number of plated GSCs and the length of culture time.
[show abstract][hide abstract] ABSTRACT: The four transcription factors Oct4, Sox2, Klf4, and c-Myc can induce pluripotency in mouse and human fibroblasts. We previously described direct reprogramming of adult mouse neural stem cells (NSCs) by Oct4 and either Klf4 or c-Myc. NSCs endogenously express Sox2, c-Myc, and Klf4 as well as several intermediate reprogramming markers. Here we report that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs. These one-factor induced pluripotent stem cells (1F iPS) are similar to embryonic stem cells in vitro and in vivo. Not only can these cells can be efficiently differentiated into NSCs, cardiomyocytes, and germ cells in vitro, but they are also capable of teratoma formation and germline transmission in vivo. Our results demonstrate that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency.
[show abstract][hide abstract] ABSTRACT: Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility of generating patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4 (also known as Pou5f1), Sox2, c-Myc and Klf4 (refs 1-11). Considering that ectopic expression of c-Myc causes tumorigenicity in offspring and that retroviruses themselves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here we show that adult mouse neural stem cells express higher endogenous levels of Sox2 and c-Myc than embryonic stem cells, and that exogenous Oct4 together with either Klf4 or c-Myc is sufficient to generate iPS cells from neural stem cells. These two-factor iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germ line, and form chimaeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors.
[show abstract][hide abstract] ABSTRACT: Mammalian oocytes are thought to be neutral as for X- or Y-bearing sperm selection is concerned, and penetration of an oocyte by an X- or a Y-bearing sperm is considered a random event. This assumption is mainly based on a posteriori evidences of a nearly equal sex ratio at birth, but it has never been experimentally demonstrated. We have designed a simple experiment, which allowed the penetration of an oocyte by more than one sperm and the further sexing by PCR of each single pronucleus present within the ooplasm. For the first time, we provide experimental evidence that mammalian oocytes do not play a selecting role since a single oocyte may be simultaneously fertilised by both X- and Y-bearing sperm.
Molecular Reproduction and Development 07/2005; 71(2):245-6. · 2.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mammalian embryos obtained by somatic nuclear transfer (NT) struggle to survive throughout development, encountering a number of hurdles leading to wrong functional reprogramming of the donor genome. However, despite these obstacles, some of these embryos continue their development, as if the required transcriptional functions are somehow satisfied. The amount of information gathered on the kinetics and quantitative profile of gene expression in NT pre-implantation embryos is still scarce and limited to a handful of genes described in two species, bovine and mouse. Using a single-cell sensitive semi-quantitative RT-PCR, we have compared the onset and profile of abundance of Hprt, Tsx, Bex1, Bax, Cpt2, and Oct4 genes, in in vitro fertilised and NT-derived mouse 1-cell, 2-cell, 4-cell embryos, morulae, and blastocysts. The genes analysed were activated in NT embryos at approximately the correct time compared to control embryos, indicating that the reprogramming phenomenon is developmentally regulated and that the somatic genome is quickly rearranged towards an embryonic-type of expression during the early stages of segmentation. Despite the right timing of genes onset, the high degree of variability in the number of transcripts found in NT embryos at the latest stages of pre-implantation development, suggests that genome reprogramming is incomplete and inaccurate.
Molecular Reproduction and Development 03/2005; 70(2):146-54. · 2.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: Very little is known about oocyte nuclear architecture during folliculogenesis. Using antibodies to reveal centromeres, Hoechst-staining to detect the AT-rich pericentromeric heterochromatin (chromocenters), combined with confocal microscopy for the three-dimensional analysis of the nucleus, we demonstrate that during mouse folliculogenesis the oocyte nuclear architecture undergoes dynamic changes. In oocytes isolated from primordial and primary follicles, centromeres and chromocenters were preferentially located at the periphery of the nucleus. During oocyte growth, centromeres and chromocenters were initially found spread within the nucleus and then progressively clustered around the periphery of the nucleolus. Our results indicate that the oocyte nuclear achitecture is developmentally regulated and they contribute to a further understanding of the role of nuclear organization in the regulation of genome functioning during differentiation and development.
Journal of Molecular Histology 09/2004; 35(6):631-8. · 1.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fatty acids represent an important energy source for preimplantation embryos. Fatty acids oxidation is correlated with the embryo oxygen consumption which remains relatively constant up to the 8-cell stage, but suddenly increases between the 8-cell and morula stages. The degradation of fatty acids occurs in mitochondria and is catalyzed by several carnitine acyl transferases, including two carnitine palmitoyl transferases, CPT-I and CPT-II. We have carried out a study to determine the relative number of transcripts of Cpt1b and Cpt2 genes encoding for m-CPT-I and CPT-II enzymes, during mouse preimplantation development. Here we show that Cpt1b transcripts are first and temporally detected at the 2-cell stage and reappear at the morula and blastocyst stage. Cpt2 transcripts decrease following fertilization to undetectable levels and are present again later at the morula stage. These results show that transcription of both Cpt1b and Cpt2 is triggered at the morula stage, concomitantly with known increasing profiles of oxygen uptake and fatty acids oxidation. Based on the number of Cpt2 transcripts detected, we could discriminate the presence of two groups of embryos with high and low number of transcripts, from the zygote throughout preimplantation development. To further investigate if the establishment of these two groups of embryos occurs prior to fertilization, we have analyzed the relative number of transcripts of both genes in antral and ovulated MII oocytes. As for preimplantation embryos, MII oocytes show two groups of Cpt2 expression. Antral oocytes, classified according to their chromatin configuration in SN (surrounded nucleolus, in which the nucleolus is surrounded by a rim of Hoechst-positive chromatin) and NSN (not surrounded nucleolus, in which this rim is absent), show three groups with different numbers of Cpt2 transcripts. All NSN oocytes have a number of Cpt2 transcripts doubled compared to that of the group of MII oocytes with high expression. Instead, SN oocytes could be singled out into two groups with high and low numbers of Cpt2 transcripts, similar to those found in MII oocytes. The results of this study point out a correlation between the timing of fatty acids oxidation during preimplantation development and the expression of two genes encoding two enzymes involved in the oxidative pathway. Furthermore, although the biological meaning for the presence of two groups of oocytes/embryos with different levels of Cpt2 transcripts remains unclear, the data obtained suggest a possible correlation between the levels of Cpt2 expression and embryo developmental competence.
Cytogenetic and Genome Research 02/2004; 105(2-4):215-21. · 1.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: The metabolism of fatty acids represents an important energy source for early embryonic development. Our knowledge of the
molecular basis that regulate this metabolism is very poor. In this study we have analysed the pattern of expression ofCpt-1 andCpt-2 genes, that encode the enzymes CPT-1 and CPT-2, which play a role in long chain fatty acids transport inside the mitochondria.
The data obtained from the RT-PCR analysis show a rapid reduction in the number of transcripts from antral oocyte to ovulated
MII oocyte and a significant difference between the expression profiles ofCpt-1 andCpt-2 genes during preimplantation development.Cpt-1 is first temporarily expressed at the G2 phase of 2-cell embryo, it disappears at the 4-cell stage and reappears consistently
between the 8-cell and morula stage;Cpt-2 is expressed from the MII oocyte, decreases to very low level at the 8-cell stage and is actively transcribed from morula,
where it abruptly increases its expression to reach its maximum at the blastocyst stage. Interestingly, the high expression
of bothCpt-1 andCpt-2 at the morula stage is correlated with an increasing profile of oxygen uptake and fatty acid oxidation found at this stage
of development. Furthermore, this rise of transcription is concomitant with other important epigenetic changes occurring prior
to embryo cavitation and involving a reorganisation of chromatin structure and DNA methylation.
Il metabolismo degli acidi grassi rappresenta una importante fonte di energia per l’embrione nelle prime fasi di sviluppo
preimpianto. Le conoscenze delle basi molecolari che regolano questo metabolismo sono molto scarse. In questo studio abbiamo
analizzato il profilo di espressione dei geniCpt-1 eCpt-2, che traducono per gli enzimi CPT-1 e CPT-2, coinvolti nel trasporto degli acidi grassi a lunga catena all’interno dei mitocondri.
I dati, ottenuti dall’analisi molecolare mediante la tecnica della RT-PCR, mostrano una notevole riduzione del numero dei
trascritti tra oocita antrale e oocita ovulato in MII, ed una notevole differenza tra i profili di espressione diCpt-1 eCpt-2 durante lo sviluppo preimpianto.Cpt-1 risulta essere temporaneamente espresso allo stadio di embrione a 2 cellule, è silente nell’embrione a 4 cellule e ricompare
tra l’embrione a 8 cellule e lo stadio di morula;Cpt-2 è invece già espresso nell’oocita in MII, la sua espressione si riduce gradualmente fino a bassi livelli nell’embrione a
4 cellule, per poi tornare a essere attivamente trascritto nella morula, dove il numero di trascritti incrementa notevolmente
fino a raggiungere il picco di espressione nella blastocisti. Significativo è il fatto che l’intensificazione dell’espressione
genica diCpt-1 eCpt-2 allo stadio di morula è correlata con l’incremento nel consumo di ossigeno legato alla β-ossidazione degli acidi grassi in
questo stesso stadio di sviluppo. Inoltre, questo aumento è concomitante con importanti fenomeni epigenetici che precedono
la cavitazione dell’embrione ed implicano la riorganizzazione della struttura della cromatina e la metilazione del DNA.
Rendiconti Lincei. Scienze Fisiche e Naturali 08/2003; 14(3):217-229. · 0.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: The study of gene expression during preimplantation embryonic development is useful to understand which are the molecular
mechanisms involved in the progressive passage from a maternal to a zygotic regulation of gene expression. It is our laboratory
main concern to develop techniques that would allow a more precise characterisation of the timing of transcription and amount
of transcripts of genes that are well known to play an important role in the early events of preimplantation development.
In this study we illustrate the use of a very sensitive protocol of semiquantitative single-cell RT-PCR that we have developed.
This technique allows the simultaneous analysis of up to eight genes on a single cell. Here we show the experiments performed
to validate this novel assay. As an example we have analysed the quantitative expression ofHprt, Bax, Bex-1, Cpt-1, Cpt-2, Oct-4, Tsix andXist on single blastocysts.
La determinazione del profilo di espressione genica durante lo sviluppo embrionale preimpianto permette di comprendere meglio
quali sono i meccanismi molecolari coinvolti nel passaggio dal controllo materno al controllo embrionale della regolazione
genica. Uno dei principali obiettivi del nostro laboratorio è quello di sviluppare metodologie di analisi per una precisa
caratterizzazione sia temporale che quantitativa dell’espressione di quei geni che svolgono un ruolo cruciale nelle prime
fasi dello sviluppo preimpianto. Nel presente lavoro mostriamo l’utilizzo di un protocollo estremamente sensibile di RT-PCR
semiquantitativa su singola cellula. Questa tecnica permette di analizzare contemporaneamente l’espressione di otto geni diversi
sulla singola cellula. In questo lavoro vengono mostrati gli esperimenti compiuti per la validazione della tecnica e i risultati
di un’analisi, compiuta su singole blastocisti murine, dell’espressione dei geniHprt, Bax, Bex-1, Cpt-1, Cpt-2, Oct-4, Tsix edXist.
Rendiconti Lincei. Scienze Fisiche e Naturali 05/2003; 14(2):117-126. · 0.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: During folliculogenesis, the oocyte undergoes changes in chromatin organization that are functional to the acquisition of the developmental competence. In the antral compartment of the mouse ovary, two types of oocytes have been described on the basis of the presence or absence of a ring of Hoechst-bright fluorescence around the nucleolus. It is not known whether these changes of chroma- tin organization entail changes in the localization of chromosomal domains. Making use of CREST serum against centromeric proteins and confocal microscopy, here we describe the three-dimensional localiza- tion of the centromeres within the nuclear volume of mouse antral oocytes. Our data reveal that centro- meres change localization during oocyte maturation showing a dramatic coalescence around the nucleo- lus surface at the stages preceding ovulation. These results further our knowledge on the nuclear organi- zation of the mouse oocyte and represent a first and necessary contribution for future studies on the role that nuclear architecture may exert as an epigenetic regulator of genome functioning. RIASSUNTO. — Cambiamenti della localizzazione dei centromeri in nuclei di oociti durante la follicologe- nesi di topo. Durante la follicologenesi, la cromatina degli oociti va incontro a numerose trasformazioni necessarie all'acquisizione della corretta competenza al successivo sviluppo embrionale. Nel comparti- mento antrale dell'ovario di topo, sono stati descritti due tipi di oociti caratterizzati rispettivamente dalla presenza o assenza, attorno al nucleolo, di un anello di cromatina Hoechst-positivo. Ad oggi non è ancora chiaro se i cambiamenti a carico dell'organizzazione della cromatina comportino cambiamenti nella loca- lizzazione di domini cromosomici. Nel presente lavoro viene descritta la localizzazione tridimensionale, utilizzando un microscopio confocale, dei centromeri all'interno del volume nucleare di oociti antrali di topo rilevati utilizzando anticorpi anticentromero presenti nel siero di pazienti affetti da CREST. I dati ottenuti rivelano che la localizzazione dei centromeri cambia durante la maturazione degli oociti, for- mando, al termine del processo di accrescimento, un anello attorno al nucleolo. I risultati ottenuti pon- gono le basi per una ricostruzione dell'organizzazione nucleare degli oociti durante il processo di follico- logenesi e rappresentano un contributo necessario per lo studio dell'eventuale ruolo che l'architettura nucleare può svolgere come modulatore epigenetico del funzionamento del genoma.
[show abstract][hide abstract] ABSTRACT: During folliculogenesis, the oocyte undergoes changes in chromatin organization that are functional to the acquisition of
the developmental competence. In the antral compartment of the mouse ovary, two types of oocytes have been described on the
basis of the presence or absence of a ring of Hoechst-bright fluorescence around the nucleolus. It is not known whether these
changes of chromatin organization entail changes in the localization of chromosomal domains. Making use of CREST serum against
centromeric proteins and confocal microscopy, here we describe the three-dimensional localization of the centromeres within
the nuclear volume of mouse antral oocytes. Our data reveal that centromeres change localization during oocyte maturation
showing a dramatic coalescence around the nucleolus surface at the stages preceding ovulation. These results further our knowledge
on the nuclear organization of the mouse oocyte and represent a first and necessary contribution for future studies on the
role that nuclear architecture may exert as an epigenetic regulator of genome functioning.
Durante la follicologenesi, la cromatina degli oociti va incontro a numerose trasformazioni necessarie all’acquisizione della
corretta competenza al successivo sviluppo embrionale. Nel compartimento antrale dell’ovario di topo, sono stati descritti
due tipi di oociti caratterizzati rispettivamente dalla presenza o assenza, attorno al nucleolo, di un anello di cromatina
Hoechst-positivo. Ad oggi nonè ancora chiaro se i cambiamenti a carico dell’organizzazione della cromatina comportino cambiamenti
nella localizzazione di domini cromosomici. Nel presente lavoro viene descritta la localizzazione tridimensionale, utilizzando
un microscopio confocale, dei centromeri all’interno del volume nucleare di oociti antrali di topo rilevati utilizzando anticorpi
anticentromero presenti nel siero di pazienti affetti da CREST. I dati ottenuti rivelano che la localizzazione dei centromeri
cambia durante la maturazione degli occiti, formando, al termine del processo di accrescimento, un anello attorno al nucleolo.
I risultati ottenuti pongono le basi per una ricostruzione dell’organizzazione nucleare degli oociti durante il processo di
follicologenesi e rappresentano un contributo necessario per lo studio dell’eventuale ruolo che l’architettura nucleare può
svolgere come modulatore epigenetico del funzionamento del genoma.
Rendiconti Lincei. Scienze Fisiche e Naturali 14(2):109-115. · 0.92 Impact Factor