Valeria Merico

Maternal Oct-4 is a potential key regulator of the developmental competence of mouse oocytes

BackgroundThe maternal contribution of transcripts and proteins supplied to the zygote is crucial for the progression from a gametic to an embryonic control of preimplantation development. Here we compared the transcriptional profiles of two types of mouse MII oocytes, one which is developmentally competent (MIISN oocyte), the other that ceases development at the 2-cell stage (MIINSN oocyte), with the aim of identifying genes and gene expression networks whose misregulated expression would contribute to a reduced developmental competence.ResultsWe report that: 1) the transcription factor Oct-4 is absent in MIINSN oocytes, accounting for 2) the down-regulation of Stella, a maternal-effect factor required for the oocyte-to-embryo transition and of which Oct-4 is a positive regulator; 3) eighteen Oct-4-regulated genes are up-regulated in MIINSN oocytes and are part of gene expression networks implicated in the activation of adverse biochemical pathways such as oxidative phosphorylation, mitochondrial dysfunction and apoptosis.ConclusionThe down-regulation of Oct-4 plays a crucial function in a sequence of molecular processes that leads to the developmental arrest of MIINSN oocytes. The use of a model study in which the MII oocyte ceases development consistently at the 2-cell stage has allowed to attribute a role to the maternal Oct-4 that has never been described before. Oct-4 emerges as a key regulator of the molecular events that govern the establishment of the developmental competence of mouse oocytes.

Chromatin organisation and nuclear architecture in growing mouse oocytes

Although the female gamete is blocked at the dictyate stage of the first meiotic prophase during the whole folliculogenesis, many important epigenetic changes occur to organise the genome to attend early embryonic development. In this paper, we will describe the results of a number of studies aimed to improve our understanding of the nuclear organization of the mouse oocyte during folliculogenesis. Using silver methods that stain NOR, centromeres and heterochromatin, as well as, the use of specific antibodies for the demonstration of centromeres, we have described the changes to the chromatin organisation and to the spatial localisation of chromocenters and centromeres during oocyte growth; these changes have been correlated to the developmental competence of the resulting antral and metaphase II (MII) oocyte.

Epigenomic differentiation in mouse preimplantation nuclei of biparental, parthenote and cloned embryos

Chromosomes, sub-chromosomal regions and genes are repositioned during cell differentiation to acquire a cell-type-specific spatial organization. The constraints that are responsible for this cell-type-specific spatial genome positioning are unknown. In this study we addressed the question of whether epigenetic genome modifications may represent constraints to the acquisition of a specific nuclear organization. The organization of kinetochores, pericentric heterochromatin and the nucleolus was analysed in pre-implantation mouse embryos obtained by in-vitro fertilization (IVF), parthenogenetic activation (P) and nuclear transfer (NT) of differentiated somatic nuclei, which possess different epigenomes. Each stage of pre-implantation embryonic development is characterized by a stage-specific spatial organization of nucleoli, kinetochores and pericentric heterochromatin. Despite differences in the frequencies and the time-course of nuclear architecture reprogramming events, by the eight-cell stage P and NT embryos achieved the same distinct nuclear organization in the majority of embryos as observed for IVF embryos. At this stage the gametic or somatic nuclear architecture of IVF or P and NT embryos, respectively, is replaced by a common embryonic nuclear architecture. This finding suggests that the epigenome of the three types of embryos partially acts as a constraint of the nuclear organization of the three nuclear subcompartments analysed.

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

BackgroundOct4 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.ResultsBy 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.ConclusionsThese 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.

Single-cell quantitative RT-PCR analysis of Cpt1b and Cpt2 gene expression in mouse antral oocytes and in preimplantation embryos.

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.

Cytogenetics of a new cytotype of African Mus (subgenus Nannomys) minutoides (Rodentia, Muridae) from Kenya: C- and G- banding and distribution of (TTAGGG) n telomeric sequences

We present the results of a cytogenetic study on Mus (Nannomys) minutoides from Kenya by means of C- and G- banding and in-situ fluorescence hybridization (FISH) to localize the telomeric sequences. The karyotype is characterized by the occurrence of several Rb chromosomes Rb(1.X), Rb(1.Y). Rb(2.17), Rb(3.13), Rb(4.10), Rb(5.11), Rb(6.7), Rb(8.12), not previously described for this species. This finding suggests a high level of chromosomal diversification, which means it is possible to consider this cytotype as a new, well-differentiated, chromosomal lineage within the subgenus. The C-banding of the metaphases illustrated conspicuous blocks of centromeric heterochromatin at the paracentromeric regions of all telocentric chromosomes. Centromeric heterochromatin is not visible on all biarmed chromosomes. Following hybridization with telomeric probes, bright interstitial telomeric sequence (ITS) fluorescence signals are evident at the pericentromeric area of all Rb chromosomes, with the exception of Rb(2.17). Considering the localization of the C-positive heterochromatin and of the telomeric sequences, the events leading to the Kenyan cytotype from an all-telocentric condition probably included two steps: first, fusion without loss of heterochromatin and pericentromeric telomeric sequences; second, the reduction of the C-positive satellite DNA followed by the amplification of telomeric sequences in the C-negative paracentromeric region of Rb chromosomes. The presence of a single Rb(2.17) without ITS indicates possible variations of this mechanism.

A mitochondrial mechanism is involved in apoptosis of Robertsonian mouse male germ cells

The aim of this study was to determine whether the intrinsic mechanism of apoptosis is involved in the death of germ cells in Robertsonian (Rb) heterozygous adult male mice. Testes from 5-month-old Rb heterozygous CD1 x Milano II mice were obtained and compared with those from homozygous CD1 (2n=40) and Milano II (2n=24) mice. For histological evaluation of apoptosis, TUNEL labelling and immunohistochemistry were used to localise Bax and cytochrome c. Expression of calbindin D(28k) (CB), an anti-apoptotic molecule, was also analysed by immunohistochemistry and immunoblotting. Testicular ultrastructure was visualised by electron microscopy. Morphology and cell associations were abnormal in the Rb heterozygous seminiferous epithelium. An intense apoptotic process was observed in tubules at stage XII, mainly in metaphase spermatocytes. Metaphase spermatocytes also showed Bax and cytochrome c redistributions. Mitochondria relocated close to the paranuclear region of spermatocytes. CB was mainly expressed in metaphase spermatocytes, but also in pachytene spermatocytes, spermatids and Sertoli cells at stage XII. The co-localisation of CB and TUNEL labelling was very limited. Sixty per cent of metaphase spermatocytes were apoptotic and calbindin negative, while 40% were calbindin positive without signs of apoptosis. Ten per cent of the Bax- and cytochrome c-positive cells were also calbindin positive. These data suggest that apoptosis of the germ cells in heterozygous mice occurs, at least in part, through a mitochondrial-dependent mechanism. Calbindin overexpression might prevent or reduce the apoptosis of germ cells caused by Rb heterozygosity, which could partially explain the subfertility of these mice.

Chromosomal speciation in mice: a cytogenetic analysis of recombination

Within species, populations differing by chromosomal rearrangements (“chromosomal races”) may become reproductively isolated in association with reduced hybrid fertility due to meiotic aberrations. Speciation is also possible if hybridizing chromosomal races accumulate genetic differences because of reduced meiotic recombination in the heterozygous configuration in hybrids. Here, we examine recombination in pure races and hybrids within a model system for chromosomal speciation: the hybridization of the Poschiavo (CHPO) and Upper Valtellina (IUVA) chromosomal races of house mouse in Upper Valtellina, Italy. These races differ by Robertsonian fusions/whole-arm reciprocal translocations, such that hybrids produce a pentavalent meiotic configuration. We determined the number and position of the recombination points (using an antibody against the MutL homolog 1 [MLH1] protein) on synaptonemal complexes at pachytene in laboratory-reared CHPO, IUVA, and hybrid males, analyzing at least 112 spermatocytes per karyotypic group, up to a total of 534 spermatocytes. The mean ± standard deviation numbers of MLH1 foci per spermatocyte were 22.2 ± 3.2, 20.1 ± 2.9, 20.7 ± 2.3, and 21.9 ± 2.9 for CHPO, IUVA, CHPO × IUVA, and IUVA × CHPO, respectively. Altogether, 10,146 chromosome arms were examined, allowing multiple comparisons. Overall, recombination events were more frequently distal than proximal or interstitial. The average number of proximal MLH1 foci per chromosome arm decreased going from telocentric to metacentric bivalents to pentavalents (when present), which (together with other factors) influenced the average number of MLH1 foci per cell between CHPO, IUVA, and hybrid mice. The low frequency of proximal recombination in pentavalents of CHPO–IUVA hybrids may promote reproductive isolation between the CHPO and IUVA races, when coupled with reduced hybrid unfitness.

Role of Oct-4 during acquisition of developmental competence in mouse oocyte.

Knowledge of what determines the developmental competence of oocytes during folliculogenesis is poor. This review, through the analysis of the expression profile of developmentally competent or incompetent mouse oocytes, summarizes the results of recent studies showing that the Oct-4 transcription factor regulating the expression of Stella and Foxj2 at the Nanog locus could play a pivotal role in the establishment of the oocytes developmental competence.

Transcriptome based identification of mouse cumulus cell markers that predict the developmental competence of their enclosed antral oocytes

BackgroundThe cumulus cells (CCs) enveloping antral and ovulated oocytes have been regarded as putative source of non-invasive markers of the oocyte developmental competence. A number of studies have indeed observed a correlation between CCs gene expression, embryo quality, and final pregnancy outcome. Here, we isolated CCs from antral mouse oocytes of known developmental incompetence (NSN-CCs) or competence (SN-CCs) and compared their transcriptomes with the aim of identifying distinct marker transcripts.ResultsGlobal gene expression analysis highlighted that both types of CCs share similar transcriptomes, with the exception of 422 genes, 97.6% of which were down-regulated in NSN-CCs vs. SN-CCs. This transcriptional down-regulation in NSN-CCs was confirmed by qRT-PCR analysis of CC-related genes (Has2, Ptx3, Tnfaip6 and Ptgs2). Only ten of the 422 genes were up-regulated with Amh being the most up-regulated in NSN-CCs, with an average 4-fold higher expression when analysed by qRT-PCR.ConclusionsThe developmental incompetence (NSN) or competence (SN) of antral oocytes can be predicted using transcript markers expressed by their surrounding CCs (i.e., Has2, Ptx3, Tnfaip6, Ptgs2 and Amh). Overall, the regulated nature of the group of genes brought out by whole transcriptome analysis constitutes the molecular signature of CCs associated either with developmentally incompetent or competent oocytes and may represent a valuable resource for developing new molecular tools for the assessment of oocyte quality and to further investigate the complex bi-directional interaction occurring between CCs and oocyte.