70 research outputs found
The Maternal legacy to the embryo: cytoplasmic components and their effects on early development
No full textRNA molecules and proteins are accumulated in the oocyte cytoplasm during its growth phase and are used to sustain the early phases of embryonic development before embryo DNA transcription begins. This makes the oocyte a very special cell, quite different from somatic cells where RNA and proteins usually undergo a rapid turnover. To enable the storage and timely use of such stored molecules, various mechanisms are effective in the oocyte and are gradually being elucidated. Our understanding of such mechanisms is important for constantly improving therapy for human and animal reproductive disorders as well as for understanding the process of nuclear reprogramming during cloning procedure or stem cell generation. This review focuses on the various aspects of these regulatory processes in an attempt to give an overview of the present knowledge on post-transcriptional and post-translational mechanisms taking place during oocyte maturation and early development. Mechanisms such as cytoplasmic regulation of the poly(A) tail, RNA localization and protein phosphorylation are described in some detail. Because most data are available from lower species these are presented together with appropriate reference to the mammalian oocyte when data are known, or when important differences have been described
Effetti cellulari e molecolari dei perturbatori endocrini sulla capacità di sviluppo degli ovociti
No full textEffects of endocrine disrupters on the oocytes and embryos of farm animals
No full textCurrently, approximately 60 chemicals have been identified as endocrine disruptors (EDs): exogenous agents that interfere with the synthesis, secretion, transport, metabolism, binding, action, or elimination of natural blood-borne hormones. Farm animals ingest these substances with food and drinking water. Their stability and lipid solubility has led to increased concern that these substances may compromise the reproductive health of both humans and animals. Oocytes are a permanent cell population established before birth which is exposed to environmental stimuli for a period that, in farm animals, can be as long as several years. Oocyte competence is acquired within the ovary during the developmental stages that precede ovulation and its role is critical during the interval between fertilization and the so-called maternal to embryonic transition, when the transcriptional activity of the embryonic genome becomes fully functional. Any perturbation of these delicate process is likely to reduce oocyte developmental competence and, therefore, to cause an arrest of embryonic development at any given stage. A critical analysis of the doses and time of exposure is presented together with a description of the effects of different EDs on farm animal oocytes and early embryonic development. Finally some of the mechanisms mediating EDs effects on the oocytes will be described. In particular the role of arylhydrocarbon receptor, maternal mRNA stability and cytoplasmic remodelling during oocyte maturation will be discussed in some details
Defective developmental capacity of bovine oocytes is accompanied by a shorte poly-(A) tail of maternal trascripts
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Different ability to form outgrowth between in vitro produced porcine embryos and parthenote
No full textThe establishment of porcine embryonic stem cell (pESC) lines would be a novel tool for animal biotechnology and it would represent a useful model for biomedical research as well as developmental biology studies. In the last years parthenotes received particular attention because they represent an alternative ethical source to obtain ESC lines. However little is known about the biological differences between parthenotes and embryos and further studies are needed in order to better elucidate these aspects. Here we produced in vitro fertilized (IVF) and parthenogenetic embryos and isolated ICMs in order to compare their ability to form outgrowths.
More in detail, blastocysts were subjected to immuno-surgery, using pronase 0.5% (w/v), monkey anti-porcine serum (supplied by Istituto Zooprofilattico Sperimentale Lombardia ed Emilia- Romagna) and Guinea pig complement. ICMs were isolated from lysed trophoblast cells by pipetting and plated onto inactivated STO feeder cells and outgrowth formation was monitored.
We observed that parthenogenetic ICMs generated a significantly higher number of outgrowths than IVF ones (22.16% vs 4.82%). In order to better understand this result we compared the expression levels of the cell adhesion molecules beta integrin-1and vitronectin in ICMs obtained from the two sources.
A statistically significant difference for the two genes was detected. In particular, the expression of beta integrin-1 was 5,6-fold higher in parthenotes than in IVF ICMs (ΔCt values: 68±0.5 vs 12±0.1). Similarly vitronectin displayed an up regulation 11,7-fold in parthenogenetic embryos (ΔCt values: 82±0.7 vs 7.3±0.2).
Our findings provide the first molecular explanation for the higher efficiency to generate outgrowths exhibited by parthenogenetic blastocysts, as compared to regular IVF embryos
Morphological and molecular changes of pig skin fibroblasts exposed to 5-AZA cytidine and addressed to subsequent pancreatic differentiation
No full textRecent experiments demonstrate that terminally differentiated cells can be induced to de-differentiate in vitro and increase their plasticity in response to synthetic molecules capable of reverting cells from their lineage commitment to a more pluripotent state. In response to specific conditions de-differentiated cells can then be re-addressed to a different cell type. However only scattered information are available on the morphological changes and the ultrastructural remodelling required when cells transit along the differentiation pathways, adopting the modifications needed in order to adequately adapt to a different and specific state.
In the present study we prepared skin fibroblast primary cultures and exposed them to 5-aza-cytidine (5-aza), an inhibitor of DNA methylation, to increase cell plasticity. We then investigated the ability of 5-aza treated cells (5-azatC) to be re-addressed to pancreatic beta cell-like cells (r-betaC), in response to specific differentiation conditions. Ultra-structural modifications were evaluated in parallel with molecular analysis for the expression of differentiation stage-specific markers.
Elucidation of the morphological changes that take place in cells undergoing de-differentiation events is essential for a better understanding of the biology of these process paving the way to the successful use of stem cells in regenerative medicine and tissue replacement therapies
Intercellular bridges functionally connect parthenogenetic cells
No full textWe previously reported that parthenogenetic cells display abnormal centrosome and spindle formation resulting in chromosome malsegregation and a high incidence of hypoaploidy. Unexpectedly this is not accompanied by a correspondingly high rate of apoptosis. We hypothesize that a series of adaptive mechanisms make this possible. The presence of intercellular bridges may represent one of such mechanism and would provide a strategy for mutual exchange of missing cell products, alleviating the unbalanced chromosome distribution.
The presence of intercellular bridges was investigated in pig parthenogenetic cells by transmission and scanning electron microscopy. For the former, cells were fixed in 2% glutaraldehyde and post-fixed in 1% osmic acid. After standard dehydration, samples were embedded in an Epon-Araldite 812 mixture, sectioned and observed with a Jeol 1010 electron microscope. For scanning electron microscopy cells were covered with a 9 nm gold film by flash evaporation of carbon and examined with a SEM-FEG Philips XL-30 microscope. Functional trafficking activity was demonstrated with fluorescent 10-kDa dextran. The tracking molecule was injected into the cytoplasm of a single cell with FemtoJet Microinjector and its movement was monitored.
Ultra-structural analysis of parthenogenetic cells showed the presence of intercellular bridges that ensured cytoplasmic continuity among cells. Furthermore extensive movement of 10-kDa dextran demonstrated functional intercellular trafficking through these canals suggesting their use for transfer of mRNAs, proteins and ribosomes among cells.
Our results demonstrate that parthenogenetic cells present a wide network of functional intercellular bridges that may constitute an adaptive mechanism to support normal cell functions
Mechanisms involved in inter-lineage conversion and differentiation of porcine fibroblasts
No full textWe previously demonstrated that porcine fibroblasts exposed to 5-aza-cytidine (5-aza-CR), an inhibitor of DNA methylation, increase their plasticity and can be re-addressed to pancreatic lineage.
Here we investigate the mechanisms involved in the acquisition of a higher plasticity state by 5-aza-CR treated fibroblasts. Furthermore we characterized the cellular and molecular events driven by the differentiation protocol following thereafter.
Cells were analyzed at different time points: untreated fibroblasts, after 5-aza-CR exposure and then on day 1-2-3-4-5-6-7 of pancreatic induction. DNA global methylation modifications were evaluated with an antibody against 5-Methylcytidine used for DNA dot blot analysis and immunolocalization studies. Cells were also immuno-characterized with primary antibodies against Vimentin, Oct4, Nanog, Sox17 and Hnf4, and gene expression level changes were evaluated in parallel.
Our results show that 5-aza-CR induced a decrease in 5- Methylcytidine positivity that gradually returned to the levels observed in untreated fibroblasts within 3 days. Consistently, a down-regulation of vimentin was observed, together with an increase of Oct4 and Nanog, which remained clearly expressed until day 4 and were down-regulated thereafter. At the same time Sox17 and Hnf4, involved in the induction of definitive endoderm and primitive gut tube, respectively, displayed a reverse trend with a signal becoming gradually more intense.
Our observations demonstrate that 5-aza-CR effect on DNA methylation is transient and initial levels are restored within 3 days. The combined and sequential action of the molecule with an induction protocol enables efficient inter-lineage conversion and controlled cell differentiation
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