Alternative strategies for nuclear reprogramming in somatic cell nuclear transfer (SCNT)

Twenty years passed by since the production of Dolly the sheep, but despite significant technical progress has been achieved in the manipulation procedures, the proportion of offspring following transfer of SCNT embryos has remained almost unchanged in farm animals. Remarkable progress has been obtained instead in laboratory animals, particularly by Japanese Groups, in the mouse. However, the nuclear reprogramming strategies tested in mouse do not always work in farm animals, and others are difficult to be implemented, for require complicated molecular biology tools unavailable yet in large animals. In this review we put in contest the previous work done in farm and laboratory animals with recent achievements obtained in our laboratory, and we also indicate a road map to increase the reliability of SCNT procedures

Ultrastructural analysis reveals abnormal mitochondria in cloned blastocysts

Somatic cell nuclear transfer (SCNT) is a powerful technique, but still very inefficient despite 20 years passed by since the cloned mammal was born. We have recently shown that the major cause of abnormalities observed in cloned fetuses are mitochondrial dysfunctions in placenta collected from cloned sheep. Investigations on mitochondria in SCNT are limited to the mtDNA hetero/homoplasmy in cloned offspring, whereas no data is available for an eventual role of mitochondria dysfunction on the developmental failure of cloned animals. Here we wanted to know whether mitochondrial abnormalities are observed already in cloned blastocysts since mitochondrial replication does not occur after the hatched blastocysts stage. SCNT and in vitro processed (IVP) blastocysts were produced and analysed for mitochondrial structure and functionality. First, embryos were analysed using transmission electron microscope (TEM). Drastic differences in mitochondrial structure between SCNT and IVP blastocysts were observed. Decrease density of mature mitochondria, very high degree of cytoplasmic vacuolisation, numerous cytoplasmic vesicle and autophagosomes were observed in SCNT blastocysts. Moreover, statistically lower expression of major mitochondrial, autophagic and apoptotic proteins were observed in SCNT embryos. Obtained results clearly shown that mitochondrial abnormalities are already observed in blastocysts stage embryos. It is important to point out that activity of mitochondria are strictly control by nuclear signals, thus, obtained results may suggest that incomplete nuclear reprogramming in cloned nucleus might be responsible also for the impaired mitochondrial function in cloned embryos/fetuses

Polychlorinated biphenyls (PCBs) alter DNA methylation and genomic integrity of sheep fetal cells in a simplified in vitro model of pregnancy exposure

Polychlorinated biphenyls (PCBs) are persistent organic pollutants ubiquitously detectable in the environment and in the food chain. Prenatal exposure to PCBs negatively affects fetal development and produces long-term detrimental effects on child health. The present study sought to evaluate the cytotoxic and genotoxic effects of chronic PCB exposure on fetal cells during pregnancy. To this aim, sheep embryonic fibroblasts (SEF) and amniocytes (SA) were cultured in vitro in the presence of low doses of PCBs for a period of 120 days, comparable to the full term of ovine pregnancy. Cellular proliferation rates, global DNA methylation, chromosome integrity, and markers of DNA damage were evaluated at different time points. Moreover, SEF treated with PCBs for 60 days were left untreated for one further month and then examined in order to evaluate the reversibility of PCB-induced epigenetic defects. PCB-treated SEF were more sensitive than SA treated with PCBs, in terms of low cell proliferation, and increased DNA damage and global DNA methylation, which were still detectable after interruption of PCB treatment. These data indicate that chronic exposure of fetal cells to PCBs causes permanent genomic and epigenetic instability, which may influence both prenatal and post-natal growth up to adulthood. Our in vitro model offer a simple and controlled means of studying the effects of different contaminants on fetal cells - one that could set the stage for targeted in vivo studies

Evidence of placental autophagy during early pregnancy after transfer of in vitro produced (IVP) sheep embryos

Pregnancies obtained by Assisted Reproductive Technologies (ART) are associated with limited maternal nutrient uptake. Our previous studies shown that in vitro culture of sheep embryos is associated with vascularization defects in their placentae and consequent reduction of embryo growth. Autophagy is a pro-survival cellular mechanism triggered by nutrient insufficiency. Therefore, the goal of our present study was to determine if autophagy is involved in early placental development after transfer of in vitro produced (IVP) embryos. To do this, placentae obtained following transfer of IVP sheep embryos were compared with placentae obtained after natural mating (control-CTR). The placentae were collected on day 20 post-fertilization and post-mating, respectively, and were analyzed using molecular (qPCR), ultrastructural and histological/immunological approaches. Our results show drastically increased autophagy in IVP placentae: high levels of expression (p<0.05) of canonical markers of cellular autophagy and a high proportion of autophagic cells (35.08%; p<0.001) were observed. We conclude that high autophagic activity in IVP placentae can be a successful temporary counterbalance to the retarded vasculogenesis and the reduction of foetal growth observed in pregnancies after transfer of IVP embryos

Nuclear quiescence and histone hyper-acetylation jointly improve protamine-mediated nuclear remodeling in sheep fibroblasts

Recently we have demonstrated the possibility to replace histones with protamine, through the heterologous expression of human protamine 1 (hPrm1) gene in sheep fibroblasts. Here we have optimized protaminization of somatic nucleus by adjusting the best concentration and exposure time to trichostatin A (TSA) in serum-starved fibroblasts (nuclear quiescence), before expressing Prm1 gene. To stop cell proliferation, we starved cells in 0.5% FBS in MEM (“starved”—ST group), whereas in the Control group (CTR) the cells were cultured in 10% FBS in MEM. To find the most effective TSA concentration, we treated the cells with increasing concentrations of TSA in MEM + 10% FBS. Our results show that combination of cell culture conditions in 50 nM TSA, is more effective in terminating cell proliferation than ST and CTR groups (respectively 8%, 17.8% and 90.2% p<0.0001). Moreover, nuclear quiescence marker genes expression (Dicer1, Smarca 2, Ezh1 and Ddx39) confirmed that our culture conditions kept the cells in a nuclear quiescent state. Finally, ST and 50 nM TSA jointly increased the number of spermatid-like cell (39.4%) at higher rate compared to 25 nM TSA (20.4%, p<0.05) and 100 nM TSA (13.7%, p<0.05). To conclude, we have demonstrated that nuclear quiescence in ST cells and the open nuclear structure conferred by TSA resulted in an improved Prm1-mediated conversion of somatic nuclei into spermatid-like structures. This finding might improve nuclear reprogramming of somatic cells following nuclear transfer

Nuclear quiescence and histone hyper-acethylation jointly improve protamine-mediated nuclear remodeling in sheep fibroblasts.

Recently we demonstrated the possibility of the direct histone-protamine exchange in somatic cells by the exogenous induction of protamine 1 (Prm1) gene expression. Here we have further advanced our protocol, by mimicking the nuclear remodelling taking place in spermatogenesis. The spermiogenesis starts by cell quiescence and the open of nuclear chromatin (by histone-hyperacetylation) of post-meiotic round spermatids. Our aim was to test if protaminization of somatic nucleus increases through the correct union of the induction of G0 stage and exposure to Trichostatin A (TSA). To stop the cell proliferation, the cells were cultured in 0.5% FBS in MEM (G0 group) whereas in the control group (CTR) the cells were culture in 10% FBS in MEM. To check the proper TSA concentration, we treated the cells with 25 nM (25 TSA), 50 nM (50 TSA) and 100 nM (100 TSA) in MEM + 10% FBS. Our results showed that combination of the cell culture condition in G0 and 50 TSA stopped the cell proliferation vs CTR (respectively 17.8%, 8% and 90.2%, p<0.0001). Moreover, quiescent markers gene expression analysis (Dicer1, Smarca 2, Ezh1, Ddx39, H2afz and Pink1) demonstrated that our cell culture condition drive the cell in a quiescent state. Finally, the union of G0 and 50 TSA produced a higher number of spermatid-like cell (39.4%) than the 25 TSA (20.4%, p<0.05) and 100 TSA (13.7%, p<0.05). To conclude, we have demonstrated that the open chromatin structure conferred by G0 stage and TSA, resulted in a more efficient Prm1-mediated conversion of somatic nuclei into spermatid-like structures

Assessing the resistance of sheep fibroblasts to increasing concentration of Trehalose

Trehalose is a non-reducing disaccharide commonly used as a cryo-protectant for deep freezing of cells. In this preliminary work, we have assayed the feasibility of its use for inducing reversible drying in somatic cells (fibroblasts), using sheep as a model. Here we have assayed the tolerance of somatic cells to increasing concentration of trehalose. To this extends, sheep fibroblasts were incubated using a growing concentration gradient of trehalose 50mM, 100mM, 250mM, 400mM and 500mM in Minimum Essential Medium (MEM) for 24hours in an incubator set at 38.5°C. On the basis of preliminary trials, the highest concentrations were excluded because they were found to be toxic to the cells and we have focused on the following: 50mM, 100mM and 200mM. The final osmolarity of the three concentrations were 376 mOsm/kg, 462 mOsm/kg and 711 mOsm/kg respectively. Cells have been incubated in MEM with trehalose for 3 hours in a stove set at 37°C and 12% humidity in lack of gas. Every hour cells were assayed for morphology and viability through Trypan Blue exclusion test. After one hour, live cells were about 71%, 63.15% and 52.85% respectively for 50mM, 100mM and 200mM explaining a negative correlation between the percentage of live cells and the sugar concentration. However, after 2 and 3 hours, death of cells was observed within each concentration as well as morphological changes which was however reversible upon washing cells from trehalose and further culture

Starvation improves sheep fibroblast chromatin remodeling in spermatide-like structure

We have recently demonstrated that the chromatin of somatic cells can be converted into spermatid-like structures, by the transient expression of human protamine 1 gene Prm1. Here we have further advanced our protocol, by mimicking the nuclear remodelling taking place in spermatids. Since nuclear maturation in spermatids occurs in G0, our first aim was to test if protaminization of somatic nucleus increases when G0-stage fibroblasts are trasfected with Prm1 gene. Protamine deposition on DNA is anticipated by a genome-wide histone acethylation. Thus, our second aim was to induce a genome hyper-acethylation of G0 cells, by optimizing timing of exposure/concentration of Histone De-Acethylase Inhibitor (HDAI) Tricostatin A. Results: G0-stage fibroblasts cultured for 24h pre-transfection with medium containing 0.5% FBS showed an higher proportion of spermatid-like cells, compared to the control (CTR: 10% FBS) (p<0.05). Furthermore, Bromodeoxyuridine incorporation demonstrated that starved somatic cells were effectively in G0-stage during protaminization (p<0.0001). Finally, we have found a greater number of spermatid-like cells with TSA concentration between 25 and 50 nM, comparing to 100 nM (p<0.05). To conclude, we have demonstrated that G0 stage, and the open nuclear structure conferred by TSA resulted in a more efficient Prm1-mediated conversion of somatic nuclei into spermatid-like structures