Time filter

Source Type

Luciano A.M.,University of Milan | Franciosi F.,University of Milan | Lodde V.,University of Milan | Corbani D.,University of Milan | And 8 more authors.
Reproductive Toxicology | Year: 2010

The new European chemicals policy for the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) will most probably impose a dramatic increase in the number of animals required for reproductive toxicity testing. For this purpose, the development and validation of alternative methods is urgently needed in order to reduce the use of laboratory animals. The present study describes the inter-laboratory variability and the transferability assessment of an in vitro test able to identify chemical effects during the process of oocyte maturation in a bovine model. The test was developed/optimised within ReProTect, an integrated research project funded by the European Union, joining together 35 partners with complementary expertise in reproductive toxicology. Eight chemicals with well-known toxic properties were tested (benzo[a]pyrene, busulfan, cadmium chloride, cycloheximide, diethylstilbestrol, ketoconazole, methylacetoacetate, mifepristone/RU-486 and DMSO as solvent) on the in vitro maturation (IVM) assay in two well-trained laboratories using the established Standard Operating Procedures. The statistical analysis demonstrated the concordance of results across the laboratories and the reproducibility of the test. We therefore conclude that the IVM test could advance toward the process of validation as alternative in vitro method that, in combination with additional in vitro tests, can become part of an integrated testing strategy in order to predict chemical hazards on mammalian fertility. © 2010 Elsevier Inc.


Colleoni S.,Laboratorio Of Tecnologie Della Riproduzione | Galli C.,Laboratorio Of Tecnologie Della Riproduzione | Galli C.,University of Bologna | Giannelli S.G.,San Raffaele Scientific Institute | And 4 more authors.
Experimental Cell Research | Year: 2010

In this study we demonstrated that neural rosettes derived from human ES cells can give rise either to neural crest precursors, following expansion in presence of bFGF and EGF, or to dopaminergic precursors after exposure to ventralizing factors Shh and FGF8. Both regionalised precursors are capable of extensive proliferation and differentiation towards the corresponding terminally differentiated cell types. In particular, peripheral neurons, cartilage, bone, smooth muscle cells and also pigmented cells were obtained from neural crest precursors while tyrosine hydroxylase and Nurr1 positive dopaminergic neurons were derived from FGF8 and Shh primed rosette cells. Gene expression and immunocytochemistry analyses confirmed the expression of dorsal and neural crest genes such as Sox10, Slug, p75, FoxD3, Pax7 in neural precursors from bFGF-EGF exposed rosettes. By contrast, priming of rosettes with FGF8 and Shh induced the expression of dopaminergic markers Engrailed1, Pax2, Pitx3, floor plate marker FoxA2 and radial glia markers Blbp and Glast, the latter in agreement with the origin of dopaminergic precursors from floor plate radial glia. Moreover, in vivo transplant of proliferating Shh/FGF8 primed precursors in parkinsonian rats demonstrated engraftment and terminal dopaminergic differentiation.In conclusion, we demonstrated the derivation of long-term self-renewing precursors of selected regional identity as potential cell reservoirs for cell therapy applications, such as CNS degenerative diseases, or for the development of toxicological tests. © 2010 Elsevier Inc.


PubMed | Laboratorio Of Tecnologie Della Riproduzione and Free University of Brussels
Type: | Journal: Cell transplantation | Year: 2016

Porcine islets have notoriously low insulin secretion levels in response to glucose stimulation. While this is somehowexpected in the case of immature islets isolated from fetal and neonatal pigs, disappointingly low secretory responses are frequently reported in studies using in vitro-maturated fetal and neonatal islets and even fully-differentiated adult islets. Herein, we show that beta-cell specific expression of a modified glucagon-like peptide 1 (GLP-1) and of a constitutively activated type 3 muscarinic receptor (M3R) efficiently amplifies glucose-stimulated insulin secretion (GSIS). Both adult and neonatal isolated pig islets were treated with adenoviral expression vectors carrying sequences encoding for GLP-1 and/or M3R. GSIS from transduced and control islets was evaluated during static incubation and dynamic perifusion assays. While expression of GLP-1 did not affect basal or stimulated insulin secretion, activated M3R produced a 2-fold increase of both first and second phases of GSIS. Coexpression of GLP-1 and M3R caused an even greater increase of the secretory response which was amplified 4-fold compared to controls. In conclusion, our work highlights pig islet insulin secretion deficiencies and proposes concomitant activation of cAMP-dependent and cholinergic pathways as a solution to ameliorate GSIS from pig islets used for transplantation.


PubMed | Laboratorio Of Tecnologie Della Riproduzione and University Utrecht
Type: Journal Article | Journal: Reproduction, fertility, and development | Year: 2016

Advanced maternal age and in vitro embryo production (IVP) predispose to pregnancy loss in horses. We investigated whether mare age and IVP were associated with alterations in mitochondrial (mt) DNA copy number or function that could compromise oocyte and embryo development. Effects of mare age (<12 vs 12 years) on mtDNA copy number, ATP content and expression of genes involved in mitochondrial replication (mitochondrial transcription factor (TFAM), mtDNA polymerase subunit B (mtPOLB) and mitochondrial single-stranded DNA-binding protein (SSB)), energy production (ATP synthase-coupling factor 6, mitochondrial-like (ATP-synth_F6)) and oxygen free radical scavenging (glutathione peroxidase 3 (GPX3)) were investigated in oocytes before and after in vitro maturation (IVM), and in early embryos. Expression of TFAM, mtPOLB and ATP-synth-F6 declined after IVM (P<0.05). However, maternal age did not affect oocyte ATP content or expression of genes involved in mitochondrial replication or function. Day 7 embryos from mares 12 years had fewer mtDNA copies (P=0.01) and lower mtDNA:total DNA ratios (P<0.01) than embryos from younger mares, indicating an effect not simply due to lower cell number. Day 8 IVP embryos had similar mtDNA copy numbers to Day 7 in vivo embryos, but higher mtPOLB (P=0.013) and a tendency to reduced GPX3 expression (P=0.09). The lower mtDNA number in embryos from older mares may compromise development, but could be an effect rather than cause of developmental retardation. The general down-regulation of genes involved in mitochondrial replication and function after IVM may compromise resulting embryos.


Galli C.,Laboratorio Of Tecnologie Della Riproduzione | Galli C.,University of Bologna | Perota A.,Laboratorio Of Tecnologie Della Riproduzione | Brunetti D.,Laboratorio Of Tecnologie Della Riproduzione | And 3 more authors.
Xenotransplantation | Year: 2010

Background: Techniques for genetic engineering of swine are providing genetically modified animals of importance for the field of xenotransplantation, animal models for human diseases and for a variety of research applications. Many of these modifications have been directed toward avoiding naturally existing cellular and antibody responses to species-specific antigens. Methods: A number of techniques are today available to engineering the genome of mammals, these range from the well established less efficient method of DNA microinjection into the zygote, the use of viral vectors, to the more recent use of somatic cell nuclear transfer. The use of enzymatic engineering that are being developed now will refine the precision of the genetic modification combined with the use of new vectors like transposons. Results: The use of somatic cell nuclear transfer is currently the most efficient way to generate genetically modified pigs. The development of enzymatic engineering with zinc-finger nucleases, recombinases and transposons will revolutionize the field. Nevertheless, genetic engineering in large domesticated animals will remain a challenging task. Conclusions: Recent improvements in several fields of cell and molecular biology offer new promises and opportunities toward an easier, cost-effective and efficient generation of transgenic pigs. © 2010 John Wiley & Sons A/S.


Schubert T.,Catholic University of Louvain | Poilvache H.,Catholic University of Louvain | Galli C.,Laboratorio Of Tecnologie Della Riproduzione | Galli C.,University of Bologna | And 2 more authors.
Biomaterials | Year: 2013

Pig adipose mesenchymal stem cells (AMSCs) could be proposed for the improvement of bone substitute. However, these xenogenic cells retain a galactosyl (Gal) epitope that elicits xenorejection. Our work aims to use Gal-Knock-Out (Gal-KO) pig AMSCs to associate cellular immunomodulation, humoral down-elicitation of Gal-KO cells and osteogenic capacity of AMSCs. Human and pig AMSCs were compared for proliferation/differentiation kinetics and bone neoformation in vivo. Humoral reaction against pig Gal+ vs. Gal-KO AMSCs and immunomodulation properties of Gal+ vs. Gal-KO AMSCs were assessed in vitro. Humoral/cellular reactions against Gal+ vs. Gal-KO osteogenic differentiated pig AMSC xenografts were assessed in an immunocompetent rodent model. Expansion/differentiation/bone neoformation was significantly improved with differentiated pig AMSCs compared with human cells. Based on immunohistochemistry and cell-based ELISA, Gal+ AMSCs had higher sensitivity to preformed/induced anti-pig antibodies than Gal-KO AMSCs. In vitro cellular immunomodulation was similar between Gal+ and Gal-KO AMSCs. In vivo, a significant reduction of anti-pig IgG was found at 1 month in rats implanted with Gal-KO AMSCs compared with those implanted with Gal+ AMSCs. Lymphocyte/macrophage infiltration of osteogenic differentiated pig AMSC xenografts was significantly lower at post-operative day (POD) 7 in recipients of Gal-KO vs. Gal+ pig cells. No significant difference was found at POD 28. The combination of the cellular immunomodulation with the Gal-KO phenotype of AMSCs can significantly improve the cellular engraftment of pig osteogenic cells by delaying xenorejection. © 2013 Elsevier Ltd.


Galli C.,Laboratorio Of Tecnologie Della Riproduzione | Galli C.,University of Bologna | Lagutina I.,Laboratorio Of Tecnologie Della Riproduzione | Perota A.,Laboratorio Of Tecnologie Della Riproduzione | And 4 more authors.
Reproduction in Domestic Animals | Year: 2012

Contents: Somatic cell nuclear transfer (SCNT) was first developed in livestock for the purpose of accelerating the widespread use of superior genotypes. Although many problems still exist now after fifteen years of research owing to the limited understanding of genome reprogramming, SCNT has provided a powerful tool to make copies of selected individuals in different species, to study genome pluripotency and differentiation, opening new avenues of research in regenerative medicine and representing the main route for making transgenic livestock. Besides well-established methods to deliver transgenes, recent development in enzymatic engineering to edit the genome provides more precise and reproducible tools to target-specific genomic loci especially for producing knockout animals. The interest in generating transgenic livestock lies in the agricultural and biomedical areas and it is, in most cases, at the stage of research and development, with few exceptions that are making the way into practical applications. © 2012 Blackwell Verlag GmbH.


Lazzari G.,Laboratorio Of Tecnologie Della Riproduzione | Colleoni S.,Laboratorio Of Tecnologie Della Riproduzione | Duchi R.,Laboratorio Of Tecnologie Della Riproduzione | Galli A.,Instituto Sperimentale Italiano Lazzaro Spallanzani | And 3 more authors.
Reproduction | Year: 2011

Infertility in cattle herds is a growing problem with multifactorial causes. Embryonic genotype and level of inbreeding are among the many factors that can play a role on reproductive efficiency. To investigate this issue, we produced purebred and crossbred bovine embryos by in vitro techniques from Holstein oocytes and Holstein or Brown Swiss semen and analyzed several cellular and molecular features. In the first experiment, purebred and crossbred embryos, obtained from abattoir oocytes, were analyzed for cleavage, development to morula/blastocyst stages, amino acid metabolism and gene expression of developmentally important genes. The results indicated significant differences in the percentage of compacted morulae, in the expression of three genes at the blastocyst stage (MNSOD, GP130 and FGF4) and in the utilization of serine, asparagine, methionine and tryptophan in day 6 embryos. In the second experiment, bovine oocytes were collected by ovum pick up from ten Holstein donors and fertilized with the semen of the respective Holstein sires or with Brown Swiss semen. The derived embryos were grown in vitro up to day 7, and were then transferred to synchronized recipients and recovered on day 12. We found that purebred/inbred embryos had lower blastocyst rate on days 7-8, were smaller on day 12 and had lower expression of the trophoblast gene PLAC8. Overall, these results indicate reduced and delayed development of purebred embryos compared with crossbred embryos. In conclusion, this study provides evidence that embryo genotype and high inbreeding can affect amino acid metabolism, gene expression, preimplantation development and therefore fertility in cattle. © 2011 Society for Reproduction and Fertility.


PubMed | Laboratorio Of Tecnologie Della Riproduzione
Type: Journal Article | Journal: Cellular reprogramming | Year: 2013

Embryologists working with livestock species were the pioneers in the field of reprogramming by somatic cell nuclear transfer (SCNT). Without the Dolly experiment, the field of cellular reprogramming would have been slow and induced plutipotent cells (iPSCs) would not have been conceived. The major drive of the work in mammalian cloning was the interest of the breeding industry to propagate superior genotypes. Soon it was realized that the properties of oocytes could be used also to clone endangered mammalian species or to reprogram the genomes of unrelated species through what is known as interspecies (i) SCNT, using easily available oocytes of livestock species. iSCNT for cloning animals works only for species that can interbreed, and experiments with taxonomically distant species have not been successful in obtaining live births or deriving embryonic stem cell (ESC) lines to be used for regenerative medicine. There are controversial reports in the literature, but in most cases these experiments have underlined some of the cellular and molecular mechanisms that are incomplete during cell nucleus reprogramming, including the failure to organize nucleoli, silence somatic cell genes, activate the embryonic genome, and resume mitochondrial replication and function, thus indicating nucleus-cytoplasmic incompatibility.


PubMed | Laboratorio Of Tecnologie Della Riproduzione
Type: | Journal: Reproduction in domestic animals = Zuchthygiene | Year: 2012

Somatic cell nuclear transfer (SCNT) was first developed in livestock for the purpose of accelerating the widespread use of superior genotypes. Although many problems still exist now after fifteen years of research owing to the limited understanding of genome reprogramming, SCNT has provided a powerful tool to make copies of selected individuals in different species, to study genome pluripotency and differentiation, opening new avenues of research in regenerative medicine and representing the main route for making transgenic livestock. Besides well-established methods to deliver transgenes, recent development in enzymatic engineering to edit the genome provides more precise and reproducible tools to target-specific genomic loci especially for producing knockout animals. The interest in generating transgenic livestock lies in the agricultural and biomedical areas and it is, in most cases, at the stage of research and development, with few exceptions that are making the way into practical applications.

Loading Laboratorio Of Tecnologie Della Riproduzione collaborators
Loading Laboratorio Of Tecnologie Della Riproduzione collaborators