Time filter

Source Type

Monti M.,Research Center for Regenerative Medicine | Redi C.A.,Research Center for Regenerative Medicine | Redi C.A.,University of Pavia
Journal of Visualized Experiments | Year: 2016

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis. Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest. © 2016 Journal of Visualized Experiments.

Monti M.,Research Center for Regenerative Medicine | Redi C.,Research Center for Regenerative Medicine | Redi C.,University of Pavia
Molecular Reproduction and Development | Year: 2013

The egg, a fantastic little laboratory of molecular biology, has played a crucial role in redefining modern biology by moving it from the description of living things to the synthesis of living things (synthetic biology). Over the centuries, many hypotheses have been advanced concerning the egg's role in reproduction-from the preformation theory until von Baer's discovery to the present, with the 2012 Nobel Prize for Physiology or Medicine celebrating the egg as a totipotent stem cell able to reprogram fully differentiated somatic nuclei. The molecular dissection of its cytoplasmic components makes the egg an ideal bioreactor for several biotechnological applications, including pharmacological and food production sciences. In addition to its ubiquitous contribution to the worldwide diet, the egg, a powerful symbol, pervades philosophy, art, religion, and idiomatic expressions. © 2013 Wiley Periodicals, Inc.

Monti M.,Research Center for Regenerative Medicine | Zanoni M.,University of Pavia | Calligaro A.,University of Pavia | Ko M.S.H.,U.S. National Institute on Aging | And 3 more authors.
Biology of Reproduction | Year: 2013

The antral compartment in the ovary consists of two populations of oocytes that differ by their ability to resume meiosis and to develop to the blastocyst stage. For reasons still not entirely clear, antral oocytes termed surrounded nucleolus (SN; 70% of the population of antral oocytes) develop to the blastocyst stage, whereas those called not-surrounded nucleolus (NSN) arrest at two cells. We profiled transcriptomic, proteomic, and morphological characteristics of antral oocytes and observed that NSN oocyte arrest is associated with lack of cytoplasmic lattices coincident with reduced expression of MATER and ribosomal proteins. Cytoplasmic lattices have been shown to store maternally derived mRNA and ribosomes in mammalian oocytes and embryos, and MATER has been shown to be required for cytoplasmic lattice formation. Thus, we isolated antral oocytes from a Matertm/tm mouse and we observed that 84% of oocytes are of the NSN type. Our results provide the first molecular evidence to account for inability of NSN-derived embryos to progress beyond the two-cell stage; these results may be relevant to naturally occurring preimplantation embryo demise in mammals. © 2013 by the Society for the Study of Reproduction, Inc.

Huang H.,Graduate School | Liu Q.,Research Center for Regenerative Medicine | Liu Q.,Medical and Scientific Research Center | Liu L.,Research Center for Regenerative Medicine | And 3 more authors.
Experimental and Therapeutic Medicine | Year: 2015

In autologous chondrocyte implantation (ACI) to restore defective cartilage, limited cell numbers and dedifferentiation of chondrocytes are the major difficulties. An alternative is the use of growth factors, but their high cost and potential for tumorigenesis are major obstacles. To ensure successful ACI therapy, it is important to find an effective substitute pro-chondrogenic agent. Epigallocatechin-3-gallate (EGCG), one of the green tea catechins, has been widely investigated in studies of interleukin-1β-induced chondrocytes. In the present study, the effects of EGCG on rabbit articular chondrocytes were investigated through the examination of cell proliferation, morphology, glycosaminoglycan synthesis and cartilage-specific gene expression. The results showed that EGCG could effectively promote chondrocyte growth and enhance the secretion and synthesis of the cartilage extracellular matrix by upregulating expression levels of aggrecan, collagen II and Sox9 genes. Expression of the collagen I gene was downregulated, which showed that EGCG effectively inhibited the dedifferentiation of chondrocytes. Hypertrophy, which may lead to chondrocyte ossification, was also unde tectable in the EGCG groups. In conclusion, the recommended dose of EGCG was found to be in the range of 5 to 20 µM, with the most marked response observed with 10 µM. The present study may provide a basis for the development of a novel agent as a substitute for growth factors in the treatment of articular cartilage defects. © 2015, Spandidos Publications. All rights reserved.

Discover hidden collaborations