Our objective is to produce in vitro high quality mammalian oocytes for use in biotechnology, in assisted reproduction techniques, in preservation of genetic diversity, in production of high value added products through transgenesis, in farm animal breeding and in toxicology-teratology. The mammalian oocyte is the Nature's foundation for the success of these new technologies. Ovaries contain a wide pool of oocytes but a considerable wastage occurs: most (>99%) of them are lost during the physiological female cycle and very few complete the capacitation process that leads to further maturation, fertilisation and embryo development. This is the most limiting factor to a wider use of technologies involving oocytes. We will design a new culture system that will allow us to rescue these oocytes and to produce oocytes of uniform quality to be used by the new reproduction-based biotechnologies.
The main objective of the project is to produce in vitro high quality mammalian oocytes for use in biotechnology, in assisted reproduction, in preservation of genetic diversity, in production of high value added products through trans-genesis, in farm animal breeding and in toxicology-teratology.
We intend to rescue the oocyte capital by developping a culture system approaching in vivo situation. This new system will allow us to mimic in vitro the block of meiotic resumption provided in vivo by follicular environment and to address to them proper stimulation sequence to induce capacitation. Oocytes will then be allowed to resume meiosis and another stimulation sequence will be addressed. This will produce oocytes of uniform quality to be used by the new reproduction-based biotechnologies.
Description of the work
Four workpackages have been defined.
WP1. Reversible meiosis inhibition:
We will allow in vitro capacitation by controlled inhibition of meiotic resumptio, which is regulated by specific kinases. New kinase inhibitors will be selected to reversibly block meiosis.
WP2. Identification of markers of developmental competence :
Oocytes will be collected in vivo at various stages of the capacitation-maturation process. Their morphological (ultrastructure, kinetics of developement) as well as biochemical (mRNAs, carbohydrates and proteins) characteristics will be used as quality markers.
WP3. Setting up the 2-step culture system :
Stimulation of capacitation and of maturation will be performed sequentially during the block of meiosis (first step) and during the resumption of meiosis (second step). We will finally propose a completely defined two-step culture system allowing the obtention of a homogenous group of fully competent oocytes from heterogeneous populations.
WP4. Application of the 2-step culture system :
The culture method will be applied to various developmentally incompetent categories of oocytes to test its efficiency. The value of these embryos in toxicity testing will be evaluated. Tests will be performed to foresee any necessary modifications for an application to other species such as sheep, pig, and deer.
Selection of a reversible inhibitor of oocyte meiotic resumption
Identification of markers of oocyte capacitation quality
Identification of markers of oocyte maturation quality
Identification of the best stimulating protocol for oocyte capacitation
Identification of the best stimulating protocol for oocyte maturation
Transmission of technology for applications
Funding SchemeCSC - Cost-sharing contracts
277 21 Libechov