Subprojeto 28 – Chromosome evaluation, embryo and gamet preimplantation caryotyping and the study of spermatozoid chemotaxis
Participantes: Ricardo Pimenta Bertolla, Edson Guimarães Lo Turco, Departament of Human Reproduction, Research Institute in Urology, UNIFESP and Carlos Lenz Cesar CEPOF IFGW-UNICAMP.
The international scientific community is searching for ways to evaluate and cure cellular alterations using the biophotonic techniques. The main objective of this sub-project is to determine and standardize the results of chromosome individual analysis using photonic techniques and to develop a chromosome diagnostic method for embryos and gametes. This method would be based in Infrared and Raman microspectroscopy of the cells fixed in a slide in the metaphase phase of the cellular cycle, in order to allow the posterior standardization of the spectral profile of the euploid cells in the interphase of the cell cycle of non-fixed material. The development of such method would change completely the present cytogenetic evaluation. With the preliminary results it would be possible to create a system to be used in common caryotyping that are present limited only to cells in the metaphase cycle and requires an evaluator trained specifically to subjectively evaluate the chromosomes, a time consuming procedure. The technique development would allow an objective evaluation and the automatization to obtain high throughput. Later, then, the process could be enlarged and improved to incorporate cells and blastomers in interphase, that, nowadays, can only be visualized after DNA probes conjugated to fluorescent markers, an invasive process that makes the cell not viable. The photonic techniques would have the advantage of being less invasive and to allow the evaluation of the cell in its real condition. For this sub-project to be possible, we will use the Raman and NIR (near infra-red) spectroscopy and microscopy, based on the preexisting knowledge and on the new knowledge we shall develop by learning with the use of the biophotonic techniques. The use of CARS shall amplify the Raman signal without the necessity of SERS. Moreover, the TERS would allow us to reach nm resolution spectroscopy.The second Project under this sub-project is the use of Optical Tweezers to evaluate the chemotaxis and the chemotatic proteins of human spermatozoids. Today, in our laboratory we only perform proteomic techniques to discover which proteins can be used for the diagnostic and therapeutic intervention for the treatment of conjugal infertility. The discovery of proteins that participate at various events, from the spermatozoid pathway to the oocyte and to the fertilization would allow per se the development of new products to decrease the necessity of costly and or invasive treatments. In this respect, our second Project is to evaluate how different proteins or lipids can influence upon the spermatozoid chemotaxis. In this case, two optical tweezers would be used in a system with two perpendicular axis and the resulting vectors of forces of spermatozoids submitted to different stimuli would measured. Integration with CARS and multiphoton FLIM will allow the simultaneous visualization and manipulation of the proteins and lipids. By the end of the first Project we expect to develop a more efficient and less invasive technology to perform genetic evaluation of embryos and cells, eventually allowing the scanning of large numbers of cells and tissue in a short time. The second project will allow us to determine which peptides and lipids shall be used to select spermatozoids or for future therapeutic intervention.