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Responsabile: prof. Giuseppe Ricci
Studi ultrastrutturali degli spermatozoi possono fornire informazioni rilevanti circa le patologie e possono essere d’aiuto per la diagnosi e il trattamento dell’infertilità. Lo studio delle proprietà meccaniche (quali elasticità e viscoelasticità) delle membrane può fornire informazioni importanti circa l’influenza della stimolazione ovarica degli oociti maturi e la potenziale fertilità. L’investigazione ultrastrutturale dei gameti crioconservati può dare informazioni determinanti per la scelta del miglior processo di crioconservazione e il modo migliore di predire il risultato finale della fertilizzazione in vitro.
Background and Significance
Recently, particular attention has been paid to cryopreservation procedures of human gametes. Despite the rapid progress, these procedures have major limitations. Currently, there are different methods of cryopreservation: vitrification is the most recent. Since this technique is the latest, there is no adequate knowledge on the changes induced during freezing and thawing. It is essential to clarify which technique has less impact, since the structural and ultrastructural changes may strongly affect cellular vitality, function and especially the outcome of in vitro fertilization procedures. For this purpose, various methods of microscopy such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM), nanoscopy such as atomic force microscopy (AFM) and scanning near-field microscopy (SNOM) can provide a detailedultrastructural characterization of cells with very high resolution (nanometer level). A better understanding of the functional aspects as the study of micromechanical properties of cells would be of fundamental importance. The characterization of the mechanical properties (such as elasticity and viscoelasticity) of oocytes could play an important role in assessing quality, vitality and cellular defects of oocytes subjected to cryopreservation. Techniques such as microfluidic or indentation could be used to investigate the mechanical properties of the gamete membranes.
Specific aims
Aim 1: The first aim of the project is to evaluate if the atomic force microscopy (AFM) and the scanning near-field microscopy (SNOM) can provide a detailed ultrastructural characterization of sperm with very high resolution (nanometer level).
Aim 2: The second aim of the project is to evaluate if sperm and oocyte vitrification produces more ultrastructural changes than the classic slow freezing method, by using transmission electron microscopy (TEM) and scanning electron microscopy (SEM).
Aim 3: The third aim of the project is to evaluate if techniques such as microfluidic or indentation could be exploited to create micro-force sensors to be used for the selection of oocytes based on the mechanical properties of their membrane.
Hypothesis
Ultrastructural investigations of sperm may provide fundamental information about sperm pathologies that may be helpful in diagnosing and treating male infertility. Investigation of the mechanical properties (such as elasticity and viscoelasticity) of membranes may provide valuable information about the influence of ovarian stimulation on the oocyte maturity and fertilization potential. Ultrastructural investigations of cryopreserved gametes may provide valuable information to choose the best cryopreservation method and to better predict the in vitro fertilization outcome.
Preliminary data
Using specific antibodies-based immunolabeling and diaminobenzidine staining (DAB), important results have been already achieved, such as the identification and localization of a specific intracytoplasmatic protein next to certain cell surface details. Tubulines have been labeled, but the technique may virtually identify every protein of the cell. A method to observe cells in physiological conditions with SNOM, in liquid environment, have been already developed and will be soon implemented.
Trevisan E, Zweyer M., et al. Neuroimage. Novel approaches for scanning near-field optical microscopy imaging of oligodendrocytes in culture. 2010 49:517-24.
Ricci G., Zweyer M. et al. A comparative analysis of human spermatozoa by scanning near field optical microscope (SNOM), with optical, atomic force and electron miscoscopy? 63° National Congress of Anatomy and Histology Society, Torino 10-12 sett 2009.
Materials and Methods
Ultrastructural and biomechanical studies will be carried out on supernumerary oocytes and sperm from couples undergoing assisted reproductive treatment. Animal models will be also used to develop method for ultrastructural and biomechanical investigation of gametes. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM), atomic force microscopy (AFM) and scanning near-field microscopy (SNOM) will be used to evaluate sperm and oocyte morphology . Stress-Relaxation Indentation Test, microsystem and microfluidic devices will be used for mechanical characterization of the membranes. Standard slow freezing and vitrification procedures will be used for sperm and oocyte cryo-oreservation. Standard protocol for in vitro fertilization will be applied.
Impact and Translational Implications
The causes of infertility are still unknown in many couples. Ultrastructural sperm and/or oocyte defects may prevent fertilization. The low success rate of gamete cryopreservation procedures requires significant improvement of methods. The availability of high tech methos to investigate human gametes may be helpful in diagnosing and treating couple infertility. The identification of an efficient method to cryopreserve sperm and oocyte may improve in vitro fertilization pregnancy rate.
