I-8: Sperm Chemotaxis towards Progesterone,A Guiding Mechanism That May Be Used to Select The Best Spermatozoa for Assisted Reproduction

Background: Spermatozoa are able to sense an attractant molecule gradient and as a consequence, orient their movement towards the source of the attractant. This mechanism is known as sperm chemotaxis. In recent years, our laboratory contributed to the knowledge of several features of mammalian sperm chemotaxis. These include the size and physiological state of the chemotactic sperm population, the biological sources of attractants, the identity of a physiological attractant candidate, the species specificity of the phenomenon, some signal transduction pathways by which chemotaxis is induced, the chemotactic pattern of movement, and the verification of sperm chemotaxis under vivo conditions. The aim of the project was to characterize the sperm chemotactic response and its potential applications for assisted reproduction. Materials and Methods: In order to study mammalian sperm chemotaxis we first developed a method to objectively assess sperm directionality and additional kinetic parameters. This method consists on a device (a chemotaxis chamber) and a videomicroscopy and image analysis system. The chamber has two wells (W1 and W2) that are connected by a bridge over which a capillary space filled with culture medium is formed by adding a coverslip. The cells are loaded in W1, while the attractant in W2, which immediately diffused from W1 to W2 forming a unidirectional long-lasting gradient across the bridge. Cells freely swimming over the bridge are digitally recorded and the tracks evaluated by computer image analysis which includes a software developed in our lab. Results: We first characterized the sperm chemotactic response under in vitro conditions. We described for the first time the chemotactic response in animal sperm from mouse, rabbit and bovine. In addition, spermatozoa respond to several biological sources of chemoattractants like follicular fluid, oviductal fluid and conditioned medium of the egg-cumulus complex. To elicit a chemotactic response spermatozoa must have accomplished capacitation, whereas only a small subpopulation of spermatozoa (~10% of the cells) are chemotactic at any given time. Follicular fluid that has been obtained from one species may attract spermatozoa of other species, suggesting that chemotaxis may not be a species-specific phenomenon. Several sperm attractants have been reported, however, we observed that progesterone seems to be of physiological importance. Thus, a small quantity of progesterone, which is secreted by the cells surrounding the egg, is able to attract spermatozoa. Moreover, the chemotactic response was suppressed when either egg conditioned medium was depleted of progesterone or the sperm progesterone receptor was blocked. We next investigated the molecular mechanisms that lead to the sperm chemotactic response towards progesterone.Thus, the transmembrane adenylyl cyclase-cAMP-protein kinase A pathway and soluble guanylate cyclasecGMP- protein kinase G pathway, calcium mobilization and protein tyrosine phosphorylation appear to be involved. Chemotactic spermatozoa swimming towards an attractant source are indistinguishable from non chemotactic sperm, but we observed that they showed a unique pattern of movement when they returned towards the source of a chemotactic concentration of progesterone. Next we observed under in vivo conditions that the chemotactic mechanism helps to transport sperm to the fertilization site, thus validating the in vitro observations. As whole, the results obtained along years lead us to design a new sperm selection assay based on sperm chemotaxis towards progesterone. Conclusion: Human spermatozoa are able of chemotactic response to very low levels of progesterone. This steroid has been considered a physiological attractant since by the time of ovulation it is secreted by the cells surrounding the egg. Only capacitated spermatozoa (those ready to fertilize the egg) may show chemotactic behavior. Therefore, a sperm population enriched with capacitated spermatozoa by means of chemotaxis may be considered a physiological selection procedure that may be useful for assisted reproduction technologies.