Conventional semen analysis - the initial method to evaluate male fertile potential - presents low correlation with fertility, and is insufficient as a diagnostic method of male infertility. It is important to have a better understanding of the molecular mechanisms that lead to changes in the sperm and, consequently to fertility disorders. Among the molecules that are related to sperm function are seminal plasma proteins, which bind to sperm and act on their survivability, in the regulation of motility and capacitation, in immune response in the female reproductive tract and on fusion of sperm with the oocyte. Some previous studies of our research group allowed us to understand the proteomic environment of seminal plasma in male infertility. Among the proteins found, ELSPBP1 protein (epididymal sperm-binding protein 1) is overexpressed in sperm samples with high DNA fragmentation. ELSPBP1 is secreted to the epididymal lumen by specific microvesicles (epididymossomes), and its function is regulated by matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). We hypothesize that ELSPBP1 present in seminal plasma is transferred to the membrane of damaged sperm, such as sperm with fragmented DNA. Moreover, we hypothesize that this protein mediates the binding of these sperm with collagen type I, leading to the formation of sperm-collagen clusters, removing them from the healthy population of sperm after ejaculation. We aim, therefore, to determine the use of ELSPBP1 protein as a biomarker of DNA fragmentation in human sperm under different conditions of infertility. The specific objectives of the four sub-studies involved in this project are: (i) determining the expression of ELSPBP1 protein in the ejaculated population of sperm and in viable sperm selected through semen processing; (ii) determining the expression of ELSPBP1 protein in seminal plasma in different causes of male infertility; and (iii) to verify the expression of seven proteins involved in the ELSPBP1 pathway (ELSPBP1, TIMP1, 2, 3 e 4 e MMP1, 2, e 7), to determine its effect on semen quality in different causes of male infertility; (iv) to verify if the ELSPBP1 protein is capable to remove sperm with low DNA quality from the sample. To this, we will use immunocytochemistry, Western blotting of sperm and seminal plasma proteins and protein multiplex assays (MagPix®), to measure ELSPBP1 protein levels, as well as levels of proteins in its pathway, under different biological conditions, and Magnetic-activated cell sorting (MACS) to evaluate if the ELSPBP1 protein can separate sperm with low DNA quality from the sample.
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