Esfingolípidos involucrados en la inducción fisiológica de la exocitosis acrosomal del espermatozoide humano
During fertilization, the spermatozoon penetrates the zona pellucida to reach the oolema. Only sperm that have completed the acrosome reaction (AR) can accomplish this task. The AR is a calcium-regulated exocytosis where the membrane of the acrosome fuses to the plasma membrane. Exocytosis is a comp...
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| Autores principales: | , , , , , |
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| Publicado: |
2019
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| Materias: | |
| Acceso en línea: | https://bdigital.uncu.edu.ar/fichas.php?idobjeto=14466 |
| Sumario: | During fertilization, the spermatozoon penetrates the zona pellucida to reach the oolema. Only sperm that have completed the acrosome reaction (AR) can accomplish this task. The AR is a calcium-regulated exocytosis where the membrane of the acrosome fuses to the plasma membrane. Exocytosis is a complicated and finely tuned process involving a large number of components. The general goal of this proposal is to identify and characterize sphingolipids´ role and elucidate the sequence of the reactions they take part in during the physiological induction of the AR. We will focus in the signaling cascade triggered by sphingosine 1-phosphate (S1P) and ceramide, described for the first time in our laboratory. Human zona pellucida (ZP) matrix, a delicate network of thin interconnected filaments, is primarily composed of four glycoproteins, namely, ZP1, ZP2, ZP3, and ZP4. Recombinant human ZP3 and ZP4 lead to dose-dependent increase in acrosome reaction. Even that, the molecular mechanisms involved in ZP-induced AR still need to be unveiled. Further, the sphingolipids involved in ZP-triggered exocytosis are largely unknown as well as if a cross talk between ZP and S1P or ceramide pathways exist. To solve these issues we propose: I) to identify the presence and activity of sphingolipid metabolism enzymes in human sperm; II) to determine which S1PRs are present and involved in the AR, ERK participation in S1P signaling, and how the S1P synthesized intracellularly reaches the extracellular media; III) to elucidate the signaling network triggered by endogenous and exogenous ceramides; IV) to evaluate if there is a connection between the signaling pathway induced by human recombinant ZP and that induced by S1P or ceramides by using exocytosis assays. To assess these aims we will perform exocytosis assays in sperm (evaluated by flow cytometry or epifluorescence microscopy) in combination with other biochemical approaches namely Western blot, immunofluorescence, confocal microscopy, thin layer chromatography, calcium measurements in life cells, in real time with high spatial and temporal resolution, and spectrofluorometry among others. We hope this project will allow significant advances in our understanding of sperm AR at the physiological level unveiling a functional cooperation between signaling pathways triggered by ZP and sphingolipids. |
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