Proteasome role during proximal centriole release at fertilization in mammals: Implications for fertilization failure in humans

VANESA Y. RAWE; PETER SUTOVSKY; SILVINA DIAZ; ROGER AGDELMASSIH; PATRICIO MORALES; HÉCTOR E. CHEMES

Baltimore USA

Congreso; 61 reunion de la American Society of Reproductive Medicine; 2006

American Society of Reproductive Medicine

Proteasome role during proximal centriole release at fertilization in mammals: Implications for fertilization failure in humans Vanesa Y. Rawe1, Peter Sutovsky2, Silvina Diaz4, Roger Agdelmassih3, Patricio Morales4, and Héctor E. Chemes5 1 Centro de Estudios en Ginecología y Reproducción (CEGyR), Buenos Aires, Argentina, 2 Departments of Animal Science, Obstetrics and Gynecology, University of Missouri–Columbia, USA, 3 Clínica e Centro de Pesquisa em Reprodução Humana Roger Abdelmassih, Sao Paulo, Brazil, 4 Department of Biomedicine, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile. 5Laboratory of Testicular Physiology and Pathology. Center for Research in Endocrinology. National Research Council (CONICET). Endocrinology Division. Buenos Aires Children´s Hospital, Argentina. Objectives: Cytoskeletal organization during fertilization is similar in bovine and humans in that they require a paternally derived centriole. After sperm penetration, the centrosome organizes a microtubule array that is responsible for bringing the parental genomes together at first mitosis. For this purpose, the paternal proximal centriole must be disassembled from the surrounding dense structures of the sperm-connecting piece. The proteasomal machinery has been shown to take part in important events during fertilization in different species. The purpose of our study was to explore their possible participation in the unmasking of the sperm centriole during sperm aster formation after mammalian sperm penetration. Materials and methods: Proteasome localization and enzyme activity were studied in normal and pathologic spermatozoa by immunocytochemistry and specific enzyme assays respectively. The roles of proteasome during early zygote development was studied in recently fertilized bovine and human oocytes under specific pharmacologic inhibition with MG132 or after oocyte/sperm proteasome block by anti proteasome antibodies intracellularly delivered using the Chariot reagent system. Pertinent findings in human zygotes discarded after ICSI failure are also presented and discussed. In all cases the material was analyzed under Confocal microscopy. Results: Evidence is presented on the existence of two different groups of proteasomes in bovine and human spermatozoa: in the acrosome and in a distinct area of the neck region overlapping the location of the sperm centriole. In head-neck defective human spermatozoa, enzymatic proteasomal activity (chymotrypsin-like, trypsin-like and peptidylglutamylpeptidase-like) was found decreased compared to normal samples. Strong nuclear and cytoplasmic proteasome localization was detected during normal fertilization in bovine and human oocytes. Absent or seriously impaired sperm aster formation and failed pronuclear apposition were consistently found after pharmacologic and immunologic block of proteasomes in human spermatozoa, bovine and human oocytes. Interestingly enough, we report similar findings in a number of human zygotes after ICSI fertilization failure. Conclusion: These studies allow us to better understand the events during the disassembly of the connecting piece, which leads to the release of a functional centriole that can act as a microtubule-organizing center of the zygote. This release seems to be due to selective proteolysis by proteasomes. Human spermatozoa with dysfunctional centriolar and/or pericentriolar structures have decreased proteasome enzyme activity and specific proteasome block disrupts sperm aster formation and pronuclear apposition in bovine and human zygotes. Taken together these findings suggest an important role of gamete proteasomes in the early events of zygote development.