New insights into the involvement of ubiquitin-proteasome system (UPS) in boar sperm capacitation

0482725 - BTÚ 2018 US eng C - Konferenční příspěvek (zahraniční konf.)
Zigo, Michal - Kerns, K. - Šutovsky, M. - Šutovský, P.
New insights into the involvement of ubiquitin-proteasome system (UPS) in boar sperm capacitation.
Programm and Abstract Book of the 50th Annual meeting SSR. Washington: Society for the Study of Reproduction, 2017.
[50th Annual meeting of the Society for the Study of Reproduction. Washington (US), 13.07.2017-16.07.2017]
Grant CEP: GA MŠMT(CZ) ED1.1.00/02.0109
Institucionální podpora: RVO:86652036
Klíčová slova: ubiquitin * proteasome * sperm capacitation
Obor OECD: Biochemical research methods

Ubiquitination is a stable, reversible posttranslational modification of target proteins by covalent ligation of the small chaperone protein ubiquitin. Ubiquitination targets proteins for degradation/recycling by the 26S proteasome. Participation of UPS in fertilization came to light only recently. This study represents expanded, ongoing research with emphasis on the changes in proteasome compartmentalization, subunit composition, and activity during in vitro capacitation of fresh boar spermatozoa.
Parallel and sequential treatments of ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions were used to isolate putative sperm proteasome-associated sperm proteins in a compartment-specific manner. Treated spermatozoa were screened with state of the art imaged based FC, to assess changes in acrosome integrity, and the quantity and electrophoretic migration patterns of candidate UPS substrate-proteins and proteasomal subunits. Resultant protein fractions were screened by WB for i) proteasomal subunits, and ii) candidate proteasomal substrate/interactor proteins that co-purify with sperm proteasomes.
Employing differential sperm compartment fractionation and protein isolation approaches and conditions, proteins were isolated from the ejaculated and in vitro capacitated spermatozoa under various UPS-modulating conditions. MS analysis revealed accumulation of proteins previously reported such as acrosin, cathepsin F, alpha subunit of ATP synthase. Image-based FC revealed differential protein localization patterns. PNA labeling revealed the capacitation-induced reorganization of outer acrosomal membrane, hindered in the presence of proteasomal inhibitors. Proteasomal inhibitors also prevented the detachment of acrosomal shrouds in non-fixed spermatozoa that is typically observed in a subpopulation of in vitro capacitating spermatozoa. These results further support the proposed role of UPS in the sperm capacitation.

Trvalý link: http://hdl.handle.net/11104/0278122