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Tytuł pozycji:

Sperm-Binding Assay Using an In Vitro 3D Model of the Mammalian Cumulus-Oocyte Complex.

Tytuł:
Sperm-Binding Assay Using an In Vitro 3D Model of the Mammalian Cumulus-Oocyte Complex.
Autorzy:
Hamze JG; Department of Cell Biology and Histology, School of Medicine, University of Murcia, Campus Mare Nostrum and IMIB-Arrixaca, Murcia, Spain.
Jiménez-Movilla M; Department of Cell Biology and Histology, School of Medicine, University of Murcia, Campus Mare Nostrum and IMIB-Arrixaca, Murcia, Spain.
Romar R; Department of Physiology, Faculty of Veterinary Medicine, University of Murcia, Campus Mare Nostrum and IMIB-Arrixaca, Murcia, Spain.
Źródło:
Current protocols in toxicology [Curr Protoc Toxicol] 2020 Dec; Vol. 86 (1), pp. e100.
Typ publikacji:
Journal Article; Research Support, Non-U.S. Gov't
Język:
English
MeSH Terms:
In Vitro Techniques*
Sperm-Ovum Interactions*
Animals ; Male ; Oocytes ; Spermatozoa ; Swine
References:
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Contributed Indexing:
Keywords: 3D model; gamete interaction; magnetic beads; sperm
Entry Date(s):
Date Created: 20201217 Date Completed: 20210901 Latest Revision: 20210901
Update Code:
20240105
DOI:
10.1002/cptx.100
PMID:
33331693
Czasopismo naukowe
We have recently described a new model to study gamete interaction in mammalian species. The model recreates the spherical surface of the oocyte by using magnetic Sepharose beads coated with a layer of a recombinant protein involved in gamete interaction (such as ZP2, or the IZUMO1 receptor JUNO) and an external layer of cumulus oophorus cells, thus mimicking, to some extent, a native cumulus-oocyte complex. Once generated, this 3D model can be used in a sperm-binding assay to obtain valuable information about the molecular basis of gamete interaction, since different recombinant proteins can be used to coat the bead surface, thus generating a variety of models to be used for several species. Furthermore, thanks to the ability of the model to decoy sperm, the physiological status of the bound sperm can be studied, making this a powerful tool to select sperm with high fertilizing capacity, to unmask subfertile animals in livestock breeding centers, or for toxicological studies. Here, we describe how to generate and use this model for sperm-binding assays, using porcine sperm as an example, and ZP2, a protein from zona pellucida, as the recombinant protein of interest. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of the in vitro 3D model Alternate Protocol 1: Binding cumulus oophorus cells to the model Basic Protocol 2: Quality control of the model by SDS-PAGE electrophoresis and western blot Support Protocol 1: Immunochemistry to confirm proper protein distribution on surface of beads Support Protocol 2: Elution of recombinant conjugated proteins Basic Protocol 3: Sperm-binding assay Alternate Protocol 2: Sperm preparation by the swim-up method.
(© 2020 Wiley Periodicals LLC.)

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