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ZP PROTEINS CONJUGATED WITH MAGNETIC BEADS TO GENERATE 3D MODELS TO STUDY GAMETE INTERACTION.
Autores:
JULIETA GABRIELA HAMZE ARAUJO, ANALUCE CANHA,
BLANCA ALGARRA OÑATE
,
MARÍA CONCEPCIÓN OLIVARES SÁNCHEZ
,
RAQUEL ROMAR ANDRÉS
,
MARIA JIMENEZ MOVILLA
,
Grupos de investigación:
[GI/IMIB/C072/2011] Fisiología Reproductiva y Reproducción Asistida
[GI/IMIB/C012/2011] Integración morfofuncional de células y tejidos
[GI/IMIB/C010/2011] Control molecular de la proliferación y diferenciación
Comunicación:
Antecedentes:
The oocyte is encapsulated by a glycoprotein matrix called zona pellucida (ZP) formed by 3 or 4 proteins (ZP1, ZP2, ZP3 and ZP4) and it is known that this matrix is involved in gamete interaction, in particular, processing of ZP2 at N terminal position (LADEN) mediates recognition between gametes (Avella M, J Cell Biol. 205(6):801-9, 2014). The study of these molecular mechanisms is very limited mainly due to ethical problems, the difficulty of obtaining mature oocytes in many mammalian species, the high cost of genetically modified mice and the difficulty to transfer this knowledge to other species. Thus, we propose an in vitro model that mimics the oocyte shape with the ZP proteins attached. This model will allow us to study gamete binding in depth and will increase our knowledge of the oocyte-sperm interaction; it could also be industry-wide implemented as an evaluator of mammalian sperm quality.
Métodos:
To develop the model we combined magnetic beads (His Mag Sepharose™ Excel) conjugated with porcine ZP recombinant proteins (ZP2, ZP3 and ZP4). These proteins were expressed in mammalian cells (CHO) and once secreted identified by electrophoresis and Western Blot. Porcine ZP2, ZP3 and ZP4 were marked with a Flag, HA and V5 tag respectively, and with a histidine tag for easy identification and adhesion to the beads. To prove that the conjugation of the ZP proteins with the beads remained in time, we repeated the Western Blot one week after conjugation. Groups of 40-45 ZP proteins conjugated-beads and beads raised with growth CHO-cell medium (Control group) were coincubated for 2hr with boar spermatozoa (heterospermic dose) in TALP medium at a final concentration of 200.000 spermatozoa/ml. After coincubation period, the beads were washed in PBS and fixed.
Resultados:
Secreted proteins ZP2, ZP3 and ZP4 were identified by electrophoresis and western blot with anti-Flag, anti-HA and Anti-V5 antibodies, respectively. Porcine ZP2 showed a molecular weight of 100 kDa, ZP3 a molecular weight of 55 kDa and ZP4, 65 kDa. Adhesion of secreted proteins to the beads was confirmed by western blot and the ZP proteins-bead conjugation is long-lasting. Bound sperm to the beads was visualized by fluorescent microscopy and scanning electron microscopy (SEM).
Conclusiones:
In conclusion, a novel in vitro model combining magnetic beads with recombinant ZP proteins was developed in order to study the role of these proteins on sperm-oocyte interaction. Future studies could increase our knowledge of the oocyte-sperm interaction and could be also implemented as an in vitro selection and quality evaluation technique of potentially fertile mammalian spermatozoa.
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