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Analysis of different cell death processes of prepubertal rat oocytes in vitro

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Abstract

The processes of cell death were studied in vitro in populations of oocytes isolated from prepubertal rats. In order to identify apoptosis, the externalized phosphatidylserine was recognized with Annexin-V coupled to FITC and the fragmentation of DNA was demonstrated by means of electrophoresis. Oocytes were tested for autophagy by means of the incorporation of monodansylcadaverine and monitoring Lc3-I/Lc3-II by western blot. The expression of mRNA marker genes of autophagy and of apoptosis was studied by means of RT–PCR in pure populations of oocytes. Some oocytes expressed at least one of the following markers: caspase-3, lamp1 and Lc3. Some oocytes were positive to Annexin-V or to monodansylcadaverine. However, most of them were simultaneously positive to both markers. The relative frequency of oocytes simultaneously positive to markers of apoptosis and autophagy did not change in the different ages studied. The transformation of Lc3-I in Lc3-II was present in all populations of oocytes studied. The mRNAs for caspase-3, lamp1 and Lc3 were present in all populations of oocytes analyzed. Our results demonstrate that oocytes of rats from new born to prepubertal age are eliminated by means of three different cell death processes: apoptosis, autophagy and a mixed event in which both routes to cell death participate in the same cell.

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Acknowledgments

We would like to thank Dr. Juan Manuel Hernández Castellanos (Departamento de Fisiología, Facultad de Medicina, UNAM) for glass micropipette manufacturing. We thank the technical assistance of Carmen Mondragón, Ma. José Gómora, and Silvia Reyes. This work was supported by PAPIIT-203308; CONACYT and PAPIME-PE204609.

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Authors and Affiliations

  1. Laboratorio de Microscopía Electrónica, Facultad de Ciencias, Departamento de Biología Celular, Universidad Nacional Autónoma de México, Mexico, DF, CP 04510, Mexico

    M. L. Escobar, O. M. Echeverría & G. H. Vázquez-Nin

  2. Laboratorio de Biología Molecular del Cáncer, Facultad de Estudios Superiores Zaragoza, UMIEZ, Universidad Nacional Autónoma de México, Mexico, DF, CP 04510, Mexico

    L. Sánchez-Sánchez

  3. Facultad de Medicina, Departamento de Embriología, Universidad Nacional Autónoma de México, Mexico, DF, CP 04510, Mexico

    C. Méndez & E. Pedernera

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  1. M. L. Escobar
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  2. O. M. Echeverría
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  3. L. Sánchez-Sánchez
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  6. G. H. Vázquez-Nin
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Correspondence to G. H. Vázquez-Nin.

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Escobar, M.L., Echeverría, O.M., Sánchez-Sánchez, L. et al. Analysis of different cell death processes of prepubertal rat oocytes in vitro. Apoptosis 15, 511–526 (2010). https://doi.org/10.1007/s10495-009-0448-1

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