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Calpain inhibition prevents flotillin re-ordering and Src family activation during capacitation

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Abstract

Prior to fertilization, mammalian sperm undergo several molecular, biochemical and physiological changes in a process termed capacitation. However, the mechanisms explaining the involvement of cytoskeletal remodeling and membrane re-ordering in each process prior to fertilization remain poorly understood. We found that the migration of both flotillin microdomains and Src family kinases towards the apical ridge of guinea pig sperm occurs under capacitating conditions. This re-ordering is associated with spectrin cleavage by calpain. Moreover, Src, Fyn, Lyn and Hck interact with flotillin-1; this interaction increases in a capacitation-dependent manner and the increased autophosphorylation of these kinases is linked to flotillin-1 association. The aforementioned results are prevented by the inhibition of calpain by calpeptin. Thus, spectrin cytoskeleton cleavage during capacitation seems to precede the reorganization of flotillin microdomains and Src family kinases towards the apical ridge of the sperm head in order to initiate the signaling cascade required for proper capacitation and further acrosome reaction. The significance of the Src family kinase reorganization for capacitation is demonstrated by the inhibition of calpain during capacitation also preventing the Src-family-kinase-dependent phosphorylation of FAK at Tyr576/577. Our work further highlights the scaffolding properties of flotillin microdomains and reveals the importance of their large-scale segregation during capacitation.

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Acknowledgements

We thank the staff of Unidad de Microscopía Confocal (Dpto. Biología Celular, CINVESTAV-IPN) for providing confocal facilities. We also thank Dr Liora Shoshani Z. and Dr. Dominique Mornet for their generous antibody donations. This work was supported by a Consejo Nacional de Ciencia y Tecnología (CONACYT) grant (79921) to Enrique O. Hernández-González, and with doctoral scholarships to Deneb Maldonado-García (322154), Tania Reyes-Miguel (262875) and Monica L. Salgado-Lucio (263011).

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Correspondence to Enrique O. Hernández-González.

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The authors declare that no conflict of interest exists that could be perceived as prejudicing the impartiality of this scientific work.

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Supplementary Figure 1

Chlortetracycline (CTC) staining patterns after capacitation of guinea pig sperm. Sperm were incubated under capacitation conditions and, after 90 min, were stained with CTC. Fluorescence images corresponding to three different CTC staining patterns of the guinea pig sperm. The patterns scored were: F corresponds to non-capacitated sperm, B corresponds to capacitated sperm, AR corresponds to acrosome-reacted sperm. Means ± SEM, n = 3 independent experiments. (JPEG 33.4 kb)

Supplementary Figure 2

Immunoprecipitation of Rac-1 as a negative control. Co-immunoprecipitation assays with Rac-1 were performed with total sperm extracts (500 μg) from non-capacitated and 90-min capacitated sperm. The precipitated proteins were subjected to SDS–PAGE and Western blot analysis probed for Rac-1 as a positive control and probed with anti-flotillin-1 (Flot-1), anti-flotillin-2 (Flot-2), anti-spectrin and anti-SFKs antibodies to determine its associations. Western blot images are representative of three independent experiments. (JPEG 34.1 kb)

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Maldonado-García, D., Salgado-Lucio, M.L., Roa-Espitia, A.L. et al. Calpain inhibition prevents flotillin re-ordering and Src family activation during capacitation. Cell Tissue Res 369, 395–412 (2017). https://doi.org/10.1007/s00441-017-2591-2

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Keywords

  • Flotillin microdomain aggregation
  • Spectrin and sperm membrane domains
  • Src family kinases
  • Acrosome reaction
  • Role of spectrin in capacitation