Comparative proteomic analysis of spermatozoa isolated by swim-up or density gradient centrifugation

Stefania Luppi, Monica Martinelli, Elisa Giacomini, Elena Giolo, Gabriella Zito, Rodolfo C. Garcia, Giuseppe Ricci

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Background: Reports about the morphologic and functional characteristics of spermatozoa prepared by density gradient centrifugation (DC) or swim-up (SU) have produced discordant results. We have performed a proteomic comparison of cells prepared by DC and SU providing a molecular insight into the differences between these two methods of sperm cell isolation. Methods: Protein maps were obtained by 2-dimensional (2-D) separations consisting of isoelectrofocusing (IEF) from pI 3 to 11 followed by SDS-polyacrylamide gel electrophoresis. 2-D gels were stained with Sypro Ruby. Map images of DC and SU spermatozoa were compared using dedicated software. Intensities of a given spot were considered different between DC and SU when their group mean differed by >1.5-fold (p <0.05, Anova). Results: No differences were observed for 853 spots, indicating a 98.7% similarity between DC and SU. Five spots were DC > SU and 1 was SU > DC. Proteins present in 3 of the differential spots could be identified. One DC > SU spot contained lactate dehydrogenase C and gamma-glutamylhydrolase, a second DC > SU spot contained fumarate hydratase and glyceraldehyde-3-phosphate dehydrogenase-2, and a SU > DC spot contained pyruvate kinase M1/M2. Conclusions: The differences in protein levels found on comparison of DC with SU spermatozoa indicate possible dissimilarities in their glycolytic metabolism and DNA methylation and suggest that DC cells may have a better capacitation potential.

Original languageEnglish
Article number36
JournalReproductive Biology and Endocrinology
Issue number1
Publication statusPublished - Apr 19 2015


  • Density gradient centrifugation
  • Protein 2-dimensional maps
  • Sperm cells
  • Swim-up

ASJC Scopus subject areas

  • Developmental Biology
  • Endocrinology
  • Reproductive Medicine


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