Comparative proteomic analysis of spermatozoa isolated by swim-up or density gradient centrifugation.
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.
Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Via dell'Istria 65/1, 34137, Trieste, Italy. stefania.luppi@burlo.trieste.it.
Stefania
S
Luppi
Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Via dell'Istria 65/1, 34137, Trieste, Italy. monica.martinelli@burlo.trieste.it.
Monica
M
Martinelli
Department of Medicine, Surgery and Health Sciences, University of Trieste, 34149, Trieste, Italy. elisa.giacomini@inwind.it.
Elisa
E
Giacomini
Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Via dell'Istria 65/1, 34137, Trieste, Italy. elena.giolo@burlo.trieste.it.
Elena
E
Giolo
Department of Medicine, Surgery and Health Sciences, University of Trieste, 34149, Trieste, Italy. gabriellazito3@virgilio.it.
Gabriella
G
Zito
International Centre for Genetic Engineering and Biotechnology (I.C.G.E.B.), Area Science Park, 34149, Trieste, Italy. garciazucconi@gmail.com.
Rodolfo C
RC
Garcia
Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Via dell'Istria 65/1, 34137, Trieste, Italy. ricci@burlo.trieste.it.|Department of Medicine, Surgery and Health Sciences, University of Trieste, 34149, Trieste, Italy. ricci@burlo.trieste.it.
Giuseppe
G
Ricci
D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism / Q000502:physiology*
D011506:Proteins