MACS -(Magnetic Activated cell sorting ) helps separate apoptotic sperms as externlized PS bind with annexin beads and beads are retained in the magnetic field. What if the sperms having fragmented DNA and membrane is intact - PS is not externalized and MACS helpless.
Thanks in advance for the replies.
When spermatozoa are capacitated, PS are flipped. Spz then obviously become annexin V positive although they certainly are not apoptotic ! Annexin V therefore is not a good tool for identifying apoptosis in spz.
Hello Hetal.
Cell dying or cell death assay using PS and annexin V is inappropriate for spermatozoa assay. Sperm cells are unique in motility and a complex sperm head membrane structures enclosed by the nuclear membranes, inner acrosomal membranes, hydrolytic enzymes, outer acrosomal membranes and the plasma membranes in a manner of layer by layer. As Serge mentioned above, acrsome-reacted sperm cell are still exposing the inner acrosomal membranes. It is also necessary for the isolation of sperm cells to kill them all or motionless entirely in applying MACS. At present, motile spermatozoa are considered as healthy sperm cells in biological and clinical terms. If sperm DNA fragmentation is in question, then it is reasonable to think that DNA fragmentation may occurs during whole process of spermatogenesis. Therefore, all the sperm cells may have DNA fragmentation at a similar certain extent all together regardless of motile or immotile.
Regards.
Thank you Serge and Sang for your very useful tips on Annexin and PS and DFI. More discussion from other researchgate forum welcome.
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