Dear Valter

FITC-PSA stain will differentiate the intact and acrosome reacted sperm while CTC could tell the capacitation like changes in sperm acrosome before it gets ruptured.

The CTC stain is much less well understood, even if it is related to calcium. There are dramatic calcium fluxes during capacitation, and a large influx triggering the acrosome reaction. These occur within seconds, so a calcium dye such as Fura2 is a better way to monitor calcium. You don't state which species you are studying, and there are differences for CTC staining. On the other hand, PSA (Pisum sativum agglutinin) is a lectin with specific carbohydrate binding properties. It recognizes glycoconjugates in the acrosomal matrix of some species including human. Antibodies such as SP10 can be used for similar staining. The sperm plasma and outer acrosomal membranes must be removed (eg, using cold ethanol) to visualize the PSA binding sites. When acrosome reaction has occurred, only the equatorial segment will be stained, as opposed to the entire acrosome in non-acrosome reacted sperm. You are right about membrane-disrupted sperm. They must be excluded from the assay either by detecting these with a dye such as Hoescht, or be eliminating dead sperm by purification prior to the assay.

Dear Mushtaq,

I appreciate your response, however, I would like a more in-depth discussion on the biological importance of these techniques.

Thank you very much anyway.

Dear Charles,

I'm working with bovine sperm. I have used both techniques (CTC and PSA) in the routine. I think the PSA recognizes when the acrosomal membrane is not intact, and this makes me believe that it can be caused by two things (acrosome reaction or damage). Because of this I had doubts about the biological significance of the two techniques. Thanks for your response, it was very enlightening.

Regards to the two researchers,

Valter

Hi Valter! I don't know if is too late for the discussion but I aready worked with CTC and FITC-PSA in my laboratory also with bovine spermatozoa. I agree with your interpretation about FITC-PSA: a spermatozoa positive for PSA means that it has a acrosome membrane alteration that could be reflect acrosome reaction (such as the end of capactation process) or damage (such as cryodamage). However, I believe that CTC is more a "complete" test, once you can see 3 different sperm populations: intact, capacitated and reacted, based on the distribution of CTC fluorescent in the sperm head. Also, many studies believe that this evaluation is the only one that we can really identify the capactation process. Based on that, if you need to choose one of them to perform, I guess that CTC is a better test for both capacitation and acrosome reaction. However, you only can access by epifluorescent microscope, and not by flow cytometry like FITC-PSA.

Letícia

Hi Letícia, thank you very much for your reply. My question was exactly that. I have worked with both evaluations, always with epifluorescence microscope. But in a paper we were working, we used the two assessments together, the CTC for the capacitation pattern and the FITC-PSA to assess the damage to the membranes. At the time, the discussion was whether the two evaluations fit into the same experiment. Thank you again for bringing this discussion to light.

Kind regards,

Valter.

Hi Valter Luiz Maciel Júnior

Interesting topic. As Charles mentioned, in my lab including me have some concerns about the CTC staining. Regarding this, since you performed both methodologies (Lectin and CTC staining) I would kindly ask you if you can tell about your experience, does the results were comparable?

Best regards

Arturo