Figure 4: Effect of FK506 hyperactivation of sperm motility and acrosome reaction.

A) Sperm were incubated (3 hrs) under non capacitating conditions or capacitating conditions in the absence or presence of 20 nM FK506 followed by motility analysis by CASA using parameter settings to detect hyperactivation as described in the materials and methods. FK506 prevents increase of sperm average path velocity (VAP), curvilinear velocity (VCL) (beyond certain threshold values as indicated) and lateral head amplitude (ALH), seen in control sperm incubated under capacitating conditions. B) Depiction of sperm movement in capacitated and FK506 treated sperm cells; green tracings denote low-amplitude movements, while blue tracings indicate high-amplitude movements of the sperm; red dots indicate stationary sperm cells; arrow indicates high-lateral head amplitude movements of sperm signifying hyperactivation. C) Sperm cells were analyzed using acrosome specific probe PNA-Alexa fluor 488 and propidium iodide (PI). Sperm populations incubated under capacitating medium with or without FK506, were used for IVF with oocytes collected from super-ovulated mice. Post IVF, spermatozoa were collected and fixed using 4% para-formaldehyde solution, stained with PNA-Alexa fluor 488 and PI and, subsequently used for flow-cytometric analysis. Capacitated sperm population that has undergone acrosome reaction showed decrease in both numbers and mean fluorescence intensity (MFI) in PNA positive/PI negative cells indicating loss of the acrosomes; however, this was not the case in the FK506 treated sperm which largely resembled the non-capacitated sperm population. Values are from 4 different mice presented as mean±SEM.