Figure 6. Effect of calcineurin inhibition on GSK3α phosphorylation in epididymal mouse spermatozoa.

A) Caput sperm were incubated with or without FK506 (20 nM) and calyculin A (5nM). Extracts were subject to western blot analysis with GSK3α/β-pSer21/pSer9 antibodies. There was clear increase in GSK3α phosphorylation in sperm treated with FK506, but both GSK3α and β increases with calyculin A; only GSK3α phosphorylation band intensities were quantified. B) Mouse caudal epididymal spermatozoa were incubated for 90 min under capacitating or non-capacitating conditions with or without FK506 (20nM) as described under materials and methods. Western blot analysis of sperm extracts following this incubation were subject to western blot analysis developed with GSK3αβ-pSer9/21 antibodies. Capacitation results in lowered GSK3α phosphorylation which is reversed in the presence of FK506 as indicated by the calculated average band intensities (n=3) provided on top of the corresponding blots. C) Lowered GSK3α phosphorylation is seen in sperm incubated in a capacitating medium in the presence but not in the absence calcium. Requirement of extracellular calcium for changes in GSK3 phosphorylation and activity. D) Spermatozoa were treated with the indicated concentrations of FK506 for 90 min; western blot developed with GSK3α-pSer21 antibodies show a FK506 dose dependent increase in the phosphorylation. E) GSK3 activity was measured using a GS2 peptide substrate and ³²ATP as described for Figure 3C and under materials and methods.