Figure 1.
SACH inhibits capacitation-associated PTP in rat caudal epididymal spermatozoa.
(A) A time course of increases of PTP in rat sperm during capacitation. Sperm were incubated in BWW at 37°C for 0, 1, 3, 4.5 and 6 hours, and cell lysates subjected to SDS-PAGE and Western Blotting with anti-PTP antibody as described in Materials and methods. The molecular mass (kDa) of the protein standard is indicated on the left. (B) Quantification of PTP levels of 52 kDa and 85 kDa bands of four independent immunoblots of Fig. 1 A and data are presented as mean ± SEM. n = 4, ** p<0.01, *** p<0.001vs. 0 hour group. (C) SACH inhibited PTP in rat caudal sperm in a dose dependent pattern. Sperm were exposed to SACH at the indicated concentrations for 6 hours in BWW at 37°C. (D) Relative intensities of bands of 52 kDa and 85 kDa in Fig. 1 C, n = 4, * p<0.01, *** p<0.001 vs. control.
Figure 2.
SACH inhibits P-PKAs, which increases in rat caudal sperm during capacitation.
(A and B) Rat sperm were incubated in BWW at 37°C for 0, 1, 3, 4.5 and 6 hours, before immunoblotting of P-PKAs. Mean ± SEM of triplicate independent experiments are shown, * p<0.05, ** p<0.01, *** p<0.001 vs. 0 hour group. (C and D) Rat sperm were exposed to various concentrations of SACH for 6 hours in capacitating condition and immunoblotted with anti-P-PKAs antibody. n = 3, * p<0.05, ** p<0.01, *** p<0.001 vs. control.
Figure 3.
The role of depletion of cAMP in inhibition of PTP and P-PKAs by SACH.
(A) cAMP levels in rat sperm decreased by SACH after incubation in capacitating conditions for 6 hours, * p<0.05 vs. control. (B C, D and E) Rat sperm were incubated in complete BWW supplemented with 50 µM SACH, 100 µM SACH and 10 µM H89 or in BWW-HCO3− in the presence and absence of 0.25 mM dbcAMP and 0.1 mM IBMX as indicated at the bottoms of the figures. After 6 hours of incubation, PTP (B) and P-PKAs (D) were identified by Western Blot, and levels of phosphorylation were quantitated by densitometry analysis of representative bands (C, E). n = 3, * p<0.05, ** p<0.01, *** p<0.001 vs. control.
Figure 4.
Glycerol rescues glycolysis, PTP and PKA after inhibition by SACH.
Rat spermatozoa were exposed to a series of concentrations of SACH in capacitating conditions for 6 hours and both GAPDS activity (A) and ATP level (B) were significantly reduced. n = 3, * p<0.05 vs. control. (C and D) Rat sperm were exposed to a serial of concentrations of glycerol in the presence of 100 µM SACH for 6 hours and GAPDS activity and ATP were measured. n = 3, a p<0.01, b p<0.001 vs. the 100 µM SACH group. (E, F, G and H) With the same treatment as in (C and D), sperm were subjected to Western Blot analyses with anti-PTP (E and F) and anti-P-PKAs (G and H) antibodies. n = 2, * p<0.05, ** p<0.01 vs. control.
Figure 5.
The model by which SACH inhibits cAMP/PKA mediated protein phosphorylation through blocking glycolysis in spermatozoa during capacitation.
The inhibition of GAPDS by SACH results in depletion of ATP and then depresses of P-PKAs and PTP. Glycerol overcomes these effects through restoring glycolysis, and IBMX and dbcAMP, the activators of PKA, reverse in part the inhibitions of P-PKAs and PTP.