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First published online 19 April 2005
doi: 10.1242/jcs.02326

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Evidence of the presence of calcium/calmodulin-dependent protein kinase IV in human sperm and its involvement in motility regulation

¹ Instituto de Biología y Medicina Experimental (IBYME) CONICET, Vuelta de Obligado 2490, (1428) Buenos Aires, Argentina
² Center for Research in Contraception and Reproductive Health (CRCRH), Department of Cell Biology, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA 22908, USA
³ Laboratorio de Estudios en Reproducción, Av. Córdoba 2077, (1120) Buenos Aires, Argentina
⁴ Department of Veterinary and Animal Sciences, University of Massachusetts, 161 Holdsworth Way, Amherst, MA 01003, USA

View larger version (12K): [in a new window]   Fig. 1. Ca2+ requirement for the maintenance of human sperm motility and viability. Motile sperm selected using glass wool columns were incubated for up to 18 hours at 37°C in medium not supplemented with CaCl2 (–Ca2+, ), or containing different concentrations of CaCl2: 0.22 mM (), 0.58 mM (), 1.5 mM () or 2.5 mM (). The percentages of progressively motile (A) and live (B) cells after 1, 2, 4, 6 and 18-hour incubation were determined as described in Materials and Methods. The results are expressed as mean±s.e.m., n=7. *P<0.05 when compared to sperm motility under all other conditions.

View larger version (13K): [in a new window]   Fig. 2. Concentration curve of CaM kinase inhibitors effect upon human sperm motility and viability. Immediately after selection using glass wool columns, motile sperm were resuspended in medium not supplemented with CaCl2, and in the presence or absence of the CaM kinase inhibitors KN62 () or KN93 (); or their inactive analogues KN04 () and KN92 () at the following final concentrations: 1, 3, 10, 30, 60, 100 µM. After 30 minutes, aliquots were supplemented with CaCl2 (final concentration: 2.5 mM) and cells were incubated for 18 hours in capacitating conditions. Aliquots incubated in medium not supplemented with CaCl2 (– Ca2+, ), in the absence of the inhibitors (+ Ca2+, ), or in the presence of 0.5% DMSO (DMSO, ) served as controls. The percentages of progressively motile (A) and live (B) cells were determined. The results are expressed as mean±s.e.m., n=6. *P<0.01 when compared to sperm motility under all other conditions.

View larger version (14K): [in a new window]   Fig. 3. Time curve of the effect of CaM kinase inhibitor (KN62) and its inactive analogue (KN04) upon human sperm motility and viability. Immediately after selection with glass wool columns, motile sperm were resuspended in medium not supplemented with CaCl2, and 60 µM KN62 () or KN04 () were added. After 30 minutes, aliquots were supplemented with CaCl2 (final concentration: 2.5 mM) and cells were incubated for up to 18 hours in capacitating conditions. Aliquots incubated in medium not supplemented with CaCl2 (–Ca2+, ) and in the absence of the inhibitors (+Ca2+, ) served as controls. The percentages of progressively motile (A) and live (B) cells after 1, 2, 4, 6 and 18-hour incubations were determined. The results are expressed as mean±s.e.m., n=7. *P<0.01 when compared with sperm motility under control conditions.

View larger version (23K): [in a new window]   Fig. 4. Effect of KN62 on the Ca2+-associated decrease in protein tyrosine phosphorylation. Immediately after selection using PercollTM gradients, motile sperm were resuspended in medium not supplemented with CaCl2, and 60 µM KN62 or KN04 were added. After 30 minutes, aliquots were supplemented with CaCl2 (final concentration: 2.5 mM) and cells were incubated for 18 hours under capacitating conditions. Aliquots incubated in medium not supplemented with CaCl2 (–Ca2+) and in the absence of the inhibitors (+Ca2+) or in the presence of 0.3% DMSO (DMSO) served as controls. Sperm protein extracts (2 x106 cells per lane) were analysed by PAGE, immunoblotted and probed using a monoclonal antibody against phosphotyrosine (clone 4G10, Upstate Biotechnology). Molecular weight standards (x10–3) are indicated on the left. Panel A, 2-minute exposure; Panel B, 10-minute exposure. A typical experiment is shown. This experiment was performed three times obtaining similar results.

View larger version (12K): [in a new window]   Fig. 5. Effect of CaM kinase inhibitors upon hFF-induced acrosome reaction. After selection using glass wool columns, motile sperm were resuspended in medium not supplemented with CaCl2 in the presence or absence of 60 µM KN62 or KN93. 30 minutes later, aliquots were supplemented with CaCl2 (final concentration: 2.5 mM). Aliquots incubated in media not supplemented with Ca2+ (–Ca2+) and in the absence of the inhibitors (+Ca2+) served as controls. After an 18-hour incubation period, the sperm were exposed to 10% human follicular fluid (hFF) or buffer for 45 minutes and the acrosomal status determined using FITC-Pisum sativum agglutinin. The results are expressed as mean±s.e.m., n=4. *P<0.001 compared to acrosomal status of cells with no hFF added under all conditions and cells with hFF but no Ca2+.

View larger version (58K): [in a new window]   Fig. 6. Presence and localization of CaM kinase IV in human sperm. Human sperm extracts were subjected to one-dimensional (A) and two-dimensional (B) gel electrophoresis, followed by western immunoblotting using a monoclonal anti-CaMKIV antibody (Transduction Laboratories). Both, pI and relative molecular mass (x10–3) are indicated. This experiment was performed three times obtaining similar results. A typical experiment is shown. (C) Localization of CaMKIV in human sperm by indirect immunofluorescence. Non-capacitated sperm were fixed and processed for indirect immunofluorescence using monoclonal anti-CaMKIV antibody (IF). The corresponding bright field photomicrograph is also shown. Bar, 10 µm.

View larger version (14K): [in a new window]   Fig. 7. CaM kinase IV activity in human sperm. CaMKIV activity was measured in sperm treated immediately after resuspension in the Ca2+-containing medium (T0) or in sperm incubated in the same medium for 18 hours in the absence (T18) or presence of 60 µM KN62 [T18 (KN62)]. Sperm subjected to these different experimental conditions were washed twice with PBS, cell concentration was adjusted to 107 cells/ml, and aliquots were stored at –20°C until use. Enzyme activity was measured in these extracts using peptide gamma and [-32P]ATP as substrates. The results are expressed as mean±s.e.m., n=7. *P<0.001 compared to CaMKIV activity in T0 and T18 (KN62) cells; **P<0.01 compared to CaMKIV activity in T0 and T18 cells.

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