Protein kinase C is present in human sperm: possible role in flagellar motility.

Abstract

We report the presence of protein kinase C (PKC) in ejaculated human sperm as revealed by enzymatic activity assay and indirect immunohistochemistry. PKC is localized in the equatorial segment and in the principal piece of the tail. Addition of phorbol 12-myristate 13-acetate resulted in increased flagellar motility that was blocked by known PKC inhibitors such as sphingosine, staurosporine, and 1-(5-isoquinoylinylsulfonyl)-2-methylpiperazine. A very good correlation (r = 0.9, P < 0.001) was found between the percentage of PKC-stained sperm cells and motility. We propose that PKC is involved in the regulation of flagellar motility in human sperm.

Identification of Extracellular Signal-regulated Kinase 1/2 and p38 MAPK as Regulators of Human Sperm Motility and Acrosome Reaction and as Predictors of Poor Spermatozoan Quality

“…The human semen was liquefied for 60 min at 36°C. Sperm was washed twice with Ham's F-10 medium containing bovine serum albumin (BSA, 0.3%) and incubated with the medium for 3 h for capacitation (20). PMA or progesterone (Sigma) were added with or without the PKC inhibitor GF109203x (Calbiochem) or the MEK1/2 inhibitors PD98059 and U0126, or the p38 inhibitors SB203580 and PD169316 (Biomol), or the Ca 2ϩ inhibitors EGTA, nifedipine, and BAPTA/AM (Sigma) to the above medium as described in the legends.…”

“…The cells were fixed and permeabilized by cold methanol (10 min), followed by cold acetone (10 min) (20). The cells were treated with the respective anti-general MAPK (ERK, JNK, and p38) antibodies (Sigma) or antibodies against mSos, MEK1/2, Raf-1, or tubulin (Santa Cruz Biotechnology) (1:25) for 18 h at 4°C.…”

“…Assessment of Sperm Motility-Progressive flagellar motility was determined manually (20) or by using Computer-aided Sperm Analysis (CASA) (Sperm Analysis System version 12-IVOS, Hamilton Thorne Biosciences, Beverly, MA), which was also used to measure hyperactivation (24). Hyperactivation parameters were as follows: curvilinear velocity Ͼ100 m/s, linearity Ͻ60%, amplitude of lateral head Ͼ5 m.…”

“…Washed cells (10 7 cells/ml) were capacitated for 3 h at 37°C and 0.5% CO 2 in F-10 medium supplemented with BSA 3 mg/ml (20). The inhibitors were added for the last 10 min of incubation, and then PMA (100 nM) was added for another hour.…”

“…Cells were then treated with biotinylated second antibody (1:100) for 30 min. After washing in PBS the cells were further treated for 30 min at room temperature using the avidin-biotin complex method as described previously (20). Visualization was performed in solutions containing diaminobenzidine (0.25 mg/ml) and 0.01% H 2 O 2 in PBS.…”