Human spermatozoa were collected from healthy, fertile men. Semen was liquefied and analyzed for volume, sperm concentration and percent and progressive motility. Only those semen samples that had sperm concentration of >50x10⁶sperm/ml, percent motility of >60%, progressive motility of >+3 (on a scale of 0 to +5), and contamination of immature germ cells and immune cells of <1% were used to collect a pure swim-up sperm population (22). The study was approved by the West Virginia University-Institutional Review Board (IRB) for Human Studies.

Mouse sperm were collected from cauda epididymis and vas deferens of mature BALB/c or CD-1 males. Motile sperm were isolated by the swim-up procedure and washed by centrifugation (500 g, 10 min) with Ham's F-10 medium supplemented with human serum albumin (5 mg/ml) or Modified Sperm Washing Medium™ (Irvine Scientific, Santa Ana, CA, USA). Motile sperm were analyzed for sperm concentration and motility (9). The study was approved by the West Virginia University-Animal Care and Use Committee (ACUC) for animal studies.

The highly purified form of curcumin (cat. #C27727) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Curcumin was dissolved in dimethylsulf-oxide (DMSO) (25-50 mM stock) and then diluted in medium to the desired concentrations. Sperm treated with medium or equivalent volume of DMSO served as the controls. The effect of curcumin on sperm forward motility was examined by incubating 10-100 μl of sperm suspension (100-250x10⁶ motile sperm/ml) with various concentrations of curcumin (50-400 μM, final concentration) up to 1 hr. The percentage of forward moving sperm was recorded every 5-20 min, before and after incubation. Sperm treated with an equivalent volume of medium or DMSO served as the controls. Each treatment in each experiment was tested in duplicate. Each experiment, including various treatment groups, was performed 3-5 times using sperm from 3-5 men or mice on different days to take care of the inter-individual variation among various sperm specimens from different men and mice.

The intracellular pH (pHi) of human and mouse sperm was measured by fluorescent pH-indicator 2,7-bicarb-oxyethy l-5, 6-carboxyfluorescein- acetoxymethylester (BCECF-AM) (Molecular Probes, Eugene, OR, USA) following the manufacturer's protocol as described by Hamamah et al. (23). BCECF is a neutral lipophilic form of bis-carboxyfluorescein which diffuses freely through the plasma membrane. In the cell, it is hydrolyzed by esterases, releasing the BCECF which is retained within the cytoplasm. The fluorescence intensity of BCECF is dependent upon the pH. The motile sperm, isolated by the swim-up procedure, were centrifuged and the pellet was washed and resuspended in 1 ml of phosphate-buffered saline (PBS, pH=7.4). Sperm (8-15x10⁶/ml) were then loaded with 2 μM BCECF (final concentration) and incubated (37°C, 35 min) in dark. Following incubation, sperm were centrifuged, washed (x2) and resuspended in PBS. For the intracellular pH calibration curve, sperm were loaded with BCECF at various extracellular pHs (pHe; 6.8, 7.0, 7.2, 7.4, 7.6 and 7.8) and then treated with 0.1% Triton-X100. Subsequently, the fluorescence intensity was measured as described below, and the calibration curve was constructed by plotting fluorescence intensity versus extracellular pH (24). Using calibration curve, the fluorescence intensity value for each treatment was converted to its respective intracellular pH.

The fluorescence intensity of BCECF is dependent upon the pH with a maximum response at an excitation of λ=535 nm (F1), while at λ=490 nm (F2), the intensity is independent of pH. The pHi was determined graphically using the ratio F1/F2 from a calibration curve obtained after permeabilization of spermatozoa with 0.1% Triton by measuring the maximum fluorescence intensity after adding NaOH and the minimum after adding HCl. The F1/F2 ratio represents a pseudo-linear function of the pH (23).

To examine the effect of curcumin, BCECF-loaded sperm were incubated (37°C, 5-10 min) with various concentrations of curcumin (50-400 μM) and then washed. The treated/control sperm were transferred into wells (200 μL/well) of a black 96-well microplate and the fluorescence intensity was measured (BioTek Synergy2 Multiplatform automated plate reader) using excitation and emission wavelengths of 490 and 535 nm, respectively. Each treatment in each experiment was tested in duplicate. Each experiment, including various treatment groups, was performed 3-5 times using sperm from 3-5 men or mice on different days to take care of the inter-individual variation among various sperm specimens from different men and mice.

All of the appropriate controls were carefully included as described by the manufacturer. Moreover, it was pertinent to examine the effect of cur-cumin per se on fluorescence intensity. Similar experiments carried out with various concentrations (50-400 μM) of curcumin without sperm did not affect the fluorescence intensity, indicating curcumin per se, without sperm, does not affect fluorescence intensity.

The changes in sperm plasma membrane polarization were examined using the fluorescence sensitive dye bis (1,3-dibarbituric acid)-trimethine oxanol [DiBAC₄(3)] (Molecular Probes, Eugene, OR, USA) following the manufacturer's protocol as described by Rossato et al. (25). The motile sperm in the swim-up fraction were centrifuged, washed (x2) with PBS and incubated (37° C, 1 hr) with 2 μM DiBAC₄(3). DiBAC₄(3) can enter depolarized cells and then bind to intracellular proteins or membrane and exhibits enhanced fluorescence. Increased depolarization results in additional influx of the anionic dye and an increase in fluorescence. Conversely, hyperpolarization is indicated by a decrease in fluorescence. This dye is excluded from mitochondria because of their overall negative charge. Sperm (8-15x 10⁶/ml) were then centrifuged and washed twice with PBS. The DiBAC₄(3)-incubated sperm were aliquoted in different tubes at equal volumes and incubated (5-10 min) with various concentrations of curcumin (100 μM, 200 μM, 300 μM, and 400 μM). Sperm treated with PBS or equivalent volume of DMSO served as the controls. After final washing with PBS, the sperm were transferred into wells (200 μL/well) of a black 96-well microplate and the fluorescence intensity was measured (BioTek Synergy2 Multiplatform automated plate reader) using excitation and emission wavelengths of 485 and 530 nm, respectively. Each treatment in each experiment was tested in duplicate. Each experiment, including various treatment groups, was performed 3-5 times using sperm from 3-5 men or mice on different days to take care of the inter-individual variation among various sperm specimens from different men and mice.

All the appropriate controls were carefully included as described by the manufacturer. It was pertinent to examine the effect of curcumin per se on fluorescence intensity. Similar experiments carried out with various concentrations (50-400 μM) of curcumin without sperm did not affect the fluorescence intensity, indicating curcumin per se, without sperm, does not affect fluorescence intensity.

The significance of difference among various groups was analyzed using analysis of variance (ANOVA) and Tukey Kramer Multiple Comparison Test. A p ≤ 0.05 was considered statistically significant.

Curcumin caused a concentration-dependent decrease in human sperm forward motility (Table 1). At 50 μM concentration, there was no apparent (p>0.05) effect on sperm forward motility in 5-10 min (with a slight decrease over time) and in 1 hr it decreased by up to ∼25% (p< 0.001). At 100 μM concentration, there was a significant (p<0.001) decrease in sperm forward motility in 5-10 min which decreased by up to ∼80% in 1 hr. At concentrations ≥200 μM, there was a complete block of sperm forward motility within 5-10 min. DMSO vehicle did not affect (p>0.05) sperm forward motility as compared to medium control.

Curcumin caused a similar effect on mouse sperm forward motility (Table 2). At 50 μM concentration, there was no apparent (p>0.05) effect on sperm forward motility in 5-10 min (with a slight decrease over time) and in 1 hr, it decreased by up to ∼40% (p<0.001). At 100 μM concentration, there was a significant (p<0.001) decrease in sperm forward motility in 5-10 min, which decreased by up to ∼70% in 1 hr. At concentrations ≥200 μM, there was a complete block of forward motility within 5-10 min. DMSO vehicle did not affect (p>0.05) sperm forward motility as compared to the medium control.

There was a linear relationship between the extracellular pH and the fluorescence intensity both in human (R²=0.9513) and mouse (R²=0.9835) sperm (Figure 1, panel A and B, respectively). With an increase in extracellular pH, there was a corresponding increase in fluorescence intensity. The pH=7.4 was selected in our subsequent experiments to examine the effect of curcumin on intracellular sperm pH (pHi).

In both human and mouse sperm, curcumin caused a concentration-dependent decrease in intracellular pH (Figure 2), in human (panel A) and mouse (panel B) sperm. In human sperm, control sperm had pHi of 7.3±0.003 which was not significantly different (p>0.05) from DMSO-treated sperm, which had a pHi of 7.3±0.105 (Figure 3, panel A). Treatment with curcumin significantly (p<0.001) decreased intracellular pH in a concentration-dependent manner as compared to control/ DMSO-treated sperm. At 50 μM curcumin concentration, pHi was 7.21±0.010, at 100 μM pHi was 7.04±0.008, at 200 μM pHi was 6.95±0.02, at 300 μM pHi was 6.88±0.0067 and at 400 μM pHi was 6.81±0.014. Comparing the change in pHi within the curcumin-treated groups, the difference was significant (p<0.05) only among 50 μM, 100 μM and 400 μM groups.

Similar results were obtained in mouse sperm (Figure 2, panel B). In mouse sperm, control sperm had pHi of 7.15±0.005 which was not significantly different (p>0.05) from DMSO-treated sperm which had a pHi of 7.15±0.00 (Figure 2, panel B). Treatment with curcumin significantly (p<0.001) decreased intracellular pH in a concentration-dependent manner as compared to control/DMSO-treated sperm. At 50 μM curcumin concentration, pHi was 7.07±0.017, at 100 μM pHi was 6.97±0.017, at 200 μM pHi was 6.91± 0.012, at 300 μM pHi was 6.88±0.02 and at 400 μM pHi was 6.83±0.017. Comparing the changes in pHi within the curcumin-treated groups, the difference was significant (p<0.05) only among 50 μM, 100 μM and 400 μM groups.

The effect of various concentrations of curcumin on plasma membrane polarization was evaluated using both human and mouse sperm. In human sperm, treatment with DMSO did not significantly (p>0.05) affect fluorescence intensity as compared to control sperm (control: 2861±43.016; DMSO: 3252± 215.860) (Figure 3, panel A). Treatment with curcumin caused significant (p<0.001) hyperpolarization of sperm plasma membrane as compared to control/DMSO-treated sperm (Figure 3, panel A). At 50 μM curcumin, fluorescence intensity were 601±51.052, at 100 μM, 597±20.664, at 200 μM, 522±72.421, at 300 μM, 458±47.576 and at 400 μM 414±29.464. Comparing the changes in fluorescence intensities within the curcumin-treated groups, the differences were not-significant.

Similar results were obtained in mouse sperm (Figure 3, panel B). In mouse sperm, treatment with DMSO did not significantly affect fluorescence intensity as compared to control (control: 5592.085±2.186; DMSO: 5130.905±130.770) (Figure 3, panel B). Treatment with curcumin caused significant (p<0.001) hyperpolarization of sperm plasma membrane as compared to control/DMSO-treated sperm (Figure 3, panel B). At 50 μM curcumin, fluorescence intensity was 997.502±22.229, at 100 μM was 877.356±41.328, at 200 μM was 624.331±42.116, at 300 μM was 578.929±43.245 and at 400 μM was 522.431±57.900. Comparing the changes in fluorescence intensity within the curcumin-treated groups, the difference was significant (p<0.05) only between 50 μM and 400 μM groups.