Fig 1.
Principle and picture of enzymatic biofuel cell.
(A) Enzymatic biofuel cells and electrical current analysis with culture media; (B) picture of enzymatic biofuel cell (EBFC) and electrical current on the 60 mm culture dish; (C) graph showing the current density of the EBFC at various enzyme concentrations (10, 20, 50, and 100 μg/mL) that generate different electrical currents in the media of the culture dish. All data are means ± standard errors of the means (SEM) of values acquired from three repeated analyses.
Fig 2.
Optimal current range of electrical stimulation nano-Ampere range induced by EBFCs for spermatozoa survival and motility.
(A) The bar graph presents the viability of the human spermatozoa with a specific electrical stimulation induced by the EBFC at currents in the range from 10 to 1000 nA/cm2. Currents in the range of 10 to 100 nA/cm2 do not induce sperm necrosis. However, currents of the order of 1000 nA/cm2 significantly decrease the sperm’s survival ratio compared to 100 nA/cm2 and in comparison, to the control group. (B) The bar graph presents the motility characterisation of human spermatozoa following electrical stimulation by EBFC at each studied time point. The open bar represents progressive (PR) motility, the gray bar represents the non-progressive (NP) motile sperm, and the black bar shows the immotile (IM) sperm with electrical stimulation. Electrical stimulation induced by the EBFC derives immotile, while none-progressive sperm derives into progressive sperm. (C) The bar graph represents the motility sperm ratios after the shutdown of electrical stimulation (1, 2 and 3 hours) ended. The open bar is the control, the gray bar corresponds to a current of 112 nA/cm2, and the black bar shows the electrical stimulation samples with a current of 250 nA/cm2. Stopping the electrical stimulation of the sperm shows decreased motility after 2 h in a manner similar to the response of the control sperm. All data are means ± SEM of values in triplicate. Significant differences are indicated by asterisks (* p < 0.05 against control).
Fig 3.
Computer-assisted sperm analysis of human spermatozoa at the initial timepoint, and at 1 h and 2 h following stimulation.
(A) The line graph presents each sample denoting the VSL (purple), VAP (green), and VCL (red) values of electrically stimulated human sperm depending on the electrical current. (B) The moving distance per second of the control and the electrically stimulated human sperm: control, 112nA and 250nA/cm2 stimulation group. Both the 112 and 250 nA/cm2 electrical stimulation groups show significant increase in the straight movement and moving distance compared to the control group. All data are means ± SEM of values in triplicate. Significant differences are indicated by asterisks (*p < 0.05 against control).
Fig 4.
Immunocytochemistry of human spermatozoa following electrical stimulation induced by EBFC.
(A) Confocal images of the electrical stimulation sperm stained with the pisum sativum agglutinin (PSA)–green fluorescent protein (FITC), p-Tyrosine protein antibody (red), and 4’6’-diamidino-2-phenylindole DAPI (blue) under 63 x magnifications. (B) The bar graph demonstrates the intact acrosomal ratios of the human spermatozoa in the electrical stimulation based on the GFP-positive signal. (C) The bar graph shows the positive p-Tyr staining signal ratios of the human spermatozoa in the electrical stimulation. The open bar denotes the samples following 1 h and 2 h (gray) of electrical stimulation. Electrical stimulation of sperm showed a strong positive signal of the tyrosine protein phosphorylation compared with the control group. In addition, the acrosomal status was not different between the control and electrical stimulation group. (D) A reprehensive confocal image indicating the intact acrosome and the p-Tyr positive sperm. All data are means ± SEM of values in triplicate. Significant differences are indicated by asterisks (* p < 0.05 against control).
Fig 5.
Quantification of tyrosine phosphorylation activity analysed by Western blot with the anti-p-Tyr antibody.
(A) Immunoblot band image of phosphorylated tyrosine following the electrical stimulation of human spermatozoa with a normalisation band (con: control, 112nA, 250nA and 250nA(S): 2hrs after electrical stimulation stop, normalization by β-actin band). (B) The open bar graph shows the immunoblot band intensity obtained from the Western blot data. Electrically stimulated sperm showed a significant increase in the tyrosine phosphorylation ratios compared with the control group. All data are means ± SEM of measurements in triplicate. Significant differences are indicated by asterisks (*,# p < 0.05 compared with control).
Fig 6.
Recovery of motility of asthenozoospermatozoa and the immotile spermatozoa following electrical stimulation in the nano-Ampere range induced by EBFC.
(A) The bar graph shows the electrically stimulated asthenozoospermatozoa motility ratios depending on the culture times which range from 0 h to 3 h. The open bar is the control group, the gray bar is the 112 nA/cm2 stimulation, and the black bar is the 250 nA/cm2 stimulation group. (B) The line graph presents the VSL (purple), VAP (green), and VCL (red) values of the electrically stimulated human asthenozoospermatozoa. (C) The moving distance per second of the control and electrical stimulated asthenozoospermatozoa sample. Control: control group, 250nA: 250 nA/cm2 stimulation group. The electrical stimulation of the EBFC to the asthenozoospermatozoa enhanced the sperm’s motility after 2 h and without a straight movement but no increased the distance moved by the sperm compared with the control groups. (D) Up side two images at high magnification at the different fields show immotile sperm morphology with neck and tail defects. And down side two images of strict morphology analysis clearly exhibits the neck and tail defect of immotile spermatozoa. Therefore, the defected immotile sperm evokes no responses following electrical stimulation. All data are means ± SEM of measurements in triplicate. Significant differences are indicated by asterisks (* p < 0.05 against control).
Table 1.
Profile of 0% motile spermatozoa and response of 250nA, 2hrs by electrical stimulation nano-Ampere range of EBFC.