Figure 1.
Structure of Isolated Compound.
Structure of desgalactotigonin (DGT).
Figure 2.
Dose-dependent sperm immobilizing activity of DGT. The percentage of motile (A) human spermatozoa and (B) rat spermatozoa were determined after 20s following exposure to the test compounds at different concentrations. All data were adjusted to a normal control motility of 95%. Each point of DGT represents the mean ± SEM. values of five independent experiments.
Figure 3.
The HOS responsiveness of the sperm, as indicated by tail coiling.
HOS responsiveness of rat sperm as tail coiling: (A) Untreated control. (B) DGT-treated rat sperm (24.18 µM) examined under a phase contrast microscope (40×). Response of control (C) and DGT-treated (D) human sperm (58.03 µM) population following exposure to hypo-osmotic solution and evaluation under a phase contrast microscope. Over 90% of control-untreated sperm exhibited HOS response typically characterized by tail coiling, where as sperm exposed to DGT at MEC examined no response under a phase contrast microscope (40×). (E): For rat sperm, (F): For human sperm. Each bar represents the mean ± SEM of five observations (P<0.05).
Figure 4.
The sperm viability, as assessed by a fluorescent staining method.
Rat Sperm viability assessment by SYBR-14/PI staining. (A) Control rat sperm appear green due to uptake of SYBR14 only; (B) DGT-treated rat sperm appear red due to uptake of PI when observed under a fluorescence microscope. Overlaid fluorescence images of (C) control and (D) DGT-treated human spermatozoa, dual stained with SYBR-14 and propidium iodide to distinguish green-fluorescing live from red-fluorescing dead spermatozoa.
Figure 5.
Irritation potential as measured by hemolytic activity and effect on Lactobacillus acidophilus in vitro.
(A) Dose-dependent effect of DGT in rat RBCs. Isolated RBCs were incubated with varying drug dilutions, Triton X-100 and PBS. The extent of hemolysis was determined spectrophotometrically. (B) Effect of desgalactotigonin (DGT) and nonoxynol-9 on Lactobacillus acidophilus. [Optical density as the measure of turbidity denoting growth of bacterial colonies during 36 h of culture in the absence (control) and presence of DGT. There was a gradual increase in the growth of colonies that reached a plateau after 24 h of culture. Irrespective of the dose of DGT (19×MEC), the growth of bacterial colony was comparable with that of the control. N-9, however, exerted constant inhibition of bacterial growth throughout the entire culture period.]
Figure 6.
The contraceptive efficacy of DGT in Sprague-Dawley rats.
Photograph of a uterus showing the presence or absence of implantation sites in the control [C] and DGT-treated [T] uterine horn of a Sprague–Dawley rat.
Figure 7.
The microscopic ultrastructural changes in the sperm.
Transmission electron micrographs of human sperm samples incubated in the absence or presence of DGT (at MEC). (A) Control spermatozoa show proper acrosomal cap with intact plasma membrane, while (B) DGT-treated spermatozoa exhibit dissolution of the acrosomal cap. High resolution scanning electron micrographs (×15000 and ×19000) of human sperm treated without and with DGT at MEC. (C) Control sperm shows intact acrosomal cap and plasma membrane around the head and neck regions, while (D) DGT-treated sperm demonstrates dissolution of the acrosomal cap.
Figure 8.
Effect of lipid peroxidation on human sperm.
Generation of malondialdehyde (MDA) as a function of lipid peroxidation by human sperm treated with or without DGT at varying concentrations. Values on Y-axis represent mean ± SEM. values of five determinations (P<0.05). The graph shows dose-dependent increase in MDA generation following exposure of motile spermatozoa to DGT.
Table 1.
Spermicidal potential, cytotoxicity and selectivity index of DGT, N-9 [51].
Figure 9.
Apoptotic changes in plasma membrane.
Representative images of apoptosis/necrosis induction by DGT in sperm cells, measured by Annexin-V/PI labelling and flow cytometry. Concentrations used were 29.01 µM, 58.03 µM and 106.38 µM of DGT against sperm for 3 h. [A. control, medium only, B. 29.01 µM DGT, C. 58.03 µM DGT, D. 106.38 µM DGT]. Cell population in four quadrants identified as: [lower left, live; lower right, apoptotic; upper right, necrotic; upper left, dead].
Figure 10.
Apoptotic changes in mitochondria.
Representative illustration of the depolarization of mitochondrial transmembrane potential of sperm cells by DGT, measured by lipophilic cationic dye JC-1 staining. Concentrations used were 29.01 µM, 58.03 µM, and 106.38 µM against sperm for 3 h. [A. control, medium only, B. 29.01 µM DGT, C. 58.03 µM DGT, D. 106.38 µM DGT, E. CCCP]. Cell population in quadrants identified as: [upper right, polarized; lower right, depolarized].