Figure 1.
Presence of p14 in caprine sperm cells and in epididymal epithelial tissue.
Rabbit polyclonal antisera were raised against p14 protein (antigen), purified by affinity purification procedure and was used for Western blotting. (A) Western blot analysis of p14 in total tissue extracts from caput (Cap), corpus (Cor) and cauda (Cau) of the epididymis and vas deferens (Vd). Blot was probed with monoclonal antibody against actin to assess protein loading. (B) Western blot analysis showing p14 in total protein extracts of sperm from caput (Cap), corpus (Cor) and cauda (Cau) regions of the epididymis and vas deferens (Vd). Actin was used as loading control. (C) The immunohistochemical staining to show the expression pattern of p14 in the epithelial cells and lumen of the caput, corpus and cauda epididymis. Preimmune serum at the same condition showed no immunoreactivity. All the analysis was performed four times.
Figure 2.
The localization of p14 on sperm surface.
The localization of p14 on the surface of spermatozoa by indirect immunofluorescence analysis. The subcellular localization of p14 was determined using anti-p14 antibody and immunofluorenscence of p14 (FITC-labeled, green) was shown by confocal microscopy. The positive p14 immunoreactivity was localized to the anterior-acrosomal region of live caput and corpus sperm whereas in cauda and vd spermatozoa, it is localized on anterior as well as post acrosomal region of sperm head. Experiments were repeated four times.
Figure 3.
The localization of p14 protein in intracellular region of caprine spermatozoa.
The localization of p14 in intracellular region of spermatozoa by indirect immunofluorescence analysis. The localization of p14 was studied as in Figure 2 except that fixed/permeabilized cells were used for analysis. Experiments were repeated four times.
Figure 4.
Flow cytometric analysis indicating the presence of p14 in sperm population.
FACS analysis showing the percent expression of p14 in caput, corpus, cauda and vd spermatozoa under live and fixed-permeabilized condition. Details are described in the text. Experiments were repeated four times.
Figure 5.
Presence of p14 in plasma membrane and soluble part of acrosome.
(A) Western blot analysis showing the expression of p14 in membrane and cytosolic fractions of sperm and whole cell lysate. (B) Expression of the p14 in the cytosolic fraction of epididymis and cauda epididymal plasma. (C) Western blot showing the presence of p14 in plasma membranes and soluble components of the acrosome, acrosomal matrix and sperm cell lysate. (D) Immunoblot with a peripheral plasma membrane protein extract prepared from washed caudal epididymis following treatment with AES solution and cell lysate. Proteins were extracted from caudal sperm with 0.625% Triton-X-100 solution for immunoblotting (details in methods). Experiments were repeated four times.
Figure 6.
Effect of capacitation on the distribution of sperm p14.
(A) Indirect immunofluorescence of anti-p14 labeled protein in live capacitated caprine spermatozoa. Upper panel showing the fluorescent and corresponding phase contrast micrographs of noncapacitated sperm (control). Lower panels showing the same in sperm capacitated for 3 hrs. Three different patterns of staining were observed. (B) Indirect immunofluorescence of anti-p14-labeled protein in fixed/permeabilized caprine sperm. Upper panels showing the fluorescence and corresponding phase contrast pattern of noncapacitated sperm. Lower panels correspond to the same but for 3 hrs capacitated spermatozoa. Two different patterns of immune-fluorescence were observed. The calculated percentages given as the mean ± SEM of at least four (n = 4) different experiments.
Figure 7.
Distribution of p14 during sperm acrosome reaction.
(A) Indirect immunofluorescence of anti-p14-labeled protein in acrosome reacted fixed caprine sperm permeabilized with paraformaldehyde. Left panel indicating the fluorescent and corresponding phase contrast photographs of sperm previously capacitated for 3 h but not acrosome reacted (control). Right panel showing the same in capacitated spermatozoa, later induced to acrosome react. (B) Indirect immunofluorescence of anti-p14-labeled protein in acrosome reacted fixed and nonpermeabilized caprine sperm. Upper panel showing the fluorescence and corresponding phase contrast pattern of sperm previously capacitated for 3 h but not acrosome reacted (control). Lower panels corresponding to the same but in capacitated spermatozoa, later induced to acrosome react. Two different patterns of immune-fluorescence were observed. The calculated percentages given as the mean ± SEM of at least four (n = 4) experiments.
Figure 8.
Expression level of p14 during different time period of capacitation and acrosome reaction.
(A) Western blot analysis showing the expression of p14 in peripheral plasma membrane (PPM) of swim-up spermatozoa incubated under capacitated condition for different time periods. (B) Immunoblot for the presence of p14 in membranes and acrosome soluble component and (C). Whole cell lysate prepared from spermatozoa capacitated for 1, 2, and 3 hrs. (D). The expression of p14 in cell lysate of acrosome reacted spermatozoa previously capacitated for 3 hrs.
Figure 9.
Effect of anti-p14 antibody on the forward motility retarding effect of p14: Microscopic analysis.
Spermatozoa (1×106) were incubated with anti-p14 antibody at different dilution (1∶500, 1∶1000) for 15 mins at room temperature (32°C±1) and forward motility of sperm was monitored. Data representing the mean of four experiments (n = 4) ± SEM. Units: nm = non-motile sperm, vm = vibrating sperm, pm = progressively motile sperm.
Table 1.
Microscopic analysis of sperm motility in the absence and presence of anti-p14 antibody.
Figure 10.
Effect of anti-p14 antibody on the forward motility retarding effect of p14: CASA.
Data showing the effect of anti-p14 antibody on different CASA parameters. Data represents mean of three experiments (n = 3) ± SEM. Units: VAP (Average Path Velocity) = µm/sec, VSL (Straight Line Velocity) = µm/sec, VCL (Curvilinear Velocity) = µm/sec.
Table 2.
Sperm motility study by CASA in the absence and presence of anti-p14 antibody.
Figure 11.
Effect of anti-p14 antibody on acrosome reaction of cauda spermatozoa monitored by Rose Bengal staining method.
Acrosome reaction was carried out under the standard assay conditions and the cells after staining with Rose Bengal were observed under microscope at 1,000× magnification. Sperm cells treated with anti-p14 antibody. (A) Data showing the percent of acrosome reacted cells at different assay conditions (without ionophore, and PBS–BSAcontrol, Pre-immune sera, anti-p14 antibody in presence of ionophore). (B) Red arrow represents acrosome unreacted (acrosome intact) and blue arrow represents acrosome reacted (acrosome not intact) sperm. The “acrosome unreacted” cell has a well defined tiny colored spot on the tip of the sperm head whereas “acrosome reacted” cell has no such colored spot.
Table 3.
Effect of anti-p14 antibody on caprine sperm acrosome reaction.