An epididymis-specific carboxyl esterase CES5A is required for sperm capacitation and male fertility in the rat

Abstract

Despite the fact that the phenomenon of capacitation was discovered over half century ago and much progress has been made in identifying sperm events involved in capacitation, few specific molecules of epididymal origin have been identified as being directly involved in this process in vivo . Previously, our group cloned and characterized a carboxyl esterase gene Ces5a in the rat epididymis. The CES5A protein is mainly expressed in the corpus and cauda epididymidis and secreted into the corresponding lumens. Here, we report the function of CES5A in sperm maturation. By local injection of Lentivirus -mediated siRNA in the CES5A -expressing region of the rat epididymis, Ces5a -knockdown animal models were created. These animals exhibited an inhibited sperm capacitation and a reduction in male fertility. These results suggest that CES5A plays an important role in sperm maturation and male fertility.

Figure 1

Identification of siRNAs targeted Ces5a effectively at the cellular level. Northern blotting analysis shows the expression of Ces5a mRNA in vitro 48 h after treatment with siRNAs. 18S RNA was used as loading control. This experiment was repeated 3 times. C5si1 and C5si2, two siRNAs specifically target the different sites of Ces5a sequence.

Figure 2

Establishment of a Ces5a knockdown rat model. (a) Expression of Cea5a in cauda epididymidal tissue of the rat at the mRNA level. (b) Expression of CES5A in cauda epididymidal tissue of the rat at the protein level. Densitometry of CES5A and its corresponding β-actin band were performed by Multi Gauge (Fujifilm, version 3.0). The densitometry ratio of CES5A to ACTIN was calculated, and this ratio was calibrated to the nontreated sample to provide the relative protein (%). (c) Detection of CES5A protein in the cauda epididymidis by western blotting. β-actin was used as loading control. The western blot is a representative of 5 independent experiments. (d) Detection of secreted CES5A protein in the cauda lumen by western blotting. Coomassie blue staining was used as internal control. Ten microgramme of total protein were loaded to sodium dodecyl sulfate polyacrylamide gel electrophoresis. In (a–c) : Csi, EGFP siRNA control; C5si1 and C5si2, two siRNAs specifically targeting the different sites of Ces5a sequence. Results represent the means ± standard error of mean of 5–7 independent experiments and were analyzed by one-way analysis of variance and subsequently by the Tukey post hoc test (SigmaPlot version 12.3 software; Systat Software, Richmond, CA, USA). **P < 0.01; ***P < 0.001 compared with the respective Csi group.

Figure 3

Sperm motility assay after Ces5a knockdown. (a) The percentage of total and progressive motility of spermatozoa from the rat cauda epididymidis treated with Ces5a siRNA. (b) Beat/cross frequency of spermatozoa from the rat cauda epididymidis after Ces5a expression had been downregulated. (c) Average path velocity, straight-line velocity and curvilinear velocity of spermatozoa from the rat cauda epididymidis after Ces5a expression had been downregulated. In (a–c): Csi, EGFP siRNA control; C5si1 and C5si2, two siRNAs specifically targeting the different sites of Ces5a sequence. Motility data were collected immediately after spermatozoa were released into the incubation medium. Results represent the means ± standard error of mean of 9 independent experiments and were analyzed by one-way analysis of variance and subsequently by the Tukey post hoc test (SigmaPlot version 12.3 software; Systat Software, Richmond, CA, USA). *P < 0.05 compared with the respective Csi group.

Figure 4

Assessment of sperm capacitation of Ces5a knockdown male rats. (a) The pattern of protein tyrosine phosphorylation of spermatozoa after Ces5a expression is inhibited. α-tubulin was used as loading control. Spermatozoa were incubated for 3 h and then collected for analysis. (b) Distinct chlortetracycline fluorescence staining patterns in an uncapacitated spermatozoon with uniform bright fluorescence over the head, F pattern, capacitated spermatozoon with a dark band in the postacrosomal region, B pattern, and acrosome-reacted spermatozoon with dark head except for the tip, which retains some weak fluorescence, AR pattern. (c–e) The changes in the percentage of uncapacitated spermatozoa, or F pattern (c), capacitated spermatozoa, or B pattern (d), and acrosome-reacted spermatzoa, or AR pattern (e) after Ces5a expression had been inhibited. In (a–d) : Csi, EGFP siRNA control; C5si1 and C5si2, two siRNAs specifically targeting the different sites of Ces5a sequence. Results represent the means ± standard error of mean of 5 independent experiments and were analyzed by one-way analysis of variance and subsequently by the Tukey post hoc test (SigmaPlot version 12.3 software; Systat Software, Richmond, CA, USA). *P < 0.05; ***P < 0.001 compared with the respective Csi group.

Figure 5

Fertility assay of Ces5a knockdown male rats. (a) In vitro fertilization by spermatozoa from Ces5a knockdown male rats. (b) Numbers of normal fetuses from normal female rats mated with control (Csi) and knockdown (C5si1 and C5si2) male rats. Csi, EGFP siRNA control; C5si1 and C5si2, two siRNAs specifically targeting the different sites of Ces5a sequence. Results represent the means ± standard error of mean of 4–9 independent experiments and were analyzed by one-way analysis of variance and subsequently by the Tukey post hoc test (SigmaPlot version 12.3 software; Systat Software, Richmond, CA, USA). *P < 0.05; **P < 0.01; ***P < 0.001 compared with the respective Csi group.