Regulation of fertilization in male rats by CatSper2 knockdown

Abstract

Interest in ion channels as drug targets for contraception has grown with the realization that certain ion channel subunits are located exclusively in sperm. Selective knockdown of ion channel subunits can lead to infertility without ill effects, and selective inhibitors and/or openers of these ion channels could interfere with sperm function. In this study, in vivo electroporation (EP) and rete testis microinjection-mediated plasmid DNA were adopted to silence CatSper2 expression, which is essential in sperm hyperactivation. The results showed that high transfection efficiency and expression were achieved by plasmid DNA that was directly injected into the rete testis. As a result of the expression of CatSper2 being blocked, the treatment group showed significantly lower (P<0.05) hyperactivation rate, fertilization rate in vitro, migration motility in viscoelastic solution and intracellular Ca(2+) peak. The low hyperactivation and fertilization rates lasted for 60 days. Meanwhile, analysis of the sperm survival rate and testis histology indicated that in vivo EP had no significant effect on the function of the testis, spermatogenesis or sperm activity. The present study demonstrated that it was feasible to achieve male contraception by silencing the expression of CatSper2, the key protein involved in sperm hyperactivation.

Figure 1

The detection of the sperm transfection rate using in vivo Imaging-200 System (IVIS-200 System) and flow cytometry. (a) The intensity of GFP expressed in rat testis as a function of the plasmid dose. (b) The intensity of GFP expressed in rat testis observed up to 60 days following plasmid transfection via in vivo testis electroporation. The GFP fluorescence intensity was detected by the IVIS-200 System (Xenogen; Caliper Life Sciences, Hopkinton, MA, USA). The pseudocolor image of the rat testis represents the relative expression of GFP. (c) The exactitude fluorescence intensity histogram description of a and b (n=3). (d) The GFP-positive epididymal-derived sperm, as detected by flow cytometry: 0.7% (704/10 000) GFP-positive sperm are detected at 5 days, while 32.7% (3274/10 000) and 61.1% (6106/10 000) are shown at 9 days and 13 days, respectively, after transfection. The maximum percentage of positive cells is observed at 22 days, which is 87.5% (8750/10 000), and 77.5% (7750/10 000) of positive cells are found at 60 days in rat epididymis (n=3). GFP, green fluorescent protein; NC, negative control.

Figure 2

Knockdown efficiency of plasmid on CatSper2 ion channel proteins in testis and epididymis. (a) The relative expression of CatSper2 mRNA in testis (left) and epididymal sperms (right). (b) The knockdown of CatSper2 protein in testis. The Western blot analysis of shRNA-CatSper2 knockdown in testis (left) and the exactitude histogram description of relative expression of CatSper2 after interference in testis (right). (c) The knockdown of CatSper2 protein in epididymal sperm. The Western blot analysis of shRNA-CatSper2 knockdown in epididymal sperm (left) and the exactitude histogram description of relative expression of CatSper2 after interference in epididymal sperm (right). NC, negative control; shRNA, short hairpin RNA.

Figure 3

Ca²⁺ imaging of fluo3-loaded sperms. Pictures were taken within 25 s of applying medium without thimerosal (a) and with thimerosal (solution containing EGTA) (b). The green spots indicated higher fluorescent intensities and increased intracellular Ca²⁺ (scale bars=10 µm).

Figure 4

Fertilizing capacity of rat epididymal sperm after electrotransfection in vivo. The motility of sperm before and after treatment was unchanged (72%). (a) The oocyte fertilization rate of rat sperm following interference in vivo, using unaltered mature oocytes. The fertilization capacity of treated sperm decreased compared to the control group at 22 and 60 days (*P<0.05). (b) The oocyte fertilization rate of rat sperm following interference in vivo, using oocytes without zona pellucida (n=3). There are no marked differences in the cleavage rate between the control group and the experimental group. NC, negative control; shRNA, short hairpin RNA.

Figure 5

Viability and motility of transfected sperm in viscoelastic solution. (a) Sperm viability of the treated and control groups at different time points after transfection. (b) The open bars represent normal medium and the filled bars represent medium containing 0.75% (w/v) long chain polyacrylamide (*P<0.05). NC, negative control.