Investigation in vitro Expression of CatSper Sub Fragment followed by Production of Polyclonal Antibody: Potential Candidate for The Next Generation of Non Hormonal Contraceptive

Abstract

Objective: CatSper is a voltage-sensitive calcium channel that is specifically expressed in the testis and it has a significant role in sperm performance. CatSper (1-4) ion channel subunit genes, causes sperm cell hyperactivation and male fertility. In this study, we have explored targeting of the extracellular loop as an approach for the generation of antibodies with the potential ability to block the ion channel and applicable method to the next generation of non-hormonal contraceptive.

Materials and methods: In this experimental study, a small extracellular fragment of CatSper1 channel was cloned in pET-32a and pEGFP-N1 plasmids. Then, subsequent methods were performed to evaluate production of antibody: 1) pEGFP-N1/CatSper was used as a DNA vaccine to immunize Balb/c mice, 2) The purified protein of pET-32a/CatSper was used as an antigen in an enzyme-linked immunosorbent assay (ELISA) and western- blot, and 3) The serum of Balb/-c mice was used as an antibody in ELISA and western-blot. The statistical analysis was performed using the Mann Whitney test.

Results: The results showed that vaccination of the experimental group with DNA vaccine caused to produce antibody with (p<0.05) unlike the control group. This antibody extracted from Balb/c serum could recognize the antigen, and it may be used potentially as a male contraception to prevent sperm motility.

Conclusion: CatSpers are the promising targets to develop male contraceptive because they are designed highly specific for sperm; although, no antagonists of these channels have been reported in the literature to date. As results showed, this antibody can be used in male for blocking CatSper channel and it has the potential ability to use as a contraceptive.

Keywords: Antibody; CatSper; Contraception; Male Fertility; Sperm Cation.

Fig 1

Topology diagram of CatSper based on the study of Ren et al. (7, 8). A unique Ca²⁺ channel is expressed exclusively in the testis. The arrow shows the situation of small segment, which was designed and cloned in this study (this figure was developed by applying 3D computer graphics software (maya)).

Fig 2

Schematic diagrams of construction the fusion vectors. (a) Digestion pET-32a by BamHI and HindIII. (b) Ligation of pET-32a vector with insertion of CatSper fragment (c) Digestion pEGFP-N1 by BamHI and HindIII. (d) Ligation of pEGFP-N1 vector with synthetic oligo. (e) Construction of fusion vector.

Fig 3

The synthetic of oligonucleotide gene resolved on 12% poly acryl amide gel electrophoresis (Lane 1: synthetic oligo of 45 bp and lane 2: synthetic oligo of 53 bp) (A). Plasmid mini preparation, double digestion with BamHI and HindIII (Lane 1: pET-32a of 5900 bp and lane 2: pEGFP-N1 of 4700 bp) and mono digestion with SalI to confirm cloning (B, C).

Fig 4

Showing the EGFP expressed from pEGFP-N1/CatSper in CHO cells (×40). The cells (1.5×105) in a 6-well tray were transiently transfected with 5 µg of pEGFP-N1/CatSper. The cells were observed under a fluorescent microscope (Olympus, CK-2, Tokyo, Japan) without fluorescent excitation in A and with blue filter in B after 48 hours (Images were taken with a ×40 objective lens).

Fig 5

SDS-PAGE of purified Thioredoxin/CatSper and His6 tag from pET-32a with Ni-NTA Sepharose (lane 1). Western blotting of purified synthetic oligo with the serum of the experimental group as the positive control (lane 2), western blotting of purified synthetic oligo with the serum of the control group as the negative control (lane 3), and western blotting of purified Thioredoxin and His6 tags without synthetic oligo with the serum of the placebo group as the second negative control (lane 4) (Marker size 45, 35, 25, 18, and 14 KD).

Fig 6

A graphical illustration of the production of antibody against CatSper1 fragment in this study.

Fig 7

The serum of the antigen-specific total IgG titer following intradermal administration of pEGPN1, pEGFP/N1/ CatSper and PBS. Each group is comprised of 5-8 mice. The results are expressed as mean ± SD.