Rabbit Anti-Human CATSPER (a.a. 551-650) Polyclonal antibody

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.

Objective: Antisperm antibodies play a main role in immunological infertility, as they impair sperm function by binding to the sperm membrane. In this study, we conducted screening experiments tried to screen sperm membrane proteins interacting with antisperm antibodies by the method of co-immunoprecipitation and liquid chromatogram mass/mass (LC-MS/MS). Methods: The serum samples from infertile persons were previously screened as positive for ASA in ELISA and reproduced with mixed antiglobulin reaction. The swim-up method was used to separate mature and motile sperm. Hypoosmotic swelling homogenization and sonication were sequentially conducted to extract sperm membranes. The purified human sperm membrane proteins were then mixed with serum from disease group (positive for antisperm antibodies) and control (not containing antisperm antibodies) serum samples. The binding proteins of antisperm antibodies were enriched using co-immunoprecipitation assay. The immunoprecipitates were separated on SDS-polyactylamide gel, then the binding proteins were cut from the gel and analyzed by LC-MS/MS after the enzymolysis. Results: The serum samples from infertile persons (39 females and 17 males) were previously screened as positive for ASA in ELISA and conformed to MAR. The healthy controls (17 females and 14 males) were ASA-negative in ELISA and already possessed healthy offspring. 107 proteins that interacted with antisperm antibodies were obtained from the study via LC-MS/MS. These proteins could be divided into three groups: 13 antigens detected by control serum samples only, 14 antigens recognized by both infertile patients and control sera, 80 antigens specific for patients with antisperm antibodies. 15 novel sperm membrane proteins are CatSper1, CatSper3, CatSper4, SPAG9, Apolipoprotein A-I, Dynein heavy chain 14, axonemal, Cylicin-2 (CYLC2), Izumo sperm-egg fusion protein 4, Thioredoxin domain-containing protein 2, IQ domain-containing protein H (IQCH), IQ domain-containing protein F1 (IQCF1), Spermatogenesis-associated protein 5 (SPATA5), Sperm acrosome membrane-associated protein 1, E3 ubiquitin-protein ligase RNF 114. Conclusions: Fifteen novel sperm membrane proteins discovered with co-immunoprecipitation coupled LC-MS/MS analysis could be referred as male immunoinfertility-related antigens. These proteins may be valuable as an indicator in the clinical diagnosis and monitoring treatment of infertility.