Analysis Of B-cell Epitope Of FSHR And Study On Its Anti-fertility Potential In Adult Male Mice

Objective Follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone required for normal spermatogenesis in males. Studies have shown that blocking FSH activity can decrease sperm counts. FSH acts through its receptor (FSHR) which expresses exclusively on the membranes of Sertoli cells of testis to regulate spermatogenesis. Moreover, the extracellular domain (ECD) of its receptor is believed to be responsible for important specific hormone-receptor contact sites. Making use of the function of FSH blocking spermatogenesis, some researchers conducted survey using peptides of the ECD of FSHR and found that infertility can be induced in different species. Priming with human recombinant FSHR protein (F140) and boosting with FSHR 32-44 amino acids peptide showed specific immune response and fertility inhibition in adult male mice in our previous study. However, it led to the reproductive organs damage. To avoid the pathological change and remain the infertility, we explored the peptide prime-boost strategy in mice. Methods 1. The peptide, comprising amino acids 32-44 of FSHR with the sequence IELRFVLTKLRVI, was linked to the promiscuous PADRE. 2. 8-week old Balb/C male mice were randomly divided into four groups (8 mice in each group) and received four subcutaneous vaccinations. The injection dose of peptide is 20?g or 80?g. The immunogens were diluted in 100?L PBS, pH 7.2, and emulsified with an equal volume of Freund's complete adjuvant for prime vaccination and Freund's incomplete adjuvant (Sigma–Aldrich) for subsequent boosts. Antibody titer was examined at different weeks after immunization. 3. When the antibody titer reached up to 1:1000 at week 10 post primary immunizations, the fertility tests were carried out to assess the effect of immunization on fertility. 4. Sperm analysis and sperm-egg binding assay were conducted following the fertility assay. 5. Histology of reproductive organs was observed after immunization.Results 1. The treatment groups displayed a moderate antibody increase in a dose dependent manner, which was not above 500 until Week 4 after prime and decreased at Week 10 after the first immunization. 2. The fertility rate in 80?g peptide group (31.25%) was significantly lower than the control group (p < 0.05). However, the fertility rate vaccinated with 20?g peptide was 68.75% and not significantly inhibited compared with the PBS control group. 3. The sperm count decreased significantly in immunized groups. Furthermore, the motility and sperm-egg binding was greatly inhibited in these groups compared with control group (p < 0.05). 4. There was no pathological change in the seminiferous tubules and interstitial cells in immunized groups compared with control group. Conclusion The immunization with the peptide prime-boost strategy has led to decreased fertility at Week 10 post vaccination, which is consistent with Balb/C mice treated with protein prime/peptide boost vaccination. In contrast to the cellular swelling and spotty necrosis in spermatogonia in the protein priming mice, the mice receiving peptide priming did not display pathological damage in seminiferous tubules and interstitial cells. Thus, the prime-boost immune regimen with the FSHR-derived peptide potenially provides a much safer candidate for a contraceptive vaccine.