Production Of Anti-sperm Multiple Peptide Antibody And Its Effects On Fertilization Of Human Sperm

There are many events in fertilization such as maturation of egg and sperm, attachment, binding, acrosomal reaction, penetration, fusion, cortical reaction, zona reaction and nuclear fusion of both gamete. It is well known that anti-sperm antibody could impair fertility in couples by resulting in immunological infertility through damaging sperm motility, reducing acrosome reaction, inhibiting sperm penetration zona pellucida, and so on. Many special sperm antigens have been utilized to prepare contraception vaccine for controlling fertilization, which is focused in immunological contraception research.Sperm protein Sp17 is one of sperm antigens on acrosomal cap, and can bind specially with ovum zona pellucida. Cyritestin, a cross-membrane protein of acrosome, as Sp17 can also effect on sperm penetration. Both Sp17 and Cyritestin play important role in process of sperm-zona pellucida or sperm-member of egg binding and fusion. This study utilized P3 multiple peptides which was synthetic polypeptide containing both epitopes of Sp17 and Cyritestin and anti-P3 multiple peptides serum of rat. Our study is aimed to investigate the biological property of anti-P3 antibody, and its efficiency against sperm-egg conjugation and anti-fertilization. This research is divided into two parts. In part one, we conjugated the P3 peptide and carrier protein KLH with EDC, and immunized BALB/C mouse with the purified compound P3-KLH. Serum were collected, anti-P3 antibody was purified using immobilized diaminodipropylamine column. The purified anti-P3 antibody was then labeled with FITC, and used to detect the sperm's positive percent by flow cytometry. Then we confirmed the binding site of antibodyagainst P3 peptide by immunofluorescence and immunocytochemistry techniques separately.In part two, after swim-up collection, the motile sperm were incubated with antiserum against P3 peptide and control serum. Parameters of sperm motility, including sperm head amplitude, linearity, straight and curvilinear velocity, motility percentage, were evaluated and quantified using Computer-aided semen analysis system. Acrosomal reaction rate was determined by FITC-PSA fluorescence stain and Coomassie's blue stain. The penetrate capacity was assessed by hamaster zona-free egg penetration assay. Brown reaction appeared on the sperm head surface by pre-incubating with antibody serum and also with the positive control. The positive rate of sperm conjugating with FITC-anti-P3 antibody is 97.7%. The conjugating positive rate of FITC-goat-anti-human IgG with sperm was reduced from 49.2% to 36.4% by acquired antiserum. After incubation with acquired antibody serum, all the parameters of sperm quality (motility) were decreased significantly, including sperm velocity, percent motility, ALH, LIN, the rate of AR, the rate of SPA. Our results suggested that: on the surface of sperm head, there is an epitope as P3 peptide, and anti-P3 antibody could conjugate with sperm specially and excellently; the anti-P3 antibody serum could reduce the motility, acrosome reaction and penetrate capacity of sperm and disturb sperm's fertilization ability. So, the potential practicability of synthetic P3 peptide needs to be further determined.