Mutual interaction of dog sperm LDHC4, PH-20, actin and tubulin proteins and their immunocontraceptive potential in bitches

摘要

In this paper, interaction of LDHC4, PH-20, alpha-actin and beta-tubulin on dog's sperm surface and their immunocontraceptive effect in bitches has been elucidate. Anti spam -1 and anti LDHC4 recognized 46/32 and 36/30/28 kDa proteins in dog sperm extracts on iimnunoblots. Whereas, proteins of 43, 36 kDa and 62, 46, 43, 30 and 28 kDa were detected on inununoblots with anti-alpha-actin and anti-beta-tubulin. In MALDI-TOF analysis, sequence of 46, 30, 28 and 43, 36 kDa antigens matched twice to beta-tubulin and alpha-actin respectively. Therefore, one sub unit each of LDHC4 and alpha-actin had same mol wt of 36 kDa and two other sub units each of LDHC4 and beta--tubulin shared the same mol wt of 30/28 kDa. Reaction of 46 kDa protein both with anti-spam-1 and beta-tubulin indicated similarity in their mol wts. Similarly the reaction of 43 kDa protein both with anti-alpha-actin and anti- beta-tubulin revealed some similarity between both. Sequence analysis revealed 19.49%, 24.28%, 21.42% and 18.75% similarity of LDHC4/ alpha-actin, PH-20/ alpha-actin, LDHC4/ beta-tubulin and LDHC4/tubulin respectively. Immunofluoresence staining of dog sperm smears also indicated similarity in localization of these four proteins. Localization of PH-20 and beta-tubulin was mainly on the entire head region. Whereas, LDHC4 is uniformly distributed on the whole sperm and actin is more concentrated on the post acrosomal cap. Purified native proteins of 46/32 and 36/30/28 kDa were injected to a group of two bitches respectively through i/m route to elucidate their immunocontraceptive potential. Immunization of bitches in both the groups resulted in suppressed heat, and inhibited natural mating. On being subjected to AI, ultrasonography of four immunized and two unimmunized (control) bitches 35 days after insemination confirmed the presence of 0 and 3-4 fetuses respectively. Therefore, it seems that LDHC4/PH-20 and alpha-actin/ tubulin share their molecular weights or interact with each other or actin/ beta-tubulin superimpose LDHC4 /PH-20 on sperm surface. In view of observed contraceptive effect, it can be concluded that LDHC4 and PH-20 collectively with alpha-actin and beta-tubulin affect estrus, process of natural mating and ultimately the fertility in bitches.