Immuno-contraceptive potential of sperm specific LDHC<sub>4</sub> and SPAM-1 (PH-20) sub units in dog

Abstract

PH-20 (Spam-1) antigen appears to be a bi-functional sperm plasma membrane protein with hyaluronidase activity and its role is related to cumulus penetration as well as zona binding. (LDH-C4) is a key enzyme, distributed specifically in testis and is highly immunogenic, function of which relates to energy metabolism and capacitation of the sperm. Therefore, in this study, we observed the effect of purified native PH-20 and LDH-C4 antigens on sperm function and morphology in dog. Native PH-20 (46, 32 kDa) and LDH-C4 (36, 30, 28) sub units, purified from dog sperm SDS- extracts were used to test as immunocontraceptive candidates in stray dog. Dogs 1, 2, 3, 4, 5/6 and 7/8 were immunized with 46, 32, 36, 30/28, 46/32, 36/30/28 kDa sub units respectively by i/m route and semen was analysed at weekly intervals. High titre of 3200 - 6400 was achieved in all dogs after three boosters. Immune response in treated dogs was confirmed by increase in Ig level in blood serum and reaction of partially purified serum Ig of immunized dogs with respective antigens in double immune diffusion and immunoblots. Immunization of dogs either with individual PH-20/ LDHC sub units or pooled PH-20/LDHC<sub>4</sub> sub units showed a significant effect on sperm count, percent motility, viability, HOST and sperm morphology. All the sperm parameters declined to a significant level (p < 0.05) between 45 - 115 DPI and remained low than that of the recommended values for fertile dog semen till 175 DPI. The incubation of spermatozoa in HOS solution in the presence of anti-PH-20 and anti-LDHC Ig significantly reduced the percent HOS positive spermatozoa. A significant decline (p ≤ 0.05) in acrylamide penetration assay was also observed in the presence of anti 46 and 30/28 kDa Ig. Since percent motility, sperm count, HOST and percent normal spermatozoa in immunized dogs were significantly less than that of recommended fertile dog semen, it can be concluded that PH-20 (46/32 kDa) and LDHC<sub>4</sub> (36/30/28 kDa) sub units had an immunocontraceptive potential in dog

Contraception and immunocastration vaccines. Use in veterinary medicine

“…Despite the identification of about 10 target antigens: sperm-specific lactate dehydrogenase (LDH-C4) (22), sperm adhesion molecule SPAM-1 (PH-20) ( 23), fertilization antigen FA-1 (24), and others; the development of vaccines against sperm proteins has been a more complex endeavor. The goal is to produce antibodies that block the binding of sperm to the oocyte and its fertilization, to cause infertility in males during sperm production in the testicles and its maturation in the epididymis and in females, through interference of sperm-egg interaction in their reproductive tract and the preimplantation embryonic development (25).…”

“…In turn, SPAM-1 (24), is a glycoprotein associated to the plasmatic and internal acrosomal membranes of sperm which has hyaluronidase activity (12). Its function is to attach to the ovum and penetrate the cumulus (25). Their combination with cellular structural proteins β-actin and α-tubulin homogenized with N-acetyl-muramyl-Lalanine-disoglutamine hydrate in the presence of CFA or IFA are part of a vaccine candidate used to control fertility in bitches.…”