96 Animal Science Reporter, Volume 7, Issue 3, July, 2013 MOLECULAR CHARACTERIZATION AND IMMUNOLOCALIZATION OF SPERM SURFACE PROTEINS (SPAM-1 AND LDH-C 4 ) IN DOG Ranjna Sandhey Cheema 1 , Nisha Vashisht 2 , Gaurav Bhakri 3 , Vinod Kumar Gandotra 4 ABSTRACT Search for immunogenic contraceptives has demonstrated that two sperm surface proteins, viz., sperm adhesion molecule-1 (SPAM-1/ PH-20) and lactate dehydrogenase-C 4 (LDH-C 4 ) possessed immunocontraceptive properties. The molecular configuration and expression of these two proteins have been amply studied in humans, non-human primates, and laboratory animals, but least studied in dogs. The present study conducted on ejaculated semen depicts molecular characterization of SPAM-1 and LDH-C 4 , and their expression in epididymis tissue and epididymis fluid in dog. Anti SPAM-1 raised in rabbit against human sperm SPAM-1 and anti LDH-C 4 raised in rabbit against a 54 amino acid long synthetic peptide sequence of LDH-C 4 were reacted with sodium dodycyl sulphate (SDS) and lithium di-iodosalicyalate (LDIS) sperm extracts (SESDS-SE & LDIS-SE), epididymal tissue extracts (ETE), and epididymal luminal fluid proteins (EFP) on immunoblots to characterize SPAM-1 and LDH-C 4 . Anti SPAM-1 and anti LDH-C 4 reacted strongly with 46 kDa and 32 kDa bands both in SDS-SE and LDIS-SE. Anti LDH-C 4 identified a strong band of 36 kDa, both in SDS-SE and LDIS-SE, two weak bands of 30 kDa and 28 kDa in SDS-SE, and one weak band of 30 kDa in LDIS-SE. Anti SPAM-1 cross reacted to proteins of 46 kDa of EFP and to 32 kDa of ETE. LDH-C 4 antibody strongly cross reacted to protein of 36 kDa, and weakly to 46 kDa, 32 kDa, and 30 kDa of EFP. It also cross reacted strongly to 36 kDa, and weakly to 14 kDa proteins of ETE. The cellular distribution of SPAM-1 and LDH-C 4 in sperm plasma membrane, determined by indirect immunofluorescence on whole sperm, indicated localization of SPAM-1 mainly on head surface, and LDH-C 4 mainly in the post-acrosomal region and mid- piece of spermatozoa. Our study provided evidence on the existence of two sub units of SPAM-1 (46 kDa, 32 kDa) and three sub units of LDH-C 4 (36 kDa, 30 kDa, 28 kDa) in spermatozoa, and in the tissues and luminal fluid of all the three segments, viz., caput, corpus, and cauda of epididymis in dog. KEY WORDS Dog, Epididymis, Immunocontraception, LDH-C 4 , SPAM-1, Author attribution: 1 Physiologist (Reprod), 4 Professor, Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India- 141004, 2 Assistant Professor, Department of Zoology, Punjab Agricultural University, Ludhiana, Punjab, India- 141004, 3 PhD Scholar, Department of Biochemistry, National Dairy Research Institute, Karnal, Haryana, India- 132001. 1 Corresponding author : ranjna.cheema@gmail.com. Date of Receipt: 08/01/2013, Acceptance: 18/04/2013. pp. 96-106.