CLONING AND SEQUENCE-ANALYSIS OF RAT FERTILIN-ALPHA AND FERTILIN-BETA- DEVELOPMENTAL EXPRESSION, PROCESSING AND IMMUNOLOCALIZATION

Fertilin alpha and beta are members of the MDC (metalloproteinase-like , disintegrin-like, cysteine-rich) protein family and are expressed on the sperm surface where they have been proposed to play a role in mam malian fertilization. Inhibition of sperm-oooyte binding and sperm-ooc yte fusion make fertilin an attractive target for the development of a n immunocontraceptive vaccine. Full-length cDNAs encoding alpha and be ta fertilin subunits were isolated from a rat testis cDNA library and sequenced. Using reverse transcription-polymerase chain reaction (RT-P CR), the developmental expression of fertilin alpha and beta was deter mined in pre-pubertal and mature rat testes. Fertilin alpha mRNA was p resent at all stages of development, suggesting that it is not exclusi vely expressed in post-meiotic germ cells. In contrast, fertilin beta mRNA was first identified in day 19 testes, coincident with the presen ce of pachytene spermatocytes. Polyclonal antisera raised against a 28 -residue peptide (corresponding to part of the disintegrin domain) and two recombinant fusion proteins identified a 90 kDa protein in testic ular sperm extracts and a 60 kDa protein in caput and cauda epididymid al sperm extracts, the predicted sizes for rat fertilin beta precursor and mature protein respectively. Indirect immunofluorescence using th e anti-peptide antisera stained the acrosomal cap of permeabilized tes ticular, caput and caudal spermatozoa and elongating spermatids in tes ticular sections.