Expression of the Be-20 Epididymal Protein Gene: In Situ Hybridization

Abstract

A protein designated as BE-20 was purified from cauda epididymal fluid of male rabbits and the amino acid sequence of the N-terminus was determined. A 23-mer oligonucleotide coding the N-terminal eight amino acids of the BE-20 protein was synthesized. The oligonucleotide was used as sense primer with rabbit epididymal mRNA as template in the RT-PCR system. The BE-20 cDNA consisted of 499 bp with an open reading frame of 285 bp encoding a deduced polypeptide composed of 95 amino acids. Digoxigenin-labeled BE-20 cDNA was prepared and used as a hybridization probe to detect the specific mRNA. The probe interacted with a 1.2-kb mRNA prepared from rabbit epididymis; mRNAs prepared from rabbit testis gave negative reaction. Using tissue sections, the BE-20 mRNA was located in the epithelial cells of the cauda epididymis and proximal segment of the ductus deferens by in situ hybridization method. Sections of the corpus and caput epididymis, testis, and liver gave negative reaction. Polyclonal anti-BE-20 antibodies were raised and found to inhibit in vitro the capacity of human sperm to penetrate zona-free hamster ova. The results suggest that BE-20 protein may influence maturation of spermatoza during its movement through the epididymis and/or the capacity of sperm to fertilize ova.

Key Words:

• capacitation
• epididymal protein gene expression
• epididymis
• fertilization
• mRNA
• sperm maturation