Transcriptional Regulation Of A Novel Epididymis-Specific Protein LCN6 In Mice And Its Polyclonal Antisera Preparation

The epididymis is a male accessory sex organ that is responsible for sperm maturation, storage, and protection. When sperm flow through epididymis, they consecutively react with the microenvironment, which is created by the expression and secretion of the epididymis-specific proteins, until the sperm acquire the ability of motility and fertilization. mLCN6 (mouse lipocalin6) is the homologous gene of rat LCN6, being first isolated and cloned in our laboratory. It exclusively expressed in the caput of the mouse epididymis and belongs to the lipocalin family, according to its amino acid sequence and predicted secondary structure. Our preliminary data suggested that mLCN6 has two isoforms of transcript, named mLCN6αand mLCN6β. In-situ hybridization showed that mLCN6 was specifically expressed in the initial segment and proximal caput of epididymis; mLCN6 was not detected before 3 weeks postnatal, and increased from 3 to 4 weeks, reaching a plateau through adulthood, but decreased in the old ages. This ontogenetic pattern indicated that this gene may be regulated by androgen and is closely related to sperm maturation and quality of reproduction.The thesis is composed of two parts. The first part of our work was about the expression and regulation of mLCN6 in the mouse epididymal epithelia on the mRNA level. mLCN6 mRNA responding to castration and testosterone supplementation, as well as efferent duct ligation was observed by Northern blot analysis. mLCN6 mRNA expression was positively correlated with serum testosterone level. Expression of mLCN6 decreased after castration, testosterone administration could only partially restore the gene expression. The ligation of efferent duct that prevents the testicular factors from entering into the epdidymis but does not affect the circulating androgen level down regulated the expression of mLCN6 in the first day after ligation and remained low thereafter, as determined with Northern blot analysis, The RT-PCR indicated that mLCN6αand mLCN6βwere both regulated by androgen in the same fashion. These results showed that mLCN6 expression is synergistically regulated by androgen and testicular factors.The second part of the present study was the preparation of the antisera against mLCN6 for providing the important tool for the future research into the function of mLCN6 in sperm maturation and the different expression pattern between the two isoforms on the protein level. Two recombinant proteins, a 118 amino acid residue fragment in N-termini which was the common sequence of mLCN6αand mLCN6βnamed mLCN6C, and a duplicated sequence which only existed in mLCN6αas the C-terminal 58 amino acid residues fragment named mLCN6D, were expressed in the E.coli expression system. These two proteins were purified by electro-elution from SDS-PAGE gel and were used to immunize New Zealand rabbits. Two polyclonal antisera were obtained and the titer and specificity were detected by ELISA and Western blotting. The two highly specific antisera provide useful tools for further functional studies of mLCN6.