Endocervical glycoproteins are central to fertility/infertility regulation as hormone dependent changes in their viscoelastic properties control sperm and bacterial access to the upper reproductive tract. The ultimate objective of this research is to understand how estrogen regulates glycoprotein biosynthesis in the mammalian endocervix. The rheological properties of the glycoproteins are determined by the structure and composition of the oligosaccharide chains and their interaction with the polypeptide backbone. We now know that N-acetylgalactosaminyl (GalNAc) transferase, the first enzyme in the pathway for 0-linked oligosaccharide chain assembly, and the glycosylation site binding protein (GSBP) component of oligosaccharyltransferase, the first enzyme of the pathway for N-linked oligosaccharide chain assembly, are estrogen regulated. As a result, the specific aim of this proposal is to define hormone-dependent changes in the mucin protein(s). This goal will be achieved by the following three-step approach: 1) isolation, purification and deglycosylation of cervical mucin protein, and the preparation of a polyclonal antibody to the protein. 2) Protein A-gold and the mucin protein antibody will be combined with stereology and Western (immuno) blot analysis to quantify the subcellular location and the amount of mucin protein during postnatal differentiation, and in long-term ovariectomized rabbits treated with specific hormone regimens. The same techniques will be used to quantify changes in the subcellular location/amount of mucin in subpopulations of endocervical secretory cells isolated by flow cytometry. 3) The anti-mucin antibody will be used to screen a rabbit endocervical lambdagt11 cDNA expression library for cDNA clones that code for a mucin protein. These clones will be characterized by hybridization selection, and restriction mapping to determine whether there are different species of mRNA that code for different mucin proteins. This coupled with NOrthern analysis will be used to determine whether the size and number of mucin mRNAs are altered during cervical differentiation, and/or in response to steroid hormones. The number of mucin genes per haploid genome will be estimated. One or more of the cDNAs will be sequenced to facilitate future studies using site-specific mutagenesis to correlate mucin structure with function. Collectively, these studies will allow us to further test our working hypothesis: estradiol regulates the structure and therefore the viscoelastic properties of endocervical secretions by regulating the amount of oligosaccharides coupled to the mucin core protein(s) and by effecting the amount (number ?) of the mucin core proteins.
