The long term objective of this project is the definition of the chemistry of the glycoprotein hormones, particularly the gonadotropins from the pituitary and chorion. It is proposed to continue conventional sequencing studies on eCG and eLH (in progress-Specific Aim 1), and to use this methodology to reinvestigate hFSHBeta and pFSHBeta sequences to resolve some remaining questions about their proposed sequences (Specific Aim 3). Studies of functional group proximity on LH will be done using bi-functional crosslinking reagents of specified length, enzymatic degradation, and amino acid sequencing of the isolated peptides to identify specific peptides involved in the crosslink (Specific Aim 4). This will require HPLC peptide mapping and location of the unaffected peptides for which considerable information has already been generated. Methodology for detection of sulfate esters at the nanomole level with precision and accuracy will be developed in order to compare the carbohydrate termination groups (sulfate ester or sialic acid) for equine or human pituitary and chorionic glycoprotein hormones (Specific Aim 5). These are the only species with well characterized chorionic gonadotropins. It is proposed to examine the gene sequence of the equine LH and CG beta subunit using recombinant DNA methodology (Specific Aim 2) since important differences have been shown for the gene products in the equine compared to the human. The gene sequences are known for the human in these two instances. The determination of the equine sequences will be through selection of recombinant clones for eLHBeta or eCGBeta from an equine cosmid genomic DNA library with human cDNA clones. Subsequent to the foregoing recombinant DNA study the sequence of the rabbit LHBeta gene would be determined (Specific Aim 6) since this gene product also has some unusual properties.
Specific Aim 7 involves collaborative studies to improve embryo transplants in cattle. Our role will be to produce FSH preparations free of LH.
