Date of Award
5-2011
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Legacy Department
Animal and Veterinary Sciences
Committee Chair/Advisor
Morris, James C
Committee Member
Birrenkott , Glenn P
Committee Member
Bodine , Ashby B
Committee Member
Scott , Thomas R
Abstract
Follicle stimulating hormone (FSH) and luteinizing hormone (LH) are glycoprotein gonadotropins from the anterior pituitary gland that are involved in reproductive functions. They are heterodimeric proteins that share a common α-subunit that is highly conserved amongst mammalian species, and is also conserved across this family of proteins. The β-subunit confers receptor specificity and is distinct. Recombinant gonadotropin expression would be an important advance for the bovine superovulation industry, as there are a number of disadvantages associated with the currently used pituitary-derived gonadotropins. For example, these pituitary products are contaminated with other proteins including hormones, exhibit batch-to-batch inconsistencies, and harbor the potential to spread disease-causing agents. Recombinant preparations can overcome these negative issues because they are potentially free of contaminating proteins. Further, recombinant gonadotropins can be produced so they have no potential to transmit harmful agents.
Here, two heterologous systems, bacteria and yeast, have been surveyed for their utility in the production of recombinant bovine FSH and LH (bFSH and bLH, respectively). bFSH was tested using a rat ovarian weight gain assay to determine biological activity and preliminary results indicate that the bacterial-derived bFSH had activity. The activity of yeast-derived bFSH could not be determined as a result of complications with the rat assay.
bLH was assayed using bovine granulosa cells treated in vitro with LH for one day, followed by gene transcript profile analysis to characterize receptor binding and activation. Both recombinant preparations reduced expression of FSH receptor transcript levels similar to pituitary LH, as expected. Both the pituitary LH and yeast LH decreased transcript expression of LH receptor by 7.0-fold and 2.9 fold (respectively), suggesting an autocrine down-regulatory effect. Notably, the bacterial preparation failed to yield similar results (1.2-fold decrease), potentially because of the differences in post-translational modifications that have been suggested to be important for protein bioactivity.
Recombinant bLH was also used in a rat ovarian ascorbic acid depletion bioassay to determine the impact of the compound at the whole animal level. Preliminary data suggests there was an ovarian ascorbic acid increase with both pituitary and recombinant LH treatments, rather than the expected decrease. Interestingly, the vehicle control caused an ascorbic acid increase for unknown reasons. While both the bacterial bLH and yeast bLH treatments resulted in an overall increase in relative ascorbic acid content post-LH treatment, the concentrations were less than the vehicle-only control, suggesting an overall reduction possibly as a result of activity maintained by the recombinant protein.
Recommended Citation
Hesser, Marcia, "A SURVEY OF HETEROLOGOUS EXPRESSION SYSTEMS FOR THE PRODUCTION OF BOVINE FOLLICLE STIMULATING HORMONE AND LUTEINIZING HORMONE" (2011). All Dissertations. 692.
https://open.clemson.edu/all_dissertations/692