417 REPORTER GENE BIOASSAY OF FOLLICLE-STIMULATING HORMONE ACTIVITY IN MARE SERUM

Abstract

Equine chorionic gonadotropin is secreted by the mare placenta and possesses both LH and FSH bioactivities in nonequine species. In ruminants, eCG is used commercially to induce superovulation. The production of commercial eCG is hampered by the variation in FSH and LH bioactivity of eCG between mares, and potentially results in batch-to-batch variation in eCG bioactivity. The objective of this study was to establish a cell-line-based bioassay of FSH activity in serum for use at eCG production facilities. Several cell lines were used for this study: HEK293 (kidney cells), KGN (a human granulosa cell line), and a new bovine granulosa cell line. The HEK293 and bovine granulosa cell lines did not express the FSH receptor (FSHR); therefore, the strategy was to cotransfect those cells with a FSHR expression plasmid and a cAMP reporter gene (β-galactosidase; β-Gal). The KGN cells transfected with β-Gal failed to respond to FSH and were not used further. The HEK293 and bovine cell lines responded to FSH in a dose-dependent manner, with a visible increase in β-Gal activity measured by colorimetric assay. The cells responded to eCG but not to LH, IGF1, or estradiol, demonstrating specificity for FSH activity. The minimum time of incubation required for clear bioactivity was 4 h. Activity was detected in serum from pregnant but not estrous mares. Attempts to create stable cell lines expressing both FSHR and β-Gal plasmids were not productive. We therefore attempted to create frozen batches of transiently transfected HEK293 cells. Several incubation conditions were tested and we succeeded in detecting β-Gal activity in response to eCG in thawed cells. The choice of serum during transfection had a major effect on the ability of the cells to respond to eCG after thawing, and the time interval between transfection and freezing significantly altered the magnitude of the response to eCG. The cells responded visibly to eCG treatment after 4-h incubation. In summary, we have developed a reasonably fast, colorimetric bioassay for FSH activity that can be used for serum in an on-farm setting. Supported by NSERC, AAFC, and Bioniche Animal Health.