Priming procedure and hormone preparations influence rat granulosa cell response.

The FSH activity in equine (e) FSH, eLH, eCG, and ovine LH were examined and compared to that in the standard reference preparation NIH FSH-S13 by three types of assay: the FSH radioreceptor assay with rat testicular homogenate and the stimulation of plasminogen activator production and steroidogenic activity in granulosa cells from diethylstilbestrol (DES)- or eCG-primed donor rats. The difference in the two types of granulosa cells was that the eCG-primed cells have already acquired significant aromatase activity. With the exception of oLH, which showed very little FSH activity (approximately 0.03-0.08 X NIH FSH-S13) throughout the three assays, the equine gonadotropins exhibited great variations in activity with respect to each assay. eFSH, the most active molecule in these assays, had an activity of 44 X NIH FSH-S13 in the receptor binding assay, 8.75 X NIH FSH-S13 in plasminogen activator production, and 4-5 X NIH FSH-S13 in steroid production when assayed in the DES-primed granulosa cells. In the eCG-primed cells, eFSH showed an activity of 4.2 X NIH FSH-S13 in plasminogen activator production and 8.2 X NIH FSH-S13 in progesterone production. eLH had an activity of 10 X NIH FSH-S13 in FSH radioreceptor assay, but showed very little activity and behaved like oLH in stimulation of the cellular responses of DES-primed granulosa cells. However, when eLH was assayed in the eCG-primed cells, it did show stimulating activity with respect to the production of plasminogen activator and progesterone; however, the dose-response curves were not parallel to those of eFSH and eCG. eCG had much less FSH receptor-binding activity (0.29 X NIH FSH-S13) than eLH. It behaved like a LH molecule in DES-primed granulosa cells, but did show activity (approximately 1 X NIH FSH-S13) in stimulating the production of plasminogen activator and progesterone in eCG-primed granulosa cells. From these results, we conclude that under our culture conditions, neither eLH nor eCG was active in the DES-primed granulosa cells, but both were active in the eCG-primed cells, and that the choice of assay conditions and reference standards is very important. Different types of assay may give rise to completely different comparisons for the same molecules. The equine gonadotropins provide a particularly dramatic example of such differences.