Flow cytometric separation of gonadotrophs from dispersed rat pituitaries using a fluorescent GnRH antagonist

A gonadotropin-releasing hormone (GnRH) antagonist, Ac-delta 3 Pro1,pFDPhe2,DTrp3,DLys6-GnRH, was synthesized, conjugated to tetramethyl rhodamine, and found to retain GnRH antagonist activity. The fluorescent compound was used to label dispersed pituitary cells from 14-17 day-old Sprague-Dawley female rats. A subset comprising approximately 10% of the pituitary population was specifically labeled with a mean intensity of fluorescence 4.9-fold higher than the unlabeled population. The labeled cells were larger on average than the general population as inferred by forward narrow angle light scatter intensity measurements, and more granular as inferred by right angle light scatter intensity. Cells were sorted on the basis of fluorescent intensity: gonadotrophs were found to be concentrated in the rhodamine-positive fraction 5- or 6-fold relative to unfractionated cells, and 21- or 28-fold relative to rhodamine-negative fraction cells based on LH or FSH content, respectively. Gonadotrophs comprised 73 +/- 3.9% of the rhodamine-positive fraction by immunocytochemical staining. Sorted rhodamine-positive cells were cultured and found to be fully functional with respect to subsequent challenge with 30 nM GnRH. We conclude that the use of the fluorescent GnRH antagonist in conjunction with a multi-parameter cell sorter allows the purification of gonadotrophs, indicating, as expected, that these cells have a significantly higher level of GnRH binding sites than the general pituitary population. This technology should prove generally valuable in endocrine research.