PREPARATION OF GONADOTROPH-ENRICHED CELL POPULATIONS FROM ADULT RAT ANTERIOR PITUITARY CELLS BY CENTRIFUGAL ELUTRIATION

Abstract

Separation of gonadotrophs from cell suspensions of adult female rat pituitary glands by centrifugal elutriation was applied to the preparation of gonadotropin-enriched cells. Trypsin-dispersed cells (200–300 million) were loaded into the elutriator chamber while the rotor was operating at 1960 rpm, and cells were separated and collected in fractions by increasing the flow rate of medium through the rotor in increments from 11.8 to 39.5 ml/min. Loading and elutriation were completed in 80 min; cell recovery was 78/95%. Immunocytochemical staining of the initial cell suspension (ICS) by the avidin-biotin-peroxidase complex technique demonstrated that 8% of the cells contained LH-β and 12% contained FSH-β. After elutriation, large, mature gonadotrophs were found in fraction 7, collected at 30.8/39.5 ml/min. In this fraction, 40% of the cells stained for LH-β and 47% stained for FSH-(β. Since nearly half of the cells in fraction 7 were endothelial cells, the gonadotrophs constituted at least 80% of the secretory cells present. The morphological enrichment of gonadotrophs was correlated with the cellular LH content, which increased 6-fold; with the binding of ¹²⁵I-labeled (D-Ala⁶]des-Gly¹⁰-GnRH-N-ethylamide (GnRHa), which increased 4- to 6-fold; and with GnRHa-stimulated LH release, which increased 8-fold. Sequential staining of serial sections for both hormones revealed a mixture of mature cells containing one or both gonadotropins. Gonadotrophs that were morphologically and functionally different from the mature type were found in other fractions. Most of the PRL-containing cells eluted prior to 20 ml/min, in fractions containing 50–88% mammotrophs, and a fraction enriched 2.5-fold in GH was collected at 19.8–25.6 ml/min. Centrifugal elutriation is an effective, rapid and reliable method for the isolation of gonadotrophenriched and other cell fractions for in vitro studies on pituitary hormone biosynthesis and secretion.