Heterogeneous Luteinizing Hormone and Follicle-Stimulating Hormone Storage Patterns in Subtypes of Gonadotropes Separated by Centrifugal Elutriation

Abstract

The enrichment and purification of gonadotropes has been a goal of reproductive physiologists who are studying biochemical events in the secretory cycle of these cells. The present authors have reported earlier that centrifugal elutriation will produce a fraction that is at least 50 per cent gonadotropes. Furthermore, since this fraction also contains nongranulated endothelial or follicular cells, 80 per cent of all its granulated cells are gonadotropes. The production of these gonadotrope-enriched fractions is a valuable aid to immunocytochemical studies of LH and FSH storage because it allows the rapid accumulation of data on gonadotropin storage patterns. The authors' previous report focused on the elutriation method itself and on analysis of the distribution of gonadotropes. The cytochemical data were correlated with assays of LH content, secretion, and GnRH binding. The results presented here expand the morphological analysis, averaging data from fractions collected in six elutriation experiments. Furthermore, the authors report the analysis of over 900 serially sectioned gonadotropes collected from the initial cell suspensions and four of the fractions, which indicates that gonadotropes in the cycling female rat exhibit different storage patterns with size. Six groups of pituitaries from at least 50 female rats (per group) were collected and dispersed. The cells were separated in a standard elutriation chamber, the cell fractions were counted, and 2 to 6 x 106 cells were incubated overnight. The initial cell suspension and the cell fractions were then washed in serum-free medium and resuspended in 1 per cent glutaraldehyde. The fractions were washed and formed into pellets, stained sections of which were subjected to light microscopic and electron microscopic analyses. The cell counts performed at the light microscopic level showed that the initial cell suspension contained 8.9 ± (SE) 0.4 per cent LH cells and 12.7 ± 1 per cent FSH cells (Table 1). The electron microscopic analysis revealed 10 pM 1 per cent LH cells and 12 ± 1 per cent FSH cells. The average diameters were 13.5 ± 4 μm for LH cells and 12.4 ±5 μm for FSH cells. An analysis of serial sections from the initial cell suspension demonstrated that 59.6 per cent of the gonadotropes contained both LH and FSH, 21.6 per