A technique for sorting rat gonadotropes using anti-LH or anti-FSH antibodies covalently attached to magnetic beads.

Abstract

A technique for sorting live LH- and FSH-secreting cells was developed. After enzymatic dispersion, a suspension of pituitary cells from male rats castrated 7 days earlier was incubated in potassium chloride (KCI 50 mmol/l) for 30 min and gonadotropin outflow was provoked. Then, considering either LH or FSH as temporary surface markers, we positively selected the secreting cells by means of antibodies toward either LH (anti-LH beads) or FSH (anti-FSH beads) covalently attached to magnetic beads. The spontaneous secretion of LH and FSH overnight and the release induced by KCI the following morning were calculated. A population enriched in gonadotropes (16% of the total) able to secrete both gonadotropins was selected by means of anti-LH beads; this released 7 times as much LH as non-selected cells. A similar population (14% of the total) was selected by means of anti-FSH-coated beads; this produced 3.3 times as much LH as non-selected cells. In some experiments, the cells not previously sorted with anti-LH-coated beads were further incubated in the presence of anti-FSH beads, in an attempt to isolate a population secreting only FSH. A limited number of cells were sorted (6% of the total cells), able to produce both gonadotropins, but with a lower LH/FSH ratio. Similarly, those cells excluded by the selection with anti-FSH beads were further incubated with anti-LH beads, with a view to obtaining only-LH-secreting cells. However, both gonadotropins were still secreted by these cells (8% of the total), which had the highest LH/FSH ratio. In conclusion, fractions from castrated male rats that are enriched in gonadotropes contain cells that secrete both gonadotropins in vitro. The secretion of LH is prevalent. However, differences in the LH/FSH ratio between the populations sorted and changes from spontaneous to stimulated release are observed. This suggests that some gonadotropes might 'specialize' in releasing LH and others in releasing both LH and FSH.