Melatonin Directly Inhibits Rat Gonadotroph Cells*

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Abstract
Melatonin, a secretory product of the pineal gland, can suppress the neonatal rat pituitary LH and FSH responses to LHRH in both intact animals and pituitary cultures. The present study has examined the effect of melatonin on an enriched gonadotroph cell preparation to determine whether the indole interacts directly with the gonadotroph or requires other pituitary cells to mediate the inhibition of gonadotropin release. Monodispersed anterior pituitary cells were prepared from 15- day-old female rats by a modification of the procedure of Hopkins and Farquhar. This procedure, in contrast to certain other methods tested in our laboratory for monodispersion, produced cells maximally responsive to both LHRH and melatonin and comparable to those dissociated with collagenase in our earlier studies. The dispersed cells were fractionated by velocity sedimentation on a 1–3% bovine serum albumin gradient. LH, FSH, TSH, PRL, and GH concentrations in cell lysates of each fraction were measured by double antibody RIA. The percentage of LH-, TSH-, and PRL-containing cells was determined by immunohistochemistry. In three separate gradient fractionations, both procedures revealed a highly reproducible elution profile, with peaks of gonadotroph cells enriched 3.9-, 7.2-, and 9.9-fold over the unfractionated mixture. The gonadotroph peak was clearly separated from lactotrophs and somatotrophs, but did contain some thyrotroph cells. Enriched gonadotrophs were cultured overnight and then with increasing concentrations of LHRH, either alone or in the presence of melatonin. LHRH-induced LH release by the enriched gonadotrophs was indistinguishable from that observed with unfractionated cells with regard to both dose-response relationships and the percentage of total LH released. Melatonin (1 μM) significantly suppressed the LH response to 0.1, 1, and 10 nM LHRH by 100%, 62%, and 43%, respectively. This inhibition was equivalent to results obtained with the unfractionated cells. These findings indicate that melatonin interacts directly with the gonadotroph to inhibit responsiveness to LHRH.