Estradiol-17β Inhibits Gonadotropin-Releasing Hormone-Induced Ca2+ in Gonadotropes to Regulate Negative Feedback on Luteinizing Hormone Release

In pituitary gonadotropes, estrogens have biphasic actions to cause an initial negative feedback followed by a positive feedback on LH secretion, but the mechanisms involved are not clearly understood. To investigate the feedback effects of estrogen, we used mixed ovine pituitary cell cultures (48-72 h), which were treated with 10(-9) M estradiol-17 beta (E-2) or vehicle followed by a pulse of 10(-9) M GnRH. Medium was collected for LH assay and cells extracted to determine activation of MAPK (phosphorylated ERK-1/2). E-2 treatment for 5 min reduced GnRH-induced LH release and caused phosphorylation of ERK-1/2. E-2 alone also caused phosphorylation of ERK-1/2, similar to the response evoked by GnRH alone. GnRH increased cytoplasmic intracellular free calcium concentration ([Ca2+](i)) and this was abolished by 2 min pretreatment with E-2 or E-bovine serum albumen conjugate. Blockade of Ca2+ channels with nifedipine had no effect on the initial peak of GnRH-induced increase in [Ca2+](i) but reduced its duration by 27 +/- 6%. Depletion of intracellular Ca2+ stores with thapsigargin prevented GnRH-induced increase in [Ca2+](i). Thapsigargin (10(-7) M) or nifedipine (10(-5) M) pretreatment (15 min) of cells lowered GnRH-induced LH secretion by 30 +/- 6 and 50% +/- 4%, respectively. We conclude that inhibition of the GnRH-induced increase in [Ca2+](i) in gonadotropes by E-2 is a likely mechanism for the negative feedback effect of E-2 on LH secretion involving a rapid nongenomic effect of E-2. Activation of the MAPK pathway by E-2 may be the mechanism for the time-delayed positive feedback effect on LH secretion at the level of the gonadotrope. (Endocrinology 150: 4213-4220, 2009)