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In vitro gonadotropin-releasing hormone release from hypothalamic tissues of ovariectomized estrogen-treated
Abstract:
The effects of in vivo 17 beta-estradiol (E2) treatment on in vitro GnRH release and serum LH levels were studied to determine the loci of E2 feedback actions and to examine the hypothalamic mechanisms by which this steroid may regulate LH secretion in monkeys. Ovariectomized cynomolgus macaques received sc Silastic capsule implants containing E2 and were killed 12, 36, 42, or 48 h later. At least one control (CTL) animal received a blank implant and was killed concurrently with each E2-treated monkey. Three untreated animals were used in validation experiments. Before death, each animal was anesthetized with ketamine (15 mg/kg, im), and blood samples were drawn for subsequent LH analysis by Leydig cell bioassay. A diencephalic tissue block was obtained at autopsy and immediately immersed in Krebs-Ringer-phosphate medium (KRP). Mediobasal hypothalamic (MBH) and anterior hypothalamic/preoptic (AH/POA) fragments were quickly dissected from the block and placed in separate superfusion chambers maintained at 37 C. Tissues were superfused at 50 microliter/min with KRP, and 10-min fractions were collected, acidified, and stored at -20 C for subsequent GnRH RIA. Basal immunoreactive GnRH (IR-GnRH) release was measurable from MBH (0.367 +/- 0.063 pg/min) and AH/POA (0.176 +/- 0.065 pg/min) fragments from CTL monkeys. In validation experiments, IR-GnRH release was increased 3- to 7-fold by superfusion with 60 mM K+-KRP only in the presence of Ca+2. Superfusate IR-GnRH coeluted with synthetic GnRH from a Sephadex G-25 chromatographic column, and superfusate and tissue extract GnRH showed appropriate LH-releasing capacities, as determined by rat pituitary cell culture assay. IR-GnRH release rates from MBH or AH/POA tissues varied as a function of in vivo estrogen treatment. GnRH release from both tissues was increased in the E2-treated group killed at 12 h when LH levels were suppressed. Thirty-six hours after E2 treatment, in vitro GnRH release was not significantly different from CTL values. GnRH release rates from MBH and AH/POA tissues obtained 42 h after E2 treatment were significantly greater than CTL release rates (P less than 0.01). This increased in vitro GnRH release at 42 h occurred during the apparent rising phase of the LH surge. Elevated GnRH release was not sustained at 48 h, when surge levels of LH were apparent.(ABSTRACT TRUNCATED AT 400 WORDS)