Concentrations of Gonadotropin-Releasing Hormone (GnRH) Receptor Messenger Ribonucleic Acid in Pituitary Tissue of Orchidectomized Sheep: Effect of Estradiol and GnRH1

Abstract

The effects of estradiol (E2) and E2 withdrawal on tissue concentrations of GnRH receptor mRNA were assessed in orchidectomized sheep (wethers). Wethers received E2 (in 20% ethanol-saline [vehicle]) by continuous infusion at rates of 0.31, 1.25, 5, or 20 micrograms/h (n = 12 animals per group). Control animals received vehicle alone. Anterior pituitary tissue was collected at the end of the 48-h infusion period. Steady-state levels of GnRH receptor mRNA were quantified by solution hybridization. Tissue levels of mRNA were significantly increased above control levels (0.5 +/- 0.1 pg/microgram RNA) by E2 infusion at 1.25 micrograms/h (1.4 +/- 0.2 pg/microgram RNA). Maximal tissue concentrations of GnRH receptor mRNA were induced by E2 delivery at 5 micrograms/h (2.4 +/- 0.3 pg/micrograms RNA). In a second study, wethers (n = 6 animals per group) received 5 micrograms E2/h for 24 h. Pituitary tissue was collected at the end of the infusion period or at 12, 24, or 48 h after cessation of E2 delivery. Infusion of E2 induced a 6-fold increase in GnRH receptor mRNA (0.4 +/- 0.1 and 2.9 +/- 0.6 pg/micrograms RNA in control and E2-treated groups, respectively). Cessation of E2 delivery resulted in rapid reduction in steady-state levels of GnRH receptor mRNA. Tissue concentrations of receptor mRNA returned to pretreatment levels within 12 h of E2 withdrawal (0.6 +/- 0.1 pg/microgram RNA). In a third study, the concentration of GnRH receptor mRNA was determined in pituitary tissue collected during preovulatory surge-like secretion induced in E2-infused wethers by episodic delivery of high-amplitude GnRH (1600 ng GnRH per hourly pulse). Estradiol (5 micrograms/ml in 10% ethanol-saline) was delivered as a continuous infusion. Episodic delivery of GnRH was initiated 24 h after E2 infusion was begun, and concurrent administration of E2 and episodic GnRH was continued to slaughter. Anterior pituitary tissue was collected at 0, 3, 6, 12, or 24 h after beginning circhoral delivery of GnRH (n = 6 wethers per group). As noted above, continuous infusion of E2 for 24 h significantly increased tissue concentrations of GnRH receptor mRNA. However, steady-state levels of GnRH receptor mRNA were returned to pretreatment levels after 3 h of circhoral delivery of GnRH (0.4 +/- 0.1 pg/microgram RNA). Taken together, these data demonstrate that physiological concentrations of E2 increase steady-state levels of GnRH receptor mRNA in a dose-dependent manner. In addition, continued estrogenic support is required to maintain enhanced tissue concentrations of GnRH receptor mRNA. Finally, high-amplitude GnRH stimulation induces down-regulation of tissue levels of GnRH receptor mRNA. These data suggest that the dynamic changes in tissue concentrations of GnRH receptor mRNA during the periovulatory period may be due to the inductive effects of gonadal steroids from the developing follicle and to the combined suppressive effects of the increased GnRH stimulation and E2 withdrawal that are associated with the gonadotropin surge.

Effect of Stress-Like Concentrations of Cortisol on Estradiol-Dependent Expression of Gonadotropin-Releasing Hormone Receptor in Orchidectomized Sheep1

“…Although estrogen-dependent augmentation of pituitary concentrations of GnRH receptor and receptor mRNA is well documented [27,30,31], the time course of this response has not been precisely characterized. The data presented here, and elsewhere [20], indicate that the E 2 -dependent increase in pituitary concentration of GnRH receptor is rapid and complete within 12 h of first introduction of E 2 .…”

“…Tissue concentrations of GnRH receptor mRNA were determined using the RNase protection assay described previously [27]. A plasmid containing a cDNA insert for the ovine GnRH receptor [28] was kindly provided by Dr. J. Brooks (MRC Reproductive Biology Unit, Edinburgh, UK).…”

Moderate and Severe Nutrient Restriction has Divergent Effects on Gonadotroph Function in Orchidectomized Sheep1

“…We have noted in previous studies that the concentrations of GnRH receptor and GnRH receptor mRNA in pituitary tissue of full-fed wethers are sensitive to estrogenic stimulation [16,18]. Interestingly, this estrogen-induced response is evident, and even amplified, during periods of moderate nutrient restriction.…”

“…We have recently demonstrated that estradiol (E 2 ) increases tissue concentrations of GnRH receptor and GnRH receptor mRNA in sheep fed ad libitum [16]. An objective of the present study was to examine the effect of nutritive status on the magnitude of this E 2 -induced response.…”

“…The minimum sensitivity of the LH and FSH assays was 0.1 and 0.25 nglml, respectively. Tissue concentrations of mRNA for the gonadotropin subunits and GnRH receptor mRNA were determined by use of the solution hybridization procedures described previously [16,221. Briefly, total RNA was isolated by homogenization of frozen blocks of hemipituitary tissue (150-300 mg) in 5 m14.0 M guanidinium thiocyanate.…”