The mechanism responsible for the supraphysiologic gonadotropin surge in females treated with gonadotropin-releasing hormone (GnRH) agonist and primed with GnRH antagonist

Abstract

Objective: To elucidate the physiologic mechanism responsible for the supraphysiologic gonadotropin release from the pituitary induced by gonadotropin-releasing hormone (GnRH) agonist in female rats primed with GnRH antagonist.

Design: Controlled experimental intervention.

Setting: Government research facility.

Animal(s): Forty 8-week-old Sprague-Dawley rats.

Intervention(s): Forty oophorectomized rats were randomized into four treatment groups of 10: group A, control vehicles; group B, GnRH agonist (leuprolide acetate; 1.7 microg/kg twice a day) on day 4; group C, GnRH antagonist (Nal-Lys; 3 mg/kg each day) days 1 to 4; or group D, GnRH antagonist (Nal-Lys; 3 mg/kg each day) days 1 to 4 plus GnRH agonist (1.7 microg/kg twice a day) on day 4.

Main outcome measure(s): Immunohistochemical methods, Northern and in situ hybridization to quantitate pituitary follicle-stimulating hormone beta (FSH-beta), luteinizing hormone beta (LH-beta), and GnRH receptor (GnRH-R) messenger RNA (mRNA), and receptor protein levels in all treatment groups.

Result(s): Treatment with GnRH antagonist was associated with increased storage of gonadotropin in the pituitary for FSH-beta and LH-beta, but mRNA levels were unchanged. The GnRH-R mRNA decreased after GnRH-agonist treatment but remained stable in the GnRH-antagonist treatment groups. Levels of GnRH-R were decreased after GnRH-antagonist treatment.

Conclusion(s): These data indicate that the in vivo mechanism responsible for the exaggerated release of gonadotropins in rats primed with GnRH antagonist and treated with GnRH agonist was an increase in releasable gonadotropin pools coupled with a reduction in GnRH-R, but receptor function was preserved.

Figure 1

Diagram of the experimental protocol. Eight-week-old ovariectomized Sprague-Dawley rats were randomized to 4 treatment groups as follows: the control Group A was treated with both vehicles and received a daily morning subcutaneous injection of corn oil on days 1–4 and two subcutaneous injections of saline given 12 hours apart on day 4; Group B received 2 doses of GnRH-a, leuprolide acetate (1.7μg/kg) diluted in saline 12 hours apart on day 4; Group C received subcutaneous injections of 3mg/kg GnRH-ant (Nal-Lys; Antide) dissolved in corn oil daily on days 1–4; and Group D received daily morning injections of GnRH-ant on days 1–4 and 2 doses of GnRH-a on day 4 given 12 hours apart. All animals were sacrificed on the fifth day, 12 hours after the second dose of leuprolide acetate. Black up-arrows indicate GnRH-ant, white up-arrows indicate GnRH-a and boxes represent vehicles, corn oil (C) and saline (S).

Figure 2

Figure 2A. Immunohistochemical staining for gonadotropins. Pituitary sections stained for FSH-β (A-D) from OVX rats after treatment with: GnRH-a alone (B); GnRH-ant alone (C); both (D); or neither (A). In all samples antibody-antigen interaction was visualized as brown staining. FSH-β staining was visibly increased after treatment with GnRH-ant alone (C). (A–D) Original magnification, X 400. Figure 2B. Quantification of Immunohistochemical staining for FSH and LH using computer-based density quantification software. The relative intensity of the immunoreaction was estimated by measurement of the diaminobenzidine peroxidase product from pituitaries of all treatment groups using light microscopic image analysis as described in materials and methods. Groups demonstrated statistically significant increased pituitary pools of FSH-β and LH-β in rats treated with GnRH-ant alone compared to control (p < 0.05). Dark bars represent FSH. Light bars represent LH. Y-axis=optical density units. Error bars=standard deviation. Significance, compared to the control group, was assumed at p < 0.05 (all asterisked values).

Figure 2

Figure 2A. Immunohistochemical staining for gonadotropins. Pituitary sections stained for FSH-β (A-D) from OVX rats after treatment with: GnRH-a alone (B); GnRH-ant alone (C); both (D); or neither (A). In all samples antibody-antigen interaction was visualized as brown staining. FSH-β staining was visibly increased after treatment with GnRH-ant alone (C). (A–D) Original magnification, X 400. Figure 2B. Quantification of Immunohistochemical staining for FSH and LH using computer-based density quantification software. The relative intensity of the immunoreaction was estimated by measurement of the diaminobenzidine peroxidase product from pituitaries of all treatment groups using light microscopic image analysis as described in materials and methods. Groups demonstrated statistically significant increased pituitary pools of FSH-β and LH-β in rats treated with GnRH-ant alone compared to control (p < 0.05). Dark bars represent FSH. Light bars represent LH. Y-axis=optical density units. Error bars=standard deviation. Significance, compared to the control group, was assumed at p < 0.05 (all asterisked values).

Figure 3

Northern Blot Analysis. RNA harvested from OVX rats following treatment with GnRH-a (Group B), GnRH-ant (Group C) or both (Group D) were probed with cDNA as described in the methods. Ribosomal RNA was used as a control for loading. Levels of steady state FSH and LH transcripts remained constant regardless of treatment group. Steady state level GnRH-R transcripts were reduced following GnRH-a treatment (Group B).

Figure 4

In situ hybridization of pituitaries from treatment groups (A-D) using a riboprobe generated from a cDNA encoding GnRH-R. Compared to the control group (A), GnRH-R transcript levels showed a reduction in staining in the GnRH-a treatment group (B). No reduction was observed in the GnRH-ant treatment group (C) or the combination group (D). Sense riboprobe (E) served as control.

Figure 5

Levels of GnRH-R protein in pituitary homogenates from treatment groups AD. The level of GnRH-R increased following treatment with GnRH-a (Group B) and decreased in both the GnRH-ant and combination groups (Groups C and D). Y-axis=GnRH-R binding in fentamolar/gram of pituitary lysate. Error bars=standard deviation. Significance was assumed at p < 0.05 for all treatment groups compared to the control.