Pituitary gonadotropin-releasing hormone II as a possible mediator of positive estrogen feedback

Abstract

It has previously been shown that rhesus macaques express two forms of gonadotropin-releasing hormone (GNRH1 and GNRH2) in the hypothalamus and that both forms can stimulate the release of luteinizing hormone (LH) in vivo. However, while much has been published about the role of GNRH1 in reproduction, very little is known about the hypophysiotropic function of GNRH2. To shed light on this issue, we studied the expression pattern of these two genes in different parts of the monkey hypothalamus and pituitary gland under controlled conditions of circulating estrogen levels, using qPCR, liquid chromatography with tandem mass spectrometry and RNAscope. GNRH1/GNRH1 expression was found throughout the hypothalamus and was largely unaffected by circulating estradiol levels. In contrast, GNRH2/GNRH2 expression was found to be enhanced by long-term treatment with estradiol and during the late follicular phase of the menstrual cycle, especially in the arcuate nucleus and pituitary gland. Together these findings suggest that pituitary GNRH2/GNRH2 (but not GNRH1/GNRH1) is induced by positive feedback-like levels of estradiol. This novel finding raises the possibility that GNRH2 plays a major role in triggering the preovulatory LH surge in primates, not only at the level of the hypothalamus but also the pituitary gland.

Keywords: GNRH2; LC‐MS/MS; estradiol; gonadotropin‐releasing hormone type II.

FIGURE 1

GNRH2 is expressed throughout the female hypothalamus and pituitary gland. (A) Schematic representation of the hypothalamic nuclei isolated by micro‐dissection for the quantitation of gene expression. Suprachiasmatic nucleus (SCN), supraoptic nucleus (SON), paraventricular nucleus (PVN), arcuate nucleus (ARC), median eminence (ME), optic chiasm (OC), optic tract (OT), third ventricle (3 V). (B) Heatmap of gene expression levels throughout the hypothalamus and pituitary gland (PIT). Each colored square represents the average of 3 animals. Luteinizing Hormone Subunit Beta (LHB), Arginine Vasopressin (AVP), Oxytocin (OXT), Gonadotropin Releasing Hormone 1 (GNRH1) and Gonadotropin Releasing Hormone 2 (GNRH2). (C) GNRH1 and GNRH2 mRNA expression throughout the hypothalamus and PIT. Vertical bars are means ± SEM and gray circles represent individual values (n = 3). Expression data were normalized by dividing each individual value by the highest point in the dataset.

FIGURE 2

Pituitary concentration of GNRH2, but not GNRH1, is increased by estrogen treatment. GNRH1 and GNRH2 mRNA expression determined by qRT‐PCR in the PVN, ARC and PIT in (OVX) control (C) and OVX + E2 (E) treated animals. E2 concentrations at the end of the average 2.7 years treatment where 5.2 ± 1.8 pg/mL for the C group and 64.3 ± 7.2 pg/mL in the E group, t(12) = 7.961, p < .0001. Data are expressed in arbitrary units; each data point was corrected with the expression of the housekeeping gene (GAPDH) and normalized by dividing each individual value by the average of the C group. Experimental groups were subjected to arc‐sine transformation to convert them from a binomial to a normal distribution before statistical analysis. Student t‐test was used when comparing two groups. Vertical bars are means ± SEM and gray circles represent individual values (n = 5–7). **p < .01 and ***p < .001 versus C group (Student's t‐test). All ∆CT values are available at the Table S1.

FIGURE 3

Pituitary concentration of GNRH2, but not GNRH1, is increased by estrogen treatment. GNRH1 and GNRH2 peptide levels in the ARC and PIT (expressed as pg/mg tissue) detected by LC‐MS/MS. Vertical bars are means ± SEM and gray circles represent individual values (n = 5–7). Two‐way ANOVA (main effects GNRH isoform and endocrine status) followed by Fisher's LSD test. ARC: GNRH isoform factor F(1,20) = 15.22, #p = .0009; endocrine status factor F(1,20) = 0.947, p = .342. PIT: GNRH isoform factor F(1,18) = 1.541.22, p = .2303; endocrine status factor F(1,18) = 13.50, **p = .001.

FIGURE 4

Pituitary concentrations of GNRH2/GNRH2, but not GNRH1/GNRH1, increases during the late follicular phase of the menstrual cycle. (A) GNRH1 and GNRH2 mRNA expression determined by qRT‐PCR in the PIT during the early follicular (EF), late follicular (LF) and mid‐luteal (ML) phase of the menstrual cycle. Data are expressed in arbitrary units; each data point was corrected with the expression of the housekeeping gene (GAPDH) and normalized by dividing each individual value by the average of the EF group. Experimental groups were subjected to arc‐sine transformation to convert them from a binomial to a normal distribution before statistical analysis. Vertical bars are means ± SEM and gray circles represent individual values (n = 3–4). *p < .05 and **p < .01 (One‐way ANOVA followed by Tukey's multiple comparisons test). All ∆CT values are available at the Table S1. (B) GNRH1 and GNRH2 peptide levels (expressed as pg/mg tissue) detected by LC‐MS/MS. Vertical bars are means ± SEM and gray circles represent individual values (n = 3–4). *p < .05 and **p < .01. Two‐way ANOVA (main effects GnRH isoform and endocrine status) followed by Tukey's multiple comparison test. GNRH isoform factor (F(1,16) = 8.971, #p = .0086: Menstrual phase Factor F(2,16) = 4.25, p = .033), GNRH2 peptide EF vs. LF, **p = .0056 and LF vs. ML, *p = .01. (EF): E2 = 32.33 ± 8.95 pg/mL and P4 = 0.08 ± 0.03 ng/mL; (LF): E2 = 106.5 ± 48.14 pg/mL and P4 = 0.3 ± 0.03 ng/mL; (ML): E2 = 39.25 ± 5.40 pg/mL and P4 = 4.93 ± 0.84 ng/mL.

FIGURE 5

Estrogen treatment increases GNRH2 mRNA in pituitary gonadotropes as determined by RNAscope. (A, D) GNRH2 mRNA expression in the pituitary gland is extremely low in a mid‐luteal (ML) phase female monkey, but increasing higher in ovariectomized (OVX) animals that had been treated with estradiol benzoate (EB) either 24 h (B, E) or 48 h earlier (C, F). (G) Neurons positive for GNRH2 mRNA expression in the ARC as described by us previously., , (H) Negative GNRH2 mRNA detection in the intestine demonstrates the specificity of GNRH2 RNAscope probe. (I) Signal for the positive control probe for Peptidylprolyl Isomerase B (PPIB) in the PIT. (J–M) Colocalization of GNRH2 mRNA by RNAscope followed and LHβ by immunofluorescence (IF). DAPI nuclear stain (J), GNRH2 mRNA in red (K), LHβ IF in green (L) and overlay of J–L (M). Neurohypophysis (Neuro), Adenohypophysis (Adeno), Arcuate nucleus (ARC). Scale bars are 500 μm in panels (A–C), and 100 μm in panels (D–M).