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. 1997 Dec 1;17(23):9145-56.
doi: 10.1523/JNEUROSCI.17-23-09145.1997.

Estradiol enhances prostaglandin E2 receptor gene expression in luteinizing hormone-releasing hormone (LHRH) neurons and facilitates the LHRH response to PGE2 by activating a glia-to-neuron signaling pathway

F Rage  1 , B J Lee, Y J Ma, S R Ojeda
Affiliations

Estradiol enhances prostaglandin E2 receptor gene expression in luteinizing hormone-releasing hormone (LHRH) neurons and facilitates the LHRH response to PGE2 by activating a glia-to-neuron signaling pathway

F Rage et al. J Neurosci. .

Abstract

Prostaglandin E2 (PGE2) mediates the stimulatory effect of norepinephrine (NE) on the secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling reproductive function. In rodents, this facilitatory effect requires previous exposure to estradiol, suggesting that the steroid affects downstream components in the cascade that leads to PGE2-induced LHRH release. Because astroglia are the predominant cell type contacting LHRH-secreting nerve terminals, we investigated the involvement of hypothalamic astrocytes in the estradiol facilitation of PGE2-induced LHRH release. A subpopulation of LHRH neurons was found to express the mRNA encoding the PGE2 receptor subtype EP1-R, which is coupled to calcium mobilization. The LHRH-producing cell line GT1-1 also contains EP1-R mRNA and, to a lesser extent, the three alternatively spliced forms of EP3-R mRNA (alpha, beta, and gamma) that encode receptors linked to inhibition and stimulation of cAMP formation. Hypothalamic astrocytes treated with estradiol produced a conditioned medium that when applied to GT1-1 cells resulted in a selective upregulation of EP1-R and EP3gamma-R mRNAs. The conditioned medium also enhanced the LHRH response to EP1-R and EP3-R agonists and the cAMP response to EP3-R activation. Thus, one mechanism by which estradiol facilitates the effect of neurotransmitters acting via PGE2 to stimulate LHRH release is by enhancing the glial production of substances that upregulate PGE2 receptors on LHRH neurons. The existence of such a mechanism underscores the emerging importance of glial-neuronal communication in the control of brain neurosecretory activity.

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Figures

Fig. 3.
Fig. 3.
PCR cloning of cDNAs encoding the alternatively spliced products of the EP3-R gene from GT1–1 cells. A, mRNA location of the deoxyoligonucleotide primers used for amplification of the α, β, and γ forms of EP3-R mRNA.B, Ethidium bromide staining of the PCR products derived from the amplification of GT1–1 RNA using a pair of primers common for the mRNA sequences encoding the EP3α-R and EP3β-R mRNA forms.C, Ethidium bromide staining of the single PCR product derived from the amplification of GT1–1 RNA using a 5′-primer complementary to the mRNA sequence common to all forms of EP3-R mRNA and a 3′-primer complementary to the unique 3′-sequence in EP3γ-R mRNA. C, PCR control, no RNA input.
Fig. 1.
Fig. 1.
Detection of EP1-R mRNA on LHRH neurons of the mouse brain by combined immunohistochemistry–in situhybridization. The LHRH decapeptide was first detected by immunohistochemistry using a polyclonal antibody (see Materials and Methods). After completion of this procedure, the sections were hybridized with an 35S-UTP-labeled EP1-R cRNA; the reaction was developed after a 4 week exposure to NTB-2 emulsion.A, LHRH neurons dorsal to the anterior recess of the third ventricle showing a low (short arrow) to abundant (arrow) content of EP1-R mRNA. B, LHRH neurons in the vicinity of the organum vasculosum of the lamina terminalis (OVLT) showing either moderate EP1-R mRNA levels (arrows) or an undetectable hybridization signal (arrowhead). C, An LHRH neuron in the preoptic area showing moderate levels of EP1-R mRNA (arrow with circle). D, An LHRH neuron devoid of detectable EP1-R mRNA (arrowhead) located in the vicinity of unidentified cells containing EP1-R mRNA (double arrowheads). Scale bar, 10 μm.
Fig. 2.
Fig. 2.
Detection of prostaglandin E2 receptor mRNAs in GT1–1 cells by RNase protection assay. Lane 1, Undigested probes (UP); lanes 2, 3, 4, digested EP1-R, EP3β-R, and EP4-R cRNA probes, respectively (DP); lane 5, GT1–1 RNA hybridized to the EP1-R cRNA probe alone; lane 6, GT1–1 mRNA hybridized to the EP4-R cRNA probe alone; lane 7, GT1–1 RNA hybridized to the EP3β-R cRNA probe alone; lane 8, GT1–1 RNA hybridized to both the EP1-R and EP3β-R probes;lane 9, kidney (Kd) RNA hybridized to both probes; and lane 10, lung (Lu) RNA hybridized to all three probes.
Fig. 4.
Fig. 4.
Quantitation of EP3-R mRNA alternatively spliced forms by quantitative RT–PCR. Top left, Ethidium bromide staining of PCR products obtained from the amplification of increasing amounts of in vitro transcribed EP3α-R and EP3β-R mRNAs, corresponding to the same cellular sequence targeted for amplification. Bottom, Standard curves generated by regression analysis of the PCR signals (top left) and used to estimate the content of EP3α and EP3β-R mRNAs in GT1–1 cells. Top right, Example of an RT–PCR assay demonstrating the ability of the assay to detect simultaneously three mRNAs in GT1–1 cells [in this case EP3α-R, EP3β-R, and cyclophilin (p1B15)]. Cyclophilin mRNA was used to normalize the EP3-R mRNA values detected in the assay (see Materials and Methods); in all cases the amount of total RNA reverse transcribed for PCR amplification was 100 ng. LN (OD), Natural logarithm of optical density.
Fig. 5.
Fig. 5.
Increase in EP1-R mRNA levels in GT1–1 cells by exposure to culture medium derived from astrocytes treated for 24 hr with 17β-estradiol (1 nm;CM-17β-E2).CM-17α-E2, Medium from astrocytes treated with the inactive stereoisomer 17α-estradiol;CM-ETOH, medium from astrocytes treated with ethanol. Numbers above the bars are the number of independent observations per group; error bars indicate SEM; and *p < 0.05, 17β-estradiol versus control group(s).
Fig. 6.
Fig. 6.
Selective increase in EP3γ-R mRNA levels in GT1–1 cells by exposure to culture medium derived from astrocytes treated for 24 hr with 17β-estradiol (1 nm;CM-17β-E2).CM-17α-E2, Medium from astrocytes treated with 17α-estradiol; CM-ETOH, medium from astrocytes treated with ethanol. Numbers above the barsare the number of independent observations per group; error bars indicate SEM; and *p < 0.01, 17β-estradiol versus control group(s).
Fig. 7.
Fig. 7.
A, Inability of 17β-estradiol to directly affect EP1-R and EP3γ-R mRNA levels in GT1–1 cells.B, Absence of detectable estrogen receptor (ER) mRNA in GT1–1 cells as assessed by RNase protection assay after a 19 hr film exposure. Lane 1, Radiolabeled RNA standards; lane 2, undigested ER cRNA probe; lane 3, digested probe; lane 4, GT1–1 RNA (10 μg); lanes 5, 6, RNA (10 μg) from the suprachiasmatic region of the mouse brain. C, Detection of low ER mRNA levels (arrow) in GT1–1 cells after a longer (96 hr) film exposure. Notice the presence of more abundant, unidentified protected bands of lower molecular size.
Fig. 8.
Fig. 8.
Facilitatory effect of astrocyte culture medium conditioned by a 24 hr exposure to 17β-estradiol (1 nm;CM-17β-E2) on the LHRH release induced by 17-phenyl trinor PGE2, a selective EP1-R agonist, from GT1–1 cells. CM-17α-E2, Astrocyte medium conditioned by 17α-estradiol; CM-ETOH, Astrocyte medium with diluent. The cells were treated with the different CMs for 3 hr before a 30 min exposure to the agonist.Inset, Blockade of the facilitatory effect of CM-17β-E2 on 17-phenyl trinor PGE2-induced LHRH release by the EP1-R antagonist AH-6809. Data are mean ± SEM; each group consists of 6–12 independent observations; *p < 0.05, CM-17β-E2 group versus either the untreated or the other two CM-treated control groups;inset *p < 0.02, AH-6809 group versus group treated with only 17-phenyl trinor PGE2.
Fig. 9.
Fig. 9.
Facilitatory effect of astrocyte culture medium conditioned by a 24 hr exposure to 17β-estradiol (CM-17β-E2) on cAMP formation (A) and LHRH release (B) induced by the treatment of GT1–1 cells with the EP3-R and EP1-R agonist sulprostone. CM-17α-E2, Medium from astrocytes treated with 17α-estradiol. The GT1–1 cells were pretreated with the CMs for 3 hr before a 30 min exposure to sulprostone; *p < 0.05,CM-17β-E2-pretreated groups versus control groups pretreated with CM-17α-E2and the control group not exposed to sulprostone. In the case ofCM-17α-E2-pretreated cultures versus controls not challenged with sulprostone, *p < 0.05.
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