Interactions between neurotensin and GnRH neurons in the positive feedback control of GnRH/LH secretion in the mouse

Abstract

In female mammals, increased ovarian estradiol (E(2)) secretion triggers GnRH release from neurons in the basal forebrain, which drives LH secretion from the pituitary and subsequently induces ovulation. However, the neural circuits that activate this preovulatory GnRH/LH surge remain unidentified. Neurotensin is expressed in neurons of the anteroventral periventricular nucleus (AVPV), a region thought to be critical for generating the preovulatory GnRH/LH surge. E(2) induces neurotensin (Nts) gene expression in this region, and blockade of neurotensin signaling reduces the LH surge in the rat. We postulated that neurotensin signaling plays a similar role in generating the E(2)-induced GnRH/LH surge in mice. We used in situ hybridization (ISH) to determine whether E(2) induces Nts expression in the mouse and found evidence to support this proposition. Next, we determined that the neurotensin receptor (Ntsr2) is present in many GnRH-expressing neurons. Since the kisspeptin gene (Kiss1) is expressed in the AVPV and is responsive to E(2), we predicted that some neurons in this region express both Kiss1 and Nts; however, by double-label ISH, we observed no coexpression of the two mRNAs. We also postulated that Nts mRNA expression would increase in parallel with the E(2)-induced LH surge and that the central (icv) administration of neurotensin would stimulate LH secretion and activation of GnRH neurons but found no evidence to support either of these hypotheses. Together, these findings suggest that, although neurotensin neurons in the AVPV are targets for regulation by E(2), neurotensin does not appear to play a direct role in generating the GnRH/LH surge in the mouse.

Fig. 1.

Double-label immunohistochemistry for gonadotropin-releasing hormone (GnRH; green) and neurotensin fibers (red) in the medial preoptic area (MPOA) (scale bars, 50 μm; A) and median eminence (scale bars, 100 μm; B). 3V, 3rd ventricle.

Fig. 2.

A: photomicrograph of GnRH and neurotensin receptor 1 (Ntsr1) in situ hybridization (ISH) in the rostral hypothalamus. Red arrows indicate neurons expressing GnRH mRNA. White arrows indicate neurons expressing Ntsr1 mRNA. Scale bar, 100 μm. B: number of GnRH cells coexpressing Ntsr1 mRNA in Sham- or estradiol-treated mice. Different letters indicate significant difference, P < 0.05. Data are expressed as means ± SE.

Fig. 3.

Double-label immunohistochemistry for GnRH (green) and neurotensin receptor 2 (NTR2; red) in the MPOA. White arrow indicates a cell expressing protein for both GnRH and Ntsr2. Scale bars, 50 μm. GFP, green fluorescent protein.

Fig. 4.

Number of cells expressing neurotensin (Nts) mRNA in the anteroventral periventricular (AVPV) and medial preoptic nuclei (MPN) of Sham- or estradiol-treated mice. Different letters indicate significant difference, P < 0.05. Data are expressed as means ± SE.

Fig. 5.

Number of cells expressing Nts mRNA in the AVPV and MPN of female mice in the morning (AM) and evening (PM). No significant differences were observed. Data are expressed as means ± SE.

Fig. 6.

Photomicrograph of Kiss1 and Nts ISH in the rostral hypothalamus. Red arrows indicate neurons expressing Kiss1 mRNA. White arrows indicate neurons expressing Nts mRNA. Few, if any, neurons express both mRNAs. Scale bar, 100 μm.

Fig. 7.

Serum LH levels in sham- and estradiol-treated mice after administration of vehicle, galanin-like peptide (GALP), or neurotensin. Different letters indicate significant difference, P < 0.05. Data are expressed as means ± SE.

Fig. 8.

Photomicrographs of GnRH and c-fos mRNA ISH in ovariectomized + sham mice treated with neurotensin (A) and GALP (B). Red arrows indicate cells expressing GnRH. White arrows indicate cells expressing c-fos. Scale bars, 100 μm. C: number of cells expressing c-fos mRNA in the AVPV/periventicular nucleus continuum. Different letters indicate significant difference, P < 0.05. Data are expressed as means ± SE.