Central somatostatin-somatostatin receptor 2 signaling mediates lactational suppression of luteinizing hormone release via the inhibition of glutamatergic interneurons during late lactation in rats

Abstract

Reproductive function is suppressed during lactation owing to the suckling-induced suppression of the kisspeptin gene (Kiss1) expression in the arcuate nucleus (ARC) and subsequent suppression of luteinizing hormone (LH) release. Our previous study revealed that somatostatin (SST) neurons mediate suckling-induced suppression of LH release via SST receptor 2 (SSTR2) in ovariectomized lactating rats during early lactation. This study examined whether central SST-SSTR2 signaling mediates the inhibition of ARC Kiss1 expression and LH release in lactating rats during late lactation and whether the inhibition of glutamatergic neurons, stimulators of LH release, is involved in the suppression of LH release mediated by central SST-SSTR2 signaling in lactating rats. A central injection of the SSTR2 antagonist CYN154806 (CYN) significantly increased ARC Kiss1 expression in lactating rats on day 16 of lactation. Dual in situ hybridization revealed that few ARC Kiss1-positive cells co-expressed Sstr2, and some of the ARC Slc17a6 (a glutamatergic neuronal marker)-positive cells co-expressed Sstr2. Furthermore, almost all ARC Kiss1-positive cells co-expressed Grin1, a subunit of N-methyl-D-aspartate (NMDA) receptors. The numbers of Slc17a6/Sstr2 double-labeled and Slc17a6 single-labeled cells were significantly lower in lactating dams than in non-lactating rats whose pups had been removed after parturition. A central injection of an NMDA antagonist reversed the CYN-induced increase in LH release in lactating rats. Overall, these results suggest that central SST-SSTR2 signaling, at least partly, mediates the suppression of ARC Kiss1 expression and LH release by inhibiting ARC glutamatergic interneurons in lactating rats.

Keywords: Glutamate; Kisspeptin; Lactation; Luteinizing hormone; Somatostatin.

Fig. 1.

Effects of central administration of somatostatin receptor 2 (SSTR2) antagonist (CYN) on Kiss1 mRNA expression in the arcuate nucleus (ARC) of lactating rats. (a) Representative photomicrographs showing Kiss1 mRNA expression in the ARC of non-lactating rats injected with vehicle in the third ventricle (3V) and lactating rats injected with vehicle or CYN in the 3V 1 h before brain sampling. Insets indicate Kiss1-expressing cells at higher magnification. Scale bars in the insets represent 10 µm. (b) Number of Kiss1-expressing cells in the ARC of vehicle-treated non-lactating rats (closed column), vehicle-treated lactating rats (open column), and CYN-treated lactating rats (shaded column). Values are the means ± SEM. Numbers in each column indicate the number of animals used. Values marked with different letters are significantly different (P < 0.05, one-way ANOVA followed by Bonferroni test).

Fig. 2.

Co-expression of Sstr2 (SSTR2 gene) or Grin1, an N-methyl-D-aspartate (NMDA) receptor marker gene, in the ARC Kiss1-expressing cells of non-lactating rats. (a) Representative photomicrographs showing Sstr2- (magenta) and Kiss1- (green) expressing cells in the ARC of non-lactating rats. A dual-labeled cell marked with an arrowhead is shown as magnified images showing Sstr2- and Kiss1-positive and merged cells. Scale bars in the magnified images represent 10 µm. (b) Number of cells expressing Sstr2 alone (magenta), both Sstr2 and Kiss1 (magenta/green), or Kiss1 alone (green) in the ARC were quantified and shown in a stacked bar graph. Values are the means ± SEM. (c) Representative photomicrographs showing Grin1- (magenta) and Kiss1- (green) expressing cells in the ARC of non-lactating rats. A dual-labeled cell marked with an arrowhead is shown as a magnified image showing Grin1- and Kiss1-positive and merged cells. (d) Number of cells expressing both Grin1 and Kiss1 (magenta/green) or Kiss1 alone (green) in the ARC were quantified and are shown in a stacked bar graph. Values are the means ± SEM.

Fig. 3.

Sstr2 (SSTR2 gene) and Slc17a6 (a glutamatergic neuronal marker gene) expression in the ARC of non-lactating and lactating rats. (a) Representative photomicrographs showing Sstr2- (magenta) and Slc17a6- (green) expressing cells in the ARC of vehicle-treated non-lactating rats, and vehicle- or CYN-treated lactating rats. A dual-labeled cell marked with arrowheads was shown as magnified images showing Sstr2- and Slc17a6-positive and merged cells. Scale bars in the magnified images represent 10 µm. (b) Number of cells expressing Sstr2 alone (magenta), both Sstr2 and Slc17a6 (magenta/green), or Slc17a6 alone (green) in the ARC of each group were quantified and are shown in a stacked bar graph. Values are shown as the means ± SEM. Values marked with an asterisk are significantly different (P < 0.05, one-way ANOVA followed by Bonferroni test) in the number of Slc17a6-expressing cells in the ARC from that in the non-lactating group. Values marked with different letters are significantly different (P < 0.05, one-way ANOVA followed by Bonferroni test) from each other in the number of the ARC cells co-expressing Sstr2 and Slc17a6.

Fig. 4.

Effects of the 3V injection of SSTR2 antagonist (CYN) with or without co-administration of glutamatergic NMDA receptor antagonist (MK801) on LH secretion in lactating rats on day 16 of lactation. (a) Profile of plasma LH levels in lactating rats treated with vehicle or CYN in combination with vehicle (CSF) or MK801 immediately after the onset of blood sampling (arrows). Arrowheads indicate the peaks of LH pulses detected by the PULSAR computer program. Mean LH concentrations (b) and the frequency (c) and amplitude of LH pulses (d) of lactating rats in each group. Open, shaded, and hatched bars show values (means ± SEM) in vehicle-, CYN-, and CYN/MK801-treated lactating rats, respectively. Numbers in each column indicate the number of animals used. Values marked with different letters are significantly different (P < 0.05, one-way ANOVA followed by Bonferroni test) from each other. The amplitude of LH pulses between vehicle- and CYN-treated lactating rats is significantly different (* P = 0.005, Student’s t-test).

Fig. 5.

Schematic showing the possible brain mechanism underlying suckling stimulus-induced inhibition of ARC Kiss1 expression and consequent GnRH/LH pulses in lactating rats based on the present and previous studies [16]. The suckling stimulus seems to activate the PIL SST neurons, and the resultant inhibitory SST-SSTR2 signaling may result in the inhibition of Kiss1 expression in KNDy neurons and subsequent GnRH/LH pulses via direct (on kisspeptin neurons) and indirect (on stimulatory glutamatergic neurons) pathways. Glu, Glutamatergic; GluR, Glutamatergic receptor.