Fourth ventricular alloxan injection suppresses pulsatile luteinizing hormone release in female rats

Previous studies have suggested the presence of a glucose-sensing mechanism in the hindbrain that appears to regulate reproductive function as well as feeding behavior. The ependymocytes lining the ventricular wall of the hindbrain showed immunoreactivities to pancreatic glucokinase (GK), a key enzyme for glucose sensing in pancreatic B cells. Our goal in the present study was to test whether the GK-immunopositive ependymocytes in the wall of the fourth cerebroventricle (4V) play a role in regulating gonadal activity. Our approach was to determine the effect of injecting alloxan, a GK inhibitor, into the 4V on pulsatile luteinizing hormone (LH) secretion. Estrogen-primed ovariectomized rats received an injection of alloxan (10 or 20 microg/animal) into the 4V and blood samples were collected every 6 min for 3 h for measurement of blood LH, corticosterone and glucose levels. Pulsatile LH secretion was suppressed after alloxan injection and all pulse parameters were significantly (P<0.05) inhibited by 20 microg alloxan. Plasma corticosterone levels were increased significantly (P<0.05) by 20 microg alloxan, suggesting that LH pulse suppression by alloxan may be at least partly mediated by activation of the hypothalamo-pituitary-adrenal axis. The present results suggest that acute suppression of GK activity in the hindbrain inhibits pulsatile LH secretion in female rats, and supports the idea that GK-immunopositive ependymocytes may sense glucose levels in the cerebrospinal fluid and play a role in regulation of LH secretion.     

Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats

Metastin/kisspeptin, the KiSS-1 gene product, has been identified as an endogenous ligand of GPR54 that reportedly regulates GnRH/LH surges and estrous cyclicity in female rats. The aim of the present study was to determine if metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges. We demonstrated that preoptic area (POA) infusion of the anti-rat metastin/kisspeptin monoclonal antibody blocked the estrogen-induced LH surge, indicating that endogenous metastin/kisspeptin released around the POA mediates the estrogen positive feedback effect on GnRH/LH release. Metastin/kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) may be responsible for mediating the feedback effect because the percentage of c-Fos-expressing KiSS-1 mRNA-positive cells to total KiSS-1 mRNA-positive cells was significantly higher in the afternoon than in the morning in the anteroventral periventricular nucleus (AVPV) of high estradiol (E(2))-treated females. The percentage of c-Fos-expressing metastin/kisspeptin neurons was not different between the afternoon and morning in the arcuate nucleus (ARC). Most of the KiSS-1 mRNA expressing cells contain ERalpha immunoreactivity in the AVPV and ARC. In addition, AVPV KiSS-1 mRNA expressions were highest in the proestrous afternoon and lowest in the diestrus 1 in females and were increased by estrogen treatment in ovariectomized animals. On the other hand, the ARC KiSS-1 mRNA expressions were highest at diestrus 2 and lowest at proestrous afternoon and were increased by ovariectomy and decreased by high estrogen treatment. Males lacking the surge mode of GnRH/LH release showed no obvious cluster of metastin/kisspeptin-immunoreactive neurons in the AVPV when compared with high E(2)-treated females, which showed a much greater density of these neurons. Taken together, the present study demonstrates that the AVPV metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges in female rats.     

Central injection of ketone body suppresses luteinizing hormone release via the catecholaminergic pathway in female rats

Ketosis is found in various pathophysiological conditions, including diabetes and starvation, that are accompanied by suppression of gonadal activity. The aim of the present study was to determine the role of ketone body in the brain in regulating pulsatile luteinizing hormone (LH) secretion in female rats. Injection of 3-hydroxybutyrate (3HB), a ketone body, into the fourth cerebroventricle (4V) induced suppression of pulsatile LH secretion in a dose-dependent manner in ovariectomized (OVX) rats with an estradiol (E2) implant producing diestrus plasma E2 levels. Plasma glucose and corticosterone levels increased immediately after the 4V 3HB injection, suggesting that the treatment caused a hunger response. The 3HB-induced suppression of LH pulses might be mediated by noradrenergic inputs to the hypothalamic paraventricular nucleus (PVN) because a local injection of α-methyl- p-tyrosine, a catecholamine synthesis inhibitor, into the PVN blocked 3HB-induced suppression of LH pulses and PVN noradrenaline release was increased by 4V 3HB injection in E2-primed OVX rats. These results suggest that ketone body sensed by a central energy sensor in the hindbrain may suppress gonadotropin release via noradrenergic inputs to the PVN under ketosis.     

Progesterone concentration,estradiol pretreatment,and dose of gonadotropin-releasing hormone affect gonadotropin-releasing hormone-mediated luteinizing hormone release in beef heifers

We examined whether progesterone (P₄)-induced suppression of LH release in cattle can be overcome by an increased dose of exogenous gonadotropin-releasing hormone (GnRH) or pretreatment with estradiol (E₂). In Experiment 1, postpubertal Angus-cross heifers (N = 32) had their 2 largest ovarian follicles ablated 5 d after ovulation. Concurrently, these heifers were all given a once-used, intravaginal P₄-releasing insert (CIDR), and they were randomly assigned to be given either prostaglandin F_2α (Low-P₄) or no treatment (High-P₄) at follicle ablation, and 12 h later. Six days after emergence of a new follicular wave, half of the heifers in each group (n = 8) were given either 100 or 200 μg of GnRH i.m. Plasma luteinizing hormone (LH) concentrations were higher in the Low- vs High-P₄ groups, and in heifers given 200 vs 100 μg of GnRH (mean ± SEM 15.4 ± 2.2 vs 9.1 ± 1.2, and 14.8 ± 2.1 vs 9.8 ± 1.4 ng/mL, respectively; P ≤ 0.01). Ovulation rate was higher (P = 0.002) in the Low-P₄ group (15/16) than in the High-P₄ group (6/16), but it was not affected by GnRH dose (P = 0.4). In Experiment 2, heifers (n = 22) were treated similarly, except that 5.5 d after wave emergence, half of the heifers in each group were further allocated to be given either 0.25 mg estradiol benzoate i.m. or no treatment, and 8 h later, all heifers were given 100 μg GnRH i.m. Both groups treated with E₂ (Low- and High-P₄) and the Low-P₄ group without E₂ had higher peak plasma LH concentrations compared to the group with high P₄ without E₂ (12.6 ± 1.8, 10.4 ± 1.8, 8.7 ± 1.3, and 3.9 ± 1.2 ng/mL, respectively; (P < 0.04)). However, E₂ pretreatment did not increase ovulation rates in response to GnRH (P = 0.6). In summary, the hypotheses that higher doses of GnRH will be more efficacious in inducing LH release and that exogenous E₂ will increase LH release following treatment with GnRH were supported, but neither significantly increased ovulation rate.     

Role of noradrenergic receptors in the bed nucleus of the stria terminalis in regulating pulsatile luteinizing hormone secretion in female rats

The bed nucleus of the stria terminalis (BNST) is one of the brain areas densely innervated by noradrenergic neurons originating in the brain stem. The present study aims to determine the role of noradrenergic receptors in the BNST in regulating pulsatile luteinizing hormone (LH) secretion in female rats. Ovariectomized (OVX) or estrogen-primed OVX (OVX+E2) rats received three 1-h-interval injections of 0.05 micromol of noradrenaline (NA), phenylephrine (alpha1-adrenergic receptor agonist), clonidine (alpha2-agonist), or isoproterenol (beta-agonist) into the BNST. Injection of NA or alpha1-adrenergic agonist into the BNST strongly suppressed pulsatile LH secretion in OVX+E2 rats with a significant (P < 0.05) decrease in the mean LH level for 3 h and LH pulse frequency, but alpha2-and beta-agonists did not affect any of the LH pulse parameters. In OVX animals, alpha1- and alpha2-adrenergic agonists caused a significant change in LH pulse frequency and amplitude, respectively, though the effect was not as apparent as the NA- or alpha1-agonist-induced changes in OVX+E2 animals. These results indicate that NA inputs to the BNST suppress pulsatile LH secretion via alpha1-adrenergic receptors and that estrogen enhances this suppression.     

Bovine C-terminal octapeptide of RFamide-related peptide-3 suppresses luteinizing hormone (LH) secretion from the pituitary as well as pulsatile LH secretion in bovines

Gonadotropin-inhibiting hormone (GnIH), observed in quail as a member of the RFamide neuropeptide family, suppresses luteinizing hormone (LH) secretion from the avian pituitary. Rats and cattle have an active gene of another member of the RFamide neuropeptide family, termed RFamide-related peptide-3 (RFRP-3), although bovine RFRP-3 is different from that of rats in both length and amino-acid sequence. A single injection of GnIH or RFRP-3 inhibited LH secretion in rodents, which continued for various periods. This study was conducted to evaluate the effects of bovine C-terminal octapeptide of RFRP-3 (RFRP-3-8) on LH secretion from cultured anterior pituitary (AP) cells of cattle, and the effects of RFRP-3-8 injections on pulsatile LH secretion in castrated male calves. The suppressive effect of RFRP-3-8 on LH secretion from AP cells was observed in the presence of gonadotropin-releasing hormone (GnRH), but not in the absence of GnRH in culture media. In another experiment collecting blood samples serially from castrated male calves with repeated intravenous injections of RFRP-3-8 (n = 6) or saline (n = 6), the RFRP-3-8 group showed suppressed LH pulse frequency during the injection period (P < 0.05); however, the RFRP-3-8 group showed no difference from the saline group in all measures of LH secretion in the postinjection period. In conclusion, our results suggested that RFRP-3-8 suppresses LH secretion from cultured AP cells, as well as LH pulse frequency in cattle.     

Kisspeptin‐10 stimulates the release of luteinizing hormone and testosterone in pre‐ and post‐pubertal male goats

The aims of the present study were to clarify the effect of kisspeptin‐10 (Kp10) on the secretion of luteinizing hormone (LH) and testosterone (T) in pre‐pubertal and post‐pubertal male ruminants. Four male goats (Shiba goats) were given an intravenous (i.v.) injection of Kp10 (5 µg/kg body weight (b.w.)), gonadotoropin‐releasing hormone (GnRH, 1 µg/kg b.w.), or 2 mL of saline as a control at the ages of 3 (pre‐pubertal) and 6 (post‐pubertal) months. A single i.v. injection of Kp10 significantly stimulated the release of LH and T in both groups. The area under the response curve (AUC) of LH for a 60‐min period after the i.v. injection of Kp10 was significantly greater in the pre‐pubertal goats (P < 0.05). The AUC of T for a 120 min period post‐injection did not differ between the two age groups. A single i.v. injection of GnRH also significantly stimulated the release of LH and T in both groups (P < 0.05). The secretory pattern of LH and T in response to GnRH resembled that in response to Kp10. These results show that the LH‐releasing response to Kp10 is greater in pre‐pubertal than post‐pubertal male goats. They also show that Kp10, as well as GnRH, is able to stimulate the release of T in male goats.     

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

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.