Control Of GnRH Release Research Articles

OR06-1 Melanocortins Act on Kiss1 Neurons to Regulate Reproduction in Females

Reproduction, controlled by the excitatory action of the neuropeptide kisspeptin, is an energy costly function for the organism and, therefore, it is tightly regulated by metabolic factors. Among those, hypothalamic melanocortins are essential in the control of metabolism. Inactivating mutations in the proopiomelanocortin (Pomc) gene or melanocortin receptor 4 (Mc4r) cause severe obesity and subfertility in humans and mice. However, whether the reproductive impairment is a direct consequence of the lack of melanocortin signaling, or indirect resulting from the obesity phenotype, remains unknown. α-MSH (derived from Pomc) stimulates LH release and this action has been shown to be mediated by Kiss1 neurons. We evaluated the expression of Mc4r in Kiss1 neurons, and using dual fluorescence in situ hybridization (RNAscope), we showed that Kiss1 neurons of the arcuate nucleus (Kiss1ARC) and the anteroventral periventricular/periventricular nucleus (Kiss1AVPV/PeN) express Mc4r. Mc4r colocalized with 84% of Kiss1ARC and with 74% of Kiss1AVPV/PeN, suggesting that Kiss1 neurons are a direct target of melanocortins. In order to evaluate the involvement of Kiss1 neurons in the reproductive role of melanocortins, we ablated Mc4r specifically from Kiss1 neurons by generating a specific Kiss1-Mc4rKO (Kiss1-cre+/Mc4r-lox+/+) mouse line. We found that Kiss1-Mc4rKO females exhibit disrupted estrous cycles with prolonged diestrous phases and delayed puberty onset compared to their control littermates, without developing obesity during the time of the study. The reproductive phenotype of Kiss1-Mc4rKO males was not affected. When central injections of an Mc4r selective agonist were performed on Kiss1-Mc4rKO intact males and their control littermates, LH levels were significantly increased to the same extend in both groups at 15 min post-injections, suggesting that, in the male, α-MSH controls LH levels through a Kiss1 neuron independent mechanism. Taken together, these results support a direct role of melanocortins on Kiss1 neurons in the control of GnRH release in female mice. Overall, understanding the neuroendocrine mechanisms underlying the metabolic regulation of reproduction will help improve the fertility rates in patients affected by metabolic disturbances. Keywords: Kisspeptin, α-MSH, Melanocortins, Mc4r, Reproduction

Novel Neuropeptides Involved in the Control of Puberty and Reproduction in Fish

Introduction: The findings that inactivation of kisspeptin signaling, because of mutations in the kisspeptin receptor, is associated with hypogonadotropic hypogonadism and absent or delayed puberty in man [1] stimulated the many subsequent studies that together have led to a new view of the neural control of GnRH release [2]. With this in mind, the recent observations [3] that mutations of the genes encoding neurokinin B (NKB) (TAC3) and its receptor (TAC3R; NKBR) were also associated with hypogonadotropic hypogonadism, is therefore of considerable interest. In order to study the physiological function(s) and evolutionary conservation of NKB, we cloned tac3 and tac3 receptor cDNAs from several fish species, including zebrafish. Methods: Zebrafish tac3a, tac3b, tac3Ra and tac3Rb were cloned by synteny, according to Biran et al. [4]. A phylogenetic tree of all the currently known vertebrate neurokinin genes was generated with the neighborjoining method. Real-time PCR and receptor transactivation assays were performed as previously described [4]. Results and Discussion: A comparison of the zebrafish protein coding sequences of tac3 cDNAs with those of the human and mouse TAC3 proteins showed identities of 23% and 24%, respectively. The phylogenetic tree showed that the vertebrate neurokinin genes fall into two distinct lineage groups. One lineage includes the mammalian and rodent TAC3, and all the piscine Tac3 that were cloned in the present study. The second lineage includes the mammalian and fish tac1. Nonetheless, high identity was found between different fish species, in the region encoding the NKB; all shared the common C-terminal sequence. Many genes encoding the tachykinins have been found to encode a precursor that produces more than one tachykinin. However, in mammals TAC3 is unusual in that it encodes only a single tachykinin – the NKB. Interestingly, we have found that in zebrafish, exon 3 of both tac3s encodes a well conserved additional tachykinin. Furthermore, in silico analysis revealed that this new tachykinin exists in all other fish species whose genomes are known, and it was termed NKF. NKF also possesses the common C-terminal sequence FVGLM, known as the tachykinin signature. zftac3a-expressing neurons were localized in specific brain nuclei that are known to be implicated in reproduction, whereas zftac3b-expressing neurons were more dispersed throughout the brain. Zebrafish tac3a – but not tac3b – mRNA levels gradually increased during the first few weeks of life, and peaked in fish with ovaries containing mature oocytes or with testes containing mature spermatozoa. Estrogen treatment of mature fish causes increase in tac3a, kiss2 and kiss1rb expression in males, with no significant change in females. Tac3Ra and tac3Rb transduce their activity via both PKC/Ca and PKA/cAMP pathways. Both tac3 receptor types were very sensitive to amidation of their cognate ligands. Conclusion: These results indicate that the NKB/NKBR system may participate in puberty initiation in fish. Moreover, this novel system may be involved, in parallel with the kisspeptin system, in neuroendocrine regulation of GnRH secretion.

The role of GABA<sub>A</sub> receptors in the neural systems of the medial preoptic area in the control of GnRH release during the luteal phase of the oestrous cycle in ewes

The role of GABA<sub>A</sub> receptors in the neural systems of the medial preoptic area in the control of GnRH release during the luteal phase of the oestrous cycle in ewes

Influence of different neural systems on the secretion of sex hormones in potassium deficient mice

The influence of different neural systems that modulate GnRH secretion by hypothalamic neurons was investigated in mice exposed to hypokalemic conditions, in which the pulsatile release of GnRH has been shown to be altered and associated with a significant decrease of plasma sex steroids. Our results demonstrate that the potentiation of the inhibitory pathways mediated by opiates and GABA may be implicated in the decrease of sex hormones secretion produced by hypokalemia since treatment with higher doses of naloxone or flumazenil are required to restore progesterone or testosterone levels in potassium deficient mice. The combination treatment of prazoxin and naloxone suggests that the inhibitory action of opiates take place through its action on noradrenergic neurons. It is also possible that the inhibition of GnRH release could be due to a decrease in the tonic stimulatory action of noradrenergic pathway implicated in the control of GnRH release. Our results also reveal that it is unlikely that the glutamatergic system may play any relevant direct role in the decrease of sex steroid secretion observed in potassium deficient mice. Finally, these results together with the normal pattern of estradiol levels found along the estrus cycle in potassium deficient mice indicate that factors different from estradiol and acting on neural systems implicated in the regulation of GnRH-secreting neurons participate in the generation of the preovulatory surge of GnRH.

Post-partum blood leptin concentrations and energy balance in first calving dairy heifers

Blood concentrations of leptin, the first of several recently-discovered adipocyte proteins, putatively signal the extent of fat energy reserves to the hypothalamus in mammals and help regulate food intake and reproductive activity (through control of GnRH release). In early post-partum dairy cows, the rapidly increasing milk energy output and slowly increasing feed energy intake produce a negative energy balance (-ve EB) that is met by mobilisation of adipose fat stores and the severity and/or duration of the –ve EB may delay resumption of normal ovarian activity. The objective of the current study was to compare post-partum EB and serum leptin concentration profiles in Holstein-Friesian and Norwegian Red heifers, both of high genetic merit within their breed.

Involvement of NPY Y2 receptor subtype in the control of the spontaneous NO/GnRH release at the rat median eminence

The role of nitric oxide (NO) from vascular endothelium in the control of GnRH release at the median eminence (ME) level is well established. Interactions between NPY receptor/endothelium/nitric oxide are clearly demonstrated. While several studies implicate NPY Y1 receptor in the control of GnRH/LH at the time of the preovulatory LH surge, our results also demonstrate the importance of NPY Y2 receptor in the control of GnRH release via endothelial NO. We conclude that NPY may be one of the elements implicated in the generation of the spontaneous NO/GnRH via Y2 receptor located on endothelium.

Variation of Endothelial Nitric Oxide Synthase Synthesis in the Median Eminence during the Rat Estrous Cycle: An Additional Argument for the Implication of Vascular Blood Vessel in the Control of GnRH Release

Recent studies from our laboratory suggested that the vascular endothelium of the median eminence was involved via nitric oxide secretion in the modulation of GnRH release during the estrous cycle. To further investigate that issue, we studied the variations of endothelial nitric oxide synthase protein and mRNA in the median eminence of female rats killed at different time points of the day and/or of the estrous cycle. Endothelial nitric oxide synthase protein levels were measured by Western blot, and endothelial nitric oxide synthase mRNA analysis was performed with semiquantitative RT-PCR (for each time point, n = 4). The results revealed that endothelial nitric oxide synthase synthesis varied markedly across the estrous cycle. Indeed, endothelial nitric oxide synthase protein (n = 20) and mRNA (n = 16) levels increase significantly on 0800 h and 1600 h proestrus compared with 1400 h diestrus II. In a second step, quantification analysis were made in median eminence obtained from ovariectomized and ovariectomized, E2 benzoate primed rat. The results show a significant increase in expression of endothelial nitric oxide synthase protein as well as endothelial nitric oxide synthase mRNA in ovx-E2 primed rat median eminence. Concurrently, the levels of the cav-1 protein, a specific endogenous inhibitor of endothelial nitric oxide synthase, were measured in median eminence during estrous cycle and in ME from ovx and ovx-E2 primed rats. A significant decrease of median eminence cav-1 was noted on 1600 h proestrus and in ovx-E2 primed rats when compared with 1400 h diestrus II and ovx, respectively. Altogether, these results strongly suggest that high NO release from median eminence observed on proestrus may be due to an increase of endothelial nitric oxide synthase expression and a decrease of the cav-1 protein levels. These findings demonstrate that E2 is able to modulate endothelial nitric oxide synthase and cav-1 expression both during the estrous cycle and in experimental conditions and consequently reinforce the idea that nitric oxide acting on GnRH release, is essentially endothelial in origin. These results may also imply that variations of endothelial nitric oxide synthase expression are essential for the pulsatile/cyclic nitric oxide median eminence release observed in a previous study.

Variation of endothelial nitric oxide synthase synthesis in the median eminence during the rat estrous cycle: an additional argument for the implication of vascular blood vessel in the control of GnRH release.

Recent studies from our laboratory suggested that the vascular endothelium of the median eminence was involved via nitric oxide secretion in the modulation of GnRH release during the estrous cycle. To further investigate that issue, we studied the variations of endothelial nitric oxide synthase protein and mRNA in the median eminence of female rats killed at different time points of the day and/or of the estrous cycle. Endothelial nitric oxide synthase protein levels were measured by Western blot, and endothelial nitric oxide synthase mRNA analysis was performed with semiquantitative RT-PCR (for each time point, n = 4). The results revealed that endothelial nitric oxide synthase synthesis varied markedly across the estrous cycle. Indeed, endothelial nitric oxide synthase protein (n = 20) and mRNA (n = 16) levels increase significantly on 0800 h and 1600 h proestrus compared with 1400 h diestrus II. In a second step, quantification analysis were made in median eminence obtained from ovariectomized and ovariectomized, E2 benzoate primed rat. The results show a significant increase in expression of endothelial nitric oxide synthase protein as well as endothelial nitric oxide synthase mRNA in ovx-E2 primed rat median eminence. Concurrently, the levels of the cav-1 protein, a specific endogenous inhibitor of endothelial nitric oxide synthase, were measured in median eminence during estrous cycle and in ME from ovx and ovx-E2 primed rats. A significant decrease of median eminence cav-1 was noted on 1600 h proestrus and in ovx-E2 primed rats when compared with 1400 h diestrus II and ovx, respectively. Altogether, these results strongly suggest that high NO release from median eminence observed on proestrus may be due to an increase of endothelial nitric oxide synthase expression and a decrease of the cav-1 protein levels. These findings demonstrate that E2 is able to modulate endothelial nitric oxide synthase and cav-1 expression both during the estrous cycle and in experimental conditions and consequently reinforce the idea that nitric oxide acting on GnRH release, is essentially endothelial in origin. These results may also imply that variations of endothelial nitric oxide synthase expression are essential for the pulsatile/cyclic nitric oxide median eminence release observed in a previous study.

Neuropeptides, neurotransmitters, neurosteroids, and the onset of puberty.

Puberty results from withdrawal of the "gonadostat" mechanisms and from increased gonadotropin sensitivity to GnRH. It has been hypothesized that GnRH release may be modulated by a non-steroid-mediated mechanism. Modifications of neuropeptides, neurotransmitters, and neurosteroids may underlie the onset of pubertal processes. Neuropeptides mainly involved in the control of GnRH release are opioids, neuropeptide Y (NPY), galanin, and corticotropin-releasing factor (CRF), whereas neurotransmitters are noradrenaline, dopamine, serotonin, melatonin and gamma-aminobutyric acid (GABA). Norepinephrine, epinephrine, and dopamine stimulate GnRH, whereas the effect of serotonin on hypothalamic-pituitary-ovarian axis seems to be norepinephrine-mediated. Neurosteroids are steroid hormones that bind to the GABA-A receptor, synthesized in the brain de novo or from blood-borne precursors. DHEA, a GABA-A antagonistic neurosteroid, and allopregnanolone, a GABA-A agonistic neurosteroid, may be important in the onset of gonadarche. In conclusion, the onset of puberty derives from the complex interplay among neuropeptides, neurotransmitters, and neurosteroids that occurs in the awakening of hypothalamic-pituitary-ovarian axis.

Involvement of nitric oxide in the regulation of gonadotropin-releasing hormone release from the GT1-1 neuronal cell line.

A role for nitric oxide (NO) in the regulation of hypothalamic neurohormone secretion has been suggested. The aim of the present study was to establish a direct involvement of this novel intracellular regulatory molecule in the control of GnRH release. For this purpose, the GT1-1 GnRH-secreting continuous cell line was treated with various agents that can modify the endogenous NO synthase activity or, alternatively, with substances that can liberate NO, mimicking an increased concentration of this molecule in the cell. Treatment of GT1-1 cells with increasing concentrations of L-arginine, the direct precursor of NO, produced a marked reduction of norepinephrine-stimulated GnRH release despite a lack of effect on basal secretion. Similarly, the NO donors SIN-1 and acidified NaNO2 potently reduced basal as well as KCl-stimulated GnRH secretion. Conversely, sodium nitroprusside caused a significant inhibition of KCl-stimulated, but not basal, GnRH secretion. Addition of these agents to GT1-1 cells resulted in a marked increase in intracellular cGMP accumulation. Addition of the NO synthase inhibitors N-nitro-L-arginine and N-nitro-L-arginine methyl ester stimulated basal GnRH secretion without modifying norepinephrine- or KCl-stimulated release. In addition, treatment of GT1-1 cells with both L-arginine analogs produced a significant inhibition of the basal cGMP concentration. Together, these data suggest an inhibitory role for NO in the control of GnRH secretion from GT1-1 cells.

Involvement of nitric oxide in the regulation of gonadotropin-releasing hormone release from the GT1-1 neuronal cell line

A role for nitric oxide (NO) in the regulation of hypothalamic neurohormone secretion has been suggested. The aim of the present study was to establish a direct involvement of this novel intracellular regulatory molecule in the control of GnRH release. For this purpose, the GT1-1 GnRH-secreting continuous cell line was treated with various agents that can modify the endogenous NO synthase activity or, alternatively, with substances that can liberate NO, mimicking an increased concentration of this molecule in the cell. Treatment of GT1-1 cells with increasing concentrations of L-arginine, the direct precursor of NO, produced a marked reduction of norepinephrine-stimulated GnRH release despite a lack of effect on basal secretion. Similarly, the NO donors SIN-1 and acidified NaNO2 potently reduced basal as well as KCl-stimulated GnRH secretion. Conversely, sodium nitroprusside caused a significant inhibition of KCl-stimulated, but not basal, GnRH secretion. Addition of these agents to GT1-1 cells resulted in a marked increase in intracellular cGMP accumulation. Addition of the NO synthase inhibitors N-nitro-L-arginine and N-nitro-L-arginine methyl ester stimulated basal GnRH secretion without modifying norepinephrine- or KCl-stimulated release. In addition, treatment of GT1-1 cells with both L-arginine analogs produced a significant inhibition of the basal cGMP concentration. Together, these data suggest an inhibitory role for NO in the control of GnRH secretion from GT1-1 cells.

Hypothalamic multiunit activity and LH secretion in conscious sheep.

The relationship between the pulsatile secretion of luteinizing hormone (LH) and multiunit activity (MUA) in the median eminence and retrochiasmatic area (RCh-ME) of the hypothalamus was investigated within 13 conscious, ovariectomized ewes during the anoestrous season. Eight of the ewes had been treated with oestradiol to reduce their LH pulse frequency. To test whether the activity had been recorded from sites involved in the control of GnRH release, we electrically stimulated the sites studied with the recording electrode and used the LH responses to classify the animals retrospectively for analysis of the MUA data. Following stimulation, LH secretion was either stimulated (Group STIM, n = 5 ewes), inhibited (Group INHIB, n = 4) or showed no response (Group NR, n = 4). Statistical analysis of the MUA data revealed that the onset of LH pulses was associated with an increase in cell activity in STIM ewes and a decrease in activity in both Group INHIB and Group NR ewes. Histological examination revealed that the electrodes were located near the midline in Group STIM ewes, but 1.25 or 2 mm lateral of the midline in Groups NR and INHIB, respectively. We concluded that the MUA in the RCh-ME probably reflects the activity of cells with fibres or terminals involved in the control of GnRH release. Activity which increased at the onset of LH pulses was detected in medial areas of the RCh-ME and may reflect the activity of GnRH neurones. The activity which decreased at the onset of LH pulses in lateral areas of the RCh-ME suggests that both stimulatory and inhibitory inputs may be involved in the release of GnRH pulses.