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GABA increased the intracellular calcium concentration of GnRH neurons isolated from adult GnRH-EGFP transgenic rats
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Abstracts / Frontiers in Neuroendocrinology 27 (2006) 63–79
Leptin acts through the arcuate nucleus of the hypothalamus to induce cFos immunoreactivity into the preoptic region E. Caron a, V. Pre´vot a, J.-C. Beauvillain a, S.G. Bouret b a INSERM U816, Lille, France b The Saban Research Institute, Childrens Hospital Los Angeles, University of Southern California, Los Angeles, USA The arcuate nucleus of the hypothalamus (ARH) is a critical component of the forebrain pathways that regulate energy homeostasis and reproduction and it plays a particularly important role in relaying centrally the anorectic effect of leptin. In addition to its role in the regulation of energy homeostasis, leptin has also been shown to have a stimulatory effect on the reproductive axis. However, little is known about the central mechanisms mediating leptin’s effects on reproduction. In the present study, we hypothesized that leptin activates, through an action within the ARH, hypothalamic neurons known to play a critical role in the regulation of reproduction. Immunohistochemical stainings were used to compare the induction of the protooncogene cFos (a marker of cellular activation that can be activated directly or transsynaptically) by leptin (10 mg/ kg, ip) in adult intact wild type mice or mice neonatally treated with monosodium glutamate (MSG). The results indicate that peripheral injection of leptin caused in intact animals a dramatic induction of cFos-immunoreactivity (cFos-IR) in the diagonal band of Brocca (NDB), the vascular organ of the lamina terminalis (OVLT), median preoptic nucleus (MEPO), and medial preoptic area (MPO), which have been implicated in the neural control of reproduction. The induction in the MEPO and MPO was maximal 90 min following leptin administration, whereas 180 min are required to observe a significant induction in the NDB and OVLT. No induction was found in GnRH neurons. Interestingly, lesions of the ARH by MSG severely attenuated leptin-induced cFos-IR in the NDB, OVLT, MEPO, and MPO. Taken together these results show that leptin is able to induce cFos-IR in nuclei of the preoptic region. These results also suggest that the ARH is a key component of the hypothalamic pathways that mediate leptin’s effect on neurons of the preoptic region regulating reproduction. doi:10.1016/j.yfrne.2006.03.158
Indomethacin inhibits progesterone release stimulated by fenoterol (b2 adrenergic agonist), VIP, and PACAP 38 from rat cultured ovarian granulosa cells Elzbieta Wasilewska-Dziubinska, Magdalena Chmielowska, Ewa Wolinska-Witort, Lidia Martynska, Wojciech Bik, Boguslawa Baranowska Neuroendocrinology Department, Medical Centre of Postgraduate Education, Warsaw, Poland Introduction: Progesterone release from cultured ovarian granulosa and luteal cells may be stimulated by several fac-
tors including catecholamines, neuropeptides, and prostaglandins. However, the exact processes underlying the regulation of ovarian hormone secretion remain unclear. It has been found that isoproterenol, adrenaline, VIP, and PACAP can stimulate progesterone release as well as synthesis of cAMP in cultured ovarian granulosa cells. It is well known that isoproterenol and VIP can regulate ovarian synthesis of P 450 scc enzyme complex that is responsible for initiating progesterone biosynthesis. Moreover, it has been shown that noradrenaline can affect progesterone secretion also indirectly by stimulating ovarian prostaglandins. The aim: We aimed to examine whether prostaglandins are involved in the long-term stimulatory effect of fenoterol (b2 adrenergic agonist), VIP, and PACAP 38 on progesterone release from ovarian granulosa cells in the rat. Materials and methods: In vitro study was performed on cultured ovarian granulosa cells of adult cyclic rats (diestrus) during 24 h incubation. Indomethacin (non-selective cyclooxygenase inhibitor— inhibitor of prostaglandin synthesis) in a dose of 10 6 M was added to cell cultures stimulated by fenoterol (10 7 M), VIP (10 7 M) or PACAP38 (10 7 M). Progesterone concentration in the culture media was estimated by RIA. For statistical analyses the unpaired Student’s t-test and analysis of variance were used, as appropriate. Results: Fenoterol, VIP, and PACAP 38 significantly stimulated progesterone release by 380, 130, and 140% (p < 0.001; p < 0.01; and p < 0.01), respectively. Administration of indomethacin totally abolished these effects. Conclusions: We conclude that local synthesis of prostaglandins influences the progesterone secretion in cultured ovarian granulosa cells in the adult cyclic rat. Cyclooxygenase pathways may mediate the long-term stimulatory effect of fenoterol, VIP, and PACAP 38 on progesterone release. doi:10.1016/j.yfrne.2006.03.159
GABA increased the intracellular calcium concentration of GnRH neurons isolated from adult GnRH-EGFP transgenic rats Nobuyuki Tanaka, Miho Watanabe, Yin Chengzhu, Yasuo Sakuma, Masakatsu Kato Department of Physiology, Nippon Medical School, Tokyo 113-8602, Japan Gonadotropin-releasing hormone (GnRH) neurons are essential in the reproductive neuroendocrine system, in the which the function of GnRH neurons is to control luteinizing hormone (LH) secretion from anterior pituitary by releasing GnRH in the median eminence. Circumstantial evidence indicates that GABA is a major player in the regulation of GnRH neurons. However, functional
Abstracts / Frontiers in Neuroendocrinology 27 (2006) 63–79
analysis of GABA action on rat GnRH neurons has not been performed. We therefore, examined the effects of GABA on GnRH neurons. Dispersed female neurons from adult GnRH-EGFP transgenic rats were plated on glass coverslips and cultured overnight in neurobasal A medium supplemented with B-27. Cells were loaded with the calcium-probe Fura PE/AM for calcium-imaging experiments. The cells were heavily labeled by Fura PE to avoid fluorescence contamination by EGFP at 380 nm excitation. We examined 370 GnRH neurons identified with EGFP fluorescence. GABA (100 lM) increased intracellular calcium concentration in 57.3% of GnRH neurons and decreased it in 5.4% of GnRH neurons. The remaining 37.3% showed no response to GABA. GABAA receptor antagonist bicuculline completely suppressed these responses and the agonist muscimol mimicked them. GABA-induced increase in intracellular calcium concentration was totally dependent on the presence of extracellular calcium. In addition, bumetanide (blocker of chloride transporter) attenuated the GABA-induced increase in intracellular calcium concentration, indicating that intracellular chloride concentration is kept high by chloride transporter NKCCl in those neurons responding to GABA with an increase in intracellular calcium concentration. We also examined 654 neurons emitting no EGFP fluorescence, which were classified as non-identified neurons. GABA increased intracellular calcium concentration in 0.2% of the neurons and decreased it in 4.6% of the neurons. The remaining 95.3% of the neurons produced no response to GABA. In conclusion, GABA increased intracellular calcium concentration in 60% of GnRH neurons from adult female rat by activating GABAA receptor. The results indicate that GABA depolarizes GnRH neurons via GABAA receptor, thereby activating calcium channels and facilitating calcium influx. Finally, those classified as non-responding neurons must include some neurons hyperpolarizing in response to GABA because hyperpolarization may not always produce a detectable response in calcium imaging experiment. doi:10.1016/j.yfrne.2006.03.160
Continuous subcutaneous administration of kisspeptin-54 causes testicular degeneration in rats Kevin G. Murphy a, Emily L. Thompson a, Michael Patterson a, Gavin A. Bewick a, Preeti H. Jethwa a, Gordon W.H. Stamp b, Jeannie F. Todd a, Mohammad A. Ghatei a, Stephen R. Bloom a a Department of Metabolic Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK b Department of Histopathology, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK Kisspeptin is a hypothalamic neuropeptide essential for reproduction. Kisspeptin-54 stimulates the hypothalamic-pituitary-gonadal (HPG) axis via the G protein-
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coupled receptor 54 (GPR54). Mice lacking the GPR54 and humans with GPR54 mutations are infertile. Acute peripheral administration of kisspeptin to rodents, primates or humans increases circulating gonadotrophins. We investigated the effects of chronic continuous subcutaneous kisspeptin administration on testicular histology, and on circulating gonadotrophins and testes-derived hormones. Chronic subcutaneous administration of 50 nmol kisspeptin-54 per day for 13 days in adult male rats significantly decreased testicular weight. Histological examination showed degeneration of the seminiferous tubules associated with a significant decrease in the circulating levels of the testes-derived hormone, inhibin B. The kisspeptin-54 treated group also showed a trend towards lower plasma testosterone. Interestingly, gonadotrophin concentrations were not significantly different from saline treated controls. The lack of an increase in gonadotrophins following chronic kisspeptin-54 administration suggests down-regulation of the HPG axis response to peripheral kisspeptin. These findings indicate that continuous kisspeptin administration may provide a novel tool for the manipulation of the HPG axis and spermatogenesis. doi:10.1016/j.yfrne.2006.03.161
Kisspeptin-10 stimulates luteinising hormone release from the ovine pituitary gland in vitro J.T. Smith, A. Pereira, A. Rao, I.J. Clarke Department of Physiology, Monash University, Victoria 3800, Australia Kisspeptin is the endogenous product of the KiSS-1 gene that binds to a G protein-coupled receptor called GPR54. Kisspeptin and GPR54 have recently been recognised in the neuroendocrine regulation of gonadotrophinreleasing hormone (GnRH) and gonadotrophin secretion in many species. In sheep, kisspeptin has been shown to be co-localised with GnRH producing cells of the preoptic area, suggesting that the two peptides may be co-secreted into the hypophyseal portal blood and act directly on the pituitary gland to regulate gonadotrophin secretion. To test the hypothesis that kisspeptin acts directly on the pituitary gland, we examined luteinising hormone (LH) release from ovine primary pituitary cell cultures treated with kisspeptin (10 nM), GnRH (10 nM), or GnRH in the presence of kisspeptin. After a 3 h treatment incubation, culture media were collected and LH concentration determined by radioimmunoassay. We found that kisspeptin treatment increased the concentration of LH found in culture media by more than 2-fold compared to control (P < 0.01). Not surprisingly, GnRH treatment resulted in a significant increase in LH concentration (5-fold, P < 0.01). Kisspeptin treatment in combination with GnRH did not appear to augment the increase in levels of LH when compared to
Abstracts / Frontiers in Neuroendocrinology 27 (2006) 63–79
Leptin acts through the arcuate nucleus of the hypothalamus to induce cFos immunoreactivity into the preoptic region E. Caron a, V. Pre´vot a, J.-C. Beauvillain a, S.G. Bouret b a INSERM U816, Lille, France b The Saban Research Institute, Childrens Hospital Los Angeles, University of Southern California, Los Angeles, USA The arcuate nucleus of the hypothalamus (ARH) is a critical component of the forebrain pathways that regulate energy homeostasis and reproduction and it plays a particularly important role in relaying centrally the anorectic effect of leptin. In addition to its role in the regulation of energy homeostasis, leptin has also been shown to have a stimulatory effect on the reproductive axis. However, little is known about the central mechanisms mediating leptin’s effects on reproduction. In the present study, we hypothesized that leptin activates, through an action within the ARH, hypothalamic neurons known to play a critical role in the regulation of reproduction. Immunohistochemical stainings were used to compare the induction of the protooncogene cFos (a marker of cellular activation that can be activated directly or transsynaptically) by leptin (10 mg/ kg, ip) in adult intact wild type mice or mice neonatally treated with monosodium glutamate (MSG). The results indicate that peripheral injection of leptin caused in intact animals a dramatic induction of cFos-immunoreactivity (cFos-IR) in the diagonal band of Brocca (NDB), the vascular organ of the lamina terminalis (OVLT), median preoptic nucleus (MEPO), and medial preoptic area (MPO), which have been implicated in the neural control of reproduction. The induction in the MEPO and MPO was maximal 90 min following leptin administration, whereas 180 min are required to observe a significant induction in the NDB and OVLT. No induction was found in GnRH neurons. Interestingly, lesions of the ARH by MSG severely attenuated leptin-induced cFos-IR in the NDB, OVLT, MEPO, and MPO. Taken together these results show that leptin is able to induce cFos-IR in nuclei of the preoptic region. These results also suggest that the ARH is a key component of the hypothalamic pathways that mediate leptin’s effect on neurons of the preoptic region regulating reproduction. doi:10.1016/j.yfrne.2006.03.158
Indomethacin inhibits progesterone release stimulated by fenoterol (b2 adrenergic agonist), VIP, and PACAP 38 from rat cultured ovarian granulosa cells Elzbieta Wasilewska-Dziubinska, Magdalena Chmielowska, Ewa Wolinska-Witort, Lidia Martynska, Wojciech Bik, Boguslawa Baranowska Neuroendocrinology Department, Medical Centre of Postgraduate Education, Warsaw, Poland Introduction: Progesterone release from cultured ovarian granulosa and luteal cells may be stimulated by several fac-
tors including catecholamines, neuropeptides, and prostaglandins. However, the exact processes underlying the regulation of ovarian hormone secretion remain unclear. It has been found that isoproterenol, adrenaline, VIP, and PACAP can stimulate progesterone release as well as synthesis of cAMP in cultured ovarian granulosa cells. It is well known that isoproterenol and VIP can regulate ovarian synthesis of P 450 scc enzyme complex that is responsible for initiating progesterone biosynthesis. Moreover, it has been shown that noradrenaline can affect progesterone secretion also indirectly by stimulating ovarian prostaglandins. The aim: We aimed to examine whether prostaglandins are involved in the long-term stimulatory effect of fenoterol (b2 adrenergic agonist), VIP, and PACAP 38 on progesterone release from ovarian granulosa cells in the rat. Materials and methods: In vitro study was performed on cultured ovarian granulosa cells of adult cyclic rats (diestrus) during 24 h incubation. Indomethacin (non-selective cyclooxygenase inhibitor— inhibitor of prostaglandin synthesis) in a dose of 10 6 M was added to cell cultures stimulated by fenoterol (10 7 M), VIP (10 7 M) or PACAP38 (10 7 M). Progesterone concentration in the culture media was estimated by RIA. For statistical analyses the unpaired Student’s t-test and analysis of variance were used, as appropriate. Results: Fenoterol, VIP, and PACAP 38 significantly stimulated progesterone release by 380, 130, and 140% (p < 0.001; p < 0.01; and p < 0.01), respectively. Administration of indomethacin totally abolished these effects. Conclusions: We conclude that local synthesis of prostaglandins influences the progesterone secretion in cultured ovarian granulosa cells in the adult cyclic rat. Cyclooxygenase pathways may mediate the long-term stimulatory effect of fenoterol, VIP, and PACAP 38 on progesterone release. doi:10.1016/j.yfrne.2006.03.159
GABA increased the intracellular calcium concentration of GnRH neurons isolated from adult GnRH-EGFP transgenic rats Nobuyuki Tanaka, Miho Watanabe, Yin Chengzhu, Yasuo Sakuma, Masakatsu Kato Department of Physiology, Nippon Medical School, Tokyo 113-8602, Japan Gonadotropin-releasing hormone (GnRH) neurons are essential in the reproductive neuroendocrine system, in the which the function of GnRH neurons is to control luteinizing hormone (LH) secretion from anterior pituitary by releasing GnRH in the median eminence. Circumstantial evidence indicates that GABA is a major player in the regulation of GnRH neurons. However, functional
Abstracts / Frontiers in Neuroendocrinology 27 (2006) 63–79
analysis of GABA action on rat GnRH neurons has not been performed. We therefore, examined the effects of GABA on GnRH neurons. Dispersed female neurons from adult GnRH-EGFP transgenic rats were plated on glass coverslips and cultured overnight in neurobasal A medium supplemented with B-27. Cells were loaded with the calcium-probe Fura PE/AM for calcium-imaging experiments. The cells were heavily labeled by Fura PE to avoid fluorescence contamination by EGFP at 380 nm excitation. We examined 370 GnRH neurons identified with EGFP fluorescence. GABA (100 lM) increased intracellular calcium concentration in 57.3% of GnRH neurons and decreased it in 5.4% of GnRH neurons. The remaining 37.3% showed no response to GABA. GABAA receptor antagonist bicuculline completely suppressed these responses and the agonist muscimol mimicked them. GABA-induced increase in intracellular calcium concentration was totally dependent on the presence of extracellular calcium. In addition, bumetanide (blocker of chloride transporter) attenuated the GABA-induced increase in intracellular calcium concentration, indicating that intracellular chloride concentration is kept high by chloride transporter NKCCl in those neurons responding to GABA with an increase in intracellular calcium concentration. We also examined 654 neurons emitting no EGFP fluorescence, which were classified as non-identified neurons. GABA increased intracellular calcium concentration in 0.2% of the neurons and decreased it in 4.6% of the neurons. The remaining 95.3% of the neurons produced no response to GABA. In conclusion, GABA increased intracellular calcium concentration in 60% of GnRH neurons from adult female rat by activating GABAA receptor. The results indicate that GABA depolarizes GnRH neurons via GABAA receptor, thereby activating calcium channels and facilitating calcium influx. Finally, those classified as non-responding neurons must include some neurons hyperpolarizing in response to GABA because hyperpolarization may not always produce a detectable response in calcium imaging experiment. doi:10.1016/j.yfrne.2006.03.160
Continuous subcutaneous administration of kisspeptin-54 causes testicular degeneration in rats Kevin G. Murphy a, Emily L. Thompson a, Michael Patterson a, Gavin A. Bewick a, Preeti H. Jethwa a, Gordon W.H. Stamp b, Jeannie F. Todd a, Mohammad A. Ghatei a, Stephen R. Bloom a a Department of Metabolic Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK b Department of Histopathology, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK Kisspeptin is a hypothalamic neuropeptide essential for reproduction. Kisspeptin-54 stimulates the hypothalamic-pituitary-gonadal (HPG) axis via the G protein-
77
coupled receptor 54 (GPR54). Mice lacking the GPR54 and humans with GPR54 mutations are infertile. Acute peripheral administration of kisspeptin to rodents, primates or humans increases circulating gonadotrophins. We investigated the effects of chronic continuous subcutaneous kisspeptin administration on testicular histology, and on circulating gonadotrophins and testes-derived hormones. Chronic subcutaneous administration of 50 nmol kisspeptin-54 per day for 13 days in adult male rats significantly decreased testicular weight. Histological examination showed degeneration of the seminiferous tubules associated with a significant decrease in the circulating levels of the testes-derived hormone, inhibin B. The kisspeptin-54 treated group also showed a trend towards lower plasma testosterone. Interestingly, gonadotrophin concentrations were not significantly different from saline treated controls. The lack of an increase in gonadotrophins following chronic kisspeptin-54 administration suggests down-regulation of the HPG axis response to peripheral kisspeptin. These findings indicate that continuous kisspeptin administration may provide a novel tool for the manipulation of the HPG axis and spermatogenesis. doi:10.1016/j.yfrne.2006.03.161
Kisspeptin-10 stimulates luteinising hormone release from the ovine pituitary gland in vitro J.T. Smith, A. Pereira, A. Rao, I.J. Clarke Department of Physiology, Monash University, Victoria 3800, Australia Kisspeptin is the endogenous product of the KiSS-1 gene that binds to a G protein-coupled receptor called GPR54. Kisspeptin and GPR54 have recently been recognised in the neuroendocrine regulation of gonadotrophinreleasing hormone (GnRH) and gonadotrophin secretion in many species. In sheep, kisspeptin has been shown to be co-localised with GnRH producing cells of the preoptic area, suggesting that the two peptides may be co-secreted into the hypophyseal portal blood and act directly on the pituitary gland to regulate gonadotrophin secretion. To test the hypothesis that kisspeptin acts directly on the pituitary gland, we examined luteinising hormone (LH) release from ovine primary pituitary cell cultures treated with kisspeptin (10 nM), GnRH (10 nM), or GnRH in the presence of kisspeptin. After a 3 h treatment incubation, culture media were collected and LH concentration determined by radioimmunoassay. We found that kisspeptin treatment increased the concentration of LH found in culture media by more than 2-fold compared to control (P < 0.01). Not surprisingly, GnRH treatment resulted in a significant increase in LH concentration (5-fold, P < 0.01). Kisspeptin treatment in combination with GnRH did not appear to augment the increase in levels of LH when compared to