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Expression of low-affinity p75 nerve growth factor receptor in developing mouse nasal region
Abstracts / Frontiers in Neuroendocrinology 27 (2006) 63–79
73
peptin/GPR54 can also regulate the gonadotropic axis at the pituitary level.
tion of these three peptides. (Supported by NIHHD39916.)
doi:10.1016/j.yfrne.2006.03.151
doi:10.1016/j.yfrne.2006.03.152
Kisspeptin is co-localized in a subset of dynorphin neurons in the ovine arcuate nucleus M.N. Lehman a, L.M. Coolen a, C.V.R. de Oliveira a, I.J. Clarke b, R.L. Goodman c a Department of Anatomy and Cell Biology, University of Western Ontario, London, Ont., Canada N6A 5C1 b Department of Physiology, Monash University 3800, Australia c Department of Physiology and Pharmacology, West Virginia University, Morgantown, WV 26506, USA
Expression of low-affinity p75 nerve growth factor receptor in developing mouse nasal region Franca Raucci, Susan Wray Cellular and Developmental Neurobiology Section, NINDS, NIH, Bethesda, MD, USA
Pharmacological and anatomical studies in sheep have indicated that dynorphin A-containing (DYN) opioidergic neurons of the arcuate nucleus play a critical role in mediating the central actions of progesterone. Specifically progesterone appears to increase dynorphin release, which then inhibits pulsatile secretion of GnRH and LH. The recently discovered peptide, kisspeptin, has received much attention as an important positive regulator of GnRH secretion. Moreover, a subset of kisspeptin cells in the sheep are located in the arcuate nucleus, and like DYN cells, are highly enriched in gonadal steroid receptors. Therefore, we carried out double label immunofluorescent staining of sheep hypothalamic sections to determine if DYN cells in the arcuate nucleus or elsewhere co-localize kisspeptin. Sections from ovariectomized breeding season ewes, bearing implants that maintain luteal phase levels of estradiol, were processed using antibodies recognizing kisspeptin/metastin (45–54) and DYN. Preliminary results revealed a close overlap of DYN and kisspeptin neurons in the arcuate nucleus with approximately 50% of DYN cells double-labeled for kisspeptin. Moreover, interesting differences in the regional co-localization were noted: the highest degree of co-localization was located in the middle and caudal regions of this nucleus (85–95%) and was less extensive in the dorsal, ventral, and rostral portions (25–35%). Kisspeptin was also co-localized within individual DYN fibers and terminals in the external zone of the median eminence. In contrast to cells in the arcuate nucleus, DYN cells in the preoptic area, paraventricular, and supraoptic nuclei did not co-localize kisspeptin. Since a high percentage of DYN cells of the arcuate nucleus also co-express neurokinin B (NKB), these findings suggest a possible interaction between kisspeptin, dynorphin, and NKB released by the same neuronal subpopulation in the regulation of GnRH secretion. Since kisspeptin and possibly NKB stimulate, while dynorphin inhibits GnRH release, this co-localization raises intriguing questions about the differential regula-
GnRH-1 neurons are a critical component of the hypothalamic–pituitary–gonadal axis and as such control reproductive function. GnRH-1 neurons are derived from nasal placode and thereafter migrate into the forebrain. The molecular cues directing GnRH-1 neurons to the final location are unclear. p75 nerve growth factor receptor (NGFR) is the low-affinity neurotrophin receptor that plays an important role in neuronal development and survival, mediating both neurite outgrowth and axonal extension. p75 NGFR has been identified in the developing pituitary gland as well as the olfactory system, suggesting involvement of neurotrophins in the establishment of these systems. In this study we characterize the temporal and spatial localization of the p75 NGFR with respect to the development of the GnRH-1 neuroendocrine system in mouse. Immunocytochemically studies were performed in nasal explants, an in vitro model system in which many of the in vivo characteristics of the developing GnRH-1 system and olfactory system are retained. Nasal explants were stained for p75 NGFR after three days in vitro (div), 7 div, 14 div, and 21 div. At all stages, p75 NGFR labeling was identified in two morphologically distinct cell types within the periphery of the explant. Both phenotypes directionally migrated out of the main tissue mass and showed p75 NGFR labeling of cell soma and processes. One p75 NGFR positive phenotype was small and had a simple unipolar/bipolar appearance (GnRH-1-like), with long processes strongly positive. The second p75 NGFR positive cell type was larger and multipolar with several short extensions. The density of p75 NGFR on these two cell types changed as a function of time in vitro. At 3 div the GnRH-1-like bipolar cells were strongly reactive in soma and processes. At 7 div the intensity increased in GnRH1-like cells farthest from the explant tissue mass. By 14 div the staining was still strong in GnRH-1-like cells, but the number was attenuated. At 21 div few positive bipolar elements were observed. In contrast to the GnRH-1-like cells, the multipolar cells showed a moderate p75 NGFR labeling at 3 and 7 div. From 14 to 21 div the staining became stronger and the number of multipolar cells in the periphery increased. Simultaneous localization of GnRH-1 and p75 NGFR was seen in the bipolar cells but not in multipolar cells. These results
74
Abstracts / Frontiers in Neuroendocrinology 27 (2006) 63–79
show that p75 NGFR is expressed in different manners in specific cell types in the developing nasal region. Identification of p75 NGFR on GnRH-1 neurons suggests a role for p75 NGFR in the migration and/or maturation of GnRH-1 neurons. In addition, p75 NGFR expression highlighted multipolar cellular elements that may participate in directing GnRH-1 cells and/or olfactory axons into the brain.
Endoreduplication in Caenorhabditis elegans: A positive or negative age-related event regulated by hormones? Ryan J. Haasl, Jennifer M. Marvitz, Sivan Vadakkadath Meethal, Miguel J. Gallego, Craig S. Atwood Section of Geriatrics and Gerontology, Department of Medicine, University of Wisconsin and Geriatric Research, Education, and Clinical Center, Veteran’s Administration Hospital, Madison, WI 53705, USA
doi:10.1016/j.yfrne.2006.03.153
Emerging evidence implicates neuroglial-endothelial interactions in the control of neurovascular junction formation for GnRH neurons in the median eminence of the hypothalamus. However, molecular determinants involved in such communication processes have not fully been identified. Screening for molecular cues known to play a role in the control of axon guidance indicated Semaphorin3A (Sema3A) as a candidate. Here, we used in situ hybridization and immunohistochemical stainings to examine the expression of Sema3A and its receptor, Neuropilin-1, in the adult median eminence of both mice and rats. Robust Sema3A mRNA expression was detected in capillaries of the median eminence, while only a scant hybridization signal was found in the nervous tissue. Consistent with these data, Sema3A immunoreactivity was restricted to portal blood capillaries of the median eminence. To determine whether GnRH nerve terminals may be able to sense Sema3A, we further investigated Neuropilin-1 expression in GnRH neurons. Double-labeling experiments revealed Neuropilin-1 mRNA expression in a significant fraction of GnRH neurons. The proportion of GnRH perikarya expressing Neuropilin-1 mRNA significantly varied during the rat estrous cycle and was maximal on proestrus (38.18 ± 2.47% in diestrus 2 vs. 49.99 ± 1.54% on proestrus, p < 0.05), a phase of the estrous cycle when GnRH nerve terminals have direct access to the portal vasculature. Accordingly, strong Neuropilin-1 immunoreactivity was detected in the external zone of the median eminence and was found to co-localize with GnRH-immunoreactive fibers. Taken together these results suggest that Sema3A may be a chemotropic factor secreted by endothelial cells to induce GnRH axon plasticity.
Endoreduplication is DNA replication in the absence of cytokinesis. Although somatic cells of adult Caenorhabditis elegans are non-mitotic, significant endoreduplication does occur in these cells, leading to body growth: quantification of Hoechst staining intensity revealed a 33% increase in total DNA content between 5 and 14 days after hatching; a concomitant 15% increase in body size was observed during the same time period. But what role, if any, does this age-related increase in endoreduplication serve? Does it have a positive or negative impact on the life of an organism? Relevant data offer contradictory answers to these questions. While in many plant species the extra DNA available to endoreduplicating cells allows for increased protein expression, polyploidy in mammals can be detrimental as seen in Down syndrome. This study characterized endoreduplication in C. elegans to assess its effects on longevity and potential regulation by age-related hormonal changes. We localized GnRH1 receptor (GnRH1R), and quantified nuclear DNA content of hypodermal (hyp7), intestinal, and neuronal cells in wild type (WT) worms and WT worms treated with antisense oligonucleotides specific to cyd-1 (enables G1 ! S progression) and lon-1 (TGFb-like pathway component). Six days after hatching, neuronal, hyp-7, and intestinal nuclei were diploid, 8-ploid and 64-ploid in WT worms and tetraploid, 16-ploid and 64ploid in anti-lon-1 worms (n = 20 nuclei/cell type). The increase in hyp-7 ploidy in anti-lon-1 worms was reflected by a significant 20% increase in hyp-7 nuclear size (p < 0.001, n = 20 nuclei), but no change in body size. The ploidy of anti-cyd-1 nuclei was identical to that of WT worms and no significant differences in body or nuclear size were detected. Though all three groups exhibited similar average lifespans, relative to WT, death rates varied: at 12 days after hatching 12, 22, and 36% of initial anti-cyd-1, WT, and anti-lon-1 populations remained. Immunohistochemical localization of GnRH1R in intestinal nuclei, by far the most endoreduplicative nuclei in C. elegans, is suggestive of a link between the HPG axis and endoreduplication. Future studies will examine the hormonal regulation of endoreduplication, the effect of endoreduplication on reproductive capacity, nutrient storage, and health, and whether endoreduplication serves some purposive response in maintaining the viability of nematodes.
doi:10.1016/j.yfrne.2006.03.154
doi:10.1016/j.yfrne.2006.03.155
Expression of Semaphorin3A and Neuropilin-1 in the adult median eminence C. Campagne a, S.G. Bouret a,b, J.-C. Beauvillain a, V. Prevot a a Inserm U816, Place de Verdun, 59045 Lille Cedex, France b The Saban Research Institute, Childrens Hospital Los Angeles, University of Southern California, Los Angeles, USA
73
peptin/GPR54 can also regulate the gonadotropic axis at the pituitary level.
tion of these three peptides. (Supported by NIHHD39916.)
doi:10.1016/j.yfrne.2006.03.151
doi:10.1016/j.yfrne.2006.03.152
Kisspeptin is co-localized in a subset of dynorphin neurons in the ovine arcuate nucleus M.N. Lehman a, L.M. Coolen a, C.V.R. de Oliveira a, I.J. Clarke b, R.L. Goodman c a Department of Anatomy and Cell Biology, University of Western Ontario, London, Ont., Canada N6A 5C1 b Department of Physiology, Monash University 3800, Australia c Department of Physiology and Pharmacology, West Virginia University, Morgantown, WV 26506, USA
Expression of low-affinity p75 nerve growth factor receptor in developing mouse nasal region Franca Raucci, Susan Wray Cellular and Developmental Neurobiology Section, NINDS, NIH, Bethesda, MD, USA
Pharmacological and anatomical studies in sheep have indicated that dynorphin A-containing (DYN) opioidergic neurons of the arcuate nucleus play a critical role in mediating the central actions of progesterone. Specifically progesterone appears to increase dynorphin release, which then inhibits pulsatile secretion of GnRH and LH. The recently discovered peptide, kisspeptin, has received much attention as an important positive regulator of GnRH secretion. Moreover, a subset of kisspeptin cells in the sheep are located in the arcuate nucleus, and like DYN cells, are highly enriched in gonadal steroid receptors. Therefore, we carried out double label immunofluorescent staining of sheep hypothalamic sections to determine if DYN cells in the arcuate nucleus or elsewhere co-localize kisspeptin. Sections from ovariectomized breeding season ewes, bearing implants that maintain luteal phase levels of estradiol, were processed using antibodies recognizing kisspeptin/metastin (45–54) and DYN. Preliminary results revealed a close overlap of DYN and kisspeptin neurons in the arcuate nucleus with approximately 50% of DYN cells double-labeled for kisspeptin. Moreover, interesting differences in the regional co-localization were noted: the highest degree of co-localization was located in the middle and caudal regions of this nucleus (85–95%) and was less extensive in the dorsal, ventral, and rostral portions (25–35%). Kisspeptin was also co-localized within individual DYN fibers and terminals in the external zone of the median eminence. In contrast to cells in the arcuate nucleus, DYN cells in the preoptic area, paraventricular, and supraoptic nuclei did not co-localize kisspeptin. Since a high percentage of DYN cells of the arcuate nucleus also co-express neurokinin B (NKB), these findings suggest a possible interaction between kisspeptin, dynorphin, and NKB released by the same neuronal subpopulation in the regulation of GnRH secretion. Since kisspeptin and possibly NKB stimulate, while dynorphin inhibits GnRH release, this co-localization raises intriguing questions about the differential regula-
GnRH-1 neurons are a critical component of the hypothalamic–pituitary–gonadal axis and as such control reproductive function. GnRH-1 neurons are derived from nasal placode and thereafter migrate into the forebrain. The molecular cues directing GnRH-1 neurons to the final location are unclear. p75 nerve growth factor receptor (NGFR) is the low-affinity neurotrophin receptor that plays an important role in neuronal development and survival, mediating both neurite outgrowth and axonal extension. p75 NGFR has been identified in the developing pituitary gland as well as the olfactory system, suggesting involvement of neurotrophins in the establishment of these systems. In this study we characterize the temporal and spatial localization of the p75 NGFR with respect to the development of the GnRH-1 neuroendocrine system in mouse. Immunocytochemically studies were performed in nasal explants, an in vitro model system in which many of the in vivo characteristics of the developing GnRH-1 system and olfactory system are retained. Nasal explants were stained for p75 NGFR after three days in vitro (div), 7 div, 14 div, and 21 div. At all stages, p75 NGFR labeling was identified in two morphologically distinct cell types within the periphery of the explant. Both phenotypes directionally migrated out of the main tissue mass and showed p75 NGFR labeling of cell soma and processes. One p75 NGFR positive phenotype was small and had a simple unipolar/bipolar appearance (GnRH-1-like), with long processes strongly positive. The second p75 NGFR positive cell type was larger and multipolar with several short extensions. The density of p75 NGFR on these two cell types changed as a function of time in vitro. At 3 div the GnRH-1-like bipolar cells were strongly reactive in soma and processes. At 7 div the intensity increased in GnRH1-like cells farthest from the explant tissue mass. By 14 div the staining was still strong in GnRH-1-like cells, but the number was attenuated. At 21 div few positive bipolar elements were observed. In contrast to the GnRH-1-like cells, the multipolar cells showed a moderate p75 NGFR labeling at 3 and 7 div. From 14 to 21 div the staining became stronger and the number of multipolar cells in the periphery increased. Simultaneous localization of GnRH-1 and p75 NGFR was seen in the bipolar cells but not in multipolar cells. These results
74
Abstracts / Frontiers in Neuroendocrinology 27 (2006) 63–79
show that p75 NGFR is expressed in different manners in specific cell types in the developing nasal region. Identification of p75 NGFR on GnRH-1 neurons suggests a role for p75 NGFR in the migration and/or maturation of GnRH-1 neurons. In addition, p75 NGFR expression highlighted multipolar cellular elements that may participate in directing GnRH-1 cells and/or olfactory axons into the brain.
Endoreduplication in Caenorhabditis elegans: A positive or negative age-related event regulated by hormones? Ryan J. Haasl, Jennifer M. Marvitz, Sivan Vadakkadath Meethal, Miguel J. Gallego, Craig S. Atwood Section of Geriatrics and Gerontology, Department of Medicine, University of Wisconsin and Geriatric Research, Education, and Clinical Center, Veteran’s Administration Hospital, Madison, WI 53705, USA
doi:10.1016/j.yfrne.2006.03.153
Emerging evidence implicates neuroglial-endothelial interactions in the control of neurovascular junction formation for GnRH neurons in the median eminence of the hypothalamus. However, molecular determinants involved in such communication processes have not fully been identified. Screening for molecular cues known to play a role in the control of axon guidance indicated Semaphorin3A (Sema3A) as a candidate. Here, we used in situ hybridization and immunohistochemical stainings to examine the expression of Sema3A and its receptor, Neuropilin-1, in the adult median eminence of both mice and rats. Robust Sema3A mRNA expression was detected in capillaries of the median eminence, while only a scant hybridization signal was found in the nervous tissue. Consistent with these data, Sema3A immunoreactivity was restricted to portal blood capillaries of the median eminence. To determine whether GnRH nerve terminals may be able to sense Sema3A, we further investigated Neuropilin-1 expression in GnRH neurons. Double-labeling experiments revealed Neuropilin-1 mRNA expression in a significant fraction of GnRH neurons. The proportion of GnRH perikarya expressing Neuropilin-1 mRNA significantly varied during the rat estrous cycle and was maximal on proestrus (38.18 ± 2.47% in diestrus 2 vs. 49.99 ± 1.54% on proestrus, p < 0.05), a phase of the estrous cycle when GnRH nerve terminals have direct access to the portal vasculature. Accordingly, strong Neuropilin-1 immunoreactivity was detected in the external zone of the median eminence and was found to co-localize with GnRH-immunoreactive fibers. Taken together these results suggest that Sema3A may be a chemotropic factor secreted by endothelial cells to induce GnRH axon plasticity.
Endoreduplication is DNA replication in the absence of cytokinesis. Although somatic cells of adult Caenorhabditis elegans are non-mitotic, significant endoreduplication does occur in these cells, leading to body growth: quantification of Hoechst staining intensity revealed a 33% increase in total DNA content between 5 and 14 days after hatching; a concomitant 15% increase in body size was observed during the same time period. But what role, if any, does this age-related increase in endoreduplication serve? Does it have a positive or negative impact on the life of an organism? Relevant data offer contradictory answers to these questions. While in many plant species the extra DNA available to endoreduplicating cells allows for increased protein expression, polyploidy in mammals can be detrimental as seen in Down syndrome. This study characterized endoreduplication in C. elegans to assess its effects on longevity and potential regulation by age-related hormonal changes. We localized GnRH1 receptor (GnRH1R), and quantified nuclear DNA content of hypodermal (hyp7), intestinal, and neuronal cells in wild type (WT) worms and WT worms treated with antisense oligonucleotides specific to cyd-1 (enables G1 ! S progression) and lon-1 (TGFb-like pathway component). Six days after hatching, neuronal, hyp-7, and intestinal nuclei were diploid, 8-ploid and 64-ploid in WT worms and tetraploid, 16-ploid and 64ploid in anti-lon-1 worms (n = 20 nuclei/cell type). The increase in hyp-7 ploidy in anti-lon-1 worms was reflected by a significant 20% increase in hyp-7 nuclear size (p < 0.001, n = 20 nuclei), but no change in body size. The ploidy of anti-cyd-1 nuclei was identical to that of WT worms and no significant differences in body or nuclear size were detected. Though all three groups exhibited similar average lifespans, relative to WT, death rates varied: at 12 days after hatching 12, 22, and 36% of initial anti-cyd-1, WT, and anti-lon-1 populations remained. Immunohistochemical localization of GnRH1R in intestinal nuclei, by far the most endoreduplicative nuclei in C. elegans, is suggestive of a link between the HPG axis and endoreduplication. Future studies will examine the hormonal regulation of endoreduplication, the effect of endoreduplication on reproductive capacity, nutrient storage, and health, and whether endoreduplication serves some purposive response in maintaining the viability of nematodes.
doi:10.1016/j.yfrne.2006.03.154
doi:10.1016/j.yfrne.2006.03.155
Expression of Semaphorin3A and Neuropilin-1 in the adult median eminence C. Campagne a, S.G. Bouret a,b, J.-C. Beauvillain a, V. Prevot a a Inserm U816, Place de Verdun, 59045 Lille Cedex, France b The Saban Research Institute, Childrens Hospital Los Angeles, University of Southern California, Los Angeles, USA