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Identification of a gonadotropin-releasing hormone receptor homologue in Caenorhabditis elegans
Abstracts / Frontiers in Neuroendocrinology 27 (2006) 63–79
results support our hypothesis that GPR30 plays a role in E2 action in LHRH neurons. (Supported by NIH Grants HD15344, HD11533, and RR00167.) doi:10.1016/j.yfrne.2006.03.147
Gene expression in migrating GnRH-GFP neurons harvested by flow cytometry Nadia Shivji, Delicia Karunadasa, R. John Bicknell Laboratory of Neuronal Development and Survival, Babraham Institute, Babraham Research Campus, Cambridge CB2 4AT, UK GnRH neurons are derived in the olfactory placode and migrate along a subset of pioneer vomeronasal axons into the forebrain during embryonic development. The pathway of this migration is well documented but the molecular signals which regulate the process are less well understood [1]. As a resource for gene expression studies at different stages of migration, we have used an established GnRH promoter-GFP transgenic mouse line [2] combined with fluorescence activated cell sorting (FACS) to produce highly enriched populations of GnRH neurons. At embryonic day 14.5 (E14.5) in these mice, 91% of GFP positive cells are GnRH-immunoreactive, and 62% of GnRH-immunoreactive cells express GFP when examined in sagittal sections (n = 3). Embryo heads were embedded in low melting point agarose and 500 lm sagittal sections prepared on a vibratome. Olfactory system and forebrain areas were microdissected from six to eight embryos and enzymically dissociated to produce a cell suspension from which GFP expressing cells were sorted using a Becton Dickinson FACSAria. At E13.5, and gating to sort an average 0.023% of the total population, a yield of 1032 ± 114 cells per embryo was obtained of which 89 ± 3% were GFP positive when viewed for DAPI/GFP fluorescence. In situ hybridisation of these harvested cells indicates that 40–45% of the population express GnRH mRNA. Restricting tissue collection to olfactory system and cribriform plate area only, combined with higher stringency gating to sort 0.003% of the total cells yields harvested populations of 100 cells per embryo in which up to 79% of cells express GnRH mRNA by in situ hybridisation. GnRH transcripts are detectable by standard RT-PCR procedures in RNA derived from 100 harvested cells. We are currently examining expression of genes encoding established cell–cell recognition molecules using RT-PCR.
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Identification of a gonadotropin-releasing hormone receptor homologue in Caenorhabditis elegans Sivan Vadakkadath Meethal a, Miguel J. Gallego a, a Ryan J. Haasl , Steven J. Petras a, Jean Y. Sgro b, Richard L. Bowen c, Craig S. Atwood a,d a Section of Geriatrics and Gerontology, Department of Medicine, University of Wisconsin-Madison and Geriatric Research, Education and Clinical Center, Veterans Administration Hospital, Madison, WI 53705, USA b Biotechnology Center, University of Wisconsin, Madison, WI 53705, USA c Voyager Pharmaceutical Corporation, Raleigh, NC 27615, USA d Case Western Reserve University, Cleveland, OH 44106, USA The molecular neuroendocrinology regulating reproduction in Caenorhabditis elegans has not been studied. Our sequence analyses indicated the presence of two genes, one of 401aa (AAK21401)) and another of 379aa (NP_506566) with 46.9 and 44.7% nucleotide similarity to that of human gonadotropin-releasing hormone 1 receptor (GnRH1R) and GnRH2R, respectively. Like the human homologue, structural analysis of GnRH1R predicted a rhodopsin family member with seven transmembrane domains, G-protein coupling sites and phosphorylation sites for protein kinase C. Of the functionally important amino acids in human GnRH1R, 60% were conserved in the C. elegans homologue. Remarkably, C. elegans cultured in the presence of human GnRH produced significantly more eggs (17%, P < 0.028; n = 8) and offspring (45%, P < 0.002; n = 14) compared to untreated worms. Conversely, the human GnRH1R antagonist acyline reduced the number of offspring (13%, P < 0.009; n = 17). Immunoanalyses using antibodies generated against both human and C. elegans GnRH1R indicated the presence of a 46 kDa protein, the calculated molecular mass of C. elegans GnRH1R, and its specific localization to the germ line and to three pairs of cells within the pharynx. Phylogenetic analysis supports an early evolutionary origin of GnRHR and a close relationship between C. elegans GnRHR’s and the GnRHR’s and adipokinetic hormone receptors of arthropods. The identification of an evolutionarily conserved, functional, primordial chemo-neuro-endocrine sensory system that regulates reproduction in C. elegans provides a new model system for the study of the neuroendocrinology of reproduction. doi:10.1016/j.yfrne.2006.03.149
References [1] S. Wray et al., Proc. Natl. Acad. Sci. USA 86 (20) (1989) 8132–8136. [2] D.J. Spergel et al., J. Neurosci. 19 (6) (1999) 2037–2050. doi:10.1016/j.yfrne.2006.03.148
Genomic elements of the hypothalamic-pituitary-gonadal axis in Caenorhabditis elegans Miguel J. Gallego a, Sivan Vadakkadath Meethal a, Ryan J. Haasl a, Jean Y. Sgro b, Craig S. Atwood a
results support our hypothesis that GPR30 plays a role in E2 action in LHRH neurons. (Supported by NIH Grants HD15344, HD11533, and RR00167.) doi:10.1016/j.yfrne.2006.03.147
Gene expression in migrating GnRH-GFP neurons harvested by flow cytometry Nadia Shivji, Delicia Karunadasa, R. John Bicknell Laboratory of Neuronal Development and Survival, Babraham Institute, Babraham Research Campus, Cambridge CB2 4AT, UK GnRH neurons are derived in the olfactory placode and migrate along a subset of pioneer vomeronasal axons into the forebrain during embryonic development. The pathway of this migration is well documented but the molecular signals which regulate the process are less well understood [1]. As a resource for gene expression studies at different stages of migration, we have used an established GnRH promoter-GFP transgenic mouse line [2] combined with fluorescence activated cell sorting (FACS) to produce highly enriched populations of GnRH neurons. At embryonic day 14.5 (E14.5) in these mice, 91% of GFP positive cells are GnRH-immunoreactive, and 62% of GnRH-immunoreactive cells express GFP when examined in sagittal sections (n = 3). Embryo heads were embedded in low melting point agarose and 500 lm sagittal sections prepared on a vibratome. Olfactory system and forebrain areas were microdissected from six to eight embryos and enzymically dissociated to produce a cell suspension from which GFP expressing cells were sorted using a Becton Dickinson FACSAria. At E13.5, and gating to sort an average 0.023% of the total population, a yield of 1032 ± 114 cells per embryo was obtained of which 89 ± 3% were GFP positive when viewed for DAPI/GFP fluorescence. In situ hybridisation of these harvested cells indicates that 40–45% of the population express GnRH mRNA. Restricting tissue collection to olfactory system and cribriform plate area only, combined with higher stringency gating to sort 0.003% of the total cells yields harvested populations of 100 cells per embryo in which up to 79% of cells express GnRH mRNA by in situ hybridisation. GnRH transcripts are detectable by standard RT-PCR procedures in RNA derived from 100 harvested cells. We are currently examining expression of genes encoding established cell–cell recognition molecules using RT-PCR.
71
Identification of a gonadotropin-releasing hormone receptor homologue in Caenorhabditis elegans Sivan Vadakkadath Meethal a, Miguel J. Gallego a, a Ryan J. Haasl , Steven J. Petras a, Jean Y. Sgro b, Richard L. Bowen c, Craig S. Atwood a,d a Section of Geriatrics and Gerontology, Department of Medicine, University of Wisconsin-Madison and Geriatric Research, Education and Clinical Center, Veterans Administration Hospital, Madison, WI 53705, USA b Biotechnology Center, University of Wisconsin, Madison, WI 53705, USA c Voyager Pharmaceutical Corporation, Raleigh, NC 27615, USA d Case Western Reserve University, Cleveland, OH 44106, USA The molecular neuroendocrinology regulating reproduction in Caenorhabditis elegans has not been studied. Our sequence analyses indicated the presence of two genes, one of 401aa (AAK21401)) and another of 379aa (NP_506566) with 46.9 and 44.7% nucleotide similarity to that of human gonadotropin-releasing hormone 1 receptor (GnRH1R) and GnRH2R, respectively. Like the human homologue, structural analysis of GnRH1R predicted a rhodopsin family member with seven transmembrane domains, G-protein coupling sites and phosphorylation sites for protein kinase C. Of the functionally important amino acids in human GnRH1R, 60% were conserved in the C. elegans homologue. Remarkably, C. elegans cultured in the presence of human GnRH produced significantly more eggs (17%, P < 0.028; n = 8) and offspring (45%, P < 0.002; n = 14) compared to untreated worms. Conversely, the human GnRH1R antagonist acyline reduced the number of offspring (13%, P < 0.009; n = 17). Immunoanalyses using antibodies generated against both human and C. elegans GnRH1R indicated the presence of a 46 kDa protein, the calculated molecular mass of C. elegans GnRH1R, and its specific localization to the germ line and to three pairs of cells within the pharynx. Phylogenetic analysis supports an early evolutionary origin of GnRHR and a close relationship between C. elegans GnRHR’s and the GnRHR’s and adipokinetic hormone receptors of arthropods. The identification of an evolutionarily conserved, functional, primordial chemo-neuro-endocrine sensory system that regulates reproduction in C. elegans provides a new model system for the study of the neuroendocrinology of reproduction. doi:10.1016/j.yfrne.2006.03.149
References [1] S. Wray et al., Proc. Natl. Acad. Sci. USA 86 (20) (1989) 8132–8136. [2] D.J. Spergel et al., J. Neurosci. 19 (6) (1999) 2037–2050. doi:10.1016/j.yfrne.2006.03.148
Genomic elements of the hypothalamic-pituitary-gonadal axis in Caenorhabditis elegans Miguel J. Gallego a, Sivan Vadakkadath Meethal a, Ryan J. Haasl a, Jean Y. Sgro b, Craig S. Atwood a