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Autocrine regulation of gonadotropin-releasing hormone in immortalized hypothalamic GT1-7 neurons
The existence of an ultrashort feedback mechanism regulating GnRH secretion has been supported from in vivo and in vitro studies. However, the complex synaptic connections of GnRH neurons with other neural elements made it difficult to determine whether the regulation was mediated by direct actions...
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| Format: | Thesis |
| Language: | English |
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Division of Chemical Pathology
2018
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| _version_ | 1867613235423739904 |
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| access_status_str | Open Access |
| author | Pithey, Anne Louise |
| author2 | Millar, Robert P |
| author_browse | Millar, Robert P Pithey, Anne Louise |
| author_facet | Millar, Robert P Pithey, Anne Louise |
| author_sort | Pithey, Anne Louise |
| collection | Thesis |
| description | The existence of an ultrashort feedback mechanism regulating GnRH secretion has been supported from in vivo and in vitro studies. However, the complex synaptic connections of GnRH neurons with other neural elements made it difficult to determine whether the regulation was mediated by direct actions on the GnRH neurons or through actions on other interneurons. The recent development of the GnRH-secreting neuronal cell line, GT1, provided a model system for the study of neural regulation of a pure population of GnRH neurons. The present studies utilized GT1 -7 cells to investigate whether GnRH (at the level of the nerve terminal) influences the control of its own release. Preliminary studies determined the presence of GnRH mRNA in GT1-7 cells and established a cell culture system for the analysis of secretagogue-induced GnRH release. In this system GnRH release was shown to be spontaneous and was enhanced by the addition of K⁺, L-GLU, forskolin and PMA. Furthermore, K⁺- and forskolin-induced GnRH release was dependent on extracellular Ca²⁺. For the analysis of an ultrashort feedback mechanism, GT1-7 cells were cultured in 6-well plates to near confluence and then incubated in serum-free medium in the presence (1 nM- 1 μM) or absence of GnRH antagonist, Ant 27. Basal, K⁺-and forskolin-induced secretion of GnRH was monitored with antiserum 1076 which does not cross-react with Ant 27 at> 1 μM. Ant 27 treatment increased basal, K⁺- and forskolin-stimulated GnRH release in a dose-dependent manner. Total content was unaffected by 18 h treatment of GT1-7 cells with Ant 27. This suggests that the effects of Ant 27 are at the level of release and not biosynthesis. The presence of GnRH binding sites in the cells was demonstrated with ¹²⁵I-GnRH analog. These findings support the concept that GnRH, acting via autoreceptors, negatively controls its own release. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/27030 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:32:54.720Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2018 |
| publishDateRange | 2018 |
| publishDateSort | 2018 |
| publisher | Division of Chemical Pathology |
| publisherStr | Division of Chemical Pathology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/27030 Autocrine regulation of gonadotropin-releasing hormone in immortalized hypothalamic GT1-7 neurons Pithey, Anne Louise Millar, Robert P Dutlow, Clive Chemical Pathology Autocrine Motility Factor Gonadorelin Receptors, LHRH The existence of an ultrashort feedback mechanism regulating GnRH secretion has been supported from in vivo and in vitro studies. However, the complex synaptic connections of GnRH neurons with other neural elements made it difficult to determine whether the regulation was mediated by direct actions on the GnRH neurons or through actions on other interneurons. The recent development of the GnRH-secreting neuronal cell line, GT1, provided a model system for the study of neural regulation of a pure population of GnRH neurons. The present studies utilized GT1 -7 cells to investigate whether GnRH (at the level of the nerve terminal) influences the control of its own release. Preliminary studies determined the presence of GnRH mRNA in GT1-7 cells and established a cell culture system for the analysis of secretagogue-induced GnRH release. In this system GnRH release was shown to be spontaneous and was enhanced by the addition of K⁺, L-GLU, forskolin and PMA. Furthermore, K⁺- and forskolin-induced GnRH release was dependent on extracellular Ca²⁺. For the analysis of an ultrashort feedback mechanism, GT1-7 cells were cultured in 6-well plates to near confluence and then incubated in serum-free medium in the presence (1 nM- 1 μM) or absence of GnRH antagonist, Ant 27. Basal, K⁺-and forskolin-induced secretion of GnRH was monitored with antiserum 1076 which does not cross-react with Ant 27 at> 1 μM. Ant 27 treatment increased basal, K⁺- and forskolin-stimulated GnRH release in a dose-dependent manner. Total content was unaffected by 18 h treatment of GT1-7 cells with Ant 27. This suggests that the effects of Ant 27 are at the level of release and not biosynthesis. The presence of GnRH binding sites in the cells was demonstrated with ¹²⁵I-GnRH analog. These findings support the concept that GnRH, acting via autoreceptors, negatively controls its own release. 2018-01-29T06:45:49Z 2018-01-29T06:45:49Z 1994 Master Thesis Masters MSc (Med) http://hdl.handle.net/11427/27030 eng application/pdf Division of Chemical Pathology Faculty of Health Sciences University of Cape Town |
| spellingShingle | Chemical Pathology Autocrine Motility Factor Gonadorelin Receptors, LHRH Pithey, Anne Louise Autocrine regulation of gonadotropin-releasing hormone in immortalized hypothalamic GT1-7 neurons |
| thesis_degree_str | Master's |
| title | Autocrine regulation of gonadotropin-releasing hormone in immortalized hypothalamic GT1-7 neurons |
| title_full | Autocrine regulation of gonadotropin-releasing hormone in immortalized hypothalamic GT1-7 neurons |
| title_fullStr | Autocrine regulation of gonadotropin-releasing hormone in immortalized hypothalamic GT1-7 neurons |
| title_full_unstemmed | Autocrine regulation of gonadotropin-releasing hormone in immortalized hypothalamic GT1-7 neurons |
| title_short | Autocrine regulation of gonadotropin-releasing hormone in immortalized hypothalamic GT1-7 neurons |
| title_sort | autocrine regulation of gonadotropin releasing hormone in immortalized hypothalamic gt1 7 neurons |
| topic | Chemical Pathology Autocrine Motility Factor Gonadorelin Receptors, LHRH |
| url | http://hdl.handle.net/11427/27030 |
| work_keys_str_mv | AT pitheyannelouise autocrineregulationofgonadotropinreleasinghormoneinimmortalizedhypothalamicgt17neurons |