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Title: Intracellular processing of luteinising hormone and follicle-stimulating hormone
Author: Evans, James G.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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The effect of transiently transfected granin expression on secretion of hCG (structurally related to LH and FSH) from JEG3 (human choriocarcinoma) cells was undetectable due to the high background of non-transfected cells. Expression of LH and FSH in transiently transfected JEG3 cells was demonstrated by RT-PCR and confocal microscopy. Like hCG, the pituitary gonadotrophins exhibited non-punctate distribution within the cytosol suggesting constitutive secretion. Stable transfections of JEG3 cells containing 1-2 copies of the oLHβ subunit gene expressed mRNA transcripts larger than those expressed by the ovine pituitary. oLH and oFSH secretion was undetectable by radioimmunoassay despite treatment with 1-16mM of the inducing agent butyric acid. Biochemical studies in which gonadotrophin and granin constructs were cotransfected into CHO cells were unable to detect any additional storage after 48 hours of expression. Confocal analyses revealed that gonadotrophins did not form granules when expressed in CHO cells. Chromogranin A (CgA) and chromogranin B (CgB) both formed aggregates upon expression in CHO cells. Coexpression with gonadotrophins induced formation of aggregates within the ER and Golgi cisternae and of secretory vesicles 100-300 nm in diameter. LH colocalised with CgA and CgB. Analysis of whole mouse pituitaries using confocal microscopy demonstrated the prevalence of bihormonal gonadotrophs abutting capillaries. Gonadotrophs exhibited differential localisation of LH and FSH. Colocalised LH and CgA occurred towards the periphery of the cell whereas FSH remained dispersed throughout the cytosol. CgA immunoreactivity appeared reduced in areas juxtaposed to capillaries whereas LH immunoreactivity remained. This suggests that CgA does not have a role in the final stages of LH exocytosis. Pituitaries from transgenic mice expressing stabilised oFSHβ mRNA under control of the LH promoter exhibited an approximate 2-fold increase in FSH expression in comparison to non-transgenics.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available