Intracellular processing of luteinising hormone and follicle-stimulating hormone

The pituitary glycoproteins LH and FSH are secreted from gonadotrophs via regulated and constitutive pathways under control of GnRH. Several distinct types of secretory vesicles (granules) have been previously reported to contain gonadotrophins colocalised with Ca+ + -binding aggregative granin proteins. The putative role of granins in gonadotrophin granulogenesis was investigated in this thesis using recombinant DNA technology. Mammalian expression constructs were transfected into cell lines permitting comparative expression levels for each of the proteins under investigation.Expression of LH and FSH in transiently transfected JEG3 (human choriocarcinoma) and CHO cells was demonstrated by RT-PCR and confocal microscopy. Although granin expression did not affect the degree of hCG storage in JEG3 cells, chromogranin A (CgA) and secretogranin II (Sgll) did exert an enhancement of hCG expression. oLH expression in CHO cells also appeared to be enhanced by CgA. In addition Sgll appeared to reduce storage of oLH in cotransfected CHO cells. Like hCG, LH and FSH exhibited dispersed distribution within the cytosol suggesting constitutive secretion. CgA and chromogranin B (CgB) displayed punctate expression within transfected CHO cells. Coexpression of CgB and oLH in CHO cells demonstrated their colocalisation in the ER and TGN but not within CgBonly secretory vesicles. In contrast CgA expression appeared to induce formation of CgA and LH-containing secretory vesicles (310-640nm diameter). Their apparent divergence into oLH- and CgA-only vesicles towards the periphery of the cell suggests that this granin does not have a role in LH exocytosis correlating with in vivo data. The suggested role of CgA in pre-exocytotic sorting and retention of LH is strengthened by the observation of reduced LH storage in high passage LPT2 cells (mouse gonadotrophs with regulated LH secretion) with undetectable levels of intracellular CgA