Components of the neuronal exocytotic machinery in the anterior pituitary of the ovariectamised ewe and the effects of oestrogen in gonadotropes as studied with confocal microscopy

We have investigated exocytotic proteins in ovine pituitary cells and sought to identify changes in expression of these proteins related to the effects of estrogen on luteinising hormone (LH) secretion in the ovariectomised ewe. Sheep were treated with either oestradiol benzoate, or oil (i.m.) and blood samples collected for LH assay. Pituitaries were perfusion-fixed and dual-label immunohistochemistry was performed to identify hormone-secreting cells, and colocalise synaptic proteins within different cell types. Synaptophysin, SNAP-25, VAMP-2, rab3A, Munc-18-1, α/β-SNAP, csp, and secretogranin II were detected in gonadotropes and somatotropes. Lactotropes were positive for SNAP-25 and synaptophysin (other synaptic proteins not investigated). Synaptotagmin I was detected in gonadotropes and lactotropes, but not somatotropes. Synaptophysin, SNAP-25, synaptotagmins I, II and III, VAMP-2, rab3A, Munc-18-1, α/β-SNAP, csp, and secretogranin II were detected in nerve fibres of the posterior lobe. Membrane staining for SNAP-25 and weak cytoplasmic labelling for both synaptotagmin I and secretogranin II were detected in the intermediate lobe. Syntaxin and complexin II antibodies did not label any region of the ovine pituitary. Oestrogen treatment, to induce a pre-ovulatory-like LH surge, caused migration of LH-containing secretory granules toward the plasma membrane of gonadotropes, but did not alter the percentage of gonadotropes expressing each exocytotic protein. Oestrogen treatment caused a similar redistribution of csp and secretogranin II staining in gonadotropes. We conclude that synaptic protein expression is not altered in the anterior pituitary at the time when LH secretion is maximal. The ubiquitous distribution of many exocytotic proteins suggests that all hormone-secreting cells of the pituitary gland contain the same, or similar exocytotic machinery, but distinct 'activating factors' are required to selectively trigger the secretion of individual hormones.