COMPONENTS OF THE NEURONAL EXOCYTOTIC MACHINERY IN THE ANTERIOR-PITUITARY OF THE OVARIECTOMIZED EWE AND THE EFFECTS OF ESTROGEN 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 o variectomised ewe. Sheep were treated with either oestradiol benzoate, or oil (i.m.) and blood samples collected for LH assay. Pituitaries w ere 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, Mu nc-18-1, alpha/beta-SNAP, csp, and secretogranin II were detected in g onadotropes and somatotropes. Lactotropes were positive for SNAP-25 an d synaptophysin (other synaptic proteins not investigated). Synaptotag min I was detected in gonadotropes and lactotropes, but not somatotrop es. Synaptophysin, SNAP-25, synaptotagmins I, II and III, VAMP-2, rab3 A, Munc-18-1, alpha/beta-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 secretogra nin II were detected in the intermediate lobe. Syntaxin and complexin II antibodies did not label any region of the ovine pituitary. Oestrog en treatment, to induce a pre-ovulatory-like LH surge, caused migratio n of LH-containing secretory granules toward the plasma membrane of go nadotropes, but did not alter the percentage of gonadotropes expressin g each exocytotic protein. Oestrogen treatment caused a similar redist ribution of csp and secretogranin II staining in gonadotropes. We conc lude that synaptic protein expression is not altered in the anterior p ituitary at the time when LH secretion is maximal. The ubiquitous dist ribution of many exocytotic proteins suggests that all hormone-secreti ng cells of the pituitary gland contain the same, or similar exocytoti c machinery, but distinct 'activating factors' are required to selecti vely trigger the secretion of individual hormones.