The investigations detailed in this thesis were undertaken to determine the
mechanisms regulating the post translational control of LH secretion in the ewe. LH is
stored within dense core secretory granules in the gonadotrophs of the
adenohypophysis. Studies on cellular ultrastructure and gene expression were
conducted on pituitary glands obtained from Welsh mountain ewes at points throughout
the oestrous cycle.
In order to determine if a structural priming response of gonadotrophin granules in
preparation for the preovulatory LH surge was present in the sheep, fixed pituitary
tissue was examined using immunogold labelling techniques under the electron
microscope. In the luteal phase, 20% of gonadotrophs possessed granules located in one
aspect of the cytoplasm juxtaposed to the nearest sinusoid, leading to the appearance of
a polarised cell. The percentage of polarised cells increased throughout the cycle,
reaching a peak of 90% in mid LH surge. Stereological analysis of granule diameter
showed that exocytosis occurred in a size dependant manner. Initial secretion occurred
from the 150-180nm size class with the larger granules released only during the
preovulatory LH surge. LHß mRNA abundance decreased from luteal through follicular
phase, reaching its lowest value during the surge. The replenishment of intracellular
LH stores occurred initially via the synthesis of the smaller granule size classes, first
visible in the cytoplasm 48h after an induced LH surge. As cells refilled, displaying a
morphology similar to a typical luteal phase gonadotroph at 96h post surge, the
diameter of granules synthesized increased, approaching values observed in the late
luteal phase. Throughout the refill phase, LHß mRNA abundance did not change.
These data suggest that the preovulatory LH surge, in the ewe, is not related to
increased synthesis of LH but a progressive recruitment of gonadotrophs into a
releasing state as indicated by the polarisation of secretory granules towards the
abutting capillary. Furthermore, the absence of changes in steady state LHß mRNA
suggests that post transcriptional mechanisms operate to mediate the refilling of the LH
The polarisation of gonadotrophs was prevented by the action of a GnRH antagonist.
The actions of GnRH and oestradiol, independently, were able to induce polarisation.
However, the direction of the granule translocation was incorrect in some 30% of cases
indicating that in vivo the two hormone actions synergise to ensure the correct direction
of the granule polarisation.