Hormone secretion in the corpus luteum

The aim of this thesis was to investigate the mechanism of progesterone secretion from the bovine corpus luteum: in particular, to examine whether steroid hormones are packaged into membrane-bound granules and then transported to the cell membrane where their contents are released by exocytosis. Morphological examination of bovine mid-cycle corpora lutea showed that luteal cell cytoplasm and extracellular space contained electron-dense granules, 0.2–0.4 μ in diameter. Specific cytochemical staining techniques identified microperoxisomes and lysosomes as being intracellular, and distinguished them from the putative secretory granules. Morphometric analysis of the bovine corpus luteum was carried out at low and high magnifications on days 6, 13, 15, 17 and 20 of the oestrus cycle. Luteal cells comprised 50% of all cell-types at the beginning and end of the cycle and rose to comprise 74% at mid-cycle. The analysis of luteal cell cytoplasm showed that the relative proportion of cellular organelles involved in the synthesis and secretion of progesterone varied throughout the oestrus cycle. The densely-staining granules occupied 2.5% of the luteal cell volume at day 6, but gradually rose in numbers to reach a maximum of 5.6% around day 17. By day 20 their volume had dropped again to 2%. During mid-cycle, when progesterone secretion was maximal, many of these granules were observed to have undergone exocytosis, whereas late in the cycle evidence of exocytosis was rare. Thus, the changes in the proportion of aer/ground plasm and densely-staining granules correlated with the changes in the rate of progesterone secretion from the corpus luteum. Extensive protein synthesis was indicated since the proportion of ger and Golgi organelles increased by the time progesterone synthesis began. This was consistent with the need to synthesize a binding protein to which progesterone could be bound within secretory granules. Biochemical and cytochemical techniques were used to demonstrate the association of progesterone with electron-dense granules in fractions obtained by density gradient centrifugation. Differential centrifugation of luteal tissue obtained at mid-cycle showed that up to one-third of the total progesterone could be sedimented. Most of this particulate hormone banded at a density of less than d=1.118 on sucrose gradients. Marker enzyme analysis of individual gradient fractions showed that mitochondria, lysosomes, microperoxisomes, and most of the membraneous elements banded at densities greater than d=1.118. Electron microscopy and cytochemistry of fixed fractions from three selected regions of the gradient identified microperoxisomes and lysosomes in the two regions with densities d>1.118, whilst non-staining electron-dense granules and progesterone were identified in the region with density d<1.118. The corpora lutea of cows and sheep were shown to contain a high-affinity binding protein for progesterone, which was distinct from CBG. This protein was also detected in utero-ovarian and jugular venous plasmas. The bovine corpus luteum contained another progesterone-binding protein which had a greater capacity, but slightly lower affinity, for progesterone. A role for both these proteins in the biosynthesis of progesterone and its secretion in granules from bovine luteal cells is proposed.