Peptidyl glycine α-amidating mono-oxygenase (PGA), the terminal enzyme in the pathway of oxytocin synthesis, was measured in extracts of ovine corpora lutea throughout the oestrous cycle. Activity of PGA was low early in the cycle but increased between days 2 and 10 (from 2·3 to 9·0 pmol/mg protein per h) and remained high until day 15. Thereafter, activity declined rapidly at structural luteolysis and was low in corpora albicantia collected 18 and 20 days after ovulation (1·28 and 1·07 pmol/mg protein per h respectively). Luteal concentrations of ascorbic acid, a cofactor for PGA, were high (4·7 μmol/g wet wt tissue) by day 4 after oestrus; concentrations fell rapidly after day 15 (to 2·1 μmol/g on day 16). Concentrations of ascorbic acid were also high in the pituitary gland and in the adrenal medulla and cortex. Concentrations of oxytocin in luteal tissue, which were low (0·3 nmol/g wet wt) on day 2 after oestrus, were highest (2·73 nmol/g) on day 6 and declined thereafter (0·56 nmol/g on day 10, 0·08 nmol/g on day 15 and not detectable on days 18 and 20).
Concentrations of oxytocin, progesterone, PGA and protein were measured in subcellular fractions obtained after density gradient centrifugation of extracts of corpora lutea collected on days 6, 7 and 12 of the oestrous cycle, and on day 7 from an anaesthetized ewe before and after treatment with the prostaglandin F2α analogue, cloprostenol. PGA co-localized with particle-associated oxytocin in fractions of density 1·049–1·054 g/ml. Exogenous [3H]oxytocin and [3H]progesterone and endogenous progesterone localized in fractions of density 1·035 g/ml. Oxytocin and PGA were depleted from fractions of density 1·049–1·054 g/ml following cloprostenol treatment in vivo.
Fractionation of extracts of ovine corpora lutea by high-performance liquid chromatography (HPLC) followed by radioimmunoassay and radioreceptor assay for oxytocin demonstrated the presence of at least two cross-reacting substances with elution characteristics distinct from oxytocin. Concentrations of these peptides increased as the cycle progressed. These compounds differed from the putative C-terminally extended post-translational processing intermediates, oxytocinyl-glycine, oxytocinyl-glycine-lysine and oxytocinyl-glycine-lysine-arginine, as indicated by their elution positions on HPLC and the specificities of the assays used to detect them, and no conclusions could be drawn on which post-translational processing step was rate-limiting in oxytocin synthesis.
These data are consistent with the suggestion that post-translational processing of oxytocin-neurophysin prohormone takes place in secretory granules in luteal cells. The low activity of PGA early in the cycle may account for the lag previously observed between concentrations of the oxytocin-neurophysin prohormone mRNA and the mature peptide, but post-translational processing intermediates could not be identified. The rate of α-amidation is unlikely to be controlled by availability of ascorbic acid.