The research presented in this thesis provides information on three different aspects of the regulation of cortisol biosynthesis by the sheep adrenal gland.
The first part of this work describes the efficacy of hydroxysteroid dehydrogenase inhibitors as inhibitors of adrenal cortisol biosynthesis. These inhibitors, if properly characterized, would be of great utility in studies of cortisol induced maturational events and the role of cortisol In the initiation of parturition in the sheep fetus. The effectiveness of trilostane and azastene as inhibitors of adrenal cortisol biosynthesis was compared both in vitro and in vivo.
In vitro studies were performed using adrenal slices from non-pregnant ewes incubated in medium with or without ACTH1-24, and the effect of adding trilostane or azastene on cortisol synthesis and secretion over 60 min of incubation was determined. It was found that trilostane decreased cortisol synthesis and release both in unstimulated and in ACTH-stimulated adrenal slices in vitro (70%-95% inhibition). Azastene was somewhat less effective in this system (40%-55% inhibition).
In vivo studies were undertaken to compare the effects of trilostane on plasma cortisol levels in non-pregnant and pregnant ewes. Seven cyclic ewes at days 10-12 of the cycle and 6 late pregnant ewes at days 120-130 of pregnancy were injected intravenously with 0, 1.0, 25 or 100 mg trilostane dissolved in 1 ml dimethyl sulphoxide (DMSO). Blood samples were obtained at 30 min intervals from the beginning of the experiment until 4-6 hr after injection and the plasma was assayed for cortisol. The results were analysed by comparing the levels of plasma cortisol after trilostane injection with levels in vehicle (DMSO) injected controls. It was found that administration of 1 or 25 mg trilostane to pregnant ewes caused a decrease in plasma cortisol concentration 2.5-4 hr and 0.5-2 hr after injection, respectively. In contrast, administration of these doses of trilostane to non-pregnant animals had only a marginal effect on plasma cortisol levels. However, administration of 100 mg trilostane did decrease plasma cortisol in non-pregnant animals, but only between 0.5-4 hr, whereas in pregnant animals at this dose cortisol levels were inhibited for over 6 hr. The results from both in vitro and in vivo experiments indicate that trilostane would be a useful agent for inhibiting cortisol biosynthesis in the sheep.
The second part of the thesis presents an ontogenic study of corticosteroid binding protein (CBG) in the plasma of fetal lambs. CBG binding capacity was measured by equilibrium dialysis in plasma from three normal fetuses during the 20 days before delivery, and in plasma from four fetuses during induction of premature delivery by graded increments in fetal cortisol infusion. CBG binding capacity was also determined in cortisol-infused fetuses in which delivery was delayed by maternal administration of either progesterone (4 fetuses) or medroxy progesterone acetate (MPA) (2 fetuses).
Fetuses infused with cortisol alone were delivered at 128.75 ±12.5 hr after the start of infusion. Delivery was prevented in all ewes injected with progesterone and in one of the two injectd with MPA during the time that cortisol was infused. CBG binding capacity and the concentration of both protein bound and unbound cortisol increased before term in normal and cortisol infused fetuses in a similar fashion. CBG binding capacity continued to increase in cortisol infused fetuses in which delivery was delayed, but decreased immediately post-partum. The CBG bound to unbound cortisol ratio increased in normal and cortisol infused fetuses as the total plasma cortisol concentration increased. The increase in CBG binding capacity was positively correlated with the total plasma cortisol concentration. These results suggest that the prepartum rise in the plasma cortisol concentration in normal or cortisol treated fetuses may be responsible for the increase in plasma CBG concentration. This increased CBG may play an important role in restricting cortisol action towards term on target tissues other than the placenta.
In the final part of the thesis, the effect of epidermal growth factor (EGF) on adrenal cortisol biosynthesis in fetal and non-pregnant adult sheep was studied both in vivo and in vitro. Fetuses were infused with EGF (500-800 µg/day over 3-5 days, or 200-300 µg/day, for 9-10 days) from 114-115 days of gestational age. In fetuses, plasma cortisol levels increased significantly during the first 4 days of EGF infusion, although no significant changes were found in maternal plasma cortisol concentrations during this time. Similarly, infusion of EGF (2.9 µg/kg/hr) into non-pregnant ewes over 24 hr, caused an increase in plasma cortisol concentrations within 1 hr after commencement of the infusion. Mean plasma cortisol rose to a sustained plateau of 700% of the saline control value and fell towards saline control values before the end of the infusion.
In vitro, adrenal slices taken from non-pregnant ewes synthesized and released significantly greater amounts of cortisol in the presence of either EGF or ACTH1-24. EGF and ACTH1-24 gave a maximal stimulation of cortisol biosynthesis between 10 and 100 ng/ml and above 100 ng/ml, respectively. Biologically inactive reduced S-carboxyamidomethyl EGF as well as a number of polypeptide hormones and growth factors were without effect at these dose levels. Stimulation of cortisol synthesis and secretion by EGF was dependent on protein synthesis as it was for ACTH. However, EGF did not synergise with ACTH in promoting cortisol production. Addition of the cholesterol synthesis inhibitors compactin and AY 9944 produced a total blockade of EGF-stimulated cortisol synthesis although they only partially blocked stimulation by ACTH. Conversely, ovine low density lipoprotein (LDL) and high density lipoprotein (HDL) did not enhance EGF-stimulated cortisol production although LDL did increase ACTH-stimulated cortisol production significantly. These results imply that the site of EGF action in the adrenal is upon de novo cholesterol synthesis, whereas the action of ACTH is only partially dependent on cholesterol precursor supply, in accord with other studies. Further work showed that EGF significantly stimulated the rate of incorporation of 14C-acetate into digitonin-precipitable sterols by adrenal slices from 30 to 120 min of incubation. Moreover, EGF also significantly increased the activity of the first and rate limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase. EGF was without effect on the conversion of 3H-pregnenolone to cortisol by adrenal slices and no alterations in the levels of steroidogenic intermediates between pregnenolone and cortisol were observed as a result of incubation in the presence of EGF. However, ACTH did significantly increase 3H-cortisol formation from 3H-pregnenolone from 30 to 120 min of incubation, and the amount of 3H-corticosterone in the medium was decreased significantly by ACTH, suggesting a stimulation of the 17α-hydroxylase. Thus, the results from these in vitro studies suggest that EGF increases adrenal cortisol biosynthesis by stimulaion of cholesterol precursor biosynthesis at the HKG-CoA reductase. It is possible that EGF may be a physiologically important regulatory factor affecting adrenal cortisol synthesis and secretion in the fetal and adult sheep.