Adrenalectomized Ewes Research Articles

Regulation of maternal ACTH in ovine pregnancy: does progesterone play a role?

Pregnancy is characterized by increased plasma adrenocorticotropic hormone (ACTH) and cortisol. Studies suggest that progesterone acts as an antagonist at mineralocorticoid receptors. Therefore, we tested the hypothesis that chronic progesterone, produced by treatment of nonpregnant ewes or during pregnancy, will result in increased plasma ACTH relative to the plasma cortisol concentrations. We studied three groups of ewes: ovariectomized nonpregnant, nonpregnant treated with progesterone, and pregnant ewes. In two series of studies, ewes were adrenalectomized and replaced with 0.35 mg x kg(-1) x day(-1) or 0.5 mg x kg(-1) x day(-1) cortisol. In both studies, aldosterone was infused at 3 microg x kg(-1) x day(-1). In the first study, additional infusions of cortisol over 24 h were used to increase daily replacement doses to 0.5, 1, or 1.5 mg x kg(-1) x day(-1), and intact pregnant and nonpregnant ewes were studied with infusions of cortisol at 0, 0.5, and 1 mg x kg(-1) x day(-1). In adrenalectomized ewes chronically replaced to 0.35 mg x kg(-1) x day(-1) cortisol, plasma ACTH concentrations were decreased significantly in the nonpregnant progesterone-treated ewes compared with the ovariectomized nonpregnant ewes. With 0.5 mg x kg(-1) x day(-1) cortisol, plasma ACTH levels were greater in pregnant ewes than in nonpregnant ewes with or without progesterone. Overall plasma ACTH levels at 0.35 mg x kg(-1) x day(-1) were significantly related to the plasma protein concentration, suggesting that the ACTH levels in the hypocorticoid ewes are most closely related to plasma volume. Across all steroid doses, ACTH was positively related to plasma proteins and progesterone, and negatively related to cortisol. We conclude that increased progesterone does not alter the feedback relation of cortisol to ACTH, but may modulate ACTH indirectly through plasma volume.

Adrenalectomy alters regulation of blood pressure and endothelial nitric oxide synthase in sheep: modulation by estradiol

Hypoadrenocorticism produces more severe hypotension during the peripartal period in pregnant ewes and women. We hypothesized that estradiol increases the severity of hypotension after withdrawal of corticosteroids and that this results from combined effects of adrenalectomy and estradiol to increase endothelial nitric oxide synthase (eNOS). In study I, blood pressure and eNOS mRNA and protein in aorta, uterine, renal, and mesenteric arteries were measured in intact ewes or adrenalectomized ewes 18-20 h after cessation of infusion of cortisol and aldosterone; half of each group ewes were treated with estradiol. In study II, adrenalectomized ewes were similarly studied 22-28 h after withdrawal of corticosteroids. Estradiol treatment in both studies significantly increased eNOS mRNA and protein in uterine artery, whereas corticosteroid withdrawal decreased expression of eNOS mRNA and protein in uterine artery. In both studies, adrenalectomy and steroid withdrawal decreased mean arterial pressure. In study II, four of six adrenalectomized ewes not treated with estradiol showed dramatic phasic variations in blood pressure and heart rate with a period of approximately 20 s, developing within 22-28 h after corticosteroid withdrawal. Although there was no effect of estradiol on blood pressure in study I, in study II, ewes treated with estradiol did not develop this pattern. Estradiol also slowed both the decline in plasma sodium and the rise in plasma potassium after corticosteroid withdrawal. These results disprove the hypothesis that estradiol increases the severity of hypotension during hypoadrenocorticism. However, the study reveals an important effect of corticosteroid withdrawal on blood pressure, consistent with corticosteroid modulation of baroreflex responsiveness.

Alterations in maternal corticosteroid levels influence fetal urine and lung liquid production.

This study was designed to test the hypotheses that disruption of maternal adrenal secretion in late pregnancy requires fetal adaptations in order to maintain fetal blood volume and fetal viability. Pregnant ewes were adrenalectomized at approximately 112 days, and cortisol and aldosterone were replaced to either normal pregnant levels (with 1 mg/kg per day of cortisol and 3 microg/kg per day of aldosterone) or normal nonpregnant levels of aldosterone or cortisol (0.5 mg/kg per day of cortisol or 1.5 microg/kg per day of aldosterone). Fetal blood volume, blood pressure, lung liquid production, urine production, free water clearance, and glomerular filtration rate were measured at 130 days. In a separate group, fetal organ blood flow was measured. Fetal blood volume was not significantly decreased by disruption of maternal corticosteroid secretion. However fetal urine production and free water clearance were reduced in fetuses of low cortisol or low aldosterone ewes. Fetal lung liquid secretion was also significantly reduced in the low aldosterone group. The glomerular filtration rate was reduced in fetuses of all adrenalectomized ewes, regardless of replacement dose. Fetal blood pressure was significantly reduced in the fetuses of low aldosterone ewes; blood flow to several fetal organs was increased in this group, indicating that decreased vascular resistance may contribute to the relative hypotension. Alterations in maternal adrenal corticosteroid levels resulted in fetal adaptation to maintain fetal blood volume despite relative maternal hypovolemia. These adaptations occurred at the expense of fetal urine and lung liquid production.

Pharmacology and Physiology of Ovine Corticosteroid Receptors

The aim of these studies was to characterize the ovine corticosteroid receptors (MR, mineralocorticoid receptors and GR, glucocorticoid receptors) in ovine hippocampus and brainstem. Adrenal-intact and adrenalectomized ewes were studied; adrenalectomized ewes were killed 47 ± 9 h after steroid withdrawal, when symptoms of hypotension and/or hyperkalemia became evident. RT-PCR, immunoblotting and pharmacologic studies indicated the presence of both MR and GR in hippocampus and brainstem. Competitive binding studies using ³H-cortisol in brain tissue showed that the ovine MR binds cortisol, aldosterone and progesterone with equal affinity. Differences in receptor availability in intact and adrenalectomized ewes, along with determination of the binding affinity (K_d) of MR and GR, suggested that MR occupancy is about 90%, whereas GR occupancy is about 30%, in normal animals. There was a significant increase in protein level of MR in brainstem, and the appearance of a higher molecular weight band for MR in hippocampus following steroid withdrawal, however no significant change in mRNA was detected by semiquantitative RT-PCR for either MR or GR in hippocampus or brainstem following steroid withdrawal. These studies suggest that physiological ligands of MR in the sheep brain include progesterone and cortisol, and that, as in other species, affinity of MR for cortisol is greater than that of GR.

The Normal Increase in Adrenal Secretion During Pregnancy Contributes to Maternal Volume Expansion and Fetal Homeostasis

This study was designed to test the hypothesis that the increase in maternal cortisol and aldosterone in pregnancy is critical to maternal and fetal homeostasis. Pregnant ewes were adrenalectomized (at approximately 112 days), and aldosterone and cortisol were given to either normal pregnant level (1 mg/kg per day cortisol and 3 microg/kg per day aldosterone) or nonpregnant level (half the dose of either cortisol or aldosterone). Blood samples for measurement of fetal and maternal plasma electrolytes, cortisol, and adrenocorticotropic hormone (ACTH) were collected at days 120, 125, and 130; fetal and maternal blood pressure and heart rate and maternal plasma volume were measured at days 120 and 130. Reduction of maternal cortisol or aldosterone decreased maternal plasma volume at 130 days. In the fetuses in the low cortisol group, cortisol concentrations were lower and ACTH concentrations were higher; however, there was no increase in fetal cortisol with age in the low cortisol or low aldosterone groups. Fetal arterial pressure was decreased in all groups of adrenalectomized ewes at 120 days and decreased in the low cortisol group at 130 days. Fetal arterial pressure was significantly related to maternal plasma volume at 130 days. Fetal arterial oxygen tension at 120 days was decreased in fetuses of low aldosterone ewes and was correlated with plasma volume. Overall fetal arterial oxygen tension correlated with fetal cortisol. Fetal ACTH concentrations correlated positively with fetal cortisol but negatively with fetal blood pressure. Maternal adrenal secretion maintains the normal increase in maternal plasma volume and fetal cortisol and indirectly supports fetal arterial oxygen tension, blood pressure, and adrenal secretion.

Pregnancy alters cortisol feedback inhibition of stimulated ACTH: studies in adrenalectomized ewes.

These studies test the hypothesis that pregnancy alters the feedback effects of cortisol on stimulated ACTH secretion. Ewes were sham-operated (Sham), or adrenalectomized (ADX) at approximately 108 days gestation and replaced with aldosterone (3 microg x kg(-1) x day(-1)) and with cortisol at either of two doses (ADX + 0.6 and ADX + 1 mg x kg(-1) x day(-1)); ewes were studied during pregnancy and postpartum. Mean cortisol levels produced in ADX ewes were similar to normal pregnant ewes (ADX+1) or nonpregnant ewes (ADX+0.6), respectively. Plasma ACTH concentrations in response to infusion of nitroprusside were significantly increased in the pregnant ADX+0.6 ewes (1,159 +/- 258 pg/ml) relative to pregnant Sham ewes (461 +/- 117 pg/ml) or the ADX+1 ewes (442 +/- 215 pg/ml) or the same ewes postpartum (151 +/- 69 pg/ml). Plasma ACTH concentrations were not significantly different among the groups postpartum. Increasing plasma cortisol to 20-30 ng/ml for 24 h before hypotension produced similar inhibition of ACTH in all groups. Pregnancy appears to decrease the effectiveness of low concentrations of cortisol to inhibit ACTH responses to hypotension.

Differential Effects of Pregnancy on Mineralocorticoid and Glucocorticoid Receptor Availability and Immunoreactivity in Cortisol Feedback Sites

The suppression of plasma adrenocorticotropic hormone by very low levels of cortisol is reduced in pregnant adrenalectomized ewes, suggesting that pregnancy reduces the efficacy of the high-affinity corticosteroid receptor. This study was designed to determine the effects of pregnancy on the availability, immunoreactivity, and affinity of both corticosteroid receptors: the high-affinity mineralocorticoid receptor (MR) and the lower-affinity glucocorticoid receptor (GR). Availability was measured in the hypothalamus, pituitary, hippocampus and kidney using a saturation point radioligand binding assay. GR availability was significantly decreased in hippocampal cytosols obtained from pregnant ewes, but did not significantly change in other tissues. This finding is consistent with increased GR activation due to elevated circulating concentrations of cortisol. MR availability significantly increased from undetectable levels in hippocampal cytosols obtained from nonpregnant ewes to 2.8 ± 1.6 fmol/mg protein in pregnant ewes, suggesting a reduced MR activation in the hippocampus during pregnancy. MR availability tended to be greater in other tissues during pregnancy, but these differences were not significant. The amount of immunoreactive MR (iMR) and GR (iGR) protein was estimated by quantifying Western blots. iGR significantly increased in the pituitary, but did not significantly change in other tissues. In contrast, iMR was significantly increased during pregnancy in all tissues assayed, suggesting that an increased cytosolic MR protein amount contributes to the observed increase in MR availability. Since studies suggest that progesterone is a potent anticorticosteroid, we tested for evidence of endogenous inhibition of binding to MR and/or GR during pregnancy by determining MR and GR affinity in pituitary cytosols obtained from nonpregnant and pregnant ewes. Although there was a tendency towards a decreased affinity of the MR in pregnant ewes, there was no significant change in the K_D of the pituitary MR or GR during pregnancy. We hypothesize that an alteration in activation and/or autoregulation of the MR during pregnancy, particularly in the hippocampus, may contribute to the observed changes in receptor availability and immunoreactivity and increase basal plasma cortisol levels during pregnancy.

Evidence for reset of regulated cortisol in pregnancy: studies in adrenalectomized ewes.

These studies test the hypothesis that the increased adrenocorticotropic hormone (ACTH) and cortisol in pregnancy reflect a reset of regulated plasma cortisol concentrations. Ewes were sham operated (Sham) or adrenalectomized (ADX) at approximately 108 days gestation. Adrenalectomized ewes were replaced with aldosterone (3 micrograms.kg-1.day-1) and with cortisol at either of two doses (ADX + 0.6 and ADX + 1.0 mg.kg-1.day-1); the ewes were also studied postpartum. Plasma cortisol concentrations in ADX + 0.6 ewes (5.3 +/- 1.3 ng/ml) were similar to the Sham ewes postpartum (5.5 +/- 0.6 ng/ml), whereas ADX + 1.0 concentrations (8.9 +/- 1.0 ng/ml) were similar to pregnant Sham ewes (9.5 +/- 1.9 ng/ml). Plasma ACTH concentrations were significantly increased in the pregnant ADX + 0.6 ewes (273 +/- 44 pg/ml) relative to pregnant Sham ewes (84 +/- 9 pg/ml) or the same ewes postpartum (42 +/- 9 pg/ml). Plasma ACTH concentrations were not different among the groups postpartum. Acute increases in plasma cortisol to 15-25 ng/ml produced similar inhibition in all groups. These results suggest that pregnancy resets the basal cortisol concentration required for normalization of basal ACTH concentration.

Role of increased maternal cortisol in fetal homeostasis: Studies in adrenalectomized ewes

Role of increased maternal cortisol in fetal homeostasis: Studies in adrenalectomized ewes

Effects of adrenalectomy on serum concentrations of progesterone during the luteal phase of sheep

An experiment was conducted to test the hypothesis that the adrenal gland influences luteal activity in sheep. Twelve Finnish Landrace × Southdown ewes were either bilaterally adrenalectomized (n = 6) or sham adrenalectomized (n = 6) during the breeding season. At approximately 37 and 47 d after surgery, all ewes received intramuscular injections of cloprostenol to synchronize estrus. Blood samples were taken via jugular venipuncture at 48-h intervals between 1 and 19 d after the last cloprostenol treatment. Serum concentrations of progesterone were determined in each of these samples. Analysis of variance showed that concentrations of progesterone during the luteal phase were lower (P<0.05) in adrenalectomized ewes than in sham-operated controls, but that patterns of progesterone were similar for both groups of sheep. Based on these results, we conclude that the adrenal gland does not appear to be necessary for initiation of luteal regression in ewes.

Adrenal glands alter the concentration of oestradiol-17 beta and its receptor in the uterus of ovariectomized ewes.

To investigate the effects of adrenal hormones on oestrogen activity in the uterus, ovariectomized ewes were either adrenalectomized, administered glucocorticoid-like preparations (CORT), or remained as controls. The adrenalectomized ewes were maintained with a corticoid-replacement therapy and monitored daily for plasma glucose and Na+/K+ concentrations. Blood samples were collected from all ewes at 15-min intervals for 4 h and assayed for LH and FSH. The adrenalectomized ewes were killed 9 days after adrenalectomy, while the CORT ewes were killed after 3 weeks of drug therapy. The control ewes were killed simultaneously with the CORT ewes. Uterine tissues were homogenized and the numbers of oestradiol receptors and tissue concentrations of oestradiol-17 beta were measured. The adrenalectomized ewes had significantly higher concentrations of cytosolic oestrogen receptors in the uterus than did the control ewes, which had significantly higher concentrations than the CORT ewes (0.95 +/- 0.06, 0.76 +/- 0.02 and 0.60 +/- 0.02 (S.E.M.) mumol/kg protein respectively). The concentrations of oestradiol-17 beta measured in uterine tissues were significantly lower in adrenalectomized and CORT ewes than in control ewes (37.4 +/- 5.5, 61.7 +/- 25.7 and 135.5 +/- 12.8 pmol/kg respectively). There were no significant differences between any groups in the peripheral concentrations of LH or FSH. These results indicate that the adrenal gland affects the concentrations of both oestrogen and its receptor in the uterus of ovariectomized ewes, probably by different mechanisms. These effects are not mediated by gonadotrophins.

Effects of adrenalectomy on photoperiod-induced changes in release of luteinizing hormone and prolactin in ovariectomized ewes.

Finnish Landrace x Southdown ewes were ovariectomized (OVX) and subjected to daily photoperiods of 16L:8D (Group I) or 8L:16D (Group II) for 84 days. Ewes were then either adrenalectomized (ADX) (N = 5 for Group I; N = 4 for Group II) or sham ADX (N = 6 for Groups I + II). After surgery, ewes in Group I were subjected to 8L:16D for 91 days and 16L:8D for 91 days whereas ewes in Group II were exposed to 16L:8D for 91 days and 8L:16D for 91 days. Oestradiol implants were inserted into all ewes on Day 148. Sequential blood samples were taken at 28, 56, 91, 119, 147 and 168 days after surgery to determine secretory profiles of LH and prolactin. Photoperiod did not influence LH release in Group I in the absence of oestradiol. Although photoperiod influenced frequency and amplitude of LH pulses in Group II before oestradiol treatment, adrenalectomy did not prevent these changes in patterns of LH release. However, in Group II the increase in LH pulse amplitude during exposure to long days was greater (P less than 0.01) in adrenalectomized ewes than in sham-operated ewes. Mean concentrations of LH increased in ADX ewes on Days 91 (P = 0.07) and 119 (P less than 0.05). Adrenalectomy failed to influence photoperiod-induced changes in mean concentrations of LH, amplitude of LH pulses and frequency of LH pulses in the presence of oestradiol. Concentrations of prolactin were influenced by photoperiod. In Groups I and II concentrations of prolactin increased (P less than 0.01) after adrenalectomy, but the magnitude of this effect decreased over time.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of adrenalectomy on LH release in sheep during the anoestrous season.

An experiment was conducted to measure LH pulse frequencies in bilaterally adrenalectomized Ile-de-France ewes during the mid-anoestrous season. Seven ewes were adrenalectomized under general anaesthesia and maintained on daily injections of 3 mg deoxycorticosterone acetate (DOCA). Adrenalectomy did not affect the patterns of LH release and the mean intervals between pulses in the adrenalectomized and sham-operated control ewes were 48 and 52 min respectively. Small implants of oestradiol significantly reduced the frequency of LH pulses in both groups and, in the presence of oestradiol, there were no differences in LH release between adrenalectomized and sham-operated ewes, with the mean interpulse intervals being 91 and 89 min respectively. In a second experiment, designed to assess the effects of DOCA on LH release, the mean interpulse intervals in unoperated control ewes (46 min) and unoperated ewes given daily injections of 3 mg DOCA (47 min) were similar to those observed in adrenalectomized and sham-operated ewes. In the presence of small implants of oestradiol, the combination of DOCA and oestradiol decreased the pulse frequency significantly more than oestradiol alone (mean interpulse intervals of 85 and 65 min respectively) suggesting that DOCA may act as a progestagen in sheep. Thus alternative treatments to DOCA for the maintenance of adrenalectomized sheep must be found for future studies on the role of adrenal steroids in the reproductive system. It appears that adrenal steroids do not play a major role in the seasonal changes in LH release in the ewe.

Steroid hormone requirements for the maintenance of early pregnancy in sheep: a study using ovariectomized adrenalectomized ewes.

Steroid hormone requirements for the maintenance of early pregnancy in sheep: a study using ovariectomized adrenalectomized ewes.