Microstructured magnetic materials for RF flux guides in magnetic resonance imaging.

Magnetic resonance imaging and spectroscopy systems use coils, either singly or as arrays, to intercept radio-frequency (RF) magnetic flux from regions of interest, often deep within the body. Here, we show that a new magnetic material offers novel possibilities for guiding RF flux to the receiver coil, permitting a clear image to be obtained where none might otherwise be detectable. The new material contains microstructure designed according to concepts taken from the field of photonic band gap materials. In the RF range, it has a magnetic permeability that can be produced to specification while exhibiting negligible direct-current magnetism. The latter property is vital to avoid perturbing the static and audio-frequency magnetic fields needed to obtain image and spectral data. The concept offers a new paradigm for the manipulation of RF flux in all nuclear magnetic resonance systems.

Development of the technical capabilities needed to build and position a prepolarization coil for a magnetic resonance imaging magnet.

An experiment to show that a magnetic resonance imaging (MRI) magnet could be assembled around a patient, and used as part of a prepolarization system in which substantial transient forces are applied to parts of it, is described. The paper describes the circumstances that develop as a result of the application of the large transient fields used in this type of study, and outlines the reason for the tolerances that are permissible on the alignment of the system components. It then describes a test rig used to evaluate how the various problems might be overcome, and reports on the performance achieved with this rig. On the basis of this work, it appears that a system could be developed that would allow the application of these methods in clinical MRI.

High cortisol response to adrenocorticotrophic hormone identifies ewes with reduced melanocortin signalling and increased propensity to obesity.

We have identified female sheep that have either high (HR) or low (LR) cortisol responses to adrenocorticotrophin. On a high-energy diet, HR have greater propensity to weight gain and obesity, although the underlying mechanisms remain to be determined. Hypothalamic appetite-regulating peptides (ARP) exert reciprocal effects on food intake and energy expenditure. We aimed to quantify the expression and function of ARP in LR and HR ewes (n = 4 per group). Gene expression for neuropeptide Y (NPY), agouti-related peptide (AgRP) pro-opiomelanocortin (POMC), melanin-concentrating hormone (MCH), orexin and the melanocortin receptors (MC3R and MC4R) was measured by in situ hybridisation. Expression of NPY, AgRP and POMC was similar in HR and LR, although expression of orexin, MCH, MC3R and MC4R was higher (P < 0.05) in LR. Intracerebroventricular infusions of a low dose (50 µg/h) of NPY, α-melanocyte-stimulating hormone (αMSH), orexin and MCH were performed between 10.00 h and 16.00 h in meal-fed ewes (n = 6-7 per group). Skeletal muscle and retroperitoneal (RP) fat temperatures were recorded using dataloggers. Post-prandial thermogenesis in muscle was higher (P < 0.05) in LR. There was little effect of ARP infusion on muscle or fat temperature in either group. Infusion of these doses of NPY, MCH or orexin did not stimulate food intake in meal-fed ewes, although αMSH reduced (P < 0.01) food intake in LR only. Using 24-h ARP infusions with ad lib. feeding, NPY increased (P < 0.001) food intake in both groups but αMSH was only effective in LR (P < 0.05). In summary, we show that HR are resistant to the satiety effects of αMSH and this coincides with a reduced expression of both the MC3R and MC4R in the paraventricular nucleus of the hypothalamus. We conclude that an increased propensity to obesity in HR female sheep is associated with reduced melanocortin signalling.

Differences in mitochondrial DNA inheritance and function align with body conformation in genetically lean and fat sheep.

Body weight and adiposity are determined by the balance between energy intake, energy expenditure, and nutrient deposition. We have identified differences in appetite-regulating peptides in sheep selectively bred to be either lean or fat, wherein gene expression for orexin and melanin-concentrating hormone are elevated in the lean group. Despite this, the underlying mechanisms leading to differences in body composition in the lean and fat lines remains unknown. We measured postprandial temperature in adipose tissue and muscle to ascertain whether a difference in thermogenesis is associated with the difference in body composition in genetically lean (n = 8) and fat (n = 12) ewes. Body weight was higher (P < 0.01) but percent fat mass was lower (P < 0.001) in the lean group. The percent lean mass was similar in lean and fat groups. Animals received intracerebroventricular cannulae and temperature probes implanted into the retroperitoneal fat and the hind-limb skeletal muscle (vastus lateralis). Animals were meal fed (1100-1600 h) to entrain postprandial thermogenesis. Food intake was similar between lean and fat animals. Postprandial thermogenesis was greater (P < 0.05) in the retroperitoneal adipose tissue of lean animals but not in skeletal muscle. Intracerebroventricular infusion of leptin reduced (P< 0.05) food intake by an equal extent in both groups. Postprandial expression of UCP1 mRNA was greater (P < 0.05) in retroperitoneal fat of lean animals, with similar UCP3 expression in skeletal muscle. Mitochondrial genome sequencing indicated haplotypic clustering in lean and fat animals within both the encoding and nonencoding regions. This demonstrates that differences in body composition may be underpinned by differences in thermogenesis, specifically within adipose tissue. Furthermore, thermogenic differences may be associated with specific mitochondrial DNA haplotypes, suggesting a strong genetic component inherited through the maternal lineage.

Evidence for hypothalamic control of the diurnal rhythms in prolactin and melatonin in the fetal sheep during late gestation.

We have investigated the effect of surgical disconnection of the fetal hypothalamus and pituitary (HPD) on generation of the daily rhythm in fetal plasma melatonin and PRL concentrations under long and short photoperiods. Fetal HPD or a sham operation was carried out at around 110 days gestation. Ewes carrying either HPD fetal sheep (n = 10) or intact fetal sheep (n = 12) were exposed to a long light (LL; 16 h of light and 8 h of darkness) or a short light (SL; 8 h of light and 16 h of darkness) regimen for the remainder of gestation. All ewes were subjected to a 24-h blood-sampling experiment (13 samples collected between 0900-0900 h the following day) between 135-140 days gestation, and fetal and maternal plasma melatonin and PRL concentrations were measured using specific RIAs. The hormonal data were analyzed using multifactorial analysis of variance and cosinor analysis. There was an increase in maternal melatonin concentrations during the dark phase in each lighting regimen in ewes carrying HPD or intact fetal sheep. In the SL regimen, there was also a significant increase in fetal melatonin concentrations during the dark phase in the HPD and intact groups. Under LL conditions, however, fetal melatonin concentrations were only consistently increased during the dark phase in the intact, not the HPD, group. The 24-h mean fetal plasma concentrations of PRL were significantly higher (P < 0.001) in both intact and HPD fetuses in the LL (intact, 111.0 +/- 22.0 pg/ml; HPD, 37.6 +/- 7.3 pg/ml) than in the SL regimen (intact, 37.8 +/- 18.4 pg/ml; HPD, 6.7 +/- 4.3 pg/ml). There was also a significant interaction (P < 0.001) between the effects of fetal surgical treatment and time of day on fetal PRL concentrations. In the intact group, fetal PRL concentrations were significantly higher (P < 0.05) at 1300 and 1700 h than between 0300-0700 h in both lighting conditions. Cosinor analysis also identified a significant rhythm in 8 of the 12 fetal PRL profiles in the intact group. In contrast, in the HPD group, there was no significant effect of time of day on fetal PRL in either the LL or SL regimen, and cosinor analysis only identified a significant rhythm in 2 of the 10 fetal PRL profiles in this group. We have, therefore, demonstrated that in the fetal sheep, HPD resulted in abolition of the diurnal melatonin rhythm under LL conditions and in the loss of the diurnal PRL rhythm under LL and SL conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Surgical disconnection of the hypothalamus from the fetal pituitary abolishes the corticotrophic response to intrauterine hypoglycemia or hypoxemia in the sheep during late gestation.

We have investigated the ACTH and cortisol responses to acute episodes of hypoxemia or hypoglycemia in fetal sheep in which the hypothalamus and pituitary were surgically disconnected at between 112 and 123 days gestation. Before 130 days gestation, basal plasma concentrations of ACTH were significantly greater in the hypothalamo-pituitary disconnected (HPD) fetuses than in the intact fetal sheep (126-130 days; 105.0 +/- 11.4 ng/liter, HPD group; 64.0 +/- 9.5 ng/liter, intact group). After 130 days, however, there was no difference between plasma ACTH concentrations in the HPD (136-140 days; 154.7 +/- 16.7 ng/liter HPD group; 113.6 +/- 19.1 ng/liter, intact group) and intact fetal sheep, and in both groups the mean ACTH concentrations were significantly greater after 136 days gestation than before 130 days. In the HPD group, however, while the plasma ACTH concentrations were elevated there was no prepartum increase in the plasma concentrations of cortisol. A decrease in the fetal arterial blood PO2 by approximately 50% for 30 min between 123 and 132 days, stimulated a significant increase in fetal ACTH and cortisol concentrations in the intact but not in the HPD fetuses. In the intact group, plasma ACTH concentrations were also significantly increased (P less than 0.001) above control values (98.2 +/- 21.2 ng/liter) at 120 min after the start of an iv infusion of insulin (1 IU/60 min) (517.2 +/- 160.5 ng/liter) and were still elevated at 60 min after the end of the infusion period (1248.1 +/- 643.2 ng/liter). In the HPD fetuses, however, there was no significant change in plasma ACTH concentrations during or after the insulin infusion. In both the HPD and intact groups, there was a significant increase in plasma ACTH concentrations above control values (62.5 +/- 8.5 ng/liter intact; 135.0 +/- 30.6 ng/liter, HPD) after intrafetal administration of CRF (+10 min; 117.5 +/- 8.1 ng/liter, intact; 225.3 +/- 33.1 ng/liter, HPD) indicating that the secretory capacity of the pituitary corticotrophs was not reduced by the HPD procedure. Our results demonstrate that an intact hypothalamic-pituitary connection is required to generate a normal prepartum increase in fetal cortisol concentrations and is essential for an appropriate fetal pituitary-adrenal response to intrauterine hypoxemia and hypoglycemia.

Hypothalamo-pituitary disconnection of the late-gestation ovine fetus results in profound changes in cortisol secretion that are not reflected in commensurate changes in adrenocorticotropin secretion.

A prepartum increase in fetal glucocorticoid concentrations is essential for the perinatal transition to extrauterine life for many mammalian species. In the case of the sheep, this increase in cortisol is also the trigger for parturition, and depends upon an intact hypothalamo-pituitary unit. Fetal sheep that have undergone hypothalamo-pituitary disconnection (HPD) fail to have a prepartum cortisol surge or initiate labor, despite apparently normal fetal ACTH concentrations in late gestation. We have investigated whether a defect exists in the regulation of pulsatile neurohormone secretion in the pituitaryadrenal axis of the HPD sheep fetus, by comparing immunoreactive (ir) ACTH and cortisol secretory dynamics in intact and HPD fetuses at 126 and 145 days of gestation (normal gestation length, 147 days). The fetal surgery was conducted at 115 days of gestation. Blood samples were collected at 5-min intervals for 2 h on each experimental day, and the resulting irACTH and cortisol concentrations were analyzed by multiple-parameter deconvolution and cross-correlation analysis. Basal irACTH secretion was less (P < 0.01) in HPD fetuses than intact fetuses at 126 days, but it had recovered by 145 days. There were no differences in irACTH half-life or the number or duration of irACTH secretory bursts between the two groups of fetuses or the two gestational ages (GAs). The size of the irACTH secretory bursts was not affected by the operation, but it increased with GA to a similar extent in both groups of fetuses (P < 0.01). In keeping with the observations for irACTH secretion, there was no effect of age or the operation on cortisol half-life or on the number or duration of cortisol secretory bursts. In contrast, there were dramatic age-related increases (P < 0.01) in the basal cortisol secretion rate and the size of the cortisol secretory bursts in the intact, but not the HPD, fetuses. Cross-correlation analysis revealed a significant (P < 0.01) concordance between irACTH and cortisol secretion in only the intact fetuses at 126 days; this was not apparent in the intact fetuses at 145 days, or in the young or old HPD fetuses. These findings confirm a major defect in cortisol secretion in the late-gestation HPD fetus but suggest that this is not caused by defects in irACTH secretion. Together with other observations, these data suggest that ACTH may not be the sole, or primary, regulator of adrenal cortisol secretion in the late-gestation ovine fetus.

Effect of cortisol infusion on basal and corticotropin-releasing factor (CRF)-stimulated plasma ACTH concentrations in the sheep fetus after surgical isolation of the pituitary.

Although it has been demonstrated that glucocorticoids can inhibit basal and stimulus-induced increase in fetal plasma ACTH concentrations, the site(s) of action of the glucocorticoids in the fetal hypothalamo-pituitary axis are unclear. We have investigated the ontogeny of the negative feedback effect of cortisol on basal and CRF-stimulated ACTH secretion in intact fetal sheep and in fetal sheep after surgical disconnection of the hypothalamus and pituitary (HPD). In saline-pretreated fetal sheep there was a significant increase in plasma ACTH concentrations in response to an ovine CRF (oCRF) bolus (1 microgram) in HPD and intact fetuses at 120-128 days and at 138-149 days gestation. In the intact group the ACTH response at 120-240 min after oCRF was significantly lower (P less than 0.01) after 138 days compared with before 128 days. In this group the cortisol response during the first hour after oCRF was significantly greater (P less than 0.005) after 138 days than in the younger fetuses. In the HPD group there was no significant difference between the two gestational age ranges in either the ACTH or cortisol responses to oCRF. Between 120 and 128 days gestation, increasing plasma cortisol concentrations (110-170 nmol/liter above baseline values) by exogenous infusion had no significant effect on the basal fetal plasma ACTH concentrations in HPD or in intact fetuses. In contrast, after 138 days gestation, the basal plasma ACTH concentrations were significantly decreased by 50% during the cortisol infusion period in the intact but not in the HPD group. It would appear therefore, that the development of the negative feedback action of cortisol to suppress basal fetal plasma concentrations of ACTH is dependent on the presence of a functional fetal hypothalamus. In HPD and intact fetuses cortisol infusion abolished the fetal ACTH response to the oCRF bolus from as early as 120 days gestation. Thus the negative feedback action of cortisol on the CRF-induced increases in fetal plasma ACTH concentrations can occur directly at the fetal pituitary.

Photoperiodic history and hypothalamic control of prolactin secretion before birth.

We investigated whether the fetal lamb can construct a photoperiodic history in utero. We measured the fetal PRL response to a 12-h photoperiod in intact fetal sheep and in fetal sheep after hypothalamo-pituitary disconnection (HPD), following exposure of the ewe to either a long (16 h L) or short (8 h L) photoperiod for 50 days in early pregnancy. Ewes were maintained on either a long light (LL, n = 20) or a short light (SL, n = 19) regimen from 57 days gestation until fetal HPD (pre-LL, n = 7; pre-SL, n = 7) or sham surgery (pre-LL, n = 13; pre-SL, n = 12) was performed at 99-113 days gestation. All ewes were housed in a 12-h photoperiod from surgery until 140 days gestation. In HPD fetal sheep previously exposed to SL, fetal PRL concentrations were significantly higher (P < 0.05) after 20 days in the 12-h L regimen than previously (0-5 days, 3.2 +/- 0.6 ng/ml; 21-25 days, 5.6 +/- 1.4 ng/ml). In the HPD fetal sheep previously exposed to LL, however, fetal PRL concentrations significantly decreased (P < 0.05) after 5 days exposure to the 12-h L regimen (6.7 +/- 2.9 ng/ml) and remained low throughout the remaining study period (31-35 days, 1.7 +/- 0.5 ng/ml). In contrast, in the sham group there was no effect of photoperiodic history on the gestational age profile of fetal PRL, and PRL concentrations increased significantly (F = 22.4, P < 0.001) in fetal sheep previously exposed to either SL or LL. Fetal PRL concentrations were significantly higher (P < 0.05) after 121 days gestation in the 12-h L regimen in all sham fetal sheep (<110 days, pre-SL 6.4 +/- 0.3 ng/ml, pre-LL 12.0 +/- 3.3 ng/ml; 121-125 days, pre-SL 20.0 +/- 3.9 ng/ml, pre-LL 25.9 +/- 4.4 ng/ml). TRH (50 microg) was administered i.v. to all fetal sheep at 130-134 days gestation. There was a significant fetal PRL response to TRH in both the HPD (F = 20.9, P < 0.001) and sham (F = 31.3, P < 0.001) groups. There was no difference, however, in the PRL response to TRH in fetal sheep previously exposed to SL or LL in either the HPD or sham groups. The maximum percentage changes in PRL occurred at +10 min after TRH administration in the HPD (pre-SL, 421 +/- 75%; pre-LL, 555 +/- 76%) and sham groups (pre-SL, 394 +/- 68%; pre-LL, 369 +/- 59%). In summary, therefore, we have demonstrated that there is an effect of photoperiodic history on the PRL response to an intermediate photoperiod in utero in HPD fetal sheep. It appears, however, that the effect of photoperiodic history on PRL secretion in intact fetal sheep is either masked or suppressed by the stimulatory effect of factors associated with an increase in gestational age acting at the fetal hypothalamus.

Prostaglandin E2 administered to fetal sheep increases the plasma concentration of adrenocorticotropin (ACTH) and the proportion of ACTH in low molecular weight forms.

This study investigated the effects of repeated short term (2-h) intrafetal infusions of prostaglandin E2 (PGE2) on ACTH and cortisol release in fetal sheep during late gestation (119-144 days). We compared the effects of administration of PGE2 (2 micrograms/min) into the fetal carotid artery or jugular vein. PGE2 infusion significantly (P < 0.001) increased fetal plasma immunoreactive (ir-) ACTH and cortisol concentrations regardless of the vessel used for administration. Saline infusion did not alter the concentrations of ir-ACTH or cortisol for the duration of the experiment. To compare the responses of fetal ir-ACTH and cortisol to repeated intracarotid infusions of PGE2, the hormone data were grouped into five gestational age ranges (119-125, 126-130, 131-135, 136-140, and 141-145 days). Fetal ir-ACTH was stimulated by PGE2 infusion at all gestational ages studied; the greatest response was achieved at the earliest gestational age range, 119-125 days. PGE2 infusion preferentially stimulated the release of low mol wt ACTH [ACTH-(1-39); 60 min from the start of infusion] at all gestational ages (P < 0.01), but basal low mol wt ACTH did not increase with gestational age until after 140 days. Cortisol concentrations were increased within 30 min of infusion at all gestational ages studied. These results suggest that PGE2 may play a role in maintaining elevated ir-ACTH concentrations in the face of high levels of cortisol in fetal sheep before parturition.

Chronic administration of low doses of adrenocorticotropin to hypophysectomized fetal sheep leads to normal term labor.

Parturition in the sheep is preceded by an increase in the plasma concentration of fetal ACTH and an increase in the plasma cortisol concentration. The role and importance of the increase in fetal ACTH in stimulating fetal glucocorticoid synthesis and the subsequent onset of labor require closer examination, as it has been demonstrated that the fetal adrenal becomes more responsive to ACTH in late gestation. This study sets out to determine whether the increase in plasma ACTH in the late gestation fetal sheep is essential for maturation of the fetal adrenal gland and normal delivery. Fetal sheep were either hypophysectomized (HX) and cannulated or cannulated only (intact) at 125 days gestation. Immediately after surgery, HX fetuses were infused with a constant dose of ACTH-(1-24) (ACTH/HX; 100 ng/h.kg, i.v.) or saline (SAL/HX) until uterine electromyography indicated the onset of labor or 161 days gestation was reached (term = 147 +/- 2.6 days). The mean gestational age at labor of the ACTH/HX group was 147 +/- 2.9 days, whereas none of the animals in the SAL/HX entered labor, and they were killed at 161 days gestation. The concentration of ACTH in both ACTH/HX and SAL/HX fetal plasma was less than 2.2 pg/ml throughout the study. The concentration of cortisol in ACTH/HX fetuses mimicked that in intact fetuses in late gestation, reaching 80 ng/ml at term. The concentration of cortisol in SAL/HX fetuses remained less than 5 ng/ml. This study supports the hypothesis that the ovine fetal adrenal becomes increasingly responsive to ACTH in late gestation and indicates that ACTH may only be permissive in the activation of adrenal function. In intact fetal sheep there may be endogenous inhibition of the fetal adrenal, requiring relatively high plasma concentrations of ACTH [100-250 pg/ml ACTH-(1-39)] in late gestation.

Melatonin and the light-dark cycle separately influence daily behavioral and hormonal rhythms in the pregnant ewe and sheep fetus.

We have studied the effects of independently altering the time of darkness and the phase of the daily melatonin rhythm during a 12-h photoperiod on the diurnal rhythms of fetal breathing movements (FBMs), low voltage electrocortical activity, and maternal and fetal plasma concentrations of PRL. Thirteen pregnant ewes were pinealectomized at 99-100 days gestation and held under a normal lighting regime (12-h photoperiod, lights off at 1900 h) until 135 days gestation. All ewes were then exposed to an altered lighting regime (12-h photoperiod, lights off at 1100 h) until 145 days gestation. Six of these ewes were infused with melatonin (12-15 micrograms/h iv) from 1100 h for 12 h each day between 125 and 135 days gestation while in the normal lighting regime, and from 1900 h while in the altered lighting regime. The remaining seven ewes were infused with saline at corresponding times. Infusion of melatonin produced a significant daily rhythm in maternal and fetal melatonin concentrations; concentrations were highest throughout the 12-h infusion period in each lighting regime. In the normal lighting regime (i.e. lights off at 1900 h) the peak incidence of FBM occurred at between 1000 and 1300 h in the ewes infused with saline or melatonin. Under the altered lighting conditions there was no 24-h variation in the incidence of FBMs in the saline-infused group, but a significant daily variation was present in the melatonin-infused group with peak incidence occurring at 1900-2200 h. The daily variation in maternal PRL concentrations was not affected by changes in the time of onset of melatonin infusion but was different in each of the two lighting regimes. In the normal lighting regime, PRL concentrations were highest at 2100 h (56.2 +/- 21.6 ng/ml) whereas in the altered lighting conditions the peak in maternal PRL occurred at 1700 h (90.4 +/- 25.1 ng/ml). The daily variation in fetal PRL concentrations was also not affected by changes in the time of onset of melatonin infusion but was different in the two lighting regimes; in the normal lighting regime, fetal PRL reached a peak (13.0 +/- 3.7 ng/ml) at 0100-0300 h whereas in the altered lighting regime the peak (29.2 +/- 9.8 ng/ml) occurred at 1700-2100 h. We have presented evidence therefore that the daily melatonin rhythm may entrain the daily rhythm in fetal breathing activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Continuous intrafetal infusion of prostaglandin E2 prematurely activates the hypothalamo-pituitary-adrenal axis and induces parturition in sheep.

This study has investigated the effect of continuous intrafetal infusion of PGE2 or saline on hormone concentrations and the length of gestation in sheep. Fetal and maternal vascular catheters were surgically implanted at 112.3 +/- 1.3 days (n = 10), and the infusions were started at 121 +/- 1.2 days of gestation (term = 147). Fetuses were infused with either PGE2 (n = 5; 2 micrograms/min for 48 h and then increased to 4 micrograms/min for the remainder of the experiment) or the vehicle solution (n = 5; sterile isotonic saline) via the fetal carotid artery. In the PGE2-infused group, fetal and maternal plasma PGE2 concentrations increased (P < 0.001) after the change to the higher dose rate (4 micrograms/min) and remained elevated, fetal plasma immunoreactive ACTH (ir-ACTH) concentrations dramatically increased after the start of the infusion being maximal at 11 h before decreasing to match concentrations exhibited by the saline-infused group. Fetal plasma cortisol concentrations increased after the start of the PGE2 infusion (P = 0.05) and increased further after the change to the higher dose rate of 4 micrograms/min (P < 0.001). Concentrations of PGE2, ir-ACTH, and cortisol in the saline-infused group did not change until labor. Plasma concentrations of PGE2 (P < 0.001) and ir-ACTH (P < 0.005) increased on the day of labor in both treatment groups, and fetal cortisol concentrations increased (P < 0.001) in both groups in the last few days before labor. The proportion of low molecular weight ir-ACTH in the plasma of PGE2-infused fetuses was significantly higher than that of saline-infused fetuses (P < 0.001) during the first 15 days of infusion. In the saline-infused group, the proportion of low molecular weight ir-ACTH increased in the last few days before labour (P = 0.001), whereas no change was seen in PGE2-infused fetuses at this time. Maternal plasma progesterone concentrations decreased in both groups in the last few days before labor (P < 0.001). Fetuses infused with PGE2 delivered at 138.4 +/- 2.1 days, whereas control fetuses infused with saline delivered at 148.2 +/- 0.5 days (P < 0.001). The spontaneous increase in PGE2 preceding normal labor may thus play an important role in activation and maturation of the hypothalamic-pituitary-adrenal axis in fetal sheep.

Hypophysectomy of the fetal lamb leads to a fall in the plasma concentration of insulin-like growth factor I (IGF-I), but not IGF-II.

The role of the pituitary gland in the regulation of the plasma concentrations of insulin-like growth factors (IGFs) in the late gestation sheep fetus has been examined. Singleton sheep fetuses were either hypophysectomized or sham-operated between days 110-120 of gestation. Blood samples were then collected via carotid cannulae at least three times weekly for the remainder of gestation. In some hypophysectomized fetuses T4 was administered (100 g/day) to overcome the hypothyroidism caused by hypophysectomy. Blood samples were also obtained from lambs during the perinatal period, neonatal lambs within 1-10 days after birth, and pregnant and nonpregnant adult ewes. All plasma samples were subjected to Sephadex G-50 gel filtration under acidic conditions (pH 2.3) to eliminate IGF-binding protein activity. The fractions containing the free IGF peptides were collected and assayed for IGF-I by heterologous RIA, and IGF-II by a homologous RRA. Plasma concentrations of IGF-I and IGF-II did not change with advancing gestational age in any fetal group and were not affected by the prolonged gestation that results from hypophysectomy. The mean plasma IGF-I and IGF-II concentrations in the sham fetuses were 112 +/- 8 and 1340 +/- 112 ng/ml, respectively. Hypophysectomy without thyroid hormone replacement resulted in a significant decrease in plasma IGF-I concentrations to 50 +/- 5 ng/ml, whereas IGF-II concentrations were not affected (1096 +/- 124 ng/ml). IGF-I concentrations in the hypophysectomized fetuses that received T4 were significantly increased (67 +/- 6.0 ng/ml) compared to those in the hypophysectomized fetuses that did not receive T4. The IGF-II concentrations in the hypophysectomized fetuses that received T4 were similar to those in the sham-operated fetuses (1120 +/- 112 ng/ml). At term IGF-I concentrations were increased (180 +/- 21 ng/ml) and IGF-II concentrations were decreased (264 +/- 25 ng/ml) compared to fetal values. Plasma IGF-I concentrations in the prepubertal lamb were similar to the fetal values. Pregnancy in the adult ewe was associated with a significant increase in IGF-II, but had no effect on IGF-I plasma concentrations. These data show that circulating IGF-I concentrations in the fetal lamb are under some pituitary and thyroid control, whereas IGF-II concentrations are independently of pituitary or thyroid status. We confirm, using a homologous assay, that fetal IGF-II concentrations are high and then decrease at term. These data also support the concept that a pregnancy-related factor may regulate plasma IGF-II concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of hypophysectomy with and without thyroxine replacement on growth and circulating concentrations of insulin-like growth factors I and II in the fetal lamb.

The role of the pituitary gland in the regulation of skeletal growth and plasma insulin-like growth factor (IGF)-I and IGF-II concentrations in the late gestation sheep fetus has been studied. Singleton fetuses were either hypophysectomized (n = 14) or sham operated (n = 8) between days 110 and 125. Fetal and maternal blood samples were collected three times weekly through the remainder of gestation. In some hypophysectomized fetuses (HXT4 group, n = 4), T4 was administered (100 micrograms L-T4/day) to overcome the hypothyroidism caused by hypophysectomy. The other hypophysectomized fetuses received no replacement therapy (HXNR group, n = 10). Six HXNR fetuses were allowed to remain in utero post term and were killed at day 163 of gestation. All other animals were killed at day 147. All values are group means +/- SE. Hypophysectomized fetuses had significantly shorter limbs and long bones and delayed osseous maturation at term compared to sham controls. Plasma free T4 concentrations in HXT4 fetuses were not significantly different from those measured in sham fetuses (P greater than 0.05). Bone maturation at term was normal in HXT4 fetuses although there was no improvement in limb or bone elongation. Retention of hypophysectomized fetuses in utero until 16 days past term yielded fetuses which were heavier than controls but whose limb and bone lengths were no greater than hypophysectomized fetuses killed at term. Osseous maturation was appropriate for term in five of the six postterm hypophysectomized fetuses. The plasma IGF-I and IGF-II concentrations were not significantly affected by hypophysectomy, hypophysectomy with T4 replacement, gestational age, or prolonged gestation. The plasma IGF-I concentrations in the sham, HXNR, and HXT4 fetuses were 35.4 +/- 6.6, 28.2 +/- 3.0, and 34.4 +/- 1.7 ngeq human (h)IGF-I/ml, and the IGF-II concentrations were 656.3 +/- 59.2, 635.3 +/- 56.3, and 645.5 +/- 71.9 ngeq hIGF-II/ml, respectively, and remained within these ranges throughout the experiment. Fetal IGF-I concentrations were significantly lower than mean maternal IGF-I concentrations (88.0 +/- 6.8 ngeq hIGF-I/ml, P less than 0.05), and fetal IGF-II concentrations were significantly higher than mean maternal IGF-II concentrations (362.4 +/- 24.0 ngeq hIGF-II/ml, P less than 0.05). We conclude that in the late gestation fetal sheep, elongation of the appendicular skeleton is under some direct pituitary control whereas appendicular maturation exhibits some dependence on circulating T4 concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of adrenocorticotropin-(1-39) and precursor peptide secretory responses in the fetal sheep during the last third of gestation.

Although it is known that concentrations of immunoreactive ACTH increase during late gestation in fetal sheep plasma, the nature of the ACTH has not been well characterized. We used two-site immunoradiometric assays to separately measure high mol wt ACTH precursors (POMC and pro-ACTH) and ACTH-(1-39) in plasma of fetal sheep with chronic arterial and venous catheters. We compared the ratio of these peptides as a function of gestational age under basal conditions and in response to exogenous vasopressin and/or corticotropin-releasing hormone. Under basal conditions, the concentration of precursors was not changed throughout the last third of gestation; however, ACTH-(1-39) increased significantly approaching term. The molar ratio of precursors to ACTH-(1-39), therefore, decreased from 15.8 +/- 1.0 at 110 days to 7.9 +/- 0.6 at 140 days gestation. At all gestational ages, vasopressin and corticotropin-releasing hormone increased ACTH-(1-39) and precursors, albeit with different time courses. At 120 days gestation, arginine vasopressin plus CRH produced synergistic increases in ACTH-(1-39) and precursors, whereas the response was only additive at other ages. The present results indicate that the elevation in the resting plasma immunoreactive ACTH concentration that occurs near term is constituted by an increase in the concentration of ACTH-(1-39) relative to those of POMC and pro-ACTH, which may have further physiological significance. Also, CRH and AVP are potent stimulators of both ACTH-(1-39) and ACTH precursors.

Emergence of the diurnal rhythm in plasma melatonin concentrations in newborn lambs delivered to intact or pinealectomized ewes.

We have monitored the 24-h profiles of plasma melatonin concentrations between birth and 10 weeks of age, in lambs which were delivered to, and suckled, either pineal-intact (control group) or pinealectomized (pinealectomized group) ewes. Between 0 and 2 weeks of age, plasma concentrations of melatonin in lambs suckling either intact or pinealectomized ewes were highest at 01.00 h. At this age, however, there was no significant difference in the mean plasma concentrations of melatonin between the entire dark and light phases in lambs in either the control group (dark, 39.7 +/- 6.0 (S.E.M.) pmol/l; light, 39.5 +/- 8.1 pmol/l) or the pinealectomized group (dark, 79.8 +/- 43.3 pmol/l; light, 60.9 +/- 8.7 pmol/l). Between 3 and 4 weeks of age, however, a diurnal rhythm in plasma melatonin concentrations was clearly present in the lambs in both the control and pinealectomized groups (control group: dark, 164.1 +/- 5.6 pmol/l; light 26.2 +/- 2.5 pmol/l; pinealectomized group: dark, 52.7 +/- 8.0 pmol/l; light, 19.1 +/- 5.3 pmol/l; P less than 0.001). Between 3 and 10 weeks of age, plasma concentrations of melatonin in the dark phase were significantly (P less than 0.05) lower in the lambs suckling pinealectomized ewes than in the control group. In both the control lambs and lambs suckling pinealectomized ewes, the mean plasma concentrations of melatonin in the dark and light phases increased significantly (P less than 0.05) between 3 and 6 weeks after birth. In conclusion, we have demonstrated that a clear diurnal plasma rhythm in melatonin concentrations does not emerge until 3-4 weeks of age in lambs suckling either pinealectomized or intact ewes.(ABSTRACT TRUNCATED AT 250 WORDS)

Failure of acid-ethanol treatment to prevent interference by binding proteins in radioligand assays for the insulin-like growth factors.

Acid-ethanol precipitation and gel filtration at acidic pH have been widely used to extract circulating binding proteins for insulin-like growth factor (IGF-I and IGF-II) from plasma or serum samples before radioligand assay for the respective IGFs. Gel filtration on Sephadex G-50 at neutral pH of neutralized acid-ethanol extracts of fetal and adult ovine plasma which had been incubated with 125I-labelled IGF-I or 125I-labelled IGF-II revealed that significant amounts of the IGF-binding protein activity survived the acid-ethanol extraction procedure. Radioimmunoassay for IGF-I in acid-ethanol extracts of plasma samples from fetal, neonatal and adult sheep yielded results which depended upon the method used for separation of the antibody-bound IGF-I tracer from the free IGF-I tracer. Acid gel filtration of ovine fetal and adult plasma was found to remove completely the IGF-binding protein activity. Radioimmunoassay for IGF-I in samples of fetal, neonatal and adult sheep plasma that had undergone acid gel chromatography yielded consistent results for both methods that were used to separate antibody-bound IGF-I tracer from the free tracer. Radioreceptor assays for IGF-II were similarly highly perturbed by the presence of binding protein in acid-ethanol extracts of ovine fetal and adult plasma. We conclude that acid-ethanol extraction can not be used reliably for the removal of IGF-binding proteins, and that only acid gel filtration is a completely safe and valid method.

Effects of progesterone, oestradiol-17 beta and androstenedione on follicular growth after removal of the corpus luteum during lactational and seasonal quiescence in the tammar wallaby.

The corpus luteum was removed from tammar wallabies during either lactational or seasonal quiescence, and the pouch young were replaced on the teat after lutectomy. Follicular development resumed and the animals came into oestrus 11-14 days later. Injections of oil, progesterone or androstenedione did not prevent follicular growth, although ovulation only occurred in one animal, but treatment with oestradiol-17 beta, either alone or mixed with progesterone, inhibited the development of follicles. These results suggest that oestrogen, of luteal origin, is responsible for the suppression of follicular development during lactational and seasonal quiescence.

Localisation and temporal changes in prostaglandin G/H synthase-1 and -2 content in ovine intrauterine tissues in relation to glucocorticoid-induced and spontaneous labour.

Parturition in the ewe is preceded by an increase in the synthesis of prostaglandins (PGs) by gestational tissues. To establish the uterine source of these PGs, placental cotyledons, fetal membranes and maternal uterine tissues were collected from ewes (n=6) at spontaneous parturition. Solubilised tissue extracts were prepared and analysed by Western blots using polyclonal antibodies to PG G/H synthase-1 and -2 (PGHS-1 and PGHS-2). PGHS-1 was expressed by all intrauterine tissues at term labour. Densitometric analysis of Western blot autoradiographs showed that the fetal membranes and maternal cervix contained the largest amounts of PGHS-1. PGHS-1 enzyme content of ovine amnion was significantly greater than that of either chorion or allantois (P<0.05). PGHS-1 protein content of myometrial, endometrial and cotyledonary tissue extracts was minimal. Formation of the PGHS-2 isozyme was confined to placental tissue at term labour. PGHS-2 protein levels in sheep placenta were significantly higher than those of PGHS-1 in all intrauterine tissues examined. This result supports the hypothesis that PGHS-2 is a major contributor to PG formation at term labour. To elucidate the developmental changes in PGHS-1 and PGHS-2 relative to labour onset, an experimental paradigm of glucocorticoid-induced delivery was used. Previous characterisation and validation of this labour model demonstrated that direct, transabdominal, intrafetal injection of the synthetic glucocorticoid betamethasone (5.7 mg in 1 ml aqueous vehicle) on day 131 of gestation induced labour onset in 56.6+/-0.8 h (mean+/-s.e.m.). As the latent period to induced-labour was known, the time course of enzyme formation could be ascertained. Sheep (n=20) were killed by barbiturate injection at various time intervals post-injection (0, 14, 28, 42 and 56 h). Tissue extracts collected at post-mortem examination were prepared and analysed by Western blots. PGHS-2 was induced in ovine cotyledon in a time-dependent fashion following glucocorticoid injection (P<0.05). There was a 12-fold increase in abundance between the time of betamethasone administration (0 h) and established labour (56 h). The PGHS-2 isozyme was not detected in any of the other tissues examined. In contrast, formation of the PGHS-1 isozyme did not change in relation to induced-labour in any of the intrauterine tissues. This finding is consistent with constitutive formation of PGHS-1. Previous studies have demonstrated a rise in PG production in association with glucocorticoid-induced labour and spontaneous delivery. The results of the present study indicate that this rise in PG production is due to increased formation of the PGHS-2 isozyme in ovine cotyledon. PGHS-2 appears to be induced by exogenous glucocorticoid administration and/or the mechanisms controlling ovine parturition. The role of PG formation by the fetal membranes is yet to be elucidated.