Differential lipopolysaccharide-induced immune alterations in the hippocampus of two mouse strains: effects of stress.

Immunological activation may result in the development of depressive-like symptoms in a large percentage of patients treated with cytokine-based therapies. The mechanisms underlying susceptibility to cytokine-induced depression are currently unknown; however activation of the tryptophan catabolising enzyme indoleamine 2,3-dioxygenase (IDO) is associated with the induction of cytokine-induced depression. Peripheral administration of lipopolysaccharide (LPS) is one of the most commonly used immunological challenges in animal models of cytokine-induced depression. Inbred mouse strains are useful tools in the investigation of the neurobiology of psychiatric illnesses. In this study we hypothesised that two strains which differ in stress susceptibility, namely the BALB/c and C57BL/6J mice, would respond differentially to LPS and swim-stress in cytokine profile, corticosterone concentrations and mRNA expression of genes coding for the tryptophan metabolising enzymes, IDO1, IDO2, Tph1 and Tph2. The stress-sensitive BALB/c strain exhibited increased depressive-like behaviour and enhanced corticosterone concentrations in response to LPS. Furthermore, swim-stress attenuated the LPS-induced corticosterone response in BALB/c mice only. LPS significantly increased plasma interleukin (IL)-1ß and tumour necrosis factor α (TNFα) concentrations to a greater extent in BALB/c mice. The LPS-induced increase in IL-1ß mRNA expression was significantly attenuated by swim-stress in the hippocampus of C57BL/6J but not in BALB/c mice. TNFα mRNA expression was significantly increased in BALB/c mice only; this increase was attenuated by swim-stress. Tph1 mRNA expression was upregulated in the brainstem of C57BL/6J mice post-LPS and following the combination of swim-stress and LPS in BALB/c mice. In the hippocampus Tph1 and Tph2 mRNA expression was increased in C57BL/6J but not BALB/c mice in response to LPS challenge and swim-stress. Conversely, IDO2 but not IDO1 mRNA expression was significantly altered following swim-stress and LPS, particularly in the hippocampus of BALB/c mice. These data indicate altered central mRNA expression of tryptophan metabolising enzymes and immune activation in BALB/c mice compared to the normo-sensitive C57BL/6J strain.

Cardiac responses to mild hypoxic hypercapnia in newborn babies: no effect of sleep position.

OBJECTIVE: To evaluate whether cardiac responses to a level of hypoxic hypercapnia that may be observed in rebreathing studies are altered with infant sleep position. METHODOLOGY: Eighteen healthy term infants (< 5-days-old) were studied. Heart rate (HR) and HR variability were monitored during air breathing and during 3 min exposure to a mixture of 15% O2/3% CO2 in both the prone and supine positions. Power spectral analysis of HR was performed. RESULTS: Heart rate was the only measured variable to be significantly changed in response to 15% O2/3% CO2. Hypoxic hypercapnia elicited no significant responses in power spectral HR variables. There was no effect of sleeping position on any of the measured variables. CONCLUSIONS: There are no significant differences in cardiac responses to mild hypoxic hypercapnia between sleep positions and power spectral indices of the autonomic control of HR are not altered by sleep position in newborn babies.

Infant autonomic function is altered by maternal smoking during pregnancy.

Sudden infant death syndrome (SIDS) is more prevalent in infants of smokers and may involve subtle alterations in autonomic control mechanisms. Autonomic function can be assessed using blood pressure responses to a passive head-up tilt and power spectral analysis of heart rate variability. This study aimed to determine if maternal smoking altered infants' responses to head-up tilt. Blood pressure and heart rate responses to a passive 70 degrees head-up tilt were compared in infants of smokers and non-smokers at 2-3 days and 3 months of age. There were no significant differences between groups in power spectral indices. At 2-3 days, the systolic pressure response to tilt was significantly different between groups (P<0.01). In infants of smokers, systolic pressure decreased by a mean (S.E.) of 7.7(1.1) mmHg, whereas in control infants it remained unchanged. At 3 months, systolic pressure in infants of smokers remained unchanged but increased in control infants by 6.2(2.1) mmHg (P<0.05). These results indicate that maternal smoking alters autonomically mediated cardiovascular responses in the infant.

Uterine luminal content of insulin-like growth factor (IGF)-I and endometrial expression of mRNA encoding IGF-binding proteins 1 and 2 during the oestrous cycle and early pregnancy in the ewe.

Cyclic (n = 30) and pregnant (n = 29) Merino ewes were examined (n = 3 to 5 at most time points) over Days 0-16 and 0-22 after oestrus, respectively. As IGFBP activity was detected in some plasma and ULF samples, all samples were subjected to acid-gel chromatography before assay for IGF-I. After oestrus, the overall means of both groups of ewes showed lower ULF IGF-I content (Days 3 and 12), lower plasma IGF-I concentrations (Days 3-16), higher endometrial expression of mRNA encoding IGFBP-I (Days 12-16) and lower endometrial expression of mRNA encoding IGFBP-2 (Day 8). Between Days 0 and 16 after oestrus, the pregnant ewes had lower plasma IGF-I concentrations and higher endometrial expression of IGFBP-1 mRNA than did the cyclic ewes. The presence of IGF-I in the ULF throughout the oestrous cycle and early pregnancy suggests a role of IGF-I in early pregnancy, influencing both uterine growth and embryonic survival. The concomitant endometrial expression of mRNA encoding IGFBP-1 and IGFBP-2 suggests a role of these binding proteins in the regulation of IGF-I bioavailability in the uterine environment of the ewe.

The effect of fetal hypophysectomy with or without ACTH replacement on the molecular weight profile of enkephalin-containing peptides in the adrenal medulla of the fetal sheep.

We have investigated the possible role of the fetal pituitary and ACTH in the control of the synthesis and post-translational processing of the enkephalin precursor, proenkephalin A (proEnk A), in the fetal sheep adrenal gland in late gestation. Fetal hypophysectomy (n = 8) or sham operations (n = 4) were performed between 109 and 118 days of gestation. At 138-139 days, either ACTH(1-24) (10.5 micrograms/0.24 ml saline per h, n = 4) was infused intravenously for 72 h into hypophysectomized fetal sheep or 0.9% (w/v) NaCl alone (0.24 ml/h, n = 4) was infused for 72 h into hypophysectomized fetal sheep and sham-operated animals. At the end of the infusion the pregnant ewe was killed and left or right adrenal glands (n = 12) were collected from the fetal sheep that were intact and given saline (Intact + sal; n = 4), hypophysectomized and given saline (Hx + sal; n = 4) and hypophysectomized and given ACTH (Hx + ACTH; n = 4). Each adrenal was homogenized in acid (acetic acid (1 mol/l)/HCl (20 mmol/l)/2-mercaptoethanol (0.2%)). After centrifugation, the supernatant was loaded onto a Sephadex G-75 column (2.0 x 50 cm), eluted at 80 ml/24 h and fractions were collected (5 ml, n = 42). An aliquot of each fraction (2 ml) was dried down prior to enzymatic digestion (trypsin/carboxypeptidase B) and oxidation with H2O2, and assay for methionine-O-enkephalin (immunoreactive Met-O-Enk).(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor and transforming growth factor alpha.

I have attempted here to outline the basic biochemical knowledge that we have now secured on the EGF family of proteins. In the future we will learn much more about the differential role of EGF versus TGF-alpha, about the physiological significance of amphiregulin, the newest member of this family, and about the roles of TGF-alpha and amphiregulin in cancer. Many questions remain. What is the importance of these factors in embryogenesis and fetal development? Is there an involvement of the EGF-like domains of extracellular proteins in cell-to-extracellular-matrix interactions? Do these extracellular matrix EGF-like entities function in a similar manner to fibroblast growth factor in cell growth and in mediating the relationship of cells to the extracellular matrix? What is the significance of cell membrane-bound forms of EGF and TGF-alpha as potential cell-to-cell contact regulators? What is the role of the viral EGF-like proteins in the viral infective and transforming process? These and other questions will be addressed in the next decade. The key question has already been well stated: 'what is the normal physiological role of EGF during development and homeostasis? The answers to these and a host of other questions must be found before we can fully comprehend this important regulatory system' (Cohen, 1987).

Effect of adrenalectomy or long term cortisol or adrenocorticotropin (ACTH)-releasing factor infusion on the concentration and molecular weight distribution of ACTH in fetal sheep plasma.

It is unclear whether the maturation of corticotrophs from the fetal to the adult type in the fetal sheep pituitary in late gestation is associated with changes in the sensitivity of the fetal pituitary to corticotrophic secretagogues and in the form of ACTH-containing peptides (IR-ACTH) secreted into the circulation. The maturation of the pituitary corticotroph population is known to be accelerated by intrafetal cortisol infusion and delayed by bilateral fetal adrenalectomy. We have therefore investigated the mol wt profile of IR-ACTH present in fetal sheep plasma from 110 days gestation until term (147 +/- 3 days) and determined whether intrafetal cortisol infusion between 105-117 days (2.5 mg cortisol/day), or bilateral fetal adrenalectomy can alter the mol wt profile of IR-ACTH in fetal sheep plasma. We have also investigated whether prior exposure to cortisol alters the subsequent responsiveness of the fetal pituitary to a long term infusion of ovine (o) CRF (10 micrograms oCRF/day). In the control group, the proportion of IR-ACTH which eluted in the low-mol wt (LMW) range (i.e. less than 12K) was significantly higher between 121-125 days (43.9 +/- 4.2%) than between 126-139 days (26.8 +/- 9.3%) but not different to that after 140 days gestation (29.9 +/- 5.5%). Between 110-117 days, cortisol infusion had no effect on the proportion of IR-ACTH in the LMW range (43.9 +/- 5.7%, saline infused; 44.1 +/- 2.4%, cortisol infused). Between 121-125 days, the proportion of IR-ACTH in the LMW range in the CRF-infused groups (with or without prior exposure to cortisol) was significantly lower (27.4 +/- 2.1%) than in the saline-infused control group. In contrast, after fetal adrenalectomy, the proportion of IR-ACTH in the LMW range between 126-139 days was significantly higher (48.0 +/- 6.7%) than in intact control animals (23.8 +/- 3.5%). We conclude that the change in the mol wt profile of IR-ACTH in fetal plasma after 125 days may be a consequence of changes in the morphological and/or functional characteristics of the corticotrophic cells in the fetal pituitary. Infusion of oCRF appears to accelerate the normal maturation of the fetal pituitary-adrenal relationship, and oCRF acting either directly or via secretion of cortisol may play a role in the posttranslational processing of POMC in the fetal sheep pituitary after 125 days gestation.

Different roles for the pituitary and adrenal cortex in the control of enkephalin peptide localization and cortico-medullary interaction in the sheep adrenal during development.

We have investigated the effect of adrenocorticotrophic hormone (ACTH) replacement after fetal hypophysectomy on the pattern of localization of enkephalin-containing peptides (enkephalins) and phenylethanolamine N-methyltransferase (PNMT) in the fetal sheep adrenal. We have also investigated the relative roles of the fetal pituitary and adrenal cortex in determining the extent of the interdigitation of the peripheral adrenaline (AD)-containing cells of the adrenal medulla with the inner zones of the adrenal cortex in the late gestation fetus. Fetal hypophysectomy (Hx; n = 12) or sham operations (n = 8) were performed at 109-118d. At 138 or 139d, ACTH (1-24) (10.5 micrograms/h) was infused intravenously for 72 h into 4 Hx fetuses (Hx + ACTH group). Saline was infused for 72 h into 4 Hx fetuses (Hx + Sal) and into 4 sham-operated fetal sheep (Intact + Sal). Fetal adrenal glands were collected at autopsy from 141/2d Intact + Sal, Hx + Sal and Hx + ACTH groups, from 4 intact fetal sheep at 145-147d gestation (145/7d Intact group) and 4 Hx fetal sheep at 147-164d gestation (147/64d Hx group). Adrenals were also collected from 4 newborn lambs at 10-12d after birth (10/12d Newborn group). Using the peroxidase-antiperoxidase immunocytochemical staining method, sections of adrenal glands (10-12 microns) from all groups were stained anti-PNMT. Sections of adrenal glands from the 141/2d groups were also stained separately with anti-dopamine-beta-hydroxylase (anti-D beta H) and anti-enkephalin (anti-ENK).(ABSTRACT TRUNCATED AT 250 WORDS)

The molecular weight profile of enkephalin-containing peptides in the sheep adrenal gland changes during development.

We have measured the content of enkephalin-containing peptides (ENK-containing peptides) in adrenal gland extracts from fetal sheep between 68-142 days gestation and from adult sheep. We investigated whether there are changes in the post-translational processing of ENK-containing peptides in the fetal sheep adrenal gland with increasing gestational age. ENK-containing peptides in adrenal extracts from fetal sheep (68-142 days gestation) and adult sheep were separated using gel filtration chromatography and the ENK immunoreactivity (ENK-IR) was measured using RIAs for Met-enkephalin-Arg6,Phe7 immunoreactivity (MERF-IR) and Met-o-enk immunoreactivity (MET-O-ENK-IR). The MET-O-ENK-IR in fetal and adult sheep adrenal extracts was distributed in four main peaks, which corresponded to mol wt (MW) ranges of more than 12, 7-12, 3-7, and less than 3 kDa. There was a significant increase (P less than 0.05) in the total ENK-IR content of the fetal adrenal between 68-78 (42.4 +/- 19.2 ng/adrenal) and 100-121 (320.1 +/- 142.6 ng/adrenal) days gestation. There was also a significant increase in the proportion of MET-O-ENK-IR and MERF in the less than 3 kDa range between 68-78 (MET-O-ENK, 6.8 +/- 2.7%; MERF, 10.3 +/- 1.4%) and 100-121 (MET-O-ENK, 24.4 +/- 5.1%; MERF, 26.2 +/- 5.8%) days gestation, with an associated decrease in the ratio of ENK-containing peptides in the high MW form (i.e. greater than 3 kDa) compared to those in the low MW (less than 3 kDa) forms (10.4 +/- 2.5 at 68-78 days gestation; 4.2 +/- 1.1 at 100-121 days gestation). There was also a significant decrease in the percentage of MET-O-ENK in the 12 kDa range after 139 days gestation (125-135 days gestation, 20.4 +/- 2.2%; 139-142 days gestation, 4.0 +/- 2.1%). Therefore, there is an increase in the proportion of the low MW forms of ENK-containing peptides in the fetal sheep adrenal with advancing gestational age, which may reflect changes in the post-translational processing of the precursor proenkephalin-A during development.

The fate and uptake of murine epidermal growth factor in the sheep.

125I-Labelled murine epidermal growth factor (EGF) was injected or infused into conscious ewes through the jugular vein. Its disappearance from the circulation and the pattern of its distribution in other body tissues and compartments were observed. Single bolus injections of 125I-labelled EGF resulted in a transient peak of radioactive EGF in the circulation which occurred within 1 min of the injection. This was followed by a very rapid fall in radioactivity in the plasma (t1/2 approximately 1 min) and the gradual appearance of 125I-labelled EGF in the urine. Immunoprecipitable 125I-labelled EGF could be detected in urine within 5 min of the start of the experiment. 125I-Labelled EGF accumulated in the urine for several hours following the injection, although with increasing time a substantial amount of non-immunoprecipitable iodide was also found. The rate of disappearance of the 125I-labelled EGF from the plasma of the ewe was found to be faster than the rate of disappearance of free [125I]iodide that had been injected into the ewe. 125I-Labelled EGF was also administered by a continuous infusion following an initial bolus injection. This again resulted in a rapid initial fall in radioactivity in blood, followed by a slow rise throughout the period of the infusion. When the infusion was stopped, there was a 15-min period of rapid readjustment, after which the radioactivity in the blood fell at a much slower rate (t1/2 approximately 70 min) than was seen initially. Again, intact 125I-labelled EGF was transferred to urine throughout the experiment. At autopsy, 125I-labelled EGF was increased in bile, liver, thyroid and kidney. Although most of the 125I found in the thyroid was free iodide, some EGF-like material was also present. There was also EGF-like material found in both the kidney cortex and the kidney medulla. These results indicate that complex multi-compartment pathways for the uptake, distribution and clearance of 125I-labelled EGF exist in the sheep.

Simultaneous isolation of insulin-like growth factors I and II from adult sheep serum.

Ovine insulin-like growth factors I and II (oIGF-I and oIGF-II) have been purified from adult sheep serum. oIGF-II-like receptor-binding activity and IGF-I-like immunoactivity were enriched on SP-Sephadex C-25, then purified using HPLC in the presence of a variety of counter ions. IGF-I- and IGF-II-like activities were separated using HPLC in the presence of 0.2% tetrabutylammonium phosphate at pH 7.0. The final recovery of oIGF-I was 82.6 micrograms from 3.2 litres of adult sheep serum (a yield of 17.6%), and the recovery of oIGF-II was 388 micrograms (a yield of 13.3%). Both IGF preparations were considered to be homogeneous as judged by single sharp peaks during analytical HPLC, and unique N-terminal amino acid sequences. Purified ovine IGFs had molecular weights similar to that of other IGFs (approximately 7000), and the first 30 N-terminal amino acids of both peptides were identical to their human counterparts. The isoelectric points of oIGF-I (pI approximately 8.2) and oIGF-II (pI approximately 6.8) were similar to those of human (h) IGFs (hIGF-I pI approximately 8.2; hIGF-II pI approximately 6.5), and the overall amino acid content of the ovine IGFs was also similar to that of IGFs from other species. oIGF-II preparations from fetal sheep and from adult sheep appeared to be identical. The isolation procedure represents one of general utility that can be easily modified to facilitate the isolation of recombinant IGFs from culture fluid.

Effect of fetal hypophysectomy on the localization of the catecholamine biosynthetic enzymes and enkephalins in the adrenal medulla of the fetal sheep.

We have investigated the effect of fetal hypophysectomy on the localization of dopamine B-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) and enkephalin-containing peptides in the fetal sheep adrenal, using immunocytochemical techniques. Staining with anti-DBH was observed throughout the adrenal medulla in the intact (140-146 days of gestation) and hypophysectomized fetal sheep (147-164 days of gestation) and the newborn lamb (10-12 days after birth). In the adrenal medulla of the late-gestation intact fetal sheep and newborn lamb, positive staining with anti-PNMT was observed in the peripheral rim of medullary cells adjacent to the adrenal cortex. After hypophysectomy, there was intense positive staining with anti-PNMT in the peripheral adrenal medullary cells and a small and variable proportion of central adrenal medullary cells were stained with anti-PNMT. In the adrenal gland of the intact fetal sheep and the newborn lamb, there was intense staining with anti-enkephalin in the peripheral rim of adrenal medullary cells. Staining with anti-enkephalin was less intense in the central medullary cells of the adrenal gland of the intact fetal sheep and the 10- to 12-day-old newborn lamb, and many unstained central medullary cells were present. After hypophysectomy, intense positive staining with anti-enkephalin was observed throughout the entire fetal adrenal medulla. Therefore, the fetal pituitary, either directly or indirectly through the adrenal cortex, plays a role in regulating the pattern of localization of both PNMT and enkephalin in the fetal sheep adrenal.

Growth hormone and testosterone can independently stimulate the growth of hypophysectomized prepubertal lambs without any alteration in circulating concentrations of insulin-like growth factors.

Castrated prepubertal lambs were hypophysectomized and then treated with GH and testosterone either alone or in combination over a series of 3-week treatment periods. Hypophysectomy resulted in a rapid reduction in skeletal growth rate which could be reversed by the administration of either GH (4 IU three times a week for 3 weeks) or testosterone propionate (10 mg daily for 3 weeks). When GH or testosterone treatment was withdrawn, skeletal growth fell to the post-operative rate. Combined treatment with both GH and testosterone was no more or less effective than either hormone given singly. The order of administration did not have any effect on the growth rate. Circulating concentrations of insulin-like growth factor-I (IGF-I) were reduced by hypophysectomy, but neither GH nor testosterone treatment, alone or in combination, had any effect on IGF-I concentrations. Concentrations of IGF-II rose following hypophysectomy, and again were not affected by any of the hormonal replacement treatments. In conclusion, both GH and testosterone could stimulate skeletal growth in the hypophysectomized lamb without any alteration of circulating IGF concentrations, and testosterone can clearly stimulate skeletal growth in the complete absence of GH.

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 on the growth and development of the sheep adrenal gland: a morphometric study.

We have investigated the effect of gestational age and fetal hypophysectomy on the growth and development of the adrenal gland of the fetal sheep. The area of the fetal sheep adrenal medulla increased significantly (P less than 0.05) from 3.21 +/- 0.42 mm2 at 90-107 days to 6.09 +/- 0.26 mm2 at 120-126 days of gestation and there was a further significant increase (P less than 0.05) in the newborn period to 9.48 +/- 0.85 mm2. The adrenomedullary area of the hypophysectomised fetal sheep (7.47 +/- 1.10 mm2) was not significantly different from that of the fetal sheep at 140-146 days of gestation or from that of the newborn lamb. The ratio of the area of the adrenal occupied by the cells which contained adrenaline (adrenaline zone) to the area of the adrenal occupied by the cells which contained noradrenaline (noradrenaline zone) was unchanged between 90 days of gestation and 12 days after birth. After hypophysectomy, the ratio of the adrenaline to noradrenaline zone was not significantly different from that in the adrenal medulla of the 140-146 days fetal sheep and the 10-12 days newborn lamb.

Endocrine control of fetal growth.

Fetal growth is regulated by fetal hormones in all species that have been studied. However, it is clear that strict definition of fetal growth must be applied in order that different studies and different species may be meaningfully compared. The experimental manipulation of fetal growth in vivo has been the main tool by which information in this area has been gained. Organ ablation experiments, with or without appropriate hormone replacement treatments, have been used now for nearly 50 years as a means of studying fetal growth. As the years have gone by, many of the original techniques have been refined beyond recognition, so that precise surgical or immunological approaches have now replaced the rather simpler earlier methods. However, the nature of the questions which are posed are still remarkably similar to those first formulated nearly 50 years ago. What regulates fetal growth? This article attempts to document the progress that has been made in the endocrine control of fetal growth.

Effect of gamma 3 or gamma 2 melanocyte stimulating hormone on steroidogenesis in the fetal sheep during late gestation.

We have measured circulating concentrations of gamma 3 Melanocyte Stimulating Hormone (MSH) in fetal sheep between 111 and 145 days gestation. There was no significant effect of gestational age on the fetal plasma concentrations of gamma 3 MSH throughout this period. We have examined the role of gamma-MSH related peptides in the control of fetal adrenal steroidogenesis and found no significant change in fetal plasma cortisol or pregnenolone concentrations during a 60-72 h infusion of saline, gamma 2 MSH or gamma 3 MSH in sheep between 130 and 135 days gestation. Therefore although we have demonstrated the presence of gamma MSH related peptides in fetal sheep plasma during late gestation we have failed to demonstrate a role for gamma 3 or gamma 2 MSH in the changes in fetal steroid concentrations which occur prepartum.

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.

Continuous measurement of biparietal distance in the intact and hypophysectomized fetal sheep using ultrasound.

Piezoelectric transducers were implanted into the parietal bones of intact (n = 4) and hypophysectomized (n = 8) fetal sheep of approximately 110-120 days gestational age (term 145-150 days). Intertransducer distance was determined by measuring the time taken for an ultrasonic pulse, generated by one transducer, to elicit a piezoelectric response in an opposing transducer. The limit of sensitivity of the timer was +/- 0.033 microsec. The ultrasonic velocity through fetal sheep brain tissue was 1549.6 +/- 2.2 m.s-1 (SEM; n = 33). This velocity remained constant throughout the entire period studied in both intact and hypophysectomized fetuses. At this velocity, the sensitivity of the measuring device was +/- 0.05mm. The ultrasonic transit time was measured daily between 0900 and 1100h until term in all fetuses. Three hypophysectomized fetuses were allowed to remain in utero until day 163 of gestation. The mean biparietal distance growth rate prior to day 135 for the intact and hypophysectomized fetuses was 0.25 +/- 0.03 and 0.27 +/- 0.025 mm/day respectively. These values were not significantly different (P greater than 0.05). A significant decrease (P less than 0.05) in growth rate was detected in both experimental groups between days 135 and 147 and was more pronounced in the sham (0.05 +/- 0.04 mm/day) than in the hypophysectomized (0.14 +/- 0.03 mm/day) group. However, the growth rate of the sham animals after day 135 was not significantly different from that of the hypophysectomized animals. In the three hypophysectomized fetuses killed at day 163 the biparietal distance growth was maintained at 0.12 +/- 0.005 mm/day. We conclude that fetal biparietal distance growth is pituitary independent from day 110 of gestation and that this technique for measuring distance is a valid and extremely accurate method for the continuous measurement of this parameter of fetal growth and may have further applications in other areas of growth research.