Prolonged low-dose dexamethasone, in early gestation, has no long-term deleterious effect on normal ovine fetuses.

Low-dose dexamethasone (D) treatment is used in pregnancies where the fetus is suspected to be at risk of congenital/virilizing adrenal hyperplasia. To study if this treatment had any immediate or long-term effects in normal fetuses, pregnant ewes were treated with D (20 microg/kg maternal body weight x d) or saline (S), from d 25-45 of gestation. Tissue was collected from fetuses killed at 45 d (S = 6; D = 8), 130 d (S = 8; D = 8), or lambs at 2 months of age (S = 6; D = 6) and mRNA levels measured using real-time PCR. D treatment reduced adrenal wt at 45 d (S, 12.2 +/- 0.7 mg; D, 6.3 +/- 0.4 mg) and significantly decreased adrenal mRNA for P(450scc). At 130 d, fetuses from the D treatment were growth retarded (S, 3.2 +/- 0.1 kg; D, 2.5 +/- 0.1 g), but the adrenals were appropriate for the body weight. mRNA levels of angiotensinogen, the AT(1) receptor and mineralocorticoid receptor (MR) and GR were similar in kidney and brain (hypothalamus, hippocampus, medulla oblongata) except for hippocampal expression of MR and GR, which was significantly decreased by D treatment. By 2 months, BW and hippocampal MR and GR mRNA levels were similar, and lambs were normotensive (S, 83 +/- 3 mm Hg; D, 78 +/- 3 mm Hg). Thus, there were no persistent, long-term effects of prolonged low-dose D treatment in normal ovine fetuses.

Ovine aquaporin-2: cDNA cloning, ontogeny and control of renal gene expression.

The aim of this study was to test the hypothesis that the relative insensitivity of the ovine fetal kidney to arginine vasopressin (AVP) is due to low levels of expression of the gene for aquaporin-2 (AQP2) which encodes the AVP-regulated water channel. We report the cloning of the cDNA for the ovine AQP2 which has a major transcript at 4.2 kilobases (kb) and a minor transcript at 1.5 kb, resembling the human gene transcripts. At 40-60 days' (term = 145-150 days'), mRNA levels are very low, detectable only by reverse transcription-polymerase chain reaction (RT-PCR). By Northern blot analysis AQP2 mRNA is detectable at 75 days'. The ratio of AQP2/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA increases approximately 2.4-fold between 100 and 140 days' when it is about 41% of adult values. Both glucocorticoids and the renin-angiotensin system are involved in maturation of renal function. When fetuses at 75 or 85 days of gestation were exposed to high levels of dexamethasone for 2-3 days, mRNAs for both GAPDH and AQP2 doubled, but the ratio was unchanged. Angiotensin I, infused for 3 days at 115-120 days' gestation, increased the AQP2/GAPDH mRNA ratios by twofold (major transcript) and sixfold (minor transcript), which were highly significant (P<0.001). The increasing sensitivity of the ovine fetal kidney to AVP, from 100-140 days of gestation, is largely due to increasing AQP2 gene expression over this period.

Ontogeny and regulation of the AT1 and AT2 receptors in the ovine fetal adrenal gland.

The expression and regulation of the receptors for angiotensin II (both AT1 and AT2) were examined in the ovine fetal adrenal gland by RNase protection assay (RPA), in situ hybridisation histochemistry, immunohistochemistry and Western blotting. Both mRNA and protein for the AT1 receptor were present in the zona glomerulosa and zona fasciculata of the cortex, but not in the medulla, from as early as these zonas were distinguishable (60 days of gestation; term is 145-150 days), and even present in the steroidogenic cells of the unzoned gland at 40 days. The mRNA for the AT2 receptor was present in the same locations (but never in the medulla) from 40-130 days, and declined to extremely low levels after 140 days. The infusion of ang II, 1 microg/h, for 3 days, at mid-gestation (76 +/- 2 days) caused a significant decrease in mRNA for AT1 but no change in AT2 levels. Thus, the biologically active receptor (in terms of aldosterone stimulation) is present in the ovine fetal adrenal from very early in development, and can be down-regulated by mid-gestation.

Location of ganglioside GM2 activator protein gene expression in sheep.

1. The present study was aimed at characterizing and establishing the site of production of a 'novel' protein isolated in 1988 during the course of studies on sheep renal morphology. This protein has subsequently been identified as the GM2 activator protein (GM2AP). 2. The 'novel' protein, with an apparent molecular weight of 18-22 kDa and a pI between 4.7 and 4.9, was isolated from enriched granular fractions of sheep kidney cortex using two-dimensional (2-D) polyacrylamide gel electrophoresis. Following electroelution, the N-terminal amino acid sequence was determined and, applying the preferred codon usage formula, an oligodeoxyribonucleotide probe was constructed for examination of sites of expression of this novel protein using northern analyses and hybridization histochemistry. 3. Western blots of the 2-D gels onto nitrocellulose membranes permitted us to select the appropriate spots for injection into rabbits for production of polyclonal antibodies. The antibodies were used to confirm the sites of protein production using immunohistochemistry. 4. Northern analyses revealed that GM2AP mRNA has a widespread distribution in ovine tissues. In the kidney, GM2 was expressed in all major renal arteries and arterioles. In the liver, the expression of the gene was prominent in the hepatic vein and ducts. Antibodies raised against the GM2AP confirmed that the protein was present at the same sites as the mRNA. 5. These are the first studies showing the location of GM2 activator gene expression in normal mammalian tissues. The arterial site of production has implications for local action or an important role in membrane integrity throughout the kidney.

The renin-angiotensin system and the development of the kidney and adrenal in sheep.

1. The earliest form of the kidney, the pronephros, does not really occur in the ovine embryo; instead, a giant glomerulus forms at the anterior end of the mesonephros. 2. In the sheep, the mesonephros is present from 11-38% of total gestation (150 days) and produces a dilute urine, as well as expressing the genes for erythropoietin, renin, angiotensinogen, angiotensin-converting enzyme and the angiotensin II (AngII) receptors AT1 and AT2. 3. The ovine metanephros begins to develop at 18% of gestation and nephrogenesis is complete several weeks before birth. All components of the renin-angiotensin system (RAS) are expressed from at least 27% of gestation. 4. Both AT1 and AT2 receptors are expressed by the adrenocortical cells early in gestation but, at mid-gestation, exogenous AngII does not stimulate aldosterone secretion in vivo. 5. Preliminary results suggest that AngII has important roles in renal development in the ovine foetus but the role(s), if any, in adrenal development, remains to be investigated.

Ovine AQP1: cDNA cloning, ontogeny, and control of renal gene expression.

The cDNA for the ovine aquaporin 1 (AQP1) was obtained and found to be 97%, 88%, and 85%, respectively, homologous to the bovine, human, and rat AQP1 cDNA. The level of total kidney mRNA expressed as a ratio to glyceraldehyde-3-phosphate dehydrogenase increased sevenfold from 60 days to 140 days of gestation (term=150 days) and reached adult values by 6 weeks after birth. Treatment of pregnant ewes (and their fetuses) at 64 and 74 days of gestation with dexamethasone (0.76 mg/h for 48 h) resulted in a small but statistically significant increase in AQP1 mRNA only in the 74-day fetuses. By immunohistochemistry, it was shown that the increase in AQP1 mRNA with dexamethasone resulted largely from an increase in maturity of the inner zone of the fetal renal cortex (i.e., more tubules) as well as stronger expression of AQP1 in proximal tubules and thin descending limbs of loops of Henle. A similar effect occurred in fetuses infused for 3 days with angiotensin I (6.7 microg/h) in the last third of gestation.

Regulation and function of steroid production by mid gestation ovine fetal adrenal cortex in vivo.

By mid-gestation (75-85 days, term=150 days), the ovine fetal adrenal gland is zoned into cortex and medulla. The cortex has an outer layer of cells which have the morphological characteristics of zona glomerulosa cells, containing mitochondria with lamellar cristae. It has been reported that cultured adrenal cells from mid-gestation bovine and ovine fetuses can be stimulated to increase aldosterone production, ten fold, by angiotensin II, and that this can be maintained for at least 3 days. However, the situation in vivo is unknown. In the current report we show that in chronically cannulated ovine fetuses at mid-gestation, angiotensin II (1 microg/h) does not increase aldosterone either in the short term (3 hours) or long term (3 days). However, ACTH (450 ng/h) can increase plasma aldosterone in the short but not long term. ACTH at this dose produces progressive and large increases in cortisol production. Angiotensin II is pressor and produces a modest diuresis without stimulating cortisol.

Ontogeny of angiotensin II receptors, types 1 and 2, in ovine mesonephros and metanephros.

By RNAse protection assay, hybridization histochemistry, and in vitro autoradiography it was shown that both mRNA and protein for AT1 and AT2 receptors were present in ovine fetal meso- and metanephroi at 40 days of gestation (term approximately 150 days). AT1 mRNA was localized to presumptive mesangial cells of glomeruli at 40-, 75-, 131-gestational-day-old fetuses and two-day-old lambs, in addition to being widely present in interstitial cells of the cortex and medulla, once these zones formed (60 days). By two days after birth the medullary AT1 distribution was confined to the inner stripe of the outer medulla. AT2 mRNA was present in peripheral interstitial/tissue of the mesonephros, and interstitial tissue surrounding developing glomeruli, but not the outermost nephrogenic mesenchyme in the metanephros from 40 to approximately 131 days (the period of active nephrogenesis). In addition, AT2 mRNA was localized to epithelial cells of the macula densa in metanephroi (40 to 131 gestational days) during, but not after completion, of nephrogenesis. These studies suggest that angiotensin II (Ang II) could have differentiating effects, via AT1 receptors, from very early in development. The unique epithelial site of AT2 expression in the macula densa raises the possibility that Ang II may play a role in the invariant positioning of the macula densa at the pole of its glomerulus, via this receptor.

Expression of aquaporin-1 (AQP1) in the adult and developing sheep kidney.

The distributions of aquaporin-1 mRNA and protein were studied by hybridization histochemistry with a homologous riboprobe and immunohistochemistry, in the adult sheep kidney. Heaviest labelling occurred in the thin descending limb (DTL) of the loop of Henle in the inner stripe of the outer medulla, with apparent decreasing expression in the inner medulla, outer stripe of the outer medulla and cortex, but no quantitation was performed. Only proximal tubules (PT) (convoluted and straight) and DTL labelled. The glomerulus showed no labelling, consistent with the pattern in the rat but different to that in the human. During ontogeny, no labelling occurred in the mesonephros at 27 or 41 days of gestation (term = 145-150 days) but other structures did label at 27 days (heart, lung bud, blood vessels surrounding developing spinal cord). Labelling first occurred faintly in the metanephros at 41 days of gestation and increased throughout gestation consistent with morphological development of nephrons.

Functional and expression analysis of ovine steroid 11 beta-hydroxylase (cytochrome P450 11 beta).

In this study, the ovine steroid 11 beta-hydroxylase (P450(11 beta) or CYP11B) cDNA previously reported by us (1) was transfected into COS-7 cells. Using 3H-11-deoxycorticosterone (3H-DOC) as the substrate, and paper partition chromatography for separation of steroid products, the expressed enzyme was shown to catalyse the conversion of DOC to corticosterone (B), 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), 18-hydroxy-corticosterone (18-OH-B), and aldosterone (ALDO). These results suggest that the expressed ovine cDNA exhibited 11 beta-hydroxylase, 18-hydroxylase and aldosterone synthesis activities. The enzymatic activity of the enzyme was further analysed by adding unlabelled steroids to compete with 3H-DOC. The conversion of 3H-DOC to 3H-ALDO was inhibited by the addition of excess DOC, B and 18-OH-DOC, indicating that all these steroids were potential substrates of the enzyme. The results also demonstrated that 18-hydroxylation could occur before 11 beta-hydroxylation with this enzyme. However, the addition of excess cold 18-OH-B had no significant effect on the level of 3H-ALDO that was synthesised. This result could imply that 18-OH-B is not an intermediate involved in the conversion of DOC to aldosterone, or, more likely, the enzyme substrate site is not accessible readily. Our results also indicated that DOC was preferred to 18-OH-DOC as a substrate for the enzyme. We have demonstrated by hybridisation histochemistry using specific oligonucleotide probes that the corresponding P450(11 beta) RNA transcript was present in all zones in the sheep adrenal cortex. In summary, we have shown that the enzyme encoded by the predominant P450(11 beta) cDNA isolated from the sheep adrenocortical cDNA library has all the enzymatic activities to biosynthesise ALDO from DOC. The corresponding transcript of this ovine P450(11 beta) cDNA was located throughout the adrenal cortex and thus the inability of the zonae fasciculata-reticularis to secrete ALDO remains to be understood.

Ontogeny of hormonal and excretory function of the meso- and metanephros in the ovine fetus.

Using reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry we investigated the ontogeny of renin, angiotensinogen and angiotensin converting enzyme (ACE) in the mesonephros at 27 and 41 days of gestation, and the metanephros at 41 and 64 days of gestation in ovine fetuses (term is 145 to 150 days). The volume and composition of fetal urine, stored as allantoic fluid were measured in 12 fetuses at 27 days, and 13 fetuses at 41 days. Renin, angiotensinogen and ACE were identified in both meso- and metanephroi at 41 days but not in the mesonephros at 27 to 30 days. Allantoic fluid volumes were 21 +/- 3 and 45 +/- 5 ml at 27 to 30 days and 41 days, respectively. This fluid was significantly different in composition to that of amniotic fluid or maternal plasma. The results suggest that the mesonephros can substantially modify its glomerular filtrate by 27 days of gestation, and can produce local angiotensin II by 41 days.

The erythropoietin gene is expressed strongly in the mammalian mesonephric kidney.

In the ovine fetus at 41 days of gestation (term is 150 days), there are two sets of kidneys, mesonephrol and metanephrol. We have examined the expression of the erythropoietin (Epo) gene in both types of kidneys by competitive reverse transcriptase-polymerase chain reaction and hybridization histochemistry and compared the expression to that of the 60-day fetal metanephros. At 41 days, the Epo gene was expressed in both mesonephros and metanephros, as well as in the fetal liver. The cells expressing the Epo gene in the mesonephros were interstitial cells in the vicinity of the proximal tubules.

The effect of relaxin on collagen metabolism in the nonpregnant rat pubic symphysis: the influence of estrogen and progesterone in regulating relaxin activity.

The aim of this study was to examine the effects of relaxin on collagen content, solubility, and composition in the rat pubic symphysis. Nonpregnant, female Sprague-Dawley rats were bilaterally ovariectomized and either unprimed or primed with estrogen or progesterone alone, or a combination of estrogen and progesterone. One week later these animals were given increasing doses of a synthetic human (gene-2) relaxin (0-100 micrograms) before being killed 16 h later. Their pubic symphysial tissues were then removed and analyzed for collagen content and solubility, whereas collagen composition was determined by SDS-PAGE. Relaxin administration significantly increased the length (140 +/- 6%) and weight (170 +/- 9%) of the interpubic fibrocartilage in estrogen-primed rats (n = 15). At the same time, it decreased the total collagen content by 68 +/- 6%, without altering the proportions of collagen types, which were predominantly type I (85%) and type II collagen (15%). Relaxin administered alone reduced the total collagen content by 64 +/- 4% but had no effect on collagen solubility or composition. Progesterone abolished the effects of relaxin in estrogen-primed rats. It is concluded that relaxin has a potent effect on the amount of collagen in the rat pubic symphysis that is enhanced by estrogen and antagonized by progesterone. The changes in the extracellular matrix within the pubic symphysis induced by relaxin may be important in the modifications that this tissue undergoes during pregnancy.

Structure and expression of Leydig insulin-like peptide mRNA in the sheep.

We have used PCR to isolate and characterise Leydig insulin-like peptide (Ley I-L) mRNA from sheep ovary. The deduced amino acid sequence of sheep Ley I-L has good homology with the pig and human peptides, having 93% and 77% amino acid identity, respectively. Northern blot analysis revealed abundant expression in both ovary and testis. An examination of ovarian RNA from non-pregnant and pregnant sheep showed that pregnancy did not significantly increase Ley I-L mRNA levels. However mRNA levels did alter depending on whether ovaries contained a corpus luteum. Also ovaries were examined by hybridisation histochemistry to locate the site of expression. Abundant Ley I-L mRNA levels were found in the theca interna cells of the ovary.

Studies on stanniocalcin: characterization of bioactive and antigenic domains of the hormone.

Stanniocalcin (STC) decreases branchial Ca(2+)-uptake in fish. In order to determine its bioactive domain, synthetic fragments (U amino acids (aa) 1-20; V aa 103-136; W aa 202-231) of eel STC were tested for their effect on Ca2+ uptake in tilapia (Oreochromis mossambicus). Ca2+ uptake was inhibited by an N-terminal fragment but not by a midfragment nor a C-terminal fragment of the mature hormone. We provide theoretical and experimental evidence that a midportion of STC, which is included in the synthetic fragment V, is the most antigenic site of the molecule. Polyclonal antibodies against stanniocalcin are directed against this midportion although this region of STC appears not to be essential for signal transduction. These results suggest that the currently available antibodies will recognize inactive STC fragments in the circulation. We conclude that the bioactive portion of STC does not correspond with the major antigenic portion of the hormone. The results imply that studies on plasma STC levels employing a polyclonal antiserum against STC should be interpreted with care.

Immunoreactive erythropoietin concentration and haemoglobin type in the perinatal period in sheep of various haemoglobin genotypes.

This study examines the hypotheses that (i) erythropoietin (the hormone responsible for red blood cell production) is higher in the fetus (at low PO2) than in the neonate (at high PO2); and (ii) that the level of erythropoietin in the neonate is influenced by the presence of high oxygen affinity haemoglobin. Haematocrit (PCV), PO2 and plasma immunoreactive erythropoietin were measured in 4 chronically cannulated fetal sheep (120 days to birth, n = 22) and in 7 neonatal lambs until 233 days post-conception (75-85 days after birth, n = 83). The percentage of globin chains (alpha, gamma, beta A, beta B, beta C) was quantitated by gel electrophoresis. Plasma erythropoietin values, in 4 fetuses were 9.8 +/- 1.3 mU/ml at 120-132 days of gestation, declined significantly (P less than 0.01) to 5.2 +/- 0.4 at 133 days until birth, then increased significantly (P less than 0.001) to 24.2 +/- 5.5(5), 26.3 +/- 7.3 (6), and 24.8 +/- 8.5 (6), respectively in weeks 1, 2 and 3 of postnatal life. By weeks 5-8 the erythropoietin was 13 +/- 0.6 (4) mU/ml. PO2 was 17.4 +/- 0.9 mmHg before birth and 88.0 +/- 10.7 in the first week after birth. PCV was constant until three weeks after birth and then declined. Fetal haemoglobin had virtually disappeared from the circulation by 166 days (3 weeks after birth); in the 4 heterozygotes (beta A beta B) beta C was expressed transiently, with a maximum value of 4%, whilst in the homozygote lamb (beta A beta A) the maximum beta C was 12%.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of the quinone reductase reaction of pig heart lipoamide dehydrogenase.

The relationship between the NADH:lipoamide reductase and NADH:quinone reductase reactions of pig heart lipoamide dehydrogenase (EC 1.6.4.3) was investigated. At pH 7.0 the catalytic constant of the quinone reductase reaction (kcat.) is 70 s-1 and the rate constant of the active-centre reduction by NADH (kcat./Km) is 9.2 x 10(5) M-1.s-1. These constants are almost an order lower than those for the lipoamide reductase reaction. The maximal quinone reductase activity is observed at pH 6.0-5.5. The use of [4(S)-2H]NADH as substrate decreases kcat./Km for the lipoamide reductase reaction and both kcat. and kcat./Km for the quinone reductase reaction. The kcat./Km values for quinones in this case are decreased 1.85-3.0-fold. NAD+ is a more effective inhibitor in the quinone reductase reaction than in the lipoamide reductase reaction. The pattern of inhibition reflects the shift of the reaction equilibrium. Various forms of the four-electron-reduced enzyme are believed to reduce quinones. Simple and 'hybrid ping-pong' mechanisms of this reaction are discussed. The logarithms of kcat./Km for quinones are hyperbolically dependent on their single-electron reduction potentials (E1(7]. A three-step mechanism for a mixed one-electron and two-electron reduction of quinones by lipoamide dehydrogenase is proposed.

Effects of synthetic peptide fragments of teleocalcin (hypocalcin) on calcium uptake in juvenile rainbow trout (Salmo gairdneri).

A rainbow trout fry bioassay based on 45Ca uptake was used to compare the effects of pure coho salmon teleocalcin (TC) and several synthetic peptide fragments of TC. Calcium uptake in the fry exhibited a cycle, with an amplitude variation of 3.3 to 48.8 mumol.kg-1.hr-1 and a periodicity of 8 to 21 days. The N-terminal 1-20 amino acid peptides of both eel and salmon TC significantly inhibited 45Ca uptake at the high point of the calcium uptake cycle (up to 75%), although the effective doses of the peptides on a molar basis were 20 to 200 times that of the intact molecule. In contrast, the C-terminal fragment of eel TC (amino acids 202-231) did not have an inhibitory effect on calcium uptake. Instead, it significantly enhanced 45Ca uptake in trout fry (up to sixfold) at the low point of the calcium uptake cycle.

Processing and bioactivity of the corpuscles of Stannius protein of the Australian eel.

The primary structure of the major protein from the Corpuscles of Stannius (CS) of the Australian eel was elucidated from the cDNA sequence and was found to bear close similarity to the N-terminal amino acid sequence of the presumably homologous salmon hormone, teleocalcin (TC). The cDNA sequence predicted a preproprotein of 263 amino acids. Following removal of a 17 amino acid signal peptide, specific monobasic cleavage at an Arg-Phe bond generates the 231 amino acid mature form of the protein. The isolation and sequence determination of the prosequence confirms that the precursor contains a prosegment of 15 residues. Various fragments of the protein have been synthesized chemically and their biological activity assessed. The N-terminal 1-20 fragment of the mature protein inhibits calcium uptake in fingerling trout, the effect being similar, but not equipotent to salmon teleocalcin. Further, infusion of either the N-terminal 1-20 or the 81-94 fragment at 50 µg/h into the renal artery of conscious sheep, caused significant decreases in systemic plasma potassium concentration and in potassium excretion. The 1-20 fragment also gave rise to a small but significant increase in sodium excretion. Infusion of TC at the same rate results in a significant decrease in plasma potassium and phosphate concentration as well as a significant decrease in potassium excretion. Bovine PTH (1-34) at 100 µg/h causes a small decrease in plasma potassium and phosphate and an increase in plasma calcium concentration, and was the only peptide to cause a significant decrease in calcium excretion.