Characterization of plasma membrane lipids and luteinizing hormone receptors of ovine corpora lutea during luteolysis and early pregnancy

Lipid composition of plasma membranes from luteal cells was examined to determine whether changes in this organelle occur during regression and maintenance of the corpus luteum in nonpregnant (NP) and pregnant (P) ewes, respectively. Forty ewes were assigned to be killed on Day 13 or 15 of the estrous cycle (D13-NP and D15-NP) or pregnancy (D13-P and D15-P). Purification of luteal plasma membranes on discontinuous sucrose gradients yielded two fractions, designated F1 and F2, that exhibited the greatest enrichment of 5'-nucleotidase activity (five- and fourfold, respectively) over that of the homogenate. These fractions also yielded the lowest contamination by endoplasmic reticulum as represented by nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome C reductase activity and mitochondrial membranes as indicated by succinate dehydrogenase activity. Predominant phospholipids identified in membranes obtained from all groups were phosphatidylcholine (PC, 48.9 +/- 0.6% of total phospholipid), phosphatidylethanolamine (PE, 33.3 +/- 0.4%), sphingomyelin (SPH, 9.7 +/- 0.3%), phosphatidylserine (PS, 3.5 +/- 0.2%), and phosphatidylinositol (PI, 4.0 +/- 0.5%). No changes in microgram phospholipid/mg membrane protein were observed for any luteal phospholipid on D13 and 15 of the estrous cycle or pregnancy. No significant changes in the relative percentages of major fatty acids present in PC (palmitic [16:0], oleic [18:1]), PE (stearic [18:0], 18:1 and arachidonic [20:4]), or PS (18:0, 18:1, docosatetraenoic [22:4]), nor in the ratios of unsaturated (U) to saturated (S) fatty acids in these phospholipids were observed. Significant differences in unsaturated fatty acids of chain length greater than 20 carbons present in minor quantities in PC, PE, and PS were detected between NP and P ewes as well as between days within reproductive stage. The profile of major fatty acids present in PI revealed decreases in 18:0 and 20:4 in D15-NP and increases in 22:4 and docosapentaenoic acid (22:5) in luteal membranes of both D13- and D15-NP ewes relative to the levels of these fatty acids in PI of corresponding groups of pregnant ewes. There was a general trend for 20:4 levels of PC and PI in membranes of D15-NP ewes to be inversely related to those of D15-P ewes. Collectively, these changes were reflected by an increased U:S fatty acid ratio in luteal membrane PI during the estrous cycle. Specific binding of [125I] iodo-human chorionic gonadotropin to luteal plasma membranes from NP and P ewes on D13 and 15 (6/group) revealed similar affinities and concentrations of unoccupied luteinizing hormone (LH) receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Aspects of adipose-tissue metabolism in foetal lambs.

1. The mean volume of adipocytes, the rates of fatty acid and acylglycerol glycerol synthesis from various precursors (in vitro), the rates of oxidation of acetate and glucose (in vitro) and the activities of lipoprotein lipase and various lipogenic enzymes were determined for perirenal adipose tissue from foetal lambs during the last month of gestation. 2. The fall in the rate of growth of perirenal adipose tissue during the last month of gestation is associated with a diminished capacity for fatty acid synthesis and lipoprotein lipase activity, but no change in the rate of acylglycerol glycerol synthesis was observed. There was no fall in the activities of cytosolic acetyl-CoA synthetase or the NADP-linked dehydrogenases, suggesting that the decrease in the rate of fatty acid synthesis was due to an impairment at the level of acetyl-CoA carboxylase or fatty acid synthetase. 3. The rate of fatty acid synthesis from acetate was greater than that from glucose. The rate of fatty acid synthesis from glucose per adipocyte of foetal lambs was similar to that of young sheep. The characteristic metabolism of adipose tissue of the adult sheep is thus present in the foetus, despite the relatively large amounts of glucose in the foetal 'diet'.     

Carbohydrate metabolism in liver from foetal and neonatal sheep

1. During development of the sheep, the activities of UDP-glucose–α-glucan glucosyltransferase and UDP-glucose pyrophosphorylase and the glycogen content are highest in the liver of lambs 2 weeks old and considerably lower in liver from adult sheep. 2. The activity of hexokinase and the rate of incorporation of [¹⁴C]-glucose into glycogen are much lower in liver from postnatal sheep than in rat liver. 3. The activities of hexose diphosphatase and glucose 6-phosphatase and the rates of incorporation of [¹⁴C]pyruvate and [¹⁴C]propionate into glycogen increase from low levels in the liver of foetal sheep to maxima a few weeks after birth. The activities in the liver of adult sheep are slightly lower. 4. The incorporation rate of [¹⁴C]pyruvate into glucose has been measured in liver slices from rats, sheep and chick embryos at several ages of these animals. This pathway is active in liver from foetal sheep, embryonic chicks and postnatal rats or sheep, but is absent from the liver from foetal rats. 5. Fructose metabolism, as measured by the rates of incorporation of [¹⁴C]fructose into glycogen and glucose in liver slices and by assays of liver ketohexokinase, is barely detectable in the liver of foetal sheep and appears soon after birth. 6. During development of the sheep, the incorporation rate of [¹⁴C]galactose into glycogen in liver slices is highest in foetal sheep and decreases with increasing age of the animal. 7. These findings are discussed with reference to the changing pattern of carbohydrate metabolism during neonatal development of liver in the sheep.     

Characterization of porcine osteonectin extracted from foetal calvariae.

Osteonectin, extracted from foetal porcine calvariae with 0.5 M-EDTA, was purified to homogeneity by using gel filtration and polyanion anion-exchange fast protein liquid chromatography under dissociative conditions without the need of reducing agents. The purified protein migrated with an Mr of 40,300 on SDS/polyacrylamide gels and was similar to bovine osteonectin in both amino acid composition and in its ability to bind to hydroxyapatite in the presence of 4 M-guanidinium hydrochloride (GdmCl). However, unlike the bovine protein, porcine osteonectin did not bind selectively to hydroxyapatite when EDTA tissue extracts were used. In addition, purified porcine osteonectin did not show any apparent affinity for either native or denatured type I collagen, but did bind to serum albumin. Primary sequence analysis revealed an N-terminal alanine residue, with approximately one-half of the subsequent 35 residues identified as small hydrophobic amino acids and one-quarter as acidic amino acids. The only significant difference between the N-terminal sequences of the bovine and porcine proteins was the deletion of the tripeptide Val-Ala-Glu in porcine osteonectin. In contrast with bovine osteonectin, far-u.v.c.d. of porcine osteonectin revealed considerable secondary structure, of which 27% was alpha-helix and 39% was beta-sheet. Cleavage of the molecule with CNBr under non-reducing conditions generated five fragments, of which two major fragments (Mr 27,900 and 12,400) stained blue with Stains All, a reagent that stains sialic-acid-rich proteins/phosphate-containing proteins and/or Ca2+-binding proteins blue while staining other proteins pink. The 12,400-Mr fragment bound 45Ca2+ selectively, indicating a Ca2+-binding site in this part of the molecule. The 27,900-Mr fragment did not bind Ca2+, and since biosynthetic studies with 32PO4(3-) did not show phosphorylation of porcine osteonectin, this fragment is likely to be highly acidic. The incomplete cleavage of the molecule with CNBr and the ability of the molecule to regain its secondary structure after exposure to 7 M-urea are features consistent with the molecule having a compact structure that is stabilized by numerous disulphide bridges. The chemical and binding properties of porcine osteonectin are closely similar to the recently described 'culture shock', SPARC and BM-40 proteins, indicating that these are homologous proteins.     

The time-course of ACTH stimulation of cortisol synthesis by the immature ovine foetal adrenal gland.

The aim of this study was to establish the time-course of foetal adrenal gland activation by ACTH at a period of intra-uterine development during which adrenal function is minimal (100-120 days of gestation). Blood samples for cortisol analysis were collected at 6-h intervals during the 24 h ACTH (0.05, 0.5 and 5.0 micrograms/h) infusion and during the subsequent 24-h period following cessation of the infusion. Plasma cortisol concentrations were measured using a newly developed radioimmunoassay, whose sensitivity was found to be comparable to that of the validated double-isotope dilution derivative method. There was a significant increase in foetal plasma cortisol concentration, from 3.9 +/- 1 to 17.8 +/- 1.9 nmol/l, within 12 h of commencement of the 2 higher doses of ACTH. Values are mean +/- SEM; n = 5. Following termination of the infusion, cortisol levels fell significantly by the first 6 h, returning to basal levels thereafter. An increase in plasma ACTH from 4.6 +/- 0.6 to 8.4 +/- 1.0 pmol/l was sufficient to initiate a significant increase in cortisol production. The results suggest that the normal low values of cortisol at this period of gestation result from inadequate endogenous ACTH production at this stage.     

Homocysteine metabolism and the oxidative modification of proteins and lipids

Altered homocysteine metabolism is implicated as a pathogenic factor in atherogenesis, neoplasia, and aging. Hereditary enzymatic deficiencies and nutritional deficiencies of folate, pyridoxine, or cobalamin are associated with elevated blood homocysteine, accelerated atherosclerosis, and manifestations of aging. The failure of malignant cells to metabolize homocysteine thiolactone to sulfate is attributed to deficiency of thioretinaco, a complex containing cobalamin, homocysteine thiolactone, and retinoic acid. The sulfhydryl group of homocysteine is believed to act catalytically with ferric or cupric ions in a mixed function oxidation system to generate hydrogen peroxide, oxygen radicals, and homocysteinyl radicals. These reactive species may interact with the active site of enzyme protein to cause inactivation of catalytic activity. Homocysteine thiolactone is oxidized to sulfae by a process involving ascorbate, thioretinamide, and superoxide, under the control of thyroxine and growth hormone. Thioretinaco is believed to be the active site of adenosine triphosphate (ATP) binding in oxidative phosphorylation with the participation of oxygen, ascorbate, proton gradient, and electron transport. Depletion of thioretinaco from mitochondrial and microsomal membranes may be associated with increased formation and release of radical oxygen species within neoplastic and senescent cells. Specific proposals are made for investigating the importance of homocysteine metabolism in the oxidative modification of proteins and lipids.     

Acetate and arachidonate incorporations into lipids of ovine chorioallantoic tissue at day 24 of pregnancy

Incorporation of acetate and arachidonic acid into lipid classes was examined in chorioallantoic membranes obtained from sheep at Day 24 of pregnancy. Conceptus tissues were incubated in vitro with 5 mM acetate, 0.042 mM arachidonate, 0.45 muCi [1-14C]acetate, and 5.0 muCi [5,6,8,9,11,12,14,15-3H]arachidonate for 3 and 6 h. After incubation, tissue lipid fractions were extracted, isolated, and examined for radiolabel incorporations. Medium was extracted and analyzed for radiolabeled metabolites. Metabolic pathways commonly associated with fatty acid metabolism were confirmed to be present. Acetate was utilized for de novo synthesis of free cholesterol and free fatty acid. Fatty acids containing radiolabel from both acetate and arachidonate were mainly esterified in phospholipid and triglyceride, major lipid classes found in chorioallantoic tissue. Labeled metabolites of acetate were not sufficient for analytical measurement in medium. Metabolites of arachidonic acid from lipoxygenase and cyclooxygenase pathways were determined in medium after incubation. Results suggest that, within Day 24 ovine chorioallantoic tissue, utilization of exogenous arachidonate and de novo lipogenesis from acetate function in a parallel and anabolic mode appropriate for membrane expansion.     

Long-term effects of foetal undernutrition on intermediary metabolism in growing lambs

The objective of this study was to investigate the effects of foetal undernutrition on the metabolism in growing lambs. Seven-month-old lambs whose mothers had been fed either restrictively (RN; n = 14) or adequately (AN; n = 6) in late gestation were fasted for three days. One hour before fasting and after 48 h and 72 h fasting, changes in plasma concentrations of metabolites, i.e. glucose, nonesterified fatty acids (NEFA), 3-beta-hydroxybutyrate (BOHB) and urea as well as hormones, i.e. insulin, the insulin-like growth factor (IGF-I) and leptin, were determined. Blood glucose, NEFA, urea, insulin, IGF-I and leptin were not different between the two groups of lambs. Unexpectedly, at the end of the 3 d fasting, in spite of lower NEFA concentration (1.6 +/- 0.03 vs. 1.9 +/- 0.05 mM in Groups RN and AN, respectively), the BOHB concentration in RN lambs (0.94 +/- 0.02 mM) was significantly higher than that in AN lambs (0.78 +/- 0.04 mM). This higher rate of BOHB production might be interpreted as perturbations in ketone body metabolism potentially induced by undernutrition during foetal life. However, more investigations are necessary to clarify this interrelationship.     

Long-term effects of foetal undernutrition on intermediary metabolism in growing lambs

The objective of this study was to investigate the effects of foetal undernutrition on the metabolism in growing lambs. Seven-month-old lambs whose mothers had been fed either restrictively (RN; n = 14) or adequately (AN; n = 6) in late gestation were fasted for three days. One hour before fasting and after 48 h and 72 h fasting, changes in plasma concentrations of metabolites, i.e. glucose, non-esterified fatty acids (NEFA), 3-β-hydroxybutyrate (BOHB) and urea as well as hormones, i.e. insulin, the insulin-like growth factor (IGF-I) and leptin, were determined. Blood glucose, NEFA, urea, insulin, IGF-I and leptin were not different between the two groups of lambs. Unexpectedly, at the end of the 3 d fasting, in spite of lower NEFA concentration (1.6 ± 0.03 vs. 1.9 ± 0.05 mM in Groups RN and AN, respectively), the BOHB concentration in RN lambs (0.94 ± 0.02 mM) was significantly higher than that in AN lambs (0.78 ± 0.04 mM). This higher rate of BOHB production might be interpreted as perturbations in ketone body metabolism potentially induced by undernutrition during foetal life. However, more investigations are necessary to clarify this interrelationship.     

Characterization and evolution of ovine MHC class II DQB sequence polymorphism

The second exons of OLA-DQB genes from 13 merino sheep were sequenced following amplification by the polymerase chain reaction or isolation from a cDNA library. Ten distinct exon 2 sequences, coding for 10 novel amino acid sequences, were characterized in these sheep. The single-strand conformation polymorphism technique was shown to be capable of discriminating between all sequences. This brings the total number of different OLA-DQB exon 2 sequences (nucleotide and amino acid) which have been characterized to 12, and demonstrates that the OLA-DQB region is highly polymorphic with 29% of nucleotide and 46% of amino acid sites showing variation. Evidence is presented that the OLA-DQB sequences belong to at least two lineages of DQB genes. Some ovine DQB sequences are more like bovine DQB counterparts than other ovine DQB sequences suggesting that the artiodactyl DQB gene and allele lineages predate the separation of the ovine and bovine species 20 million years ago.     

Propionate metabolism and its regulation by fatty acids in ovine hepatocytes

Propionate metabolism was studied in ovine hepatocytes. The main products of metabolism were CO2, glucose, L-lactate and pyruvate. The fatty acids, butyrate and palmitate inhibited propionate oxidation; butyrate inhibited but palmitate slightly stimulated gluconeogenesis from propionate. Butyrate and palmitate also inhibited lactate and pyruvate production from both endogenous substrates and from propionate.