Endometrial glands are required for preimplantation conceptus elongation and survival

Endometrial glands secrete molecules hypothesized to support conceptus growth and development. In sheep, endometrial gland morphogenesis occurs postnatally and can be epigenetically ablated by neonatal progestin exposure. The resulting stable adult uterine gland knockout (UGKO) phenotype was used here to test the hypothesis that endometrial glands are required for successful pregnancy. Mature UGKO ewes were bred repeatedly to fertile rams, but no pregnancies were detected by ultrasound on Day 25. Day 7 blastocysts from normal superovulated ewes were then transferred synchronously into Day 7 control or UGKO ewes. Ultrasonography on Days 25-65 postmating indicated that pregnancy was established in control, but not in UGKO ewes. To examine early uterine-embryo interactions, four control and eight UGKO ewes were bred to fertile rams. On Day 14, their uteri were flushed. The uterus of each control ewe contained two filamentous conceptuses of normal length. Uteri from four UGKO ewes contained no conceptus. Uteri of three UGKO ewes contained a single severely growth-retarded tubular conceptus, whereas the remaining ewe contained a single filamentous conceptus. Histological analyses of these uteri revealed that endometrial gland density was directly related to conceptus survival and developmental state. Day 14 UGKO uteri that were devoid of endometrial glands did not support normal conceptus development and contained either no conceptuses or growth-retarded tubular conceptuses. The Day 14 UGKO uterus with moderate gland development contained a filamentous conceptus. Collectively, these results demonstrate that endometrial glands and, by inference, their secretions are required for periimplantation conceptus survival and development.     

Heat stress-induced alterations in the synthesis and secretion of proteins and prostaglandins by cultured bovine conceptuses and uterine endometrium

Effect of in vitro heat stress on protein and prostaglandin synthesis and secretion by bovine conceptuses and endometrium was examined. Conceptuses (n = 11) and endometrium (n = 10) obtained on Day 17 of pregnancy were cultured at thermoneutral (39 degrees C, 24 h) or heat stress (39 degrees C, 6 h; 43 degrees C, 18 h) temperatures in medium supplemented with L-[4,5-3H]leucine (100 microCi) and arachidonic acid (10 micrograms/ml). Radiolabeled protein secreted into culture medium increased with time in both groups. Heat stress reduced (p less than 0.001) incorporation of [3H]leucine into intracellular and secreted proteins by conceptuses but did not alter incorporation of [3H]leucine by endometrium. In particular, heat stress reduced by 72% the secretion of bovine trophoblast protein-1, the conceptus polypeptide believed to cause extension of luteal lifespan. Two-dimensional, sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that heat stress altered the array of proteins in endometrial and conceptus tissues, as evidenced by the induction of "heat-shock proteins." Endometrial secretion of prostaglandin F (p less than 0.001) and conceptus secretion of prostaglandin E2 (p less than 0.05) increased in response to heat stress. Sensitivity of bovine conceptuses and endometrium to heat stress in vitro suggests that infertility associated with maternal heat stress may be caused, partially by alterations in signals required for maintenance of the corpus luteum during early pregnancy.     

Prostaglandins regulate conceptus elongation and mediate effects of interferon tau on the ovine uterine endometrium

In ruminants, both the endometrium and the conceptus (embryo and associated extraembryonic membranes) trophectoderm synthesizes and secretes prostaglandins (PG) during early pregnancy. In mice and humans, PGs regulate endometrial function and conceptus implantation. In Study One, bred ewes received intrauterine infusions of vehicle as a control (CX) or meloxicam (MEL), a PG synthase (PTGS) inhibitor from Days 8-14 postmating, and the uterine lumen was flushed on Day 14 to recover conceptuses and assess their morphology. Elongating and filamentous conceptuses (12 cm to >14 cm in length) were recovered from all CX-treated ewes. In contrast, MEL-treated ewes contained mostly ovoid or tubular conceptuses. PTGS activity in the uterine endometrium and amounts of PGs were substantially lower in uterine flushings from MEL-treated ewes. Receptors for PGE2 and PGF2 alpha were present in both the conceptus and the endometrium, particularly the epithelia. In Study Two, cyclic ewes received intrauterine infusions of CX, MEL, recombinant ovine interferon tau (IFNT), or IFNT and MEL from Days 10-14 postestrus. Infusion of MEL decreased PGs in the uterine lumen and expression of a number of progesterone-induced endometrial genes, particularly IGFBP1 and HSD11B1. IFNT increased endometrial PTGS activity and the amount of PGs in the uterine lumen. Interestingly, IFNT stimulation of many genes (FGF2, ISG15, RSAD2, CST3, CTSL, GRP, LGALS15, IGFBP1, SLC2A1, SLC5A1, SLC7A2) was reduced by co-infusion with MEL. Thus, PGs are important regulators of conceptus elongation and mediators of endometrial responses to progesterone and IFNT in the ovine uterus.     

In vitro age-dependent incorporation of [1-14C]acetate into lipid subclasses in rat ventral prostate

The [1-14C]acetate incorporation into different lipid subclasses by the rat prostate gland was lineal between 20 and 80 mg of wet tissue. The in vitro [1-14C]acetate incorporation into lipid subclasses was a development-dependent process. The highest values of [1-14C]acetate incorporation into triacylglycerols, free cholesterol and esterified cholesterol were observed at puberty, but radioactivity incorporation into phospholipids was similar in both prepuberty and puberty, then decreasing in maturity. The relationship between triacylglycerols, free cholesterol and esterified cholesterol with respect to total lipids was about 12, 10 and 3.5%, respectively, values being maintained during the animal development. The in vitro [1-14C]acetate incorporation into lipid subclasses in castrated rats decreased considerably as compared with normal rats.     

Uptake of 13C-Tracer Arachidonate and γ-Linolenate by the Brain and Liver of the Suckling Rat Observed Using 13C-NMR

Arachidonic acid (AA; 20:4n-6) is one of the principal components of the phosphoglycerides in neural cell membranes. During the critical period of postnatal development in mammals, AA is supplied preformed, directly from the milk or derived from precursor fatty acids such as gamma-linolenic acid (GLA; 18:3n-6). In this study, 13C-NMR spectroscopy was applied to investigate the incorporation of [1-(13)C]AA and [3-(13)C]GLA into liver and brain lipids of 7-15-day-old rats. The main objective was to establish the importance of dietary GLA for tissue AA accretion relative to the contribution from preformed dietary AA. [1-(13)C]AA and [3-(13)C]GLA were injected into the stomach of 7-day-old rats as a mixture. 13C-NMR spectroscopy of lipid extracts revealed incorporation of [1-(13)C]AA and [5-(13)C]AA (the latter derived from metabolism of the injected [3-(13)C]GLA) into phosphoglycerides and triacylglycerols. Preformed AA was 10 (liver)-17 (brain) times more efficient in contributing to tissue AA than AA derived from precursor GLA. In separate experiments, NMR spectroscopy was used to assess uptake of [1-(13)C]AA directly in living rats and intact organs. Results showed that intact liver and brain contain an appreciable amount of NMR-detectable lipids. The in vivo/in vitro information obtained from organs provided details on the mobility and turnover of tissue lipids.     

Oestrogen production by the preimplantation donkey conceptus compared with that of the horse and the effect of between-species embryo transfer

Aromatase distribution in membranes of preimplantation horse and donkey conceptuses was compared by measuring the incorporation of [3H]androstenedione into oestrone and oestradiol-17 beta. In the donkey conceptus, aromatase activity was similar in all the tissues examined (yolk sac, chorionic girdle and allantochorion), whereas in the horse it was generally lower and showed the relationship chorionic girdle greater than yolk sac greater than allantochorion. A higher proportion of labelled precursor was incorporated into oestradiol-17 beta by extra-embryonic tissues of the donkey compared with those of the horse. In contrast to previous results, aromatase in the chorionic girdle did not decline progressively before its migration into the endometrium on Day 36 to form the endometrial cups. The chorionic girdle of a donkey conceptus carried in the uterus of a mare failed to invade the surrogate horse endometrium and aromatase activity was still high in this tissue at Day 42. Aromatase distribution in 2 transferred donkey-in-horse conceptuses resembled that of the fetal, rather than the maternal, genotype indicating a lack of effect of the maternal environment.     

Dynamics of uptake and channeling of arachidonic acid in hamster alveolar macrophages and the effect of bleomycin on arachidonyl distribution.

The uptake and distribution of [14C]-arachidonic acid (AA) by primary culture hamster alveolar macrophages (AM) were examined. The macrophages were incubated for 2 to 18 hrs in RPMI-1640 medium containing 0.1% BSA and [14C]-AA. The uptake of [14C]-AA by AM was rapid with 71% and 83% of exogenous [14C]-AA taken up after 2 and 4 hrs of incubation, respectively. Initially, the uptaken [14C]-AA was equally distributed between phospholipids (PL) and neutral lipids (NL). However, by 8 hrs, 86% and 14% of [14C]-AA was found in PL and NL, respectively. This distribution pattern remained constant through 18 hrs. Within the PL pool, most of the [14C]-AA was initially incorporated into phosphatidylcholine (PC). However, with time, as the percent of [14C]-AA incorporated in PC declined, the percent incorporated in phosphatidylethanolamine increased. The incorporation of [14C]-AA into sphingomyelin, phosphatidylinositol, and phosphatidylserine remained constant with time. Within the NL, most of the [14C)-AA was initially found incorporated into triacylglycerols (TG). After 4 hrs, the percent of [14C]-AA found in TG decreased markedly, while the percent found in cholesterol esters markedly increased. Incubation of AM prelabeled with [14C]-AA with bleomycin at 0.5, 5, 50 and 500 microM for 2 and 8 hrs failed to have any effect on the distribution of [14C]-AA in PL and NL pools.     

In vitro and in vivo synthesis of long-chain fatty acids from (1-14C) acetate in the renal papillae of rats.

1. The relationship between the rate of [1-14C] acetate incorporation into the fatty acids of renal papillary lipids and the acetate concentration in the medium has been measured. 2. [1-14C] acetate was incorporated mainly into fatty acids of phospholipids and triacylglycerols. Only a few per cent of the radioactivity was found in the free fatty acid fraction. 3. The major part of the [1-14C] acetate was found to be incorporated by a chain elongation of prevalent fatty acids. The major component of the poly-unsaturated fatty acids in triacylglycerols and the major product of fatty acid synthesis from [1-14C] acetate in vitro was demonstrated by mass spectrometry to be docosa-7,10,13,16-tetraenoic acid. 4. The radioactivity of docosa-7,10,13,16-tetraenoic acid accounted for 40% of total radioactivity in triacylglycerol fatty acids (lipid droplet fraction) and 20% of total radioactivity in membrane phospholipid fatty acids.     

Incorporation of [3H]Arachidonic and [14C]Eicosapentaenoic Acids into Glycerophospholipids and Their Metabolism via Lipoxygenases in Isolated Brain Cells from Rainbow Trout Oncorhaynchus mykaiss

The incorporation of [3H]arachidonate [( 3H]AA) and [14C]eicosapentaenoate [( 14C]EPA) into glycerophospholipids was studied in isolated brain cells from rainbow trout, a teleost fish whose lipids are rich in (n-3) polyunsaturated fatty acids (PUFAs). EPA was incorporated into total lipid to a greater extent than AA, but the incorporation of both PUFAs into total glycerophospholipids was almost identical. The incorporation of both AA and EPA was greatest into phosphatidylethanolamine (PE). However, when expressed per milligram of individual phosphoglycerides, both AA and EPA were preferentially incorporated into phosphatidylinositol (PI), the preference being significantly greater with AA. On the same basis, significantly more EPA than AA was incorporated into phosphatidylcholine (PC). When double-labelled cells were challenged with calcium ionophore A23187, the 3H and 14C released from the cells closely paralleled each other, peaking at 10 min after addition of ionophore. The 12-monohydroxylated derivative was the pre-dominant lipoxygenase product from both AA and EPA with a rank order of 12-hydroxyeicosatetraenoic acid (12-HETE) greater than leukotriene B4 (LTB4) greater than 5-HETE greater than 15-HETE for the AA products and 12-hydroxyeicosapentaenoic acid (12-HEPE) greater than 5-HEPE greater than LTB5 greater than 15 HEPE for EPA products. The 3H/14C (dpm/dpm) ratios in the glycerophospholipids, total released radioactivity, and the lipoxygenase products suggested that PC rather than PI was the likely source of eicosanoid precursors in trout brain cells.     

The transfer of fatty acids across the sheep placenta

Maternal and fetal plasma concentrations of free fatty acids, triacylglycerols and phospholipids and the profile of their fatty acids were measured in three catheterized and unanaesthetized sheep. Fetal concentrations of all three lipid fractions were low and did not correlate with maternal concentrations. There were no measurable umbilical venous-arterial differences. Linoleic acid concentrations were low in both mother and fetus. The fatty acid composition of fetal adipose tissue, liver, lung and cerebellum of five animals was analysed. Again linoleic acid levels were very low, but phospholipids contained 2-8% arachidonic acid. [14C] linoleic acid and [3H] palmitic acid were infused intravenously into three ewes. Only trace amounts of labelled fatty acids were found in fetal plasma and these were confined to the free fatty acids. 14C-label was incorporated into fetal tissue lipids, but most of this probably was due to fetal lipid synthesis from [14C] acetate or other water-soluble products of maternal [14C] linoleic acid catabolism. It is concluded that only trace amounts of fatty acids cross the sheep placenta. They are derived mainly from the maternal plasma free fatty acids and might just be sufficient to be the source of the small amounts of essential fatty acids found in the lamb fetus, but are insignificant in terms of energy supply or lipid storage.     

Steroid metabolism by endometrial and conceptus tissues during early pregnancy and pseudopregnancy in gilts

Endometrial and conceptus tissues were obtained on Days 10.5, 11, 12, 16 and 25 of pregnancy and Day 25 of pseudopregnancy of gilts and incubated for 6 h in Minimal Essential Medium (5 ml) containing 35 ng [3H]progesterone. Metabolism of [3H]progesterone to oestrone, oestradiol and oestriol was determined by gas and high-pressure liquid chromatography and successive recrystallizations with unlabelled standards. Conceptuses collected between Days 10.5 and 12 were spherical, tubular or filamentous and incubated with 500 mg endometrium and [3H]progesterone. Production of oestrone by spherical conceptuses was not detected, but was 44-47 pg/tubular conceptus and 21 pg/filamentous conceptus. A similar trend was observed for oestradiol. Conceptus tissues from Days 16 and 25 (chorion) were most active in producing oestrone (123 and 520 pg/mg tissue, respectively) and oestradiol (277 and 876 pg/mg tissue, respectively). Endometrial oestrogen production was less than that for conceptus tissue for oestrone and oestradiol on Days 16 and 25 of gestation. Coincubations of endometrium and conceptus tissues had lower oestrogen production than conceptus alone. Endometrium from Day 25 of pseudopregnancy metabolized [3H]progesterone to several non-polar metabolites, but no oestrogens were detected. An unidentified phenolic metabolite of [3H]progesterone was detected in higher quantities than either oestrone or oestradiol; 445 to 461 pg/conceptus at the tubular stage. These results indicate temporal changes in the conversion of [3H]progesterone to oestrogens by conceptus and endometrial tissue from pregnant gilts, but not endometrium from pseudopregnant gilts.     

Influence of the endometrium, protease inhibitors and freezing on antiviral activity of proteins secreted by pig conceptuses

In Exp. 1, only medium from cultures containing conceptus tissue had antiviral activity (P less than 0.05). Addition of Day-15 pregnant endometrium or Day-14 cyclic uterine flush proteins to cultures containing 200 mg conceptus tissue decreased antiviral activity (conceptus x endometrial protein interaction, P less than 0.06). Effects of endometrium (-54%) and uterine flush proteins (-40%) on antiviral activity of conceptus cultures did not differ from each other (P greater than 0.10). In Exp. 2, antiviral activity was only detected in cultures containing conceptus tissue (P less than 0.06). The amount of antiviral activity in cultures of Day-15 conceptus tissue was not influenced differently (P greater than 0.10) by culture in medium conditioned by endometrium from Day 10 or Day 12 of pregnancy. However, antiviral activity was undetectable in medium conditioned by endometrium from one of the Day-12 gilts. In Exp. 3, antiviral activity was present in medium from only 1 of 3 cultures from Day-12 gilts when assayed unfrozen. Antiviral activity was lower (P less than 0.01) in cultures of conceptuses from Day 12 than Day 14 of pregnancy; however, antiviral activity increased quadratically (P less than 0.05) when cultures contained 0, 0.01, 0.1 and 1.0 units/ml aprotinin, respectively. Freezing and thawing culture medium did not reduce (P greater than 0.10) antiviral activity compared to medium assayed unfrozen (1438 vs 1354 units/ml, respectively). These results suggest a regulatory influence of the endometrium on secretion of antiviral proteins by pig conceptuses in vitro.     

Insulin-like growth factor-I expression during early conceptus development in the pig

Porcine conceptuses (embryo and associated membranes) in utero undergo developmental morphological transformations coincident with structural and biochemical changes in the uterine endometrium during early gestation. To elucidate a possible role for insulin-like growth factor-I (IGF-I) in these events, porcine endometrial (Days 8, 10, 11, 12, 14, and 30) and conceptus (Days 12, 14, and 16) tissues were characterized for the presence of IGF-I peptide and mRNAs. The corresponding uterine luminal fluids (ULF) at these stages of pregnancy were also analyzed for immunoreactive IGF-I concentration. ULF IGF-I was lowest on Day 8, highest on Day 12, and declined by Day 14. In contrast, endometrial tissue IGF-I content remained constant during this period. Conceptus tissues contained less IGF-I than endometrial tissues; however, conceptus IGF-I values were maximum on Day 12 coincident with peak values for ULF IGF-I. Dot-blot hybridization analyses revealed temporal variation in steady-state levels of IGF-I mRNAs in endometrium. Highest levels of endometrial IGF-I mRNA were detected on Day 12 and were about 4-fold greater than on Day 30 of pregnancy. IGF-I mRNA expression in conceptus tissues on Days 12, 14, and 16 was the same and was significantly less than that in endometrium on Day 12. These results demonstrate the temporal variation of IGF-I mRNA abundance in uterine endometrium and of immunoreactive IGF-I in ULF and in conceptus tissues, with the developmental processes occurring in the conceptuses at early pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arachidonic acid incorporation in cardiomyocytes,endothelial cells and fibroblast-like cells isolated from adult rat heart

The incorporation of radiolabeled arachidonic acid (3[H]-AA) in normoxic cardiomyocytes (MC), cardiac endothelial cells (EC) and fibroblast-like cells (FL) isolated from adult rat heart was studied. Deposition of 3[H]-AA in the cellular lipid pool was assessed with biochemical and autoradiographic techniques. Extraction and subsequent analysis of lipids from the three different cell types revealed that MC contained significantly more triacylglycerols than EC and FL. The proportion of (unlabeled) AA was also higher in MC triacylglycerols than in EC and FL. The quantity of phospholipids did not differ among the three cell types studied. However, the content of (unlabeled) AA in the MC phospholipid pool was twice as high as in EC and FL. The amount of 3[H]-AA incorporated in the cellular lipid pool of MC, EC and FL depended on the concentration of AA in the incubation medium and the incubation time. In EC and FL incorporation of 3[H]-AA was highest in the cellular phospholipid pool (0.01 microM AA, 30 min incubation). With increased concentration of AA and longer incubation times, the cellular triacylglycerol pool became more important as site of 3[H]-AA incorporation. In MC, comparable amounts of 3[H]-AA were incorporated in the cellular triacylglycerol and phospholipid pools (0.01 and 1 microM AA). At higher AA concentrations (10 microM) the triacylglycerol pool was the preferred site of 3[H]-AA deposition. Autoradiographic analysis at the light microscopic level revealed that the extra-nuclear space was readily stained when the three cell types were incubated with 3[H]-AA. These findings indicate that all cellular lipid pools and membranes are most likely site of deposition of radiolabeled arachidonic acid.     

Microbial metabolism of amino alcohols. Biosynthetic utilization of ethanolamine for lipid synthesis by bacteria.

1. Ten bacteria utilizing [2-14C]ethanol-2-amine as the sole or major source of nitrogen for growth on glycerol + salts medium incorporated radioactivity into a variety of bacterial substances. A high proportion was commonly found in lipid fractions, particularly in the case of Erwinia carotovora. 2. Detailed studies of [14C]ethanolamine incorporation into lipids by five bacteria, including E. carotovora, showed that all detectable lipids were labelled. Even where phosphatidylethanolamine was the major lipid labelled, radioactivity was predominantly in the fatty acid rather than the base moiety. The labelled fatty acids were identified in each case. 3. The addition of acetate to growth media decreased the incorporation of radioactivity from ethanolamine into both fatty acid and phosphatidyl-base fragments of lipids from all the bacteria except Mycobacterium smegmatis. Experiments with [3H]ethanolamine and [14C]acetate confirmed that unlabelled acetate decreased the incorporation of both radioactive isotopes into lipids, except in the case of M. smegmatis. 4. Enzyme studies suggested one of two metabolic routes between ethanolamine and acetyl-CoA for each of four bacteria. A role for ethanolamine O-phosphate was not obligatory for the incorporation of [14C]ethanolamine into phospholipids, but correlated with CoA-independent aldehyde dehydrogenase activity.     

Biosynthesis of triacylglycerols in the intestines of rainbow trout (Salmo gairdnerii) fed marine zooplankton rich in wax esters

Intestinal preparations from rainbow trout fed a diet rich in wax esters incorporated [1(-14)C]hexadecanoic acid and [1(-14)C]hexadecanol into triacylglycerols at the same rate. The ratio of the number of H atoms from C1 of hexadecanol to the number of molecules of hexadecanol incorporated into triacylglycerols was 1.6 : 3.0. [U-14C]Glucose was incorporated much faster into the glycerol moiety of triacylglycerols than was [U-14C]aspartic acid. We conclude that the oxidation of absorbed fatty alcohol to fatty acid and its subsequent incorporation into triacylglycerols is closely linked with the reductive formation of triacylglycerol-glycerol from glucose. The ability of trout intestines to metabolise fatty alcohol to triacylglycerols was the same in fish fed wax esters as in those fed triacylglycerols.     

The formation of lipid-linked sugars as intermediates in glycoprotein synthesis in rabbit mammary gland

1. The incorporation of d-[1-(14)C]mannose, d-[2-(3)H]mannose and N-acetyl-d-[1-(14)C]-glucosamine into glycoproteins and lipid-linked intermediates of mammary explants obtained from lactating rabbits was studied. The amount of radioactivity incorporated into lipid-linked intermediates was very low compared with the incorporation into protein. Most of the radioactivity incorporated into the chloroform/methanol-soluble fraction was present as neutral lipid. Radioactivity from d-[2-(3)H]mannose was incorporated mainly into the fatty acid moiety, whereas radioactivity from d-[1-(14)C]mannose and N-acetyl-d-[1-(14)C]glucosamine was present in the glycerol moiety of triacylglycerol. 2. The labelled lipid-linked intermediate that was soluble in chloroform/methanol/water (10:10:3, by vol.) was partially characterized and was found to exhibit properties characteristic of an oligosaccharide linked to lipid via a pyrophosphate bridge. It migrated largely as a single zone of radioactivity on t.l.c. and was eluted from a column of DEAE-cellulose acetate as a single peak by 50mm-ammonium acetate. 3. The oligosaccharide moiety was released from the lipid by mild acid hydrolysis. The size of the oligosaccharide was estimated by paper chromatography to be 10 or 11 monosaccharide units. 4. d-[1-(14)C]Mannose was incorporated largely into glycopeptides with molecular weights in the range 40000-80000, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Label from N-acetyl-d-[1-(14)C]glucosamine was incorporated into a glycopeptide with an electrophoretic mobility identical with that of rabbit casein (mol.wt. 32000) as well as into glycopeptides of higher molecular weight. 5. Approx. 50% of the total radioactivity in the protein labelled from N-acetyl-d-[1-(14)C]glucosamine was present as galactosamine, a component of the carbohydrate portion of rabbit casein. No labelled galactosamine was present in the lipid-linked oligosaccharide labelled from N-acetyl-d-[1-(14)C]glucosamine. It thus appears that the lipid-linked oligosaccharide is not involved in the glycosylation of casein.     

Changes in ovine conceptus and endometrial function following asynchronous embryo transfer or administration of progesterone

The secretory protein profile from conceptuses collected from naturally mated ewes on Days 10, 12, 14, and 16 was characterized by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. The presence of the anti-luteolysin ovine trophoblast protein-1 (oTP-1) in culture medium from Day 10 conceptuses was confirmed by fluorography, Western blotting, and radioimmunoassay (RIA). On each of the days studied, oTP-1 was the dominant secretory protein, and was secreted in increasing quantities as pregnancy progressed. In a second experiment, Day 6 embryos were transferred to either Day 6 (SR) or Day 4 (AR) recipients. Three mated ewes (P) received daily injections of 50 mg progesterone on Days 4-9. Controls consisted of 2 groups of pregnant ewes (D8 and D10). Conceptuses and ipsilateral endometrium were collected 4 days following transfer of conceptuses to SR and AR ewes, on Day 10 in P and D10 ewes, and on Day 8 in D8 ewes. Conceptus volume was estimated upon recovery from the uterus. Tissues were cultured with 35S-methionine, and the medium was analyzed for total and trichloroacetic acid-precipitable radiolabeled proteins. Levels of specific endometrial secretory proteins were determined after protein separation by 2D-PAGE and estimation of the radioactivity associated with discrete radiolabeled proteins on fluorographs. The concentration of oTP-1 in conceptus culture medium was estimated by RIA. Thirty endometrial proteins were investigated. All 30 proteins were present in endometrial cultures from SR, AR, D10, and P ewes, but 13 proteins were absent from D8 ewes. Levels of three proteins were higher in AR compared to D8 (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro lipid metabolism in the rat pancreas. III. Effects of carbamylcholine and pancreozymin on the turnover of phosphatidylinositols, 1,2-diacylglycerols and phosphatidylcholines

1. The turnover of phosphatidylinositols and other glycerolipids was examined in rat pancreatic fragments incubated in the presence of carbamylcholine and pancreozymin used at a concentration inducing maximal alpha-amylase hypersecretion. 2. In stimulated tissue, [1-14C]acetate-labeled fatty acids were incorporated into phosphatidylinositols, 1,2-diacylglycerols, and phosphatidic acids in preference to phosphatidylcholines, phosphatidylethanolamines, triacylglycerols, monoacylglycerols, and free fatty acids. Variations in the percent distribution of 14C among fatty acids and in specific activity of individual fatty acids in each lipid class suggested that the secretagogues reduced selection of newly synthesized 1,2-diacylglycerols which occurred in the resting state before their incorporation into phosphatidylinositols. Secretagogues also promoted recycling of endogenous 1,2-diacylglycerols (produced from hydrolysis of unlabeled glycerolipids) for the biosynthesis of phosphatidylinositols. 3. Increased rate of incorporation of [1-14C]palmitate, [1-14C]linoleate, [1-14C]arachidonate and [1(3)(n)-3H]glycerol into phosphatidylinositols was detrimental to phosphatidylcholines. 4. The lipolytic effects of carbamylcholine and pancreozymin as illustrated by the release of 1,2-diacylglycerols and free fatty acids, were markedly inhibited in calcium-free medium enriched with 1 mM EGTA but increased turnover of phosphatidylinositols as determined from incorporation of radioactive precursors was only moderately affected.     

Differential incorporation of docosahexaenoic and arachidonic acids by the yolk sac membrane of the avian embryo

During avian development, lipoproteins derived from yolk lipid are assembled in the yolk sac membrane (YSM) for secretion into the embryonic circulation. To investigate how yolk polyunsaturated fatty acids, essential for the development of certain tissues, are distributed among the lipid classes of the lipoproteins, pieces of YSM were incubated in vitro with [14C]arachidonic and [14C]docosahexaenoic acids (DHA). There was a marked difference in the partitioning of these two precursors among the lipid classes of the tissue. Of the radioactivity incorporated into total lipid from [14C]-arachidonic acid during 1 h of incubation, 67.3% was esterified as phospholipid and 29.5% as triacylglycerol. In contrast, only 14.6% of the label incorporated from [14C]-DHA was esterified as phospholipid, whereas 73.2% was recovered in triacylglycerol. This pattern of differential partitioning was observed at all time points and across a 20-fold range of fatty acid concentrations. There was no evidence for conversion of the radioactive arachidonic and DHAs to other fatty acids prior to incorporation into tissue lipids. It is suggested that the selective incorporation of yolk-derived DHA into the triacylglycerol of secreted lipoproteins represents part of a mechanism for directing this polyunsaturate to particular embryonic tissues.