Human placental 15-hydroxy-prostaglandin dehydrogenase activity is under fetal genetic control

Placental 15-hydroxy-prostaglandin dehydrogenase (PGDH type I) was measured in 33 placentae obtained from 1 trizygotic, 7 dizygotic and 8 monozygotic pregnancies. PGDH activity ranged from 0.33 to 4.62 nmol PGF_2α metabolized per mg placental protein per min, which was within the range observed in singleton pregnancies. Expressing PGDH activity per mg DNA, offered no advantage over expressing it per mg total protein. PGDH activity differed significantly between the placentae of 6 of the 9 genetically non-identical placental pairs. The placentae of genetically identical twins, on the other hand, showed no difference in PGDH activity between the pairs. The data indicate that the genetic constitution of the fetus determines placental PGDH activity. They also provide us with the first evidence that the variation in prostaglandin catabolized capacity of the human placenta is not entirely dictated by the maternal endocrine environment, but is under fetal control.     

Increase in placental 15-hydroxy-prostaglandin dehydrogenase in the first half of human pregnancy

NAD-dependent 15-hydroxy-prostaglandin dehydrogenase (PGDH) activity was measured in homogenates of 25 human placentae obtained between 7 and 17 weeks of gestation. PGDH activity, expressed in nanomoles PGF2 alpha metabolized per min, ranged from 0.2 to 5.4 nmoles per mg placental protein and from 1.5 to 80 nmoles per g wet weight. PGDH activity per mg protein and per g weight increased significantly in function of gestational age (p less than 0.001). Between 7-8 weeks' gestation and 15-16 weeks mean values increased tenfold from 0.4 to 3.0 nmoles per mg protein and from 2.7 to 36.6 nmoles per g wet weight. Per unit of weight these early placentae contained less PGDH activity than term controls, but this related mainly to their high water content. Per mg placental protein PGDH activities already equalled values found at term before the end of the first trimester. The data indicate that the development of terminal villi and the migration of trophoblast into the maternal spiral arteries is associated with a substantial increase in the placental capacity for prostaglandin metabolism.     

Increase in placental 15-hydroxy-prostaglandin dehydrogenase in the first half of human pregnancy

NAD-dependent 15-hydroxy-prostaglandin dehydrogenase (PGDH) activity was measured in homogenates of 25 human placentae obtained between 7 and 17 weeks of gestation. PGDH activity, expressed in nanomoles PGF_2α metabolized per min, ranged from 0.2 to 5.4 nmoles per mg placental protein and from 1.5 to 80 nmoles per g wet weight. PGDH activity per mg protein and per g weight increased significantly in function of gestational age (p<0.001). Between 7–8 weeks' gestation and 15–16 weeks mean values increased tenfold from 0.4 to 3.0 nmoles per mg protein and from 2.7 to 36.6 nmoles per g wet weight. Per unit of weight these early placentae contained less PGDH activity than term controls, but this related mainly to their high water content. Per mg placental protein PGDH activities already equalled values found at term before the end of the first trimester. The data indicate that the development of terminal villi and the migration of trophoblast into the maternal spiral arteries is associated with a substantial increase in the placental capacity for prostaglandin metabolism.     

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase: immunochemical characterization of the lung enzyme from pregnant rabbits

A polyclonal antibody was produced in guinea pig against the lung NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) purified from pregnant rabbits. Western blot analysis demonstrated that the protein identified by this antibody in the 105,000g supernatant fraction of lung tissue from pregnant rabbits had a molecular mass of 30 kDa and comigrated with the purified PGDH. The specific activity of the lung PGDH in pregnant rabbits (25- to 28-day gestations) was 36.7 nmol NADH formed/min/mg protein compared to 0.3 nmol NADH formed/min/mg protein in nonpregnant rabbits. Although the PGDH activity in the lung cytosol of nonpregnant rabbits was inhibited by the anti-lung PGDH antibody, the 30-kDa protein was not detected by Western blot analysis. An examination of this 30-kDa protein during the gestational period indicated that the protein was present after 10 days and the amount of the protein increased from Day 10 to Day 28. This increase in the immunochemically reactive protein correlated with the marked increase in PGDH specific activity between 10 and 28 days. An immunochemically reactive protein also was observed in the ovary of 25- to 28-day pregnant rabbits and the specific activity of the ovary PGDH was 19.3 nmol NADH formed/min/mg protein. Only trace levels of the PGDH activity were detected in the ovaries of nonpregnant rabbits. A 30-kDa protein was not detected by the anti-rabbit lung PGDH in brain, kidney, bladder, uterus, liver, and heart tissue of pregnant or nonpregnant rabbits. When rabbit or human placental cytosol was examined with the anti-rabbit lung PGDH only faint 30-kDa bands were observed by Western blot analysis. A monoclonal antibody prepared against human placental PGDH did not recognize the 30-kDa band in the pregnant rabbit lung. Localization studies indicated a marked increase in immunochemical staining in pulmonary epithelial cells of pregnant rabbits as compared to nonpregnant rabbits. Lung epithelial cells but not endothelial cells were identified as containing the PGDH.     

Ultrastructural study on human placentae from women subjected to assisted reproductive technology treatments

This study compared the ultrastructural differences of term placentae from human pregnancies resulting from assisted reproductive technology (ART) with term placentae from spontaneous human pregnancies. Term placentae were taken from women who had undergone an ART procedure (n = 8) and matched with term placentae from women who had had a spontaneous pregnancy (controls, n = 15). Using light microscopy (LM) and transmission-electron microscopy (TEM), terminal villi were evaluated with respect to the placental blood barrier, fetal capillaries, villous stroma, as well as cytotrophoblasts and syncytiotrophoblasts (ST) along with their substructures. No obvious differences were found between the ART-derived and control placentae when LM was used. With TEM, however, differences in the ultrastructural features were seen in the ART-derived placentae, specifically degenerative alterations of the terminal villi, mainly in ST, including a thicker placental barrier, decreased apical microvilli, and increased multiple vacuoles. The results demonstrate that some ultrastructural differences exist between ART-derived and control placentae with respect to the placental blood barrier, which may suggest maternofetal traffic downregulation following ART treatment. Further studies are required to understand the ultrastructural changes and their potential functional aspects in ART pregnancies.     

Eicosanoid production and transfer in the placenta of the diabetic pregnancy

The metabolism of arachidonic acid (AA) and the transfer of its metabolites was determined in in vitro perfused placental tissue from normal pregnancies and those complicated by maternal insulin-dependent diabetes mellitus (IDDM). 14C-labelled AA was recirculated in the fetal circulation for 60 min while 3H-AA was recirculated in the maternal circulation. Placental effluent was subjected to high performance liquid chromatography (HPLC) and analysis of dual-label scintillation counts. Placentae from IDDM pregnancies converted 3-6 times more radiolabelled AA to eicosanoids than did normal placentae. In addition, the transfer of eicosanoids into the opposing circulation was doubled in placentae from IDDM pregnancies compared to normal placentae. The predominant direction of eicosanoid transfer in both groups of placentae was in the fetal-to-maternal direction. The relative amounts of eicosanoids produced was also altered in placentae from IDDM pregnancies. Increased amounts of thromboxane (Tx) B2 and hydroxyeicosatetraenoic acids (HETEs) were present in both circulations of placentae from IDDM pregnancies. Levels of 6-keto prostaglandin F1a (6KPGF1a) were significantly reduced in both circulations in placentae from IDDM pregnancies. Thus, the ratio of TxA2 to PGI2 and the ratio of HETEs to PGI2 were both significantly increased in placentae from IDDM pregnancies. These results suggest an imbalance in eicosanoid production which may be relevant to abnormal placental structure and function in IDDM pregnancies.     

Increase in 15-hydroxyprostaglandin dehydrogenase activity in the ovine placentome at parturition and effect of oestrogen

Type 1 NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is the key enzyme for metabolism of active primary prostaglandins to inactive forms in gestational tissues. The present study examined the activity and immunolocalization of PGDH in the ovine placenta, fetal membranes and uterus over the latter half of pregnancy, and its potential regulation by oestradiol. Placenta, fetal membranes and myometrium were collected from sheep with known single insemination dates on days 70, 100 and 135 of gestation and in active labour demonstrated by electromyographic activity. In addition, chronically catheterized fetuses were infused with oestradiol (100 microgram kg(-1) per 24 h) (n = 5) or saline vehicle into the fetus from day 120 to day 125. PGDH activity measured in placental extracts remained constant from day 70 to day 135 of gestation, and then significantly (P < 0.05) increased by 300% in active labour. Immunoreactive PGDH was localized in the placentome at all stages and was present predominantly in the fetal component of the placentome in uninucleate, but not in binucleate, trophoblast cells. Similarly, in the fetal membranes PGDH immuno-reactivity was present in the uninucleate trophoblast but not in the binucleate cells of the chorion. PGDH immunostaining was also present in the endometrial luminal epithelium, in the smooth muscle of the myometrium, and the glandular epithelium of the cervix. Infusion of oestradiol into the fetal circulation from day 120 to day 125 of gestation had no effect on placental PGDH activity. Immunohistochemistry was used to localize oestrogen receptor alpha in intrauterine tissues to investigate further the failure of oestradiol to increase PGDH activity. Immunoreactive oestrogen receptor alpha was not present in the fetal component of the placenta, although it was expressed in adjacent maternal-derived cells. It is concluded that (1) PGDH activity increases in late gestation; (2) PGDH is expressed in uninucleate trophoblast cells in the ovine placenta and fetal membranes, and also in the maternal endometrial epithelium and stroma, myometrium and cervix; (3) oestrogen receptor alpha is not expressed in fetal cells in the placenta or fetal membranes; and (4) the increase in PGDH activity is not regulated by oestradiol administered to the fetus.     

Changes in ovarian NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase activity in pregnant and pseudopregnant rabbits.

The specific activity of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to increase in the ovaries of pregnant and pseudopregnant rabbits. The mean specific activity of cytosolic ovarian PGDH in 14- to 28-day pregnant rabbits was 24.3 +/- 8.1 nmol NADH formed/min/mg protein (n = 16) using PGE1 as substrate whereas in nonpregnant rabbits the specific activity was 1.5 +/- 0.8 nmol NADH formed/min/mg protein (n = 8). The reaction was dependent on NAD+; NADP+ did not support the reaction. In grouping the PGDH activities from pregnant rabbits into second (14-18 days) and third (2-28 days) trimester periods, no significant difference between values was found (26.1 +/- 8.9 vs 23.4 +/- 8.1 nmol NADH formed/min/mg protein, respectively). Western blot analysis of the ovarian cytosol using an antibody which was made to the purified lung PGDH of pregnant rabbits recognized an ovarian protein of identical molecular mass (30 kDa). Ovarian PGDH activities were also examined in rabbits treated with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to induce a state of superovulatory/pseudopregnancy and only on day 11 following hCG treatment was an increase in PGDH specific activity observed. On day 11, the specific activity was 14.8 +/- 4.3 nmol NADH formed/min/mg protein whereas values on days 10 and 12 were only 1.1 +/- 1.1 and 1.0 +/- 0.8, respectively. PGDH activities on days 3, 7 and 16 were also low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression pattern of tumor necrosis factor alpha in placentae of idiopathic fetal growth restriction

Tumor Necrosis Factor-Alpha (TNF-α) is one of the proinflammatory cytokines that provokes a variety of biological effects on the placenta. The increased placental exposure to TNF-α have induced impaired fetal development in experimental animals, but no data are available on the expression and localization of TNF-α in human placenta of idiopathic fetal growth restriction (FGR). The aim of this study was to characterize the immunohistochemical expression and localization of TNF-α in idiopathic FGR placentae in comparison with those of appropriate for gestational age (AGA) fetuses. 75 human placentae were collected between April, 2010 and March, 2011; 50 placentae were collected from pregnancies associated with idiopathic FGR and 25 placentae from AGA pregnancies. Histological and Immunohistochemical methodologies were employed in formalin fixed paraffin-embedded sections from the placentae of all subjects. Area percent of TNF-α immunostaining was evaluated using image analysis technique. In both AGA and idiopathic FGR placentae, cytoplasmic TNF-α was localized in the decidual and chorionic trophoblasts and in the endothelium of decidual and chorionic vessels. Trophoblast giant cells (TGC) in the decidua and chorionic villi of AGA specimens show deficient or negative TNF-α immunoexpression while those of idiopathic FGR show positive immunostaining. The mean area percent of TNF-α staining was greater in idiopathic FGR placentae (5.93 ± 0.69) compared to AGA ones (3.28 ± 0.41) (p = 0.001). Enhanced placental expression and specific cellular localization and of TNF-α are expected to contribute to impaired fetal development in idiopathic FGR and the TGCs are proposed to be an obvious source of this cytokine in such cases.     

Spermatogonial stem cell sensitivity to capsaicin: An in vitro study

Background

The placenta is an important site for iron metabolism in humans. It transfers iron from the mother to the fetus. One of the major iron transport proteins is transferrin, which is a blood plasma protein crucial for iron uptake. Its localization and expression may be one of the markers to distinguish placental dysfunction.

Methods

In the experimental study we used antibody preparation, mass spectrometric analysis, biochemical and immunocytochemical methods for characterization of transferrin expression on the human choriocarcinoma cell line JAR (JAR cells), placental lysates, and cryostat sections. Newly designed monoclonal antibody TRO-tf-01 to human transferrin was applied on human placentae from normal (n = 3) and abnormal (n = 9) pregnancies.

Results

Variations of transferrin expression were detected in villous syncytiotrophoblast, which is in direct contact with maternal blood. In placentae from normal pregnancies, the expression of transferrin in the syncytium was significantly lower (p < 0.001) when compared to placentae from abnormal ones (gestational diabetes, pregnancy induced hypertension, drug abuse).

Conclusion

These observations suggest that in the case of abnormal pregnancies, the fetus may require higher levels of transferrin in order to prevent iron depletion due to the stress from the placental dysfunction.     

3-Phosphoglycerate dehydrogenase from Corynebacterium glutamicum: the C-terminal domain is not essential for activity but is required for inhibition by L-serine

The serA gene of Corynebacterium glutamicum coding for 3-phosphoglycerate dehydrogenase (PGDH) was isolated and functionally characterized. It encodes a polypeptide of 530 aminoacyl residues (aa), which is substantially longer than the corresponding Escherichia coli polypeptide of 410 aa. The difference is largely due to an additional stretch of aa in the carboxy- (C)-terminal part of the polypeptide. Overexpression of serA in C. glutamicum results in a 16-fold increase in specific PGDH activity to 2.1 U/mg protein, with activity being inhibited by high concentrations of L-serine. A set of muteins that were progressively truncated at the C-terminal end was constructed. When overexpressed, mutein SerADelta197 showed a specific PGDH dehydrogenase activity of 1.3 U/mg protein, with the activity no longer being sensitive to L-serine. Gel filtration experiments showed that wild type PGDH is a homotetramer, whereas mutein SerADelta197 constitutes a dimer. Thus, the specific regulatory features of C. glutamicum PGDH are due to the C-terminal part of the polypeptide, which can be deleted with almost no effect on the catalytic activity of the enzyme.     

Bovine placentomes contain factors which decrease progesterone secretion

We examined the effects of 20% ammonium sulfate precipitates from cytosolic extracts of whole placental tissue collected between 100-150 days of gestation on progesterone secretion by bovine granulosa cells and dispersed bovine luteal cells. These extracts produced a dose-dependent inhibition (23-92%) of progesterone synthesis by bovine granulosa cells. However, no inhibitory activity could be demonstrated in similarly prepared extracts from term placentae. Inhibitory activity could be extracted from both maternal caruncles and fetal cotyledons. In the presence of 2 mg/ml of maternal caruncle extract, basal progesterone secretion was dramatically reduced (90%), as was steroidogenesis in the presence of bovine lutenizing hormone (bLH) and 8 bromocyclic (Br)-cAMP. Moreover, coincubation of dispersed luteal cells and dispersed fetal or maternal placental cells from 100- to 150-day placentae produced a significant (50%) reduction in progesterone content of the medium. The addition of 2 mg/ml of caruncle or fetal cotyledon extract from 100- to 150-day placentae also produced 100% and 50% inhibitions, respectively, of progesterone secretion by dispersed placental cells. Thus, the inhibitory factor appears to be produced by cells of both the maternal and fetal placenta. It is heat-stable and not extractable by ether. The inhibitory substance eluted was two distinct peaks from Sephadex G-100 columns, one with a molecular weight of about 60,000 daltons and the other about 30,000 daltons. Using isoelectric focusing, several peaks of inhibitory activity were obtained, one with a pI of 3-5, the others having pIs between 6 and 9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered downstream target gene expression of the placental Vitamin D receptor in human idiopathic fetal growth restriction

Fetal growth restriction (FGR) affects up to 5% of pregnancies and is associated with significant perinatal complications. Maternal deficiency of vitamin D, a secosteroid hormone, is common in FGR-affected pregnancies. We recently demonstrated that decreased expression of the vitamin D receptor (VDR) in idiopathic FGR placentae could impair trophoblast growth. As strict regulation of cell-cycle genes in trophoblast cells is critical for optimal feto-placental growth, we hypothesised that pathologically decreased placental VDR contributes to aberrant regulation of cell-cycle genes. The study aims were to (i) identify the downstream cell-cycle regulatory genes of VDR in trophoblast cells, and (ii) determine if expression was changed in cases of FGR. Targeted cell-cycle gene cDNA arrays were used to screen for downstream targets of VDR in VDR siRNA-transfected BeWo and HTR-8/SVneo trophoblast-derived cell lines, and in third trimester placentae from FGR and gestation-matched control pregnancies (n = 25 each). The six candidate genes identified were CDKN2A, CDKN2D, HDAC4, HDAC6, TGFB2 and TGFB3. TGFB3 was prioritised for further validation, as its expression is largely unknown in FGR. Significantly reduced mRNA and protein expression of TGFB3 was verified in FGR placentae and the BeWo and HTR-8/SVneo trophoblast cell lines, using real-time PCR and immunoblotting respectively. In summary, decreased placental VDR expression alters the expression of regulatory cell-cycle genes in FGR placentae. Aberrant regulation of cell-cycle genes in the placental trophoblast cells may constitute a mechanistic pathway by which decreased placental VDR reduces feto-placental growth.     

Distribution of mosaicism in human placentae

Traditional first trimester chorionic villus sampling (CVS) for prenatal diagnosis can be performed by cytogenetic analysis of cytotrophoblast or chorionic villous stroma. Approximately 2% of pregnancies studied by CVS show confined placental mosaicism (CPM) involving either cytotrophoblast, stroma or both. We present the results of a cytogenetic study of nine term placentae from pregnancies with prenatally diagnosed CPM. The aneuploid cell lines involved trisomies for chromosomes 7,9,16, and X. The cytotrophoblast and villous stroma from multiple biopsies of these placentae were examined using a combination of interphase and metaphase cytogenetic analysis. CPM was detected in all nine of the term placentae and both tissue-specific and site-specific patterns of mosaicism could be discerned. These results indicate that the analysis of villous stroma and cytotrophoblast from multiple placental biopsies is necessary to improve our understanding of the evolution of CPM during pregnancy and its effect on the fetus. Received: 1 May 1995 / Revised: 11 August 1995     

Molecular and biochemical characterization of D-phosphoglycerate dehydrogenase from Entamoeba histolytica. A unique enteric protozoan parasite that possesses both phosphorylated and nonphosphorylated serine metabolic pathways.

A putative phosphoglycerate dehydrogenase (PGDH), which catalyzes the oxidation of d-phosphoglycerate to 3-phosphohydroxypyruvate in the so-called phosphorylated serine metabolic pathway, from the enteric protozoan parasite Entamoeba histolytica was characterized. The E. histolytica PGDH gene (EhPGDH) encodes a protein of 299 amino acids with a calculated molecular mass of 33.5 kDa and an isoelectric point of 8.11. EhPGDH showed high homology to PGDH from bacteroides and another enteric protozoan ciliate, Entodinium caudatum. EhPGDH lacks both the carboxyl-terminal serine binding domain and the 13-14 amino acid regions containing the conserved Trp139 (of Escherichia coli PGDH) in the nucleotide binding domain shown to be crucial for tetramerization, which are present in other organisms including higher eukaryotes. EhPGDH catalyzed reduction of phosphohydroxypyruvate to phosphoglycerate utilizing NADH and, less efficiently, NADPH; EhPGDH did not utilize 2-oxoglutarate. Kinetic parameters of EhPGDH were similar to those of mammalian PGDH, for example the preference of NADH cofactor, substrate specificities and salt-reversible substrate inhibition. In contrast to PGDH from bacteria, plants and mammals, the EhPGDH protein is present as a homodimer as demonstrated by gel filtration chromatography. The E. histolytica lysate contained PGDH activity of 26 nmol NADH utilized per min per mg of lysate protein in the reverse direction, which consisted 0.2-0.4% of a total soluble protein. Altogether, this parasite represents a unique unicellular protist that possesses both phosphorylated and nonphosphorylated serine metabolic pathways, reinforcing the biological importance of serine metabolism in this organism. Amino acid sequence comparison and phylogenetic analysis of various PGDH sequences showed that E. histolytica forms a highly supported monophyletic group with another enteric protozoa, cilliate E. caudatum, and bacteroides.     

Prostaglandin dehydrogenase mRNA in baboon intrauterine tissues in late gestation and spontaneous labor

The present study was designed to characterize prostaglandin dehydrogenase (PGDH) mRNA expression in critical intrauterine tissues of pregnant baboons in late gestation and at spontaneous labor. In addition, we determined regulatory effects of betamethasone in vivo on chorionic and placental PGDH mRNA expression. PGDH mRNA was present in chorion, decidua, lower uterine segment, fundal myometrium, and cervix in late gestation but undetectable in amnion. PGDH mRNA significantly decreased in decidua and cervix during late gestation and in chorion and fundus during spontaneous labor. PGDH mRNA in lower uterine segment, decidua, cervix, and placenta was unchanged during spontaneous labor from late gestation levels. Betamethasone had no effect on chorionic and placental PGDH mRNA expression. In summary, our data suggest that PGDH mRNA expression is tightly controlled in gestation- and tissue-specific manners. Decreased chorionic and fundal PGDH abundance during labor and decreased decidua and cervical PGDH mRNA in late gestation allow local uterine prostaglandin accumulation and assist prostaglandin transfer to myometrium. Local differences in PGDH function may regulate tissue- and region-specific requirements for prostaglandins to promote and complete labor.     

Effects of iron,zinc, calcium,and vitamins on the activity and contents of human placental copper/zinc and manganese superoxide dismutases

One hundred seventy-nine pregnant women, ages 15–45 yr, were divided into three groups. Group A was orally given one spansule per day containing 150 mg dried ferrous sulfate, 61.8 mg zinc sulfate, and 500 μg folic acid, starting from the first 4 wk of pregnancy and ending at the day of delivery. Similarly, group B was given one tablet containing 625 mg calcium carbonate, 1000 mg vitamin C, 300 IU Vitamin D, 1350 mg citric acid, and 15 mg Vitamin B6. Group C was without any supplements and served as a control. Mothers who received iron/zinc supplements (group A) during pregnancy had significantly higher copper/zinc superoxide dismutase activity in their placentae than calcium/vitamin-supplemented mothers (group B) or unsupplemented mothers (group C). The enzyme activity increased with age of the mothers from 15 to 40 yr, then decreased after in both supplemented groups, whereas this increase and decrease occurred at early age in the unsupplemented group. Immunochemical quantitation of the enzyme contents showed no significant difference between the supplemented and unsupplemented groups, suggesting that the observed increase in the enzyme activity might arise from posttranslational processing of the enzyme. The placental manganese superoxide dismutase activity and contents, however, were similar in the supplemented groups, whereas they were slightly higher in the unsupplemented group; the overall superoxide dismutase-like activities in the placentae were the highest in iron-zinc supplemented group and the lowest in the unsupplemented group.     

Effects of placenta and maternal serum on prolactin secretion in vitro

The effects of the placenta and maternal sera on the secretion of prolactin (Prl) were examined in vitro. Placentae were obtained on each of Days 8-11 of pregnancy and extracted in 2.0% butanol-saline. To determine if these extracts could inhibit Prl secretion in vitro, dispersed anterior pituitary cells were incubated with placental extracts containing 1.0 placental equivalent obtained on each of Days 8-11 of pregnancy. Prl secretion was not affected by extracts of placentae obtained on Day 8 but was significantly inhibited by placental extracts obtained on Days 9-11 of pregnancy. In fact, progressively more mature placentae induced greater degrees of Prl inhibition. Extracts of placentae that were obtained on each of Days 8-11 of pregnancy, normalized on the basis of protein and tested for a 24-h period in the dispersed pituitary bioassay, caused the same degree of inhibition over Prl release. Additionally, placental protein from any given day (Days 8-11) of pregnancy induced a highly significant dose-dependent inhibition over Prl secretion. Equivalent amounts of a nonspecific protein, bovine serum albumin, had no effect. These findings indicate that the placenta does indeed contain a Prl inhibitory factor whose specific activity remains relatively constant between Days 8 and 11 of pregnancy. To determine if the inhibitory activity is humoral, maternal sera collected on each of Days 8-11 of pregnancy were placed in culture with dispersed pituitary cells at a concentration of 15.0%. Concomitant with gestational maturity, there was a progressively greater inhibition of Prl release. These findings indicate that the placenta may secrete a substance into the blood which suppresses Prl release directly at the level of the pituitary gland.     

Isolation and properties of lung 15-hydroxyprostaglandin dehydrogenase from pregnant rabbits

A NAD-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was purified to a specific activity of over 25,000 nmol NADH formed/min/mg protein with 50 microM prostaglandin E1 as substrate from the lungs of 28-day-old pregnant rabbits. This represented a 2600-fold purification of the enzyme with a recovery of 6% of the starting enzyme activity. The lungs of pregnant rabbits were used because a 42- to 55-fold induction of the PGDH activity was observed after 20 days of gestation. The enzyme was purified by CM-cellulose, DEAE-cellulose, Sephadex G-75, octylamino-agarose, and hydroxylapatite chromatography. The enzyme could not be purified by affinity chromatography using NAD- or blue dextran-bound resins. The purified enzyme was specific for NAD and had a subunit molecular weight of 29,000. The optimal pH range for the oxidation of prostaglandin E1 was between 10.0 and 10.4 using 3-(cyclohexylamino)propanesulfonic acid as the buffer. The Km and Vmax values for prostaglandin E1 were 33 microM and 40,260 nmol/min/mg protein, respectively, while the Km and Vmax values for prostaglandin E2 were 59 microM and 43,319 nmol/min/mg protein, respectively. The Km for prostaglandin F2 alpha was four times the value for prostaglandin E1. The PGDH activity was inhibited by p-chloromercuriphenylsulfonic acid but the enzymatic activity was restored by the addition of dithiothreitol. n-Ethylmaleimide also produced a rapid decline in enzymatic activity but when NAD was included in the incubation system, no inhibition was observed.     

Placental tissue metabolome analysis by GC-MS: Oven-drying is a viable sample preparation method

Abstract

Analysis of the human placenta metabolome has great potential to advance the understanding of complicated pregnancies and deleterious fetal outcomes in remote populations, but samples preparation can present unique challenges. Herein, we introduce oven-drying as a simple and widely available method of sample preparation that will facilitate investigations of the placental metabolome from remote and under-studied populations. Placentae from complicated and uncomplicated pregnancies were prepared in three ways (oven-dried at 60?°C, fresh, lyophilized) for metabolome analysis via gas chromatography-mass spectrometry (GC-MS). Multiple computer models (e.g. PLS-DA, ANN) were employed to classify and determine if there was a difference in placentae metabolome and a group of metabolites with high variable importance in projection scores across the three preparations and by complicated vs. control groups. The analyses used herein were shown to be thorough and sensitive. Indeed, significant differences were detected in metabolomes of complicated vs. uncomplicated pregnancies; however, there were no statistical differences in the metabolome of placentae prepared by oven-drying vs. lyophilization vs. fresh placentae. Oven-drying is a viable sample preparation method for placentae intended for use in metabolite analysis via GC-MS. These results open many possibilities for researching metabolome patterns associated with fetal outcomes in remote and resource-poor communities worldwide.