Immunohistochemical study on oxytocin-like substance in the human placenta

Our previous studies have demonstrated that a large quantity of oxytocin (OT)-like substance exists in human placental tissue. In the present study, the localization of the OT-like substance in the human placenta was investigated by the PAP (peroxidase and antiperoxidase complex) immunohistochemical method. The results demonstrated that the site of syncytiotrophoblast was positively stained by specific antiserum to OT, whereas the tissue was not significantly stained by normal rabbit serum (NRS). These results suggest that the OT-like substance might be localized in syncytiotrophoblast of the placental tissue.     

Nature of the pressor substance in rabbit placenta

1. The renin-like substance isolated from the placenta of the rabbit produces a prolonged increase of blood pressure in the nephrectomized rat. If incubated with renin substrate from ox serum, it forms a pressor substance that elevates blood pressure in exactly the same way as does angiotensin. 2. This angiotensin-like principle was concentrated by means of ion-exchange chromatography and compared with Val-5-angiotensins I and II in two paper-chromatography systems and in paper electrophoresis. 3. In all the three methods the unknown principle behaved like a mixture of the two reference compounds. 4. It is concluded that incubation of the renin-like substance of placental origin with substrate from ox serum gave a mixture of Val-5-angiotensins I and II. This is evidence that the enzyme isolated from the placenta is either closely related to, or identical with, renin.     

Formation of vasculo-syncytial membranes in the human placenta.

Vasculo-syncytial membranes are localised areas of the placental villous membrane where the thickness of the barrier separating the maternal and fetal circulations is reduced to as little as 1-2 microns. Consequently, they are believed to be important sites for diffusional exchange. The morphological appearances suggest that they are caused by the obtrusion of locally dilated segments of the fetal capillaries into the trophoblast layer. This study sought quantitative evidence for the hypothesis by performing stereological analyses on vasculo-syncytial membranes at the electron microscopic level. The results confirmed that a strong relationship existed between the thickness of the capillary endothelium and that of the overlying stromal and trophoblastic tissue at these sites (r = 0.47, P < 0.001), indicating that some asymmetrical stretching or remodelling of the capillary wall was involved. Comparisons were also made between the thickness of the trophoblastic, stromal and endothelial components of the villous membrane in villi obtained from the central and from the peripheral parts of placental lobules, where vasculo-syncytial membrane formation is accentuated. The mean thickness of each component was lowest in the samples from the peripheral region, although the differences only proved to be statistically significant for the stromal layer (P = 0.01). Both sets of data lend quantitative support to the hypothesis that vasculo-syncytial membrane formation is the result of obtrusion of locally dilated segments of the fetal capillaries. The way in which this may be linked to changes in the dynamics of the fetal circulation as gestation advances is discussed.     

Small GTP-binding proteins in the nuclei of human placenta.

Mitochondria and crude nuclei containing fractions from human placenta have been shown to contain proteins which bind [alpha(32)P]-GTP. Prior to this study the number of GTP-binding proteins in placental nuclei and their nucleotide specificity was not known. Also unknown was the identity of any of the GTP-binding proteins in mitochondria of human placenta. Nuclei and mitochondria were purified from human placental extracts by sedimentation. Proteins were separated by electrophoresis and transferred to nitrocellulose membranes. Overlay blot with [alpha(32)P]-GTP identified two nuclei proteins with approximate molecular weights of 24 and 27 kDa. Binding of [alpha(32)P]-GTP to the 27 and 24 kDa proteins was significantly displaced by guanine nucleotides but not by adenine, thymine or cytosine nucleotides or deoxy (d) GTP. Western blot with a specific antibody to Ran identified a band at 27 kDa in nuclei and in mitochondrial fractions. These data indicate that both nuclei and mitochondria contain 24 and 27 kDa GTP-binding proteins. The GTP-binding proteins in nuclei display binding specificity for guanine nucleotides and the hydroxylated carbon 2 on the ribose ring of GTP appears essential for binding. It will be important in future studies to determine the functions of these small GTP-binding proteins in the development and physiology of the placenta.     

Arginase from human full-term placenta.

Arginase was purified about 1800-fold from extracts of human full-term placenta; the enzyme appeared to be homogenous by disc electrophoresis and molecular-sieve chromatography. The mol. wt. determination by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis yielded a value of 70000 for the most pure and the partially purified enzyme. The human placenta arginase is a metalloenzyme with an optimum pH of 9.1. The Km for L-arginine is 27 mM. L-Ornithine and L-lysine show competitive inhibition with Ki values of 6.3 and 14 mM respectively.     

Purification of human placenta diamine oxidase.

Diamine oxidase (histaminase) is produced at very high levels by the decidual cells of the placenta. The presence of diamine oxidase has been demonstrated in human neutrophils. Purification of human placenta diamine oxidase was performed by four subfractionation steps and led to the isolation of one polypeptide whose molecular weight was 84,000, as assessed by SDS PAGE. Using polyclonal antibodies raised against the purified enzyme, we have demonstrated that the neutrophil diamine oxidase is immunochemically identical to the placental diamine oxidase. Development of immunological methods will be useful for detection and quantitation of diamine oxidase in neutrophils during the inflammation process.     

Purification of hyaluronidase from human placenta.

Hyaluronidase [EC 3.2.1.35] was isolated from human placenta and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-150. Its isoelectric point was at pH 5.2 and the molecular weight was 7 X 10(4) based on Sephadex G-200 gel filtration data. This enzyme was very stable at temperatures below 30 degree, but was almost completely inactivated at 60degree within 30 min. Its optimum pH was 3.9, a characteristic property of a lysosomal hyaluronidase. The Michaelis constant was 1.18 x 10(-1) mg per ml with purified hyaluronate. This enzyme depolymerized hyaluronate, chondroitin, chondroitin 4-sulfate and 6-sulfate, and the end product formed from hyaluronate was tetrasaccharide. Its biological diffusing activity was statistically significant on intracutaneous injection of 1.86 mU of the hyaluronidase into the back skine of a rabbit.     

Monoamine oxidase in rat placenta, human placenta, and cultured choriocarcinoma.

The activity of monoamine oxidase (MAO), an enzyme which metabolized catecholamines and indoleamines, was determined in rat placenta at various stages of gestation, in human term placenta, and in choriocarcinoma grown in culture. From Day 15 to Day 20 of gestation the specific activity (units/mg protein) of MAO in rat placenta increased at least 3-fold; from Day 20 to the time of parturition, it decreased about 50%. The specific activity of MAO in human placenta at term was about 50%. The specific activity of MAO in human placenta at term was about 8 times higher than that of rat placenta at term. No MAO activity was found in choriocarcinoma grown in culture.     

Urocortin in human placenta and maternal plasma.

Plasma immunoreactive (IR-) urocortin (Ucn) and corticotropin-releasing factor (CRF) levels in pregnant women were measured by their specific radioimmunoassays after extraction. Although plasma IR-CRF levels were increased in pregnant women as compared to men and non-pregnant women, there was no difference of plasma IR-Ucn levels among groups. Ucn mRNA was detected in cytotrophoblasts and syncytiotrophoblasts by in situ hybridization. A reverse-phase high-performance liquid chromatography (HPLC) showed the major peak of IR-Ucn in placenta and plasma that had similar chromatographic mobility to synthetic Ucn1-40. These data suggest that Ucn is produced and processed into the same form of synthetic Ucn in placenta, but not secreted into maternal blood.