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Chorion peroxidase-mediated NADH/O(2) oxidoreduction cooperated by chorion malate dehydrogenase-catalyzed NADH production: a feasible pathway leading to H(2)O(2) formation during chorion hardening in Aedes aegypti mosquitoes.

A specific chorion peroxidase is present in Aedes aegypti and this enzyme is responsible for catalyzing chorion protein cross-linking through dityrosine formation during chorion hardening. Peroxidase-mediated dityrosine cross-linking requires H(2)O(2), and this study discusses the possible involvement of the chorion peroxidase in H(2)O(2) formation by mediating NADH/O(2) oxidoreduction during chorion hardening in A. aegypti eggs. Our data show that mosquito chorion peroxidase is able to catalyze pH-dependent NADH oxidation, which is enhanced in the presence of Mn(2+). Molecular oxygen is the electron acceptor during peroxidase-catalyzed NADH oxidation, and reduction of O(2) leads to the production of H(2)O(2), demonstrated by the formation of dityrosine in a NADH/peroxidase reaction mixture following addition of tyrosine. An oxidoreductase capable of catalyzing malate/NAD(+) oxidoreduction is also present in the egg chorion of A. aegypti. The cooperative roles of chorion malate/NAD(+)oxidoreductase and chorion peroxidase on generating H(2)O(2) with NAD(+) and malate as initial substrates were demonstrated by the production of dityrosine after addition of tyrosine to a reaction mixture containing NAD(+) and malate in the presence of both malate dehydrogenase fractions and purified chorion peroxidase. Data suggest that chorion peroxidase-mediated NADH/O(2) oxidoreduction may contribute to the formation of the H(2)O(2) required for chorion protein cross-linking mediated by the same peroxidase, and that the chorion associated malate dehydrogenase may be responsible for the supply of NADH for the H(2)O(2) production.

Aedes↗

The formation of the functional chorion structure of Drosophila virilis involves inercalation of the "middle" and "late" major chorion proteins: a general model for chorion assembly in Drosophilidae.

We have shown by in vitro development of staged follicles in the presence of tritiated proline, as well as by immunoblot analysis, that the Dvs19 and DVS15 chorion proteins of Drosophila virilis are synthesized by the follicular epithelium and are incorporated in the chorion at the terminal stages of choriogenesis. Light microscopy immunolocalization has revealed that these two proteins represent structural components of the endochorion in all the specialized regions of the eggshell, while, contrary to their temporal pattern of secretion, electron microscopic immunolocalization has shown that these two proteins participate in endochorionic structures formed before the onset of their synthesis, thus suggesting an intercalation process during chorion formation. Furthermore, double immunolocalizations demonstrated that cosecreted proteins do not coexist in all secretory vesicles of the follicle cells, indicating that probably the final association of these chorionic components occurs extracellularly. On the basis of these data and those presented in the accompanying paper [Trougakos, I.P., and Margaritis, L.H. (1998) Immunolocalization of the temporally "early" secreted major structural chorion proteins, Dvs38 and Dvs36, in the eggshell layers and regions of Drosophila virilis, J. Struct. Biol. 123, 000-000.], a model for chorion assembly in Drosophilidae is proposed.

Animals↗

Gonadotropin-releasing hormone effects on placental hormones during gestation: I. Alpha-human chorionic gonadotropin, human chorionic gonadotropin and human chorionic somatomammotropin.

The release of alpha-human chorionic gonadotropin (alpha hCG), gonadotropin human chorionic gonadotropin (hCG) and human chorionic somatomammotropin (hCS) in vitro from placentas of different gestational ages was studied. In addition, the effect of gonadotropin-releasing hormone (GnRH) on these hormonal releases, as related to the gestational age of the placenta cultured and the dose of GnRH, was determined. The basal release of alpha hCG and hCG was greatest at 9-13 wk of gestation (1000-1500 ng/mg and 250-350 ng/mg, respectively). Lowest release rates were at term (28 ng/mg and 20 ng/mg, respectively). Hormonal release declined with extended culture, except from the cultures of 13- and 15-wk placentas, in which the initially high release continued throughout the 8 days of culture. The initial release of hCS was low at 6 wk, increased to maximum rates by 15 wk, and was similar to the initial rate of release at term. Gonadotropin-releasing hormone stimulated the release of alpha hCG and hCG most dramatically in cultures of 16-wk and 17-wk placentas, where as much as a 400- and 250-fold increase, respectively, on Day 6 of culture was observed (p less than 0.0001). In term placenta cultures after 6 days in vitro, a 20-fold stimulation of alpha hCG and a 10-fold increase of hCG was effected by GnRH (p less than 0.001). The largest responses of alpha hCG and hCG to GnRH were observed when estrogen levels were low. Dose-related responses were observed in some placentas, yet in some instances, maximal effects were attained with all doses utilized in these studies (0.2 to 50 micrograms/ml). These data demonstrate that human placentas of different gestational ages have varying hormonogenic capabilities in vitro. The data also establish that synthetic GnRH is capable of stimulating alpha hCG and hCG production, but the degree and pattern of response to GnRH stimulation are related to the gestational age of the placental tissue and its time in culture. The most responsive period to exogenous GnRH stimulation of alpha hCG and hCG release was on Days 5 and 6 of culture, when basal estrogen release was very low. These data support the hypothesis that hCG release might be controlled by a chorionic GnRH stimulation and suggest that local steroid levels may modulate the hCG response to GnRH stimulation.

Chorionic Gonadotropin↗

Is deglycosylated human chorionic gonadotropin an antagonist to human chorionic gonadotropin? Characterization of deglycosylated human chorionic gonadotropin action in two testicular interstitial cell fractions.

In order to determine the significance of carbohydrate residues of human chorionic gonadotropin (hCG) in receptor interaction and signal transduction leading to steroidogenesis, the effect of deglycosylated hCG (DG-hCG) was studied in vitro with two different hCG-responsive purified testicular interstitial cell fractions. Fraction I light cells, previously found to bind 125I-labeled hCG with high affinity without producing testosterone, also bound 125I-labeled DG-hCG with high affinity (Kd 7.2.10(-10) M) without stimulating testosterone production. Fraction IV heavier cells, which produced testosterone in response to hCG without detectable high-affinity hCG-binding sites, neither bound DG-hCG nor sufficiently produced cAMP and testosterone in response. With the addition of intact hCG, DG-hCG inhibited cAMP levels, although not sufficiently to inhibit testosterone production. This observation was contrary to previous studies in which DG-hCG was shown to be an antagonist to hCG action. We conclude that: (a) DG-hCG retains its binding activity in light cells and this high-affinity binding is unrelated to steroidogenesis; (b) DG-hCG does not bind to heavier cells with high affinity and loses its biological activity as result of deglycosylation; (c) DG-hCG actions in this study strengthen the concept of two different hCG-responsive cells in the rat interstitium which, if not separated, will yield misleading data supporting the coexistence of hCG high-affinity binding and biological response in the same cell; and (d) DG-hCG partially antagonizes the activation of adenylate cyclase but does not block testosterone production, thus questioning the usefulness of this analogue in antagonizing the action of native hCG in rat testis.

Animals↗

Chorionic gonadotropin receptors and immunoreactive chorionic gonadotropin in implantation of the rabbit blastocyst.

To determine the temporal relationship between immunoreactive chorionic gonadotropin, chorionic gonadotropin receptors, and implantation of the rabbit blastocyst, (1) immunoreactive chorionic gonadotropin in plasma, uterine flushings and blastocysts; (2) chorionic gonadotropin receptors on blastocysts (day 5 and 6) and embryonic and interembryonic segments of the uterus (day 7); and (3) chorionic gonadotropin receptors on the endometrium and myometrium (day 1 through 6) were measured. Binding of I125-labeled beta-subunit of human chorionic gonadotropin (hCG) by cell membranes from blastocysts increased significantly from 6.0 +/- 1.1 fmol/mg of protein (mean +/- SE) on day 5 (N = 6) to 16.1 +/- 1.3 fmol/mg of protein on day 6 (n = 6) (P less than 0.001). Immunoreactive chorionic gonadotropin levels in blastocyst fluid increased from 0.3 ng/ml of day 5 to 0.65 ng/ml on day 6. Specific binding of I125-labeled hCG was absent in endometrial and myometrial cell membranes before implantation (days 1 to 6) but was found in decidual cells from embryonic segments on day 7. Plasma immunoreactive chorionic gonadotropin or chorionic gonadotropin--like material increased from 6 ng/ml of day 1 to 52 ng/ml on day 7. Uterine flushings had chorionic gonadotropin levels of 0.4 ng/ml of day 2, which increased slowly to 1.0 ng/ml on day 7. Intrauterine instillation of hCG into nonpregnant uterine horns demonstrated transuterine absorption of hCG with plasma hCG levels showing a dose-related response. Our findings demonstrate that (1) immunoreactive chorionic gonadotropin or chorionic gonadotropin--like material is detectable in plasma, uterine flushings, and blastocyst fluid before implantation, (2) chorionic gonadotropin receptors are present on the blastocyst cells before implantation, and (3) the uterus can absorb chorionic gonadotropin from its lumen.

Absorption↗

Instruments for transcervical chorionic villus sampling for prenatal diagnosis.

BACKGROUND: The type of instrument used in chorion villus sampling could have a significant impact on the success rate of the procedure. An ability to manoeuvre the instrument within the uterine cavity without puncturing the gestational sac, and to see the tip of the instrument on ultrasound scanning are particularly important. OBJECTIVES: The objective of this review was to assess the effects of different instruments for transcervical chorionic villus sampling on perinatal outcome, quality and quantity of obtained tissue, technical difficulties during the procedure and maternal adverse effects. SEARCH STRATEGY: The Cochrane Pregnancy and Childbirth Group trials register was searched. Date of last search: October 1998. SELECTION CRITERIA: Randomised trials comparing different instruments for transcervical chorionic villus sampling. DATA COLLECTION AND ANALYSIS: Eligibility and trial quality were assessed by one reviewer. MAIN RESULTS: Three trials involving 268 women were included. None of the trials mentioned method of randomisation. An adequate sample was more likely to be obtained using small forceps than aspiration cannula (odds ratio 7.29, 95% confidence interval 3. 39 to 15.67). However small forceps were more difficult to insert than an aspiration cannula (odds ratio 0.35, 95% confidence interval 0.13 to 0.96). Use of the Portex cannula was more likely to result in an inadequate sample and a difficult or painful procedure when compared with either the silver or aluminium cannula respectively. REVIEWER'S CONCLUSIONS: Although there is some evidence to support the use of small forceps for transcervical chorionic villus sampling, it is not strong enough to support change in practice for clinicians who have become familiar with aspiration cannulas.

Chorionic Villi Sampling↗

The cellular localization of chorionic somatomammotrophin in ovine chorion.

Sections of Days 12 to 55 sheep chorion, embedded at low temperature in glycol methacrylate, were exposed to rabbit anti-ovine chorionic somatomammotrophin (oCS) to ascertain the distribution of oCS. This hormone was first detectable on Day 14, when the cytoplasm of each chorionic cell displayed a low level of fluorescence surrounding droplets which were shown to be lipid containing. At this time, all chorionic cells were uninucleate. By Day 28, binucleate chorionic cells had appeared but showed no binding of the oCS antiserum which was confined to a significant proportion of the uninucleate chorionic cells surrounding lipid droplets, as at Day 14. The same pattern of hormone distribution, although with reduced fluorescence intensity, was observed on Day 55; fluorescence indicative of antibody binding was seen only in some of the uninucleate chorionic cells. Hence, oCS was detected in chorionic tissue before the differentiation of binucleate cells (Day 14) and, at all stages, it was confined to the cytoplasm of specific uninucleate chorionic cells in close association with lipid droplets.

Animals↗

Medical Research Council European trial of chorion villus sampling. MRC working party on the evaluation pf chorion villus sampling.

First-trimester chorion villus sampling has the advantage over second-trimester amniocentesis of allowing earlier prenatal diagnosis of various genetic and cytogenetic disorders in the fetus (and therefore earlier termination in affected pregnancies) but the relative safety and diagnostic accuracy remain unclear. Between 1985 and 1989, 3248 women seeking prenatal diagnosis, principally because of their age, were recruited to an international, multicentre, randomised comparison of the safety and diagnostic accuracy of the two techniques--5% of women allocated chorion villus sampling and 8% of those allocated amniocentesis were not tested, usually because of spontaneous miscarriage. 6% and 2% were retested, in most because of sampling failure. The endpoint of a liveborn infant who survived was achieved by 86% of women allocated chorion villus sampling and 91% of those allocated amniocentesis; statistical analysis, after appropriate weighting for a centre's contribution, showed that the typical difference between the groups was 4.6% (95% confidence interval 1.6-7.5%; p less than 0.01). This difference reflected more spontaneous fetal deaths before 28 weeks' gestation (2.9% [0.6-5.3%]); more terminations of pregnancy for chromosomal anomalies (1.0% [0.0-2.1%]); and more neonatal deaths (0.3% [-0.1 to 0.7%]). The difference in neonatal deaths was due to a preponderance of very immature liveborn infants in the chorion villus sampling group, and this factor also explained that group's longer mean stay in hospital. More abnormal diagnoses followed chorion villus than amniotic fluid analyses (5.6% vs 3.9%). This difference was largely due to diagnoses of trisomy 18 and of (usually mosaic) abnormalities known to be confined to the placenta. 3 terminated pregnancies were false positives, 1 tested by chorion villus sampling and 2 by amniocentesis, and 2 other mosaic cases diagnosed by chorion villus sampling may have been false positives. There was 1 false-negative result in the chorion villus sampling group. The possibility of earlier exclusion or diagnosis of some fetal disorders afforded by first-trimester chorion villus sampling must be set against its clinical risks.

Abortion, Induced↗

Activity and expression of 15-hydroxyprostaglandin dehydrogenase in cultured chorionic trophoblast and villous trophoblast cells and in chorionic explants at term with and without spontaneous labor.

OBJECTIVE: The aim of this study was to investigate whether any changes occurred at term before and with labor in the 15-hydroxyprostaglandin dehydrogenase messenger ribonucleic acid level and in the 15-hydroxyprostaglandin dehydrogenase activity in cultured chorionic and villous trophoblast cells and in chorionic explants. STUDY DESIGN: Twelve placentas (labor group [vaginal delivery], n = 6; nonlabor group [elective cesarean delivery], n = 6) were collected. Chorionic trophoblast and villous trophoblast cells and chorionic disks were obtained, cultured, and incubated with 282-nmol/L prostaglandin F(2)(alpha). Medium was collected to measure the 13,14-dihydro-15-keto metabolite of prostaglandin F(2)(alpha), and the cells and disks were snap-frozen to quantify 15-hydroxyprostaglandin dehydrogenase messenger ribonucleic acid expression by Northern blot analysis. RESULTS: The formation of the 13,14-dihydro-15-keto metabolite of prostaglandin F(2)(alpha) was significantly lower in the labor group than in the nonlabor group for both sets of cultured cells and for chorionic explants. 15-Hydroxyprostaglandin dehydrogenase messenger ribonucleic acid expression was lower in the chorionic trophoblast cells and chorionic disks of the labor group than those of the nonlabor group. However, the 15-hydroxyprostaglandin dehydrogenase messenger ribonucleic acid level in the villous trophoblast cells did not differ between the labor and nonlabor groups. CONCLUSION: Prostaglandin metabolic activity in the chorion is reduced significantly at the time of labor.

Blotting, Northern↗

Developmental changes in the fine structure of the chorion laeve (smooth chorion) of the rhesus monkey placenta.

Developmental changes in the fine structure of the chorion laeve (smooth chorion) of the rhesus monkey were studied at two time periods during gestation: 1) Early (19-60 days of gestation), before the chorionic epithelium fuses with the parietal decidua, and 2) near term, when the fused chorioamnion has also fused with the parietal decidua. Early in gestation the chorionic epithelium consisted of columnar and cuboidal cells one or two layers thick. The apical border of the cells had microvilli and coated pits, and adjacent cells were joined by tight junctions and desmosomes. The chorionic epithelial cells during this early period contained numerous large vesicles and vacuoles of varying electron-density. The apical cytoplasm contained various small coated vesicles and tubules. Taken together these observations were interpreted as indicating a possible role for these cells in endocytosis or phagocytosis of substances from the uterine lumen; i.e., a potential role in histiotrophic nutrition during this early period. Late in gestation the trophoblastic cells were more irregular in shape. The cells contained abundant granular endoplasmic reticulum (ER) and mitochondria, and a well-developed Golgi complex, suggesting the cells were actively synthetic late in gestation. The numerous cytoplasmic vacuoles characteristic of the trophoblastic cells of early gestation were absent near term. Glycogen deposits and lipid droplets were moderately well-developed near term. Most of the cells were joined by desmosomes but wide intercellular spaces, unobstructed by any cell junctions, were frequently observed. This observation provides at least one explanation for the increase in permeability of the chorion laeve later in gestation. Cells of the parietal decidua associated with the chorion laeve were also examined. These cells generally had a well-developed granular ER and Golgi apparatus, and numerous mitochondria. Limited numbers of membrane-bounded secretory bodies, similar to those in human decidual cells, were also present.

Animals↗

Fetomaternal transfusion depends on amount of chorionic villi aspirated but not on method of chorionic villus sampling.

Transcervical and transabdominal chorionic villus sampling are believed, on the basis of indirect evidence, to result in fetomaternal transfusion. We sought to measure this phenomenon by devising a simple method that would allow us to identify variables that influence fetomaternal transfusion. We investigated patients undergoing transcervical-chorionic villus sampling (n = 15) and transabdominal-chorionic villus sampling (n = 15), restricting the sample to subjects who required only a single catheter passage or needle insertion to obtain villi. Maternal serum alpha-fetoprotein was measured before and after the procedure along with alpha-fetoprotein concentration of the transport medium into which the villi had been aspirated. We first confirmed that the change in maternal serum alpha-fetoprotein levels after chorionic villus sampling, an indirect measure of fetomaternal transfusion, was indeed correlated with the alpha-fetoprotein concentration of transport medium into which the villi were aspirated (p = 0.0350). Fetomaternal transfusion next proved to be correlated with the amount of villi obtained (p = 0.0279). However, when adjusted for the amount of villi obtained, no significant difference was observed between transcervical and transabdominal-chorionic villus sampling with respect to the change in maternal serum alpha-fetoprotein levels after chorionic villus sampling (p = 0.8512). These data suggest that the magnitude of fetomaternal transfusion depends on the amount of villi obtained but not on the chorionic villus sampling method used.

Adult↗

Lymphocyte-mediated lysis of sheep chorion: susceptibility of chorionic cells to third-party and maternal cytotoxic lymphocytes and presence of cells in the endometrium exhibiting cytotoxicity toward natural-killer cell targets.

In several species, the trophoblast is resistant to lysis by cytotoxic lymphocytes. Such resistance is believed to contribute to survival of the semiallogenic conceptus. We tested whether ovine chorionic cells are susceptible to lysis by specific and nonspecific cytotoxic lymphocytes in peripheral blood (PBL) and whether cytotoxic cells that can lyse target cells for natural-killer cells are present in the endometrium. Primary chorionic cells from pregnant ewes at Days 51-91 of gestation were labeled with 51Cr and incubated for 20 h at 50:1 and 100:1 ratios with PBL from the pregnant mother or from a third-party ewe. In the absence of interleukin-2 (IL-2), there was no killing of primary chorionic cells by third-party PBL even after infection of chorionic cells with bovine herpes virus-1. Incubation with IL-2-induced cytotoxic action in third-party PBL towards one of six primary chorionic cell preparations only. Primary chorionic cells from two of four placentae were lysed by maternal PBL. Luminal epithelial cells from cyclic ewes and from the pregnant and nonpregnant uterine horns of unilaterally-pregnant ewes were evaluated for the presence of cells capable of killing D17 target cells (a natural-killer cell target). Killing was observed but there was no difference in activity between physiological stages. In contrast, there was intense immunochemical localization of perforin in glandular and luminal endometrial epithelial cells in pregnant ewes, and less intense staining in nonpregnant animals. It is concluded that ovine chorionic cells are generally resistant to killing by natural-killer-like cells and lymphokine-activated killer cells. Generation of maternal cytotoxic lymphocytes against trophoblast can occur in some cases and may contribute to pregnancy loss.

Animals↗

Enzyme responsible for egg envelope (chorion) hardening in fish: purification and partial characterization of two transglutaminases associated with their substrate, unfertilized egg chorion, of the rainbow trout, Oncorhynchus mykiss.

From our previous studies, we have suggested that "egg envelope (chorion) hardening-enzyme" (first proposed by Zotin [J. Embriol. Exp. Morph. 6, 546-568 (1958)]) in the rainbow trout, Oncorhynchus mykiss, is transglutaminase (TGase), and that it coexists with its substrate, the unfertilized egg chorion, and forms epsilon-(gamma-glutamyl)lysine cross-links between the chorion proteins. In the present study, we extracted the TGase activity from the isolated chorions by homogenization with isotonic saline (143 mM NaCl-10 mM Tris.HCl, pH 7.2) and fractionated the extract by Toyopearl HW55S gel filtration with the isotonic saline containing 5 mM CaCl2 and 5 mM 2-mercaptoethanol (2-ME). One peak of TGase activity (P2) was obtained. When the eluates were dialyzed against 5 mM CaCl2/5 mM 2-ME/10 mM Tris.HCl (pH 7.2), another peak of the activity (P1) appeared. P1 TGase activity, which becomes apparent in a medium of low ionic strength, is involved in acceleration of the chorion hardening after egg activation in fresh water, so-called water activation. We purified the two TGases, P1 and P2, by SP-Sepharose, Q-Sepharose, and TSK-gel column chromatography. The molecular mass of the native form of P1 TGase was estimated as 103 kDa by Toyopearl HW55S gel filtration and as 100 kDa by the TSK-gel filtration. SDS-PAGE analysis showed that it consisted of heterogeneous 86- and 76-kDa proteins. However, these proteins closely resembled each other in amino acid composition, which was characterized by high content of Thr, Gly, and Pro residues as compared with P2 TGase. In contrast, the P2 TGase was isolated as a homogeneous 76-kDa protein and characterized by high content of Glx (Glu/Gln) and His residues. Neither of the chorion TGases of rainbow trout, P1 and P2, was similar to the liver-type TGase of red sea bream or the tissue-type TGase of chum salmon in amino acid composition. Examination of susceptibility to various inhibitors, reactivation by CaCl2, pH dependency, and activity of the polymerization of chorion proteins suggested that the P1 and P2 TGases were essentially similar to each other in enzymatic properties.

Animals↗

A randomized comparison of transcervical and transabdominal chorionic-villus sampling. The U.S. National Institute of Child Health and Human Development Chorionic-Villus Sampling and Amniocentesis Study Group.

BACKGROUND: Chorionic-villus sampling is done in early pregnancy to obtain fetal cells for the prenatal diagnosis of genetic and chromosomal defects. Transcervical chorionic-villus sampling has been shown to be safe and effective in national trials. Recently, an alternative transabdominal technique has been suggested as potentially easier and safer. METHODS: From April 1987 through September 1989, we prospectively compared transcervical and transabdominal chorionic-villus sampling in 3999 women with singleton pregnancies in whom the risk of a genetically abnormal fetus was increased. Women between 7 and 12 weeks of gestation underwent ultrasonographic evaluation of placental and uterine position. Those with active vaginal infections, active bleeding, or cervical polyps were excluded. If the obstetrician thought either sampling procedure was acceptable, the woman was asked to consent to random assignment to one of the two procedures. Both groups were followed to determine the outcome of pregnancy and the rate of spontaneous fetal loss after chorionic-villus sampling. RESULTS: Among the 3999 women who entered the study, sampling was attempted in 3873 (97 percent), 1944 of whom had been assigned to undergo transcervical sampling and 1929 to undergo transabdominal sampling. Of these 3873 women, sampling was eventually successful in 3863. Sampling was successful after a single insertion of the sampling instrument in 94 percent of the transabdominal procedures and 90 percent of the transcervical procedures. Among the women with cytogenetically normal pregnancies who had sampling because of maternal age, the rate of spontaneous fetal loss through 28 weeks of pregnancy was 2.5 percent in the transcervical-sampling group and 2.3 percent in the transabdominal-sampling group (difference, 0.26 percent; 95 percent confidence interval, -0.5 to 1.0 percent). CONCLUSIONS: Transabdominal and transcervical chorionic-villus sampling appear to be equally safe procedures for first-trimester diagnosis of fetal abnormalities.

Abdomen↗