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PubMed1998

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↗

PubMed2006

The dual role of chorion peroxidase in Bactrocera oleae chorion assembly.

In the present study, we reveal for the first time that the Bactrocera oleae chorion peroxidase (bPxd) participates essentially in B. oleae chorion formation and clearly represents the homologous member of Drosophila melanogaster chorion peroxidase (Pxd). Comparative sequence analysis disclosed that the bPxd cDNA semi-central region, which encodes for the putative catalytic domain of the enzyme, exhibits great homology (98%) with its Pxd counterpart. Thus, it is very likely that bPxd is highly responsible for the chorion hardening process, through protein cross-linking mediated by the formation of di- and tri-tyrosine bonds. Distinct molecular weight bPxd RNA transcripts were detected in Northern blotting analysis of total RNA extracts of adult flies (2.9 and 1.7 kb) and ovaries (2.2 kb). The ovarian-specific bPxd RNA transcript is selectively expressed in the follicle cell layer during the late stages of oogenesis 12-14, as revealed by in situ hybridization. Moreover, reverse transcription reactions confirmed the stage-specific developmental regulation of the bPxd gene, which is maximally expressed during stage 13. Western blotting with the rabbit anti-rAePO polyclonal antibody revealed three immunoreactive bands of 76, 66 and 54 kDa in crude protein extracts from adult flies, while in larva and purified chorion preparations, a unique 54 kDa band was clearly detected. Immunolocalization experiments revealed that bPxd peroxidase constitutes an essential structural chorionic component, being abundantly localized in all the successive chorionic layers and vitelline membrane as well.

Animals↗

PubMed1992

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↗

PubMed1994

Enzyme-cytochemistry of human chorion laeve at term: enzyme localization on the chorionic trophoblast.

Is the chorion laeve merely a remnant of the chorion frondosum in placental development? Or is it metabolically active, having something to do with maternofetal interactions? In order to answer these questions at least in part, we determined the ultracytochemical localizations of some important enzymes such as nonspecific phosphatase (alkaline phosphatase), specific phosphatase (Ca(++)-ATPase and 5'-nucleotidase) and adenylate cyclase in the human chorion laeve at term. Strong activities of these enzymes were localized by ultracytochemistry on the plasma membrane of the trophoblast in the chorion laeve. These enzyme activities were confirmed by a series of cytochemical-control experiments, i.e., substrate-free control, heat-stability control, and inhibition control by inhibitors of alkaline phosphatase. These observations indicate that the chorionic trophoblast is probably metabolically active and that it might play an important role in the physiology of the fetal membrane.

5'-Nucleotidase↗

PubMed1994

Enzyme-cytochemistry of human chorion laeve at term: enzyme localization on the chorionic trophoblast.

Alkaline Phosphatase↗

PubMed1987

Immunocytochemistry of human chorionic gonadotropin in human chorionic villi.

The immunocytochemical localization of human chorionic gonadotropin was investigated in chorionic villi from the seventh to twelfth week of gestation. By the light microscopic peroxidase-antiperoxidase technique, positive reactions of human chorionic gonadotropin were found exclusively in the syncytiotrophoblast. Immunoelectron microscopy by means of the protein A-gold technique reveals localization of the immunoreactive gold particles in two kinds of membrane-bound granular inclusions in this cell; one type is granules of 200 to 300 nm in diameter with moderate electron density and the other is large electron-dense bodies of 500 to 1000 nm. The former seems to be Golgi-derived secretory granules that play a role in the release of human chorionic gonadotropin from the syncytiotrophoblast. Although the origin of the latter is still uncertain, a certain amount of this hormone might be stored or treated by lysosomal digestion in the large bodies during these stages.

Antigen-Antibody Reactions↗

PubMed1991

Quantitation of whole molecule human chorionic gonadotropin and free beta-human chorionic gonadotropin subunit in the Abbott IMx analyser.

An assay for the quantitation of the serum levels of free and whole molecule-associated beta-subunit of human chorionic gonadotropin on the Abbott IMx analyzer is described. The assay detects human beta-chorionic gonadotropin with a sensitivity of approximately 0.5 IU/l while showing no cross-reactivity with follitropin (1000 IU/l) or thyrotropin (2.0 IU/l), and 0.02% cross-reactivity with lutropin (1000 IU/l). Haemoglobin (7.50 milligrams), bilirubin (0.50 milligrams), and triacylglycerols (10.6 milligrams) did not interfere with the assay. Pooled within-run, between-run, and total assay CVs were less than or equal to 5.2%, and less than or equal to 2.7%, and less than or equal to 6.3%, respectively. Values obtained with this assay correlated well (r = 0.98, n = 228) with those values obtained using the Hybritech TandemR-R HCG (Total Beta-HCG) IRMA. The normal range of the assay was found to be less than or equal to 5 IU/l (n = 311). The assay protocol provides results for up to 23 serum samples in approximately 47.5 minutes with the ability to report the human beta-chorionic gonadotropin concentrations of 5 specimens in approximately 15.5 minutes. We conclude that this is an acceptable assay for monitoring human beta-chorionic gonadotropin levels associated with normal pregnancy, reproductive pathology, and reproductive technology such as in vitro fertilization or embryo transfer.

Chorionic Gonadotropin↗

PubMed1987

Amplification of the X-linked Drosophila chorion gene cluster requires a region upstream from the s38 chorion gene.

Genomic sequences controlling follicle cell-specific amplification of the X-linked Drosophila chorion gene cluster were mapped by P element-mediated transformation. Several DNA fragments containing the s38 gene and flanking sequences induced tissue-specific amplification, although replication levels were subject to position effects. Deletion analysis identified a 467-bp region upstream from the s38 transcription start site that contained sequences essential in cis for amplification. The essential region shared 32 bp of imperfect sequence homology with a previously identified region necessary for third chromosome chorion gene cluster amplification. This homologous segment contained a repetitive motif consisting of perfect and imperfect AATAC repeats; it was localized near the boundary of the essential domain since most, but not all, the repeats could be deleted without eliminating transposon-induced amplification. The repetitive region was not required for developmentally regulated s38 transcription, therefore our results identified at least one element required for amplification but not for chorion gene transcription. The homologous repetitive sequences within the amplification-essential regions may constitute part of the replication origins used to differentially replicate the two chorion domains during oogenesis.

Animals↗

PubMed1999

A monoclonal antibody against chorion proteins of the sea bass Dicentrarchus labrax (Linnaeus, 1758): studies of chorion precursors and applicability in immunoassays.

The monoclonal antibody DLE7 was obtained against 44- to 50-kDa polypeptides solubilized from the vitelline envelope of the Mediterranean sea bass Dicentrarchus labrax. In Western blot analysis of chorion lysates it recognized cross-reactive bands at 44 kDa, 48 kDa, and 110 kDa. Previous affinity blotting with concanavalin-A showed that most of solubilized bands were glycosylated. Enzymatic deglycosylation of chorion proteins followed by Western blot analysis with DLE7 showed that the 48-kDa and 110-kDa antigens were differentially affected by endoglycosidase-F treatment. When DLE7 was employed in immunofluorescence analysis, isolated chorions and ovarian cryosections stained intensely. Positivity was also observed in liver cryosections of spawning females but not in liver of males and nonspawning females. To study the origin and delivery of chorion proteins, DLE7 was used in Western blot analysis of liver homogenates and blood serum of spawning females. Cross-reacting bands were detected in liver (90 kDa) and serum (180 kDa, 50 kDa). DLE7 was also used for the first time to set up an indirect ELISA assay to detect egg antigens in the blood of egg-producing females, raising the possibility of using DLE7 as a female-specific marker of spawning for sea bass.

Animals↗

PubMed1991

[Prenatal cytogenetic diagnosis using chromosomal preparations of the native biopsy samples of chorionic villi, cultured chorionic cells and amniocytes].

Analysis of the results of prenatal cytogenetic diagnosis carried out in the first and second pregnancy trimesters in more than 300 women permitted comparing the efficacies of two methodologic approaches, diagnostic amniocentesis and chorion sampling , with due consideration for the methodologic errors typical of these methods and of the tested biologic material. Up to 5% of the diagnoses are erroneous if the diagnosis is based on chorion sampling data, whereas in amniocentesis the share of diagnostic errors is lower by an order. The authors have given a theoretical rationale for and tried a methodologic approach, involving the employment of the 'direct' chromosomal preparations from villous chorion biopsy specimens and the so-called 'maintained' cell culture technique, that permits obtaining chromosomal preparations of higher quality and, consequently, helps improve the accuracy of chromosomal diagnosis.

Adult↗

PubMed1991

Sampling success and risk by transabdominal chorionic villus sampling, transcervical chorionic villus sampling and amniocentesis: a randomized study.

A total of 3347 women were randomized to either transabdominal chorionic villus sampling, transcervical chorionic villus sampling or amniocentesis. Unless induced or spontaneous abortion had occurred, they had all completed up to 28 weeks of pregnancy at the time of analysis. No significant difference was seen between total fetal loss in the transabdominal chorionic villus sampling group and the amniocentesis group (6.2% and 6.6%, respectively, SE difference 0.92%, p = 0.01). The total fetal loss in the transcervical group was 10.1%. When the number of failed procedures and those cases which were evaluated as infeasible for the assigned method, are compared, the overall sampling efficacy is less good transcervically than transabdominally. After pooling our data with data from the Canadian randomized study and the American non-randomized study, the difference in risk between transcervical chorionic villus sampling and amniocentesis was 2.1%, SE difference = 0.64%, p = 0.8.

Clinical Trial↗

PubMed1989

Investigations of chorionic villi after chorionic villus sampling (CVS). Correlation of morphological with clinical and laboratory data.

This report documents the first 262 cases of chorionic villus sampling (CVS) performed in parallel with cytogenetic and morphological investigations. Histomorphological examination of these CVS specimens gave suitable results in about 96% (251 cases). Of the latter, 201 samples (80.1%) exhibited villi and 176 (70.1%), maternal tissue. Viability and maturation of the chorionic villi were determined light microscopically even in cases with few villus trees. Smooth avascular villi with poorly defined margins observed under an inverted microscope, less than 10 mitoses after short-term incubation, and reduced growth of cell cultures were significantly correlated with sampling at the chorion laeve by means of histomorphologic criteria. Villi from cases exhibiting cytogenetically proved chromosomal abnormalities were characterized by molar degeneration or stromal fibrosis, or both, in 4 out of 9 cases, including 3 mosaics. In early abortions (within 3 weeks after CVS), an unexpectedly high rate of pathohistological changes within maternal tissue was evident. These results need further confirmation by investigation of a greater number of samples with immunohistochemical and morphometric methods.

Adult↗

PubMed2005

A link between high serum levels of human chorionic gonadotrophin and chorionic expression of its mature functional receptor (LHCGR) in Down's syndrome pregnancies.

Human chorionic gonadotrophin (hCG) is released from placental trophoblasts and is involved in establishing pregnancy by maintaining progesterone secretion from the corpus luteum. Serum hCG is detected in the maternal circulation within the first 2-3 wks of gestation and peaks at the end of the first trimester before declining. In Down's syndrome (DS) pregnancies, serum hCG remains significantly high compared to gestation age-matched uncompromised pregnancies. It has been proposed that increased serum hCG levels could be due to transcriptional hyper-activation of the CGB (hCG beta) gene, or an increased half life of glycosylated hCG hormone, or both. Another possibility is that serum hCG levels remain high due to reduced availability of the hormone's cognate receptor, LHCGR, leading to lack of hormone utilization. We have tested this hypothesis by quantifying the expression of the hCG beta (CGB) RNA, LHCGR RNA and LHCGR proteins in chorionic villous samples. We demonstrate that chorionic expression of hCG beta (CGB) mRNA directly correlates with high serum hCG levels. The steady-state synthesis of LHCGR mRNA (exons 1-5) in DS pregnancies was significantly higher than that of controls, but the expression of full-length LHCGR mRNA (exons 1-11) in DS was comparable to that of uncompromised pregnancies. However, the synthesis of high molecular weight mature LHCGR proteins was significantly reduced in DS compared to uncompromised pregnancies, suggesting a lack of utilization of circulating hCG in DS pregnancies.

Animals↗

PubMed1995

Culture of first-trimester and full-term human chorionic villus explants: role of human chorionic gonadotropin and human placental lactogen as a viability index.

In long-term cultures of human chorionic villus explants, the viability of the tissue must be controlled to ensure the reliability of functional studies. Ionic levels (pH), gas concentrations (pO2, pCO2) and metabolic markers (glucose, lactate) in the culture medium are often utilized. Analyses of hormone, enzyme and protein levels are also frequently used to estimate viability. The purpose of this study was to evaluate whether in vitro release and immunoreactivity of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) were correlated with the viability of first-trimester and full-term chorionic villus explants as determined by histopathology. Villus explants of first-trimester and full-term pregnancies were incubated in 6-well plates of RPMI medium which was supplemented with 10% fetal calf serum. Incubations were performed for 10 days, and the plates were kept at 37 degrees C under a water-saturated atmosphere containing 5% CO2 and 95% O2. The medium was replaced every day and samples of supernatant were frozen for later testing of hCG (first trimester) or hPL (full term), glucose consumption and lactate production. The tissue was also fixed and embedded for light-microscopic examination and immunocytochemistry. The hCG release remained stable during 6-7 days at a high level before decreasing, whereas hPL release decreased during the first 5-6 days then stabilized at a relatively low level. Only hCG kinetics were significantly different between tissue incubated with and without cycloheximide or iodoacetic acid. Both hCG and hPL immunoreactivity were not significantly different between tissue cultures with, and without, addition of cycloheximide or iodoacetic acid and even with morphological evidence of trophoblast and endothelial necrosis. The immunoreactivity for both hormones remains highly positive when the significant release has stopped, and does not reflect the tissue viability.

Chorionic Gonadotropin↗

PubMed1980

Chorion cDNA clones of D. melanogaster and their use in studies of sequence homology and chromosomal location of chorion genes.

Clones corresponding to two distinct A1 and A2 chorion genes have been isolated from a cDNA library in Drosophila melanogaster and characterized by hybrid-selected translation and blotting-hybridization analysis. These sequences detectably cross hybridize, thus indicating that at least some chorion genes in the fruit fly are homologous. According to in situ hybridization results, the A1 and A2 genes are not linked (mapping in regions 66D 10-12 and 54C-D of the third and second chromosomes, respectively). In conjunction with other evidence, these results suggest that in Drosophila, clustering of chorion genes may be limited to genes which are expressed in parallel during development.

Animals↗

PubMed1984

Complement system in abnormal pregnancy and chorionic neoplasia: immunotherapy and chemotherapy of chorionic neoplasia.

In normal pregnancy, the activity of the alternate pathway elevates latently and compensatorily for the suppression of cellular immunity. Aging of the placenta becomes a trigger for the onset of labor and production of C3-a. In cases of hydatidiform mole, abruptio placentae, threatened abortion, and chorionic malignancy, the activity of the complement system is depressed, especially that of C3-activator and alternate pathway. This may in turn result from activation of complement by the pathologic lesion of the chorionic villi. In pathologic lesions of the placenta, activation of alternate pathway can lead to a number of diseases. Immunotherapy is effective in the prevention of malignant transformation of hydatidiform mole. Immunotherapy and combination chemotherapy are markedly effective for treatment of metastatic chorionic malignancy.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1992

The possible evolutionary significance of repeat elements near and within an early chorion gene in the late chorion locus of Bombyx mori.

Three beta-type early chorion gene copies (6F6.1, 6F6.2, and 6F6.3) are dispersed in the late region of chorion locus Ch1-2. Detailed analysis of the 5'-flanking region and the intron of 6F6.1 shows that they contain sequences that are homologous to Bombyx mori Bm1 repeat elements. Interestingly, the Bm1-type segment of the intron is interrupted by the insertion of a sequence that shows significant similarities with part of an intron of B. mori and Bombyx mandarina fibroin genes, and with part of the 3'-flanking region of B. mori prothoracicotropic hormone and tRNA-Glu genes; this sequence may represent a new repetitive, possibly transposable, element of B. mori. Following the Bm1-homologous sequence of the 6F6.1 5'-flanking region and preceding the gene promoter region, a short DNA segment shows sequence motifs that are also present in the ErA.1 promoter region. The occurrence of these sequences near one end or within the Bm1 repeat element is suggestive of complex sequence transfer events. Comparative analysis of known B. mori chorion alpha-gene promoters and of Bm1 repeat elements suggests, with marginal statistical significance, that these two sets of sequences contain common elements.

Animals↗

PubMed1991

Enhancing effects of human macrophage colony-stimulating factor on the secretion of human chorionic gonadotropin by human chorionic villous cells and tPA30-1 cells.

Human macrophage colony-stimulating factor (hM-CSF) concentration-dependently enhanced the secretion of human chorionic gonadotropin (hCG) by primary cultured human cytotrophoblastic cells and a human placental cell line, 3A-SubE (tPA30-1). Since this effect appeared 12 hours after the addition of hM-CSF and disappeared when protein synthesis was inhibited, it was surmised that hCG synthesis was enhanced by hM-CSF. When anti fms (hM-CSF receptor) antibody was added, hCG secretion by cultured human cytotrophoblasts in early pregnancy markedly decreased. These findings demonstrate that hM-CSF acts on the chorionic villous cells and promotes hCG synthesis by these cells.

Cell Line↗