Biomedical subjects

G Charpigny

Publications and source records attributed to G Charpigny.

28 records · Page 2Linked to original sources

PubMed1995

Secretion of biologically active interferon tau by in vitro-derived bovine trophoblastic tissue.

Secretion of interferon tau (IFN tau) by trophoblastic tissue has been shown to be the first embryonic signal for pregnancy recognition. Therefore we tried to derive biologically active trophoblastic tissue by in vitro techniques. Since conventional in vitro conditions for bovine embryo development were not sufficient for long-term culture, we tested more complex culture conditions, including Ménézo B2 or Buffalo rat liver (BRL) cell-conditioned medium, for their ability to support proliferation and IFN tau secretion by in vitro-derived trophoblastic tissue. IFN tau activity was determined by using a biological assay based on the inhibition of the cytopathic effect of vesicular stomatitis virus on Madin-Darby bovine kidney cells. When cultures of individual hatched blastocysts were started in 60-microliters drops of BRL cell-conditioned medium, mean IFN tau secretion (antiviral units/ml/48 h) corresponded to 1200 on Day 11 and to 5000 on Day 13 (p < 0.01). To characterize trophoblast cell-specific secretions, the inner cell mass was removed from all embryos by microsurgery on Day 13. IFN tau secretion by trophoblastic tissue increased to mean levels of > 10(5) antiviral units/ml/48 h on Day 23m, stayed high for about 1 wk, and then slowly declined to levels below 10(3) antiviral U/ml/48 h. The specificity of the cytoprotective effect of IFN tau was tested by Western blot analysis and by immunoneutralization with use of a polyclonal antiserum specific to IFN tau. Our results demonstrate that viable trophoblastic tissue can be maintained entirely in vitro and secretes high amounts of IFN tau.

Animals↗

PubMed1993

Phospholipase A2 activity in endometrium from early pregnant and non-pregnant ewes.

In the ewe, synthesis of the luteolytic factor, prostaglandin F2 alpha, increases from day 13 to the end of the estrous cycle. Availability of free arachidonic acid is usually the rate-limiting step in prostaglandin biosynthesis. Phospholipase A2 (PLA2) may be the key enzyme for the hydrolysis of arachidonic acid from membrane-bound phospholipids. To investigate uterine PLA2 activity during the estrous cycle and early pregnancy, we monitored the release of [14C]oleic acid from the substrate 1-palmitoyl-2-[14C]oleoyl-phosphorylcholine by homogenates and cytosolic fractions of endometrium from ewes on days 12, 14 and 16 of the estrous cycle or pregnancy. We observed that PLA2 activity dropped by 58% (p < 0.02) in day-16 pregnant endometrium compared to day-16 non-pregnant endometrium. We then investigated whether the reduced PLA2 activity was due to induction of a specific inhibitor. The PLA2-inhibitor activity was determined by monitoring the inhibition of release of [14C]oleic acid from the radioactive substrate by porcine pancreatic PLA2. Inhibition by endometrial homogenates of pregnant animals of the control enzyme activity was 27% and only 14% by cyclic ones. Inhibition was dose-dependent and was as high as 53% (p < 0.01) with 1 mg protein from pregnant endometrial homogenates. Endometrial PLA2 behaved as a Michaëlian enzyme in the endometrium of day-16 cyclic ewes (Km = 79.4 mumol/l). Furthermore, the inhibitory activity from pregnant endometrium had characteristics of competitive inhibition. Our results suggest that inhibition of endometrial PLA2 activity could occur in early pregnant ewes.

Animals↗

PubMed1993

Recombinant ovine trophoblastin (roTP) inhibits ovine, murine and human lymphocyte proliferation.

Ovine trophoblastic protein (oTP) is a 20-kDa embryonic secretory product constitutively secreted by ovine conceptus trophoblast from days 12-22 of pregnancy. Amino acid sequencing as well as molecular cloning revealed it to bear structural analogies with interferons of the class 2 alpha subfamily, defining the tau interferon group. It is endowed with classical interferon-like biological activities. Recombinant ovine trophoblastin (roTP), produced by genetic engineering, was purified by anion exchange HPLC to a high degree of homogeneity (98%). It behaved in immunodetection and antiviral activity assays like the natural form. We show here that when assayed on PHA-driven murine, human, and ovine (sheep) lymphocyte proliferation, roTP is immunosuppressive. It also inhibits unidirectional and bidirectional murine and human mixed lymphocyte reactions (MLRs). Since natural oTP possesses (at least) 5 isoforms, we also assayed these for immunosuppressive activities. All of them inhibited PHA-driven human and ovine lymphoblastogenesis. Finally, CD4+ and CD8+ ovine T cell selection was performed by panning. In contrast with earlier observations assaying roTP activity on human lymphocytes, both ovine CD4 and CD8 T cell subsets were sensitive to roTP in a PHA-driven proliferation assay. It is therefore suggested that trophoblast interferons might have a strategic function in preventing early embryonic demise by immunologic rejection, at least in ovine species.

Animals↗

PubMed1992

Addition of a dipeptide spacer significantly improves secretion of ovine trophoblast interferon in yeast.

Yeast has been analysed for its potential to secrete an ovine member of the type-I interferon (IFN) family, trophoblastin (oTP-1). The processing potential of the yeast KEX2 gene product (KEX2p) was evaluated using gene oTP-1 fused to the pre-pro sequence encoding the pre-pro peptide of the yeast alpha-factor precursor. High-level accumulation of nonprocessed (unmatured) recombinant oTP-1 (re-oTP-1) was observed in the medium. In order to short-circuit the limiting activity of KEX2p and to obtain a fully matured re-oTP-1, secretion was directed using a pre::oTP-1 fusion, relying only on signal peptidase-dependent processing. However, secretion of oTP-1 was impaired. High-level secretion was restored when the gene product contained a peptide spacer between oTP-1 and the signal peptidase cleavage site. The oTP-1 variant was shown to have an extended N terminus. An N-extended form was examined further and shown to have the correct size. Surprisingly, the variant retained its in vitro and in vivo biological activities. This system is likely to represent a general method for high-level secretion of type-I IFNs.

Amino Acid Sequence↗

PubMed1990

Evidence for extended maintenance of the corpus luteum by uterine infusion of a recombinant trophoblast alpha-interferon (trophoblastin) in sheep.

Ovine trophoblastin (oTP) is a natural interferon of the class-II interferon-alpha subfamily. Recombinant ovine trophoblastin (r.oTP), produced by genetic engineering, was purified by anion-exchange HPLC. The product exhibited a high degree of homogeneity (greater than 98%), and similar immunological cross reaction and antiviral activity to natural oTP. Antiluteolytic activity of r.oTP was established by intrauterine injection in two groups of cyclic recipient ewes. Control group A included 10 ewes which received sterile BSA in saline twice daily for 8 days (from day 10-12 of oestrous cycle). Experimental group B included 17 ewes which received 80 micrograms (4 ewes), 170 micrograms (8 ewes) or 340 micrograms (5 ewes) r.oTP daily for 8 days. Maintenance of functional corpora lutea for 1 month or more was observed in 4 out of 5 ewes which received high doses of r.oTP. These results indicate that oTP alone extends luteal secretory activity.

Animals↗

PubMed1988

High homology between a trophoblastic protein (trophoblastin) isolated from ovine embryo and alpha-interferons.

Ovine trophoblastic protein B (oTPB), an embryonic protein, is a 20 kDa secretory protein which is synthesized by the ovine conceptus from days 12 to 22 of pregnancy. oTPB was purified by HPLC using ion-exchange chromatography on a DEAE column and was subsequently chromatographed on a reversed-phase column. Automated Edman degradation was then used to determine the N-terminal amino acid sequence up to 45 residues. The sequence data reveal a significant homology between oTPB and bovine interferons alpha of class II: 64% of the amino acids are identical and 75% are homologous. A highly conserved region including residues 23-44 exhibits 82% homology. Identity between oTPB and either HuIFN-alpha.9 or MuIFN alpha.1 is 55%. These alignments between oTPB and IFNs occur at the N-terminus of the mature proteins and proceed without deletion. These results suggest that oTPB is an embryonic interferon.

Amino Acid Sequence↗

PubMed1988

Embryo-uterine interactions during early stages of pregnancy in domestic mammals.

The first part of this paper presents data concerning our knowledge of uterine proteins during early pregnancy in domestic mammals; the second part gives results of in vitro biochemical studies on embryo-uterine interactions in the ewe. We have developed an in vitro technique of the co-culture of ovine uterine epithelial cells with the blastocyst or its secretory proteins. The effects of a specific trophoblastic protein (oTPB), involved in the maintenance of the corpus luteum, have been particularly studied by this system. The modifications of endometrial protein synthesis have been measured by incorporation of radiolabelled amino acids and analysed by electrophoresis (SDS-PAGE or bidimensional). The results show that the presence of the blastocyst, or of its total secretory proteins or oTPB alone, decreased overall protein synthesis by the endometrial cells. The nature of the secreted proteins was apparently not affected by the blastocyst, but the addition of oTPB alone increased the production of 3 polypeptides (MW = 150.10(3); 74.10(3); 50.10(3) and pI = 7-8.2; 5.4-5.2; 6.4, respectively) and decreased the synthesis of 2 others (MW = 57.10(3); 35.10(3) and pI = 7-6.7; 5.3, respectively). We also studied the effects of co-culture with uterine cells on blastocyst DNA and protein synthesis. In no cases we obtained stimulation of blastocyst development during the co-culture period, and DNA or protein synthesis decreased in the presence of uterine cells. In conclusion, the presence of specific uterine proteins has been established in some domestic mammals (pig, rabbit) but not in all of them. Although local modifications of uterine protein synthesis are induced by the embryo or its secretory products, the nature and the role of the proteins which are affected need to be determined by further studies.

Animals↗

PubMed1988

Is oxytocin of conceptus origin involved in inhibition of luteal regression in early pregnancy in ewes?

This study describes the presence in and production by the ovine conceptus of an oxytocin-like peptide during the early stages of development. Oxytocin was measured by radioimmunoassay in ovine conceptuses from days 14 to 30 of pregnancy. Tissue concentrations of oxytocin increased from day 14 (24.8 +/- 5 pg/100 mg) until day 19 (122.9 +/- 52 pg/100 mg) and then decreased (3 +/- 1 pg/100 mg). Oxytocin was released into culture medium by day-15 ovine conceptuses at a rate of 262 +/- 55 pg/24 h. Reverse-phase high-performance liquid chromatography (HPLC) analysis of peptides extracted from a pool of ovine conceptuses was conducted using chromatographic conditions developed to separate oxytocin from other nonapeptides. Radioimmunoassay of HPLC fractions for oxytocin revealed an immunoactive conceptus peptide in a single fraction at the same retention time as chromatographed authentic oxytocin. Radioimmunoassay and chromatographic data therefore suggest that this oxytocin-like peptide is similar, if not identical, to authentic oxytocin. Concentrations of oxytocin in conceptus tissue were maximal during the period of inhibition of luteal regression (days 14-19). It is proposed that conceptus oxytocin is involved in the maintenance of luteal function in early pregnancy.

Animals↗

PubMed1988

[Trophoblastic proteins].

Many studies conducted on human or animal placenta (chorion) suggest that the trophoblast is not only a passive filter between maternal and foetal blood flow, but is also endowed with complex functions. Factors of trophoblast origin involved in the mechanism of pregnancy recognition or maintenance of the progesterone environment required for the embryo survival, are reviewed. The main proteins involved in pregnancy are reported in table 1. Emphasis is laid on early signals of pregnancy which are of practical interest in human clinical medicine and animal husbandry. Among them, human chorionic gonadotropin (hCG), protein SP1 ("Schwangerschaftsprotein" 1) and some pregnancy-associated plasma proteins such as the PAPP A are very useful in the diagnosis of pregnancy, abortion, foetal abnormality or tumor in the human species. The presence of trophoblastin (presently studied in our laboratory) of a pregnancy-specific protein B and of early pregnancy factor (EPF) attest the establishment of pregnancy in domestic animals and in other mammals. The biological properties of some hormones such as placental lactogens (PL) or chorionic somatomammotropins (CS), human placental growth hormone (hPGH) contribute to a better understanding of the gestation function. Many other factors participate in the foetal development, for example, proliferin. Some proteins can display immunosuppressive properties or be responsible for the immune tolerance between the mother and the foetus. Although many placental proteins have already been defined, their biological functions have not yet been elucidated.

Animals↗

PubMed1986

Relationships between structure and function of lactogenic hormones.

Lactogenic activity of several hormone derivatives obtained by chemical modifications of lysine residues was studied by radioreceptor assay. The relationships between structure and binding to lactogenic receptors are discussed taking into account lysine residue positions liable to be involved in the location of lactogenic function.

Amino Acid Sequence↗