Biomedical subjects

J Martal

Publications and source records attributed to J Martal.

At least 37 records · Page 2Linked to original sources

PubMed1994

Detection of pregnancy by radioimmunoassay of a pregnancy serum protein (PSP60) in cattle.

The accuracy and efficiency of pregnancy diagnoses in cattle by pregnancy serum protein (PSP60) radioimmunoassay, a progesterone radioimmunoassay or oestrus detection were compared. Blood samples were taken from 349 suckling heifers and cows (1,191 inseminations) at 28, 35, 50 and 90 d post-insemination for PSP60 determination and at 22-23 d for progesterone. Females were declared nonpregnant when plasma PSP60 concentration was lower than 0.2 ng/ml at 28, 35 and 50 d and 0.5 ng/ml at 90 d. When compared with rectal palpation at 90 d, the accuracy of positive (negative) diagnoses by progesterone assay was 80% (100%) in heifers and 75% (99%) in cows. The accuracy of positive diagnoses by PSP60 assay increased with gestation stage from 90% on d 28 in heifers (74% in cows) to 100% (99% in cows) at the time of rectal palpation. This accuracy was 84% on d 28 in cows when the interval from calving to blood sampling was higher than 115 d. Whatever the stage, the accuracy of negative diagnoses was higher than 90%. Efficiency in detecting pregnant or nonpregnant females on d 28 was equivalent to the progesterone assay. The method for detecting oestrus applied in this experiment was as efficient as the PSP60 or progesterone test at any stage of gestation. The PSP60 test is very flexible, which makes its use particularly interesting in naturally mated suckling herds because of the uncertainty regarding the date of fertilization.

Animals↗

PubMed1994

Evidence that a non-steroidal factor from corpus luteum of pregnant sheep inhibits aromatase activity of ovarian follicles in vitro.

Porcine and ovine follicular tissues were used to investigate, in vitro, the effect of charcoal-treated aqueous extract from ovine corpora lutea of pregnancy on aromatase activity as determined by the conversion of [3H]testosterone to oestradiol by follicular walls and measurement of 3H2O release. Extract (500 micrograms protein) prepared from corpora lutea of day 112 of pregnancy but not extract (500 micrograms) prepared from ovine fetal cotyledonary tissue obtained at a similar time significantly decreased (P < 0.02) aromatase activity of pig follicles in the absence of FSH. These results demonstrate that a non-steroidal factor in the corpora lutea of late pregnancy directly inhibits aromatase activity. When the effects of different doses (300, 600 or 1200 micrograms) of luteal extract from corpora lutea of day 100 of pregnancy on aromatase activity of pig follicles were studied, the dose by treatment (presence or absence of FSH) interaction was not significant. Luteal extract dose at 300 micrograms did not affect aromatase activity but a significant decrease in activity occurred at 600 micrograms of luteal extract (600 versus 300 micrograms, P < 0.02). There was no further significant increase in the inhibitory effect with 1200 micrograms luteal extract. When the effects of 600 micrograms luteal extract from corpora lutea of days 15, 75 or 100 of pregnancy on aromatase activity of pig follicles were studied, a significant (P < 0.05) stage of pregnancy effect was detected, but the stage of pregnancy by treatment (presence or absence of FSH) interaction was not significant. No effect was noted with day 15 or day 75 luteal extract.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

[Cytokines and the corpus luteum].

This minireview present main findings concerning the contribution of cytokines to the regulation of some key processes of luteal functions. Data concerning the preovulatory follicles invasion by white blood cells and the migration of macrophages, granulocytes and T lymphocytes into corpus luteum suggest that local secretion of regulatory cytokines may be involved in regulating corpus luteum formation and demise as well its maintenance in early pregnancy. Several lines of evidence indicate that the pro-inflammatory cytokines such as IL-1, IL-6 and TNF-alpha modulate the steroidogenic changes which take place during luteinization. For instance, an inhibition of E2 biosynthesis is evidenced in granulosa cells in human or porcine species with IL-1, in rat with TNF-a and in bovine with IL-6. Moreover, IL-1 stimulates P4 production but to a much lower extent than LH, and PGE2 synthesis by rat thecal cells. The potential relevance of pro-inflammatory cytokines in the mechanisms controlling luteolysis is suggested by the ability of IL-1 and TNF-alpha to decrease both P4 production and the survival of bovine luteal cells. As opposed to ruminants, TNF-alpha has no effect in human luteal cells but potentiates the decrease of P4 secretion induced by IFN-gamma. Finally, data regarding the participation of trophoblast interferons in the mechanisms for maintaining the corpus luteum at the establishment of pregnancy are now available in ruminants. From these observations and others, we can consider that cytokines are involved in the regulation of the corpus luteum function.

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↗

PubMed1993

Peripheral concentrations of a 60-kDa pregnancy serum protein during gestation and after calving and in relationship to embryonic mortality in cattle.

In order to have a specific marker for studying pregnancy in cattle we examined the characteristics of a pregnancy serum protein produced by the placenta (PSP60). Its profile in peripheral blood was determined by radioimmunoassay in pregnant cows of 3 breeds after artificial insemination (AI): Charolais (n = 24), Normande (n = 24) and Holstein (n = 26). From 27 d post-AI to the end of pregnancy the plasma PSP60 concentration increased, especially during the last 2 wk, to reach a peak a few d before calving, which was higher (P < 0.001) in the Charolais (1,238 +/- 422 ng/ml) than in the other breeds (528 +/- 458 and 444 +/- 204 ng/ml). With an apparent half-life of approximately 8 d, this protein was still detectable in the maternal blood from 105, 85 and 87 d post-partum in the Charolais, Normande and Holstein breeds, respectively. Fertility results on 1,102 inseminations in the Charolais breed showed that the PSP60 concentration dropped then disappeared after embryonic mortality. Sequential assays of this protein between 28-90 d after AI are useful for studying the course of pregnancy, although they do not allow discrimination between early embryonic mortality and non-fertilization which together constitute 75% of pregnancy failures.

Animals↗

PubMed1993

Cloning and structural analysis of four genes encoding interferon-omega in rabbit.

By using an ovine interferon-tau (IFN-tau) cDNA probe, four recombinant phages were isolated from a rabbit genomic library and sequenced from nucleotides -450 to 1,300 relative to the CAP site. Each of the four rabbit genes contains an open reading frame of 595 nucleotides and code for proteins that exhibit structural characteristics of the interferon-omega (IFN-omega) family. They display more than 98% identity in their coding regions. The deduced amino acid sequences share > 96% sequence similarity. In contrast, the 5' and 3' noncoding regions have diverged considerably (approximately 50% identity). Amino acid comparisons of rabbit IFN-omega with IFN-omega of other species reveal the highest degree of identity with human (72%), followed by porcine (68%) IFN-omega. Rabbit IFN-omega displays only 57% sequence similarity with ovine IFN-tau. The coding regions of the four genes subcloned in a cytomegalovirus eukaryotic expression vector and transfected in monkey COS-7 cells direct the production of proteins that protect bovine and rabbit cells against vesicular stomatitis virus infection, thus demonstrating that these genes encode fully active IFN proteins. The expression of these genes was studied in Sendai-induced rabbit leukocytes. A single band of poly(A)+RNA hybridized with a rabbit IFN-omega probe under stringent conditions, whereas no IFN-omega transcript was detected with RNA isolated from uninduced leukocytes. Southern blot analysis suggest the existence of at least eight IFN-omega genes or pseudogenes in the rabbit genome.

Amino Acid Sequence↗

PubMed1993

Potential role for arachidonic acid and eicosanoids in modulating progesterone secretion by ovine chorionic cells.

The present study was conducted to investigate whether arachidonic acid and its metabolites can modulate progesterone (P4) secretion in ovine chorionic cells. At concentrations of 7.5 mumol/l and 12.5 mumol/l, arachidonic acid caused an increase of basal P4 secretion (about 1.8-fold (p < 0.01) and 2.5-fold (p < 0.001), respectively, over control). Such a stimulatory effect was suppressed when the concentration of arachidonic acid attained 25 mumol/l, and at 50 mumol/l the fatty acid led to a decline of basal P4 synthesis (about 35%, p < 0.01). Phospholipase A2 (PLA2) and melittin had a similar dual effect to that observed when arachidonic acid was added exogenously. In contrast, eicosatrienoic acid (a closely related fatty acid) did not stimulate P4 secretion but inhibited it at a concentration of 50 mumol/l (about 40% inhibition, p < 0.01). The possible involvement of calcium on the effects of arachidonic acid was explored. Interestingly, 3 mmol/l ethylene glycol bis(beta-aminoethyl ether)-N,N,N,N'-tetraacetic acid (EGTA) and 10 mumol/l 8-N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) further enhanced the steroidogenic effect of 12.5 mumol/l arachidonic acid (p < 0.05 and p < 0.01 vs the corresponding value in the absence of EGTA or TMB-8, respectively). In contrast, these agents failed to modify P4 secretion observed in the presence of 50 mumol/l arachidonic acid. We also tested the effect of inhibition of arachidonic acid metabolism via cyclooxygenase and lipoxygenase pathways. Indomethacin (10 mumol/l) failed to block the effects of arachidonic acid, but nordihydroguaiaretic acid (10 mumol/l) prevented the stimulatory action of this fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens↗

PubMed1993

Localization and characterization of EGF/TGF-alpha receptors on peri-implantation trophoblast in sheep.

Receptors for epidermal growth factor (EGF) have been identified on the ovine trophoblast as early as day 15 of gestation. A radioligand assay with 125I-labelled EGF was used to detect high and low affinity binding sites on the trophoblastic and placental membranes. The binding of 125I-labelled EGF was inhibited by increasing concentrations of unlabelled EGF. Competition studies with other peptide hormones including transforming growth factor alpha (TGF-alpha), insulin-like growth factor-I (IGF-I) and ovine placental lactogen confirmed the specificity of EGF/TGF-alpha for its receptor. Cross-linking experiments using disuccinimidyl suberate (DSS) revealed a radiolabelled band of relative molecular mass 170 kDa. Immunohistochemical localization of the receptors demonstrated their distribution on the epithelial layer cells. The presence of receptors for EGF/TGF-alpha suggests that these factors could be involved in the regulation of embryonic development and fetal growth.

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↗

PubMed1991

Immunoregulatory effects of ovine trophoblastin protein (oTP): all five isoforms suppress PHA-induced lymphocyte proliferation.

The immunosuppressive properties of ovine trophoblastin protein (oTP) isoforms purified to homogeneity by DEAE HPLC have been studied within and across species barriers by in vitro assays. It has been demonstrated that not only the classical oTP 1, but in fact all 5 isoforms, are immunosuppressive in a PHA-induced proliferation assay, whilst being ineffective on IL-2 dependent CTL-L2 cell replication. The significance of these findings is discussed.

Animals↗

PubMed1991

Co-expression of the proto-oncogene fos (c-fos) and an embryonic interferon (ovine trophoblastin) by sheep conceptuses during implantation.

Expression of the c-fos proto-oncogene by ovine conceptuses was analyzed by Northern and slot blots and indirect immunohistofluorescence in relation to the expression of the embryonic interferon-alpha (oTP) during implantation. c-fos was expressed initially in the trophoblast, and then in the allantois, when this tissue began to develop (day 17). In the embryonic tissues, the c-fos proto-oncogene was weakly expressed up to day 22 and increased thereafter. In the trophoblast, the expression of c-fos proto-oncogene was transient, occurring when the oTP gene was transcribed at a maximal level at the beginning of implantation (days 14-15), and decreased thereafter, following the pattern of oTP gene expression. This decline is due essentially to the arrest of c-fos and oTP gene expression by the trophoblastic cells which established cellular contacts with the uterine epithelium during the implantation process.

Actins↗

PubMed1991

Cloning and structural analysis of two distinct families of ovine interferon-alpha genes encoding functional class II and trophoblast (oTP) alpha-interferons.

Ovine trophoblast protein (oTP) is a polypeptide secreted by ovine trophectoderm from day 11 to 21, which plays a key role in maternal recognition of pregnancy. Structural analyses established that oTP shares extensive homology with class II alpha-interferon (IFN-alpha II) subfamily. Previous screening of an ovine genomic DNA library probed with an oTP cDNA incidently resulted in the isolation of a functional IFN-alpha II gene and two relevant pseudogenes, as shown by sequence analysis and study of expression in eukaryotic COS cells. The expected oTP gene together with a cognate pseudogene was successfully isolated from the series of clones selected from another genomic library probed with the oTP cDNA, using two specific oligonucleotides, each one complementary to a region of oTP cDNA with little homology with the IFN-alpha II gene and related pseudogenes. Southern blotting of ovine genomic DNA indicated the existence of at least five trophoblast IFN-alpha genes or pseudogenes. Nucleotide sequence comparisons showed that the oTP gene exhibits a higher homology (90%) with bovine trophoblast IFN gene (Stewart et al. (1990) J. Mol. Endocrinol. 4, 275-282) than with oIFN-alpha II gene (70%), thus providing evidence that embryonic IFNs constitute a distinct subfamily of IFN-alpha s.

Amino Acid Sequence↗

PubMed1991

Production of two species of interferon by Large White and Meishan pig conceptuses during the peri-attachment period.

Antiviral activities present in uterine flushings from pregnant Large White, Large White 'hyperprolific', and prolific Meishan gilts, between Days 8 and 20 of gestation were compared. Flushings (20 ml) from all gilts between Days 14 and 20 were positive in an in-vitro interferon (IFN) assay using vesicular stomatitis virus as a challenge infection. Highest antiviral activities (of up to 400,000-1,200,000 total Units/flushing) were obtained at Day 16 of gestation, i.e. clearly after the beginning of attachment. There was no major difference between breeds although, at Day 14, flushings from Meishan gilts yielded significantly higher titres than those from the other two, suggesting a correlation with the previously described earlier trophoblast elongation in Meishan gilts. Conceptus cultures contained antiviral activity, with values very close to those obtained in vivo, but the difference between breeds was not significant. Cultures from Day 20 on contained very little antiviral activity. The antiviral activity was associated with a mixture of at least two IFNs, one of which was IFN-alpha like, and the other was serologically identified as an IFN-gamma, that is an 'immune IFN', previously found to be secreted only by T lymphocytes. This finding may have implications for our understanding of the immunology of early pregnancy.

Animals↗

PubMed1990

Application of preparative high-performance liquid chromatography to the purification of a fetal ovine insulin-like growth factor II: N-terminal sequence determinations using two different carriers.

A highly efficient procedure for the purification to homogeneity of an ovine fetal insulin-like growth factor II (IGF II) is described. Fetal sheep serum was used as the source material, and the bioactivity was followed throughout purification by an IGF II radioreceptor assay. Ovine IGF II was isolated by a combination of gel permeation, ion-exchange chromatography and reversed-phase high-performance liquid chromatography. The amino-terminal sequence of the first 36 amino acid residues was compared using two supports (polyethylenimine and polybrene) as carrier for protein sequencing. Ovine fetal IGF II was found to differ from human IGF II in three residues of the C-domain, with serine, isoleucine and asparagine substituted for alanine, valine and serine, respectively, at positions 32, 35 and 36. The final yield of highly purified ovine fetal IGF II was 134 micrograms, starting from 450 ml of serum.

Amino Acid Sequence↗

PubMed1990

Cellular localization of an embryonic interferon, ovine trophoblastin and its mRNA in sheep embryos during early pregnancy.

The ovine embryo produces an interferon named ovine Trophoblastin (oTP) which is involved in the maternal recognition of pregnancy and ensures the maintenance of progesterone secretion by the corpus luteum. We have used indirect immunohistofluorescence and in situ hybridization on histological sections to investigate the fate of this protein and its mRNA in ovine embryos from days 3 to 25 of pregnancy. The level of expression was measured by image analysis of the autoradiographs after in situ hybridization. Both techniques clearly demonstrated that oTP and its mRNA were specifically localized in the extra-embryonic trophoblast. Neither the embryonic cells, nor the yolk sac or the amniotic tissues produced the protein or its mRNA. The protein could be detected by d 11 of pregnancy in the elongated blastocyst. Maximum of expression is observed at d 14 and the level decreased by d 16 of pregnancy. The arrest of expression occurred in the regions of trophoblast which have established cellular contacts with the uterine epithelium during the implantation process.

Animals↗

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↗

PubMed1989

Cloning and expression of cDNA encoding ovine trophoblastin: its identity with a class-II alpha interferon.

The cDNAs encoding ovine trophoblastin (oTP) were isolated from an ovine embryo cDNA lambda gt 11 library by screening with a synthetic 29-mer oligodeoxynucleotide corresponding to amino acid (aa) residues 34 to 43 of oTP. The cDNA contained an open reading frame of 595 bp and the deduced amino acid sequence indicates a protein precursor of 195 aa. Nucleotide and amino acid sequence comparisons establish that oTP shares extensive homology with alpha-interferon (IFN-alpha) but is more closely related to the IFN-alpha sII subfamily. When the oTP cDNA was cloned into an eukaryotic expression vector and transfected in monkey COS cells, a high level of antiviral activity was detected. RNA blot analyses of total RNA reveal that the oTP-coding gene is expressed during a relatively short period (eleven to 21 days). The abundant expression of oTP mRNA corresponds closely to the time at which the embryo acts to extend luteal lifespan. RNAs homologous to oTP were also detected in goat and cow embryos at equivalent periods of their development, but not in the pig.

Amino Acid Sequence↗