Relationship between early pregnancy factor, mouse embryo-conditioned medium and platelet-activating factor.

The effects of synthetic platelet-activating factor (PAF-acether) and mouse embryo-conditioned medium (a source of embryo-derived PAF (EPAF)) on production of early pregnancy factor (EPF) were compared. Embryo-conditioned medium, itself inactive in the EPF bioassay, stimulated ovarian production of EPF in vitro but PAF-acether did not. In vivo, embryo-conditioned medium induced EPF activity in serum of oestrous female, but not in male, mice in contrast to PAF-acether, which induced activity in serum of both male and female mice. This PAF-induced activity was transitory, declining significantly by 2 h and disappearing by 3 h after injection. Activity induced by embryo-conditioned medium was first evident at 2 h after injection, serum concentrations increasing up to 6 h after injection. By discriminating between the behaviour of PAF-acether and EPAF, these studies reinforce the conclusions of other workers that the molecule produced by the embryo is not PAF. Further investigations into the mechanism of action of PAF-acether revealed that it is a potent inducer of activity in the EPF bioassay, with an absolute requirement for platelets in the spleen cell suspension used in the assay. This platelet-derived active species was bound specifically by an anti-EPF monoclonal antibody, indicating that it is EPF-like. This is consistent with parallel studies showing that platelets are not required for induction of activity by either pregnancy serum or purified EPF. These studies were applied to the PAF-induced leukotriene-like species, which had been found by others to be active in the EPF bioassay. Pregnancy serum induced the appearance of this substance from the spleen cell suspension used in the assay; thus the leukotriene-like substance may be regarded as an effector molecule in vitro or mediator of the initiating stimulus of EPF in the bioassay.     

Investigation of the immunocompetent cells that bind early pregnancy factor and preliminary studies of the early pregnancy factor target molecule

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. It has been shown to suppress the delayed-type hypersensitivity response in mice as well as acute and chronic forms of experimental autoimmune encephalomyelitis in rats and mice, respectively. In previous studies, we have demonstrated that EPF binds to a population of lymphocytes and we hypothesized that it mediates its suppressive effects by binding to CD4+ T cells. In the present study, we isolated monocytes and subpopulations of lymphocytes and labelled them with fluoresceinated EPF in order to determine which populations bind EPF. We demonstrated that EPF binds specifically to CD4+, CD8+, CD14+ (monocytes) and CD56+ NK cells but not to CD19+ B cells. The identity of the molecule(s) on the cell surface that is targeted by EPF is unknown, but as EPF is an extracellular homologue of the intracellular protein chaperonin 10 (Cpn10), we examined the possibility that the EPF receptor is a membrane-associated form of chaperonin 60 (Cpn60), the functional associate of Cpn10 within the cell. The EPF target molecule on lymphocytes was visualized by chemical cross-linking of exogenous iodinated Cpn10 to cells and probed with anti-Cpn60. The effect of anti-Cpn60 on activity in the EPF bioassay, the rosette inhibition test, was also examined. In both instances, no specific interaction of this antibody and the putative receptor was observed. It was concluded that the cell surface molecule targeted by EPF is unlikely to be a homologue of Cpn60.     

Purification and characterization of early pregnancy factor from human pregnancy sera

Early pregnancy factor (EPF) is a pregnancy-associated protein detected in the maternal serum by using the rosette inhibition assay and by evaluating the suppression of adoptive transfer of contact sensitivity. Because of its inhibitory effect on the functional reactivity of immunocompetent cells, EPF is thought to be involved in immunoregulation of the maternal immune system during early pregnancy. EPF was purified six million-fold from the serum of pregnant women between 5 and 12 weeks of gestation. The specific activity of purified EPF was approximately 8 x 10(8) units/mg. The purification scheme involved sequential DEAE-cellulose chromatography, S-Sepharose chromatography, concanavalin A-Sepharose chromatography, heparin-Sepharose chromatography, Mono S fast protein liquid chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein has an apparent molecular weight of 21,500 as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 28,000 by gel permeation high pressure liquid chromatography. The isoelectric point of purified EPF moiety is 6.5. The biological activity was susceptible to the proteolytic enzyme trypsin, acidic pH conditions, organic solvents, and sodium dodecyl sulfate, but stable to heat treatment at 56 degrees C for 30 min and the reducing agent dithiothreitol. The biological and physicochemical properties of EPF appear to be distinct from other pregnancy-associated and immunoregulatory proteins.     

Effect of monoclonal antibodies to early pregnancy factor (EPF) on the in vivo growth of transplantable murine tumours

Summary Neutralisation studies with monoclonal antibodies (mAbs) specific for early pregnancy factor (EPF) have shown it to be essential for the continuation of pregnancy in mice and the growth of some tumour cells in vitro. These studies report that the mAbs are also able to limit the growth of two murine tumour lines transplanted s. c. The development of MCA-2 tumours in CBA mice was unaffected by the injection of 1 mg anti-EPF IgM at the time of tumour cell inoculation. However, four doses of 500 µg anti-EPF, injected one dose per day for 4 days after tumour cell inoculation, significantly retarded tumour development such that no tumours were palpable on day 13. A similar dose regimen of control IgM had no effect on tumour size. Dose/response studies revealed that lower doses of anti-EPF administered after tumour cell inoculation were effective in retarding the growth of the MCA-2 tumours. The effect of anti-EPF mAb administration on the growth rate of palpable B16 tumours established s. c. in C57BL/6 mice was also determined. Tumours injected with 6 mg anti-EPF 5/341 or anti-EPF 5/333 mAbs showed significant decrease in the uptake of [³H]thymidine into tumour tissue, measured 16 h after injection. Furthermore, titration of sera for active EPF showed that a significant reduction in the EPF titre was associated with a significant inhibition of tumour DNA synthesis. Thus it appears that neutralisation of EPF retards tumour growth both in vitro and in vivo. In vitro the effects must be due to anti-EPF mAb interfering with a direct mechanism that contributes to the maintenance of cells in the active growing phase. However, in vivo host immunological mechanism that are modified to allow tumour survival may also be affected. The presence of EPF-induced suppressor factors curculating in the serum of tumour-bearing mice has been confirmed and the contribution of such factors to tumour progression must now be investigated.     

Production of a recombinant form of early pregnancy factor that can prolong allogeneic skin graft survival time in rats

Early pregnancy factor (EPF), an extracellular chaperonin 10 homologue, has immunosuppressive and growth factor properties. In order to carry out more extensive studies on the in vivo characteristics of EPF, a recombinant form of the molecule has been prepared. Recombinant human EPF (rEPF) was expressed in Escherichia coli using the plasmid pGEX-2T expression system. Potency of rEPF in vitro in the rosette inhibition test, the bioassay for EPF, was equivalent to that of native EPF (nEPF), purified from human platelets, and synthetic EPF (sEPF). However, the half-life of activity (50% decrease in the log value) in serum, following i.p. injection, was significantly decreased (3.2 h, compared with nEPF 6.2 days, sEPF 5.8 days). This was thought to be due to modification of the N-terminus of the recombinant molecule inhibiting binding to serum carrier proteins. Because EPF can modify Th1 responses, the ability of the recombinant molecule to suppress allogeneic graft rejection was investigated. Following skin grafts from Lewis rats to DA rats and vice versa, rEPF was delivered locally at the graft site and the effect on survival time of the allografts noted. Results demonstrated that rEPF treatment significantly prolonged skin graft survival time by as much as 55% in stringent models of transplantation across major histocompatibility barriers.     

In-vivo and in-vitro determination of components of rabbit early pregnancy factors

Two types of rabbit early pregnancy factor (EPF) components, prepared from the in-vitro perfused ovary and oviduct and from serum ammonium sulphate fractions, were investigated to elucidate the source of EPF production. The state of the animals, i.e. pregnant, pseudopregnant, unstimulated or platelet activating factor (PAF)-treated, and order of addition of the components to the assay lymphocytes were varied to characterize conditions of production and expression of EPF activity. Although each component alone had no EPF activity, combination of two components, i.e. ovary and oviduct components, or serum precipitate and supernatant components, expressed EPF activity. The oviduct and serum supernatant components were produced in pregnancy and pseudopregnancy but the ovary and serum precipitate components were produced only in pregnancy. The similarity of the production pattern and ability of the perfusate and serum components to yield EPF activity when combined suggests that they are similar or the same. The sources of stimulation of EPF production did not appear to affect component production because the activity produced by the perfused ovary and oviduct in pregnancy or in response to PAF stimulation appeared similar. Oviduct and supernatant components apparently bound directly to lymphocytes. These results suggest that EPF components are produced in the ovary and oviduct individually and that the combination of the two components expresses EPF activity in the rabbit.     

THE ROLE OF ALLELOPATHIC SUBSTANCES IN THE “SMOTHER CROP” BARLEY

The high degree of success of barley as a “smother crop” generally has been attributed to physical competition for nutrients and water. However, it was found that even in the absence of such competition, barley still inhibits germination and growth. This occurred both in mixed cultures receiving adequate nutrients and water and in germination tests. Aqueous leachates of seeds and roots of barley caused similar inhibition of germination and growth, thereby indicating an inhibitory allelopathic substance. A specificity of reaction was found, with the greatest inhibition occurring with Stellaria media (L.) Cyr., less with Capsella bursa-pastoris (L.) Medic. and tobacco (Nicotiana tabacum L.), and no significant effect with wheat (Triticum aestivum L.). A concentration effect and possible periodic production of the inhibitor were indicated. Living plants and aqueous leachates of living roots were more inhibitory than dead ones, thereby supporting the hypothesis of an active metabolic secretion of the allelopathic substance. Preliminary attempts to identify the active inhibitory components demonstrated the presence of alkaloids, with a much greater concentration of substance in the living than in the dead root leachates. The alkaloid, gramine, known to occur in barley, was found to have an inhibitory effect on the growth of Stellaria media and it is suspected as an active component of the root leachates. These results suggest that factors other than the previously assumed physical competition are involved in the mechanism of the “smother crop” barley.     

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E. coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding approximately 42 and 36mg EPF from 300ml bacterial and 1L Sf9 cultures, respectively. The preparations were highly purified (#10878;99% purity on SDS-PAGE for the bacterial products and #10878;97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immunosuppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity.     

Some Properties of Inhibitor β from Solanum tuberosum Compared to Abscisic Acid

The growth-inhibiting effect of inhibitor β, extracted from the potato variety Majestic, was compared with the effect of abscisic acid by means of the Avena coleoptile straight-growth test. Dose-response curves showed that total growth inhibition is attained by inhibitor β, but this is never caused by abscisic acid even in high unphysiological doses. By chromatographic methods inhibitor β can be separated into three active components. One of these is a phenolic substance with weak growth-inhibiting effect. The two others contribute equally to the inhibitory effect, and one of them is probably abscisic acid. Beside these inhibiting substances there are components without inhibitory effect, many of phenolic character, included in the inhibitor β complex.     

Measurement of early pregnancy factor activity for monitoring the viability of the equine embryo

The viability of embryos before flushing from donor mares (n = 5) and after transfer to recipient mares (n = 7) was monitored in mare serum by detecting early pregnancy factor (EPF) using the rosette inhibition test (RIT). The EPF activity was measured in donor mares before and after natural mating at natural estrus; after ovulation on Days 2, 5 and 8; and after embryo flushing (Day 8) on Days 8, 9, 10 and 13 after ovulation. The collected embryos were transferred immediately after flushing. The EPF activity in recipient mares were measured on the day of transfer and after embryo transfer on Days 1, 2, 3 and 5. Pregnancy was confirmed on Day 12 to 14 after embryo transfer. The mean EPF activity of donor mares was increased to the pregnant level (> an RI titer score of 10) on Day 2 after ovulation. Two days after flushing the embryos, the EPF activity of donor mares had decreased to the nonpregnant level. Among the 7 recipient mares, 3 mares were diagnosed pregnant on Day 12 after embryo transfer with ultrasound. The EPF activity of the pregnant recipient mares was increased above the minimum level observed in pregnant mares on Days 2 to 3 after transfer. However, among the nonpregnant recipient mares after embryo transfer, the EPF activity of 3 mares remained at the pregnant level only 2 to 3 d and then declined to the nonpregnant level. In one recipient mare, EPF activity did not reach the pregnant level throughout the sample collection. The results of this study indicated that equine EPF can be detected in serum of pregnant mares as early as Day 2 after ovulation. From our observation, we conclude that the measurement of EPF activity is useful for monitoring the in vivo viability of equine embryos and early detection of embryonic death.     

Identification of molecules involved in the 'early pregnancy factor' phenomenon.

An isolated preparation from ovine placental extracts which was active in the rosette inhibition assay mimicking the activity of the so-called 'early pregnancy factor' (EPF) has been shown to contain a 12 kDa polypeptide which could be partially resolved from low-molecular-weight active moieties. N-terminal amino acid sequence analysis of the polypeptide indicated that it was ovine thioredoxin, an identification confirmed by isolation and complete sequence analysis of the corresponding cDNA. The cDNA for human thioredoxin was expressed in Escherichia coli and the recombinant protein isolated and purified. Pure recombinant thioredoxin alone did not induce the expression of increased rosette inhibition titres (RITs) when tested in the rosette inhibition assay; but, when tested in combination with cell stimuli such as platelet-activating factor (PAF) or serum, it allowed the expression of increased RITs where none was achieved in its absence. Thioredoxin acted in the assay to reverse a refractory state normally induced by these stimuli, allowing lipoxygenase-dependent moieties also induced by the stimuli to exert their effects, resulting in the expression of increased RITs. Antibodies to recombinant thioredoxin removed from pregnancy sera the capacity to induce increased RITs, i.e. to express EPF activity, thus establishing a role for thioredoxin or thioredoxin-like proteins and associated molecules in the mechanisms which allow pregnancy sera to induce increased RITs. Based on a consideration of these and other results, a new model for the study of the EPF phenomenon is presented and discussed.     

The inhibitory effect of cytosolic fraction of human decidua on prostaglandin synthesis

To elucidate the mechanism of suppression of prostaglandin (PG) production in decidua in early pregnancy, the PG synthetase activity of decidua and mid-secretory endometrium was studied. The microsomal fractions and their supernatants were prepared from the tissue by ultracentrifugation at 105,000 g. The standard incubation mixture consisted of the microsomal fraction and 14C arachidonic acid with cofactors, with incubation being carried out for 10 minutes at 37 degrees C. After extraction, the radioactivity of PGE2 was measured and PG synthetase activity was assayed. The apparent Km value for PG synthetase in decidua was 4.6 +/- 0.14 x 10(-6) M (n = 4), whereas that in endometrium was 4.6 +/- 1.18 x 10(-6) M (n = 3). Subsequently, kinetic studies on PG synthetase inhibitor in decidua were carried out. When sheep seminal vesicle was used as an enzyme source, the decidual supernatant showed competitive inhibition. The inhibitory substance in decidua was inactivated after incubation for 15 minutes at 65 degrees C. It seems likely that the suppression of PG biosynthesis in human decidua in early pregnancy is not due to the difference in PG synthetase found in decidua and in endometrium, but due to the existence of PG synthetase inhibitor in decidua.     

Isolation of a cigarette smoke fraction responsible for the inhibition of benzo[a]pyrene metabolism in the isolated perfused rabbit lung

Among the several thousand components of cigarette smoke is a substance or substances capable of inhibiting pulmonary metabolism of nicotine and altering the metabolite profile of procarcinogens such as benzo[a]pyrene (BP). This substance(s) inhibits BP metabolism in the lung in amounts present in a few puffs of cigarette smoke. By a series of extractions and chromatographic methods an active subfraction containing only 1% of the total cigarette smoke condensate (CSC), was isolated. This fraction demonstrated the same inhibition of BP metabolism in the isolate perfused lung (IPL) as the whole smoke. The inhibitor(s) present in this fraction possess amphoteric characteristics. The acidic function is believed to be a phenolic one.     

早孕因子研究进展

早孕因子既是一种微量蛋白又是一种妊娠相关蛋白,其纯品的获得尤为重要。简要论述了早孕因子的本质和功能,并就早孕因子在超早期妊娠诊断中的实际功能价值进行了深入探讨;结合早孕因子的国内外研究进展和生理生化特点提出了2种蛋白质提纯方案;针对当前早孕因子提纯及检测方法中存在的问题进行了讨论。     

Cytostatic peptide isolation from culture media of mouse peritoneal exudate cells

It was found that the supernatant of mouse PEC culture medium (MCM) (both resident and casein-elicited cells) has an inhibitory effect in vitro on the incorporation of [3H]TdR into DNA of mouse spleen cells. The inhibitory effect in the MCM appears in the first 24 hr and also reaches its maximum value within this time. The inhibitory effect of this factor could not be demonstrated in the extract of freshly harvested M phi cells. The factors responsible for inhibition proved to be heat stable at 80 degrees C for longer than 30 min. Following heat treatment, the crude extract was separated into four fractions absorbing uv light at 280 nm using Sephadex G-25 column chromatography, and the most potent biologically active inhibitory factor was eluted in the last fraction. This fraction could also be obtained with a more effective permeation volume using Trysacryl GF 05 gel chromatography, and the active B fraction from this chromatography could be separated into four subfractions by isotachophoresis (ITP). The active fraction, which was obtained by Trysacryl GF 05 gel chromatography and further separated by ITP, was found to be highly inhibitory. It contained a peptide-like substance with a molecular mass of approximately 2.0 kDa and had an anionic character at pH 4.0. The inhibitory effect of MCM cannot be influenced either by inhibitory compounds of protein synthesis or by proteolysis blocking agents. Furthermore, the inhibitory effect is shown to be reversible and is more pronounced on B cells than on T lymphocytes.     

Presence of an intracellular endometrial inhibitor of prostaglandin synthesis during early pregnancy in the cow

Previous studies have detected reduced endometrial secretion of prostaglandins during pregnancy in cattle. The present experiment tested the hypothesis that reduced secretion of prostaglandins is caused by induction of an intracellular endometrial inhibitor of prostaglandin synthesis. The microsomal fraction of parturient bovine cotyledons was utilized as a source of enzymes for prostaglandin synthesis. Endometrial tissues collected at Day 17 of the estrous cycle (n = 12) and pregnancy (n = 12) were homogenized and subjected to differential centrifugation for preparation of microsomes and a high-speed (100,000 x g) cytosolic supernatant. Endometrial intracellular preparations were then examined for the ability to modulate prostaglandin synthesis by cotyledonary microsomes from parturient cows. Endometrial intracellular preparations from cyclic cows decreased (P less than 0.05) PGF synthesis by cotyledonary microsomes to a slight extent (supernatant, 21% reduction; microsomes, 11% reduction), while preparations from pregnant cows markedly decreased (P less than 0.01) PGF synthesis (supernatant, 63% reduction; microsomes, 28% reduction; supernatants vs microsomes, P less than 0.01). Regardless of the amount of arachidonic acid available as substrate (25-400 micrograms) endometrial supernatant from pregnant cows (pooled sample) caused a 50% inhibition (IC50) of prostaglandin synthesis at a tissue equivalent of 270 +/- 9.1 mg. The mechanism of inhibition by endometrial high-speed supernatant from pregnant cows appears to be non-competitive with respect to arachidonic acid. The inhibitor(s) may be proteinaceous (70-75 kDa and 25-35 kDa) and can be precipitated by 20% saturated ammonium sulfate. In conclusion, early pregnancy in cattle appears to be associated with increased amounts of an intracellular endometrial inhibitor of prostaglandin synthesis.     

Measurement of EPF for detection of cow pregnancy using rosette inhibition test

Early embryonic death of calves due to sub-fertility in cows is of great economic concern to dairy industry. Early pregnancy factor (EPF) is a secretory protein with pregnancy associated immunosuppressive properties. Rosette inhibition test (RIT) was used to detect EPF in inseminated dairy cows. Blood samples were collected at two intervals, 1-3 and 5-7 days after insemination from 23 inseminated and 18 non-inseminated control cows for RIT and pregnancy diagnosis performed between 42 and 45 days on palpation. The study indicates that RIT (P<0.05) has the potential to distinguish pregnant from non-pregnant dairy cows in the first week of pregnancy.     

Detection of early pregnancy factor in swine: A need for dialogue

The rosette inhibition test, which has been used to detect early pregnancy factor (EPF) and other immunosuppressive factors in the serum of pregnant mice, women, and sheep, was adapted for use in swine. Since the initial methods described for use of the assay were cumbersome and time consuming, our first efforts were to simplify the technique. Substitution of microtiter plates for test tubes and treatment of sheep erythrocytes with AET to increase numbers and stability of the rosettes were successful. Although consistently higher rosette inhibition titers were obtained after incubation of lymphocytes in the serum of pregnant sows compared to controls, the variability in numbers of rosettes obtained on a day to day basis and the cumbersome nature of the test have impeded progress in firmly establishing the presence of EPF in pregnant swine and demonstrating the homology between it and the EPF reported in other species.