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.     

Identification of a putative inhibitor of early pregnancy factor in mice

Previous studies have indicated that early pregnancy factor (EPF) produced in the pre- and peri-implantation stage of pregnancy appears to consist of inactive components which combine to produce the active species. This is in contrast with EPF produced later in gestation which appears to consist of a single active species. The original studies on ammonium sulphate fractionation of mouse serum and in-vitro culture of mouse ovaries and oviducts have been repeated but tested in the bioassay for EPF, the rosette inhibition test, over an extended range of dilutions. This revealed that the two components in early pregnancy can be understood as EPF and an inhibitor(s). Once this inhibitor is removed, the active fractions in both early and late pregnancy sera exhibit similar behaviour in the above assay. It was shown also that the ovary alone is the source of activity but that this is modulated by an inhibitory substance(s) from the oviduct. Reversed-phase HPLC studies on purified 'early' EPF confirm that active and inhibitory components are present and demonstrate that the active component exhibits an identical elution pattern to 'late' EPF. Thus as pregnancy proceeds, it is not EPF that alters but rather the inhibitor(s), which disappears from the circulation soon after implantation. This substance(s) is under hormonal control, being present during oestrus as well as the early stages of pregnancy; it may be an important biological regulator of EPF. Its action in the rosette inhibition test has profound implications for further study using this bioassay.     

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.     

Platelet-activating factor induces the expression of early pregnancy factor activity in female mice

When synthetic platelet-activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was injected into mature female mice during dioestrus, pro-oestrus or oestrus, it induced the expression of early pregnancy factor (EPF) activity in the sera of these animals within 1 h of injection. The sera of similarly injected males, metoestrous or immature females did not display any EPF activity. The results suggest that embryo-derived PAF may be the ovum factor responsible for triggering the generation of serum EPF activity during the preimplantation stages of pregnancy.     

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.     

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。     

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.     

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.     

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.     

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.     

The early conception factor (ECF) lateral flow assay for non-pregnancy determination in the mare

The horse early conception factor (ECF) test is designed for qualitative determination of the ECF glycoprotein in the mare that has conceived. The objectives of this study were to determine the performance of the horse ECF test for the detection of the non-pregnant mare, and to determine the agreement among subjects or "readers" regarding the interpretation of the test. Blood samples from 60 mares were collected on Days 0, 5, 8, 11 and 18 following ovulation. Pregnancy status diagnosed with the ECF test was compared (2 x 2 table) to pregnancy status diagnosed by palpation per rectum and ultrasound examination on Day 18 following ovulation. Three readers interpreted the ECF test outcome independently. Two laboratories independently interpreted the ECF test outcome from the same serum samples. Agreement was tested by kappa coefficient. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy ranged from 0.74 to 0.84, 0.14 to 0.33, 0.62 to 0.66, 0.33 to 0.44 and 0.57 to 0.60, respectively. Agreement between readers was substantial (0.60相似文献   

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.     

Dominant-lethal test results with known mutagens in two laboratories

During investigations of the potential dominant lethal activity of various chemicals, concurrent controls were run to check the sensitivity and the reproducibility of the test systems. These experiments were carried out over an 18-month period in two laboratories using similar protocols.Negative control substances used were maize oil, dispersol, tween 80 and water. Positive control substance used were cyclophosphamide, ethyl methane-sulphonate and mechlorethamine hydrochloride (nitrogen mustard). The substances were administered either intraperitoneally or by gavage.The results were analysed using, principally, a hierarchical analysis of variance of the total implants per pregnancy and the transformed early deaths per pregnancy data.Pregnancy frequency generally did not fall below 80%. The total number of implantations per pregnancy was usually about 11.8 in negative controls and was variably reduced by the mutagens used. With cyclophosphamide or ethyl methanesulphonate (EMS), there were some quantitative variations in the results obtained but qualitative agreement was good in the results both between experiments in the same laboratory and between the two laboratories. It was demonstrated that EMS is a useful positive control substance in experiments with orally administered materials. Sensitivity of the system was indicated by positive effects with a relatively low dose of EMS and consistent positive results with mechlorethamine for which a dominant lethal effect has not been demonstrated previously.It is concluded that the dominant lethal test gives reproducible data and it is, thus, possible to have confidence in the results and to compare findings in different laboratories using similar protocols.     

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.     

Prospects and limitations of the rosette inhibition test to detect activity of early pregnancy factor in the pig

After preincubation of lymphocytes in serum of non-pregnant pigs and using a standard anti-pig-lymphocyte serum the upper limit of the 99.9% confidence interval of rosette inhibition titres (RIT) for pig serum was calculated to be 11.4 and all titres greater than 12 were defined as a proof of early pregnancy factor (EPF)-activity. The reproducibility of the RITs with serum samples of pregnant pigs was considered satisfactory (interassay coefficient of variation 23.8%), whereas their reproducibility was considered good with sera taken from non-pregnant animals (interassay coefficient of variation 6.5%). Problems of the test were particularly evident due to the absence of increased RITs after previous incubation of lymphocytes in serum of pregnant pigs. Therefore, all EPF-negative samples were reanalyzed up to two times; pregnancy was then correctly diagnosed for 88.7% of sows. There were 8.6% false positive results (N = 70) and 12.2% false negative results (N = 205). During the first half of pregnancy RITs displayed periodic fluctuations which resembled the physiological cycle interval of the pig. Between Weeks 5 and 9 of pregnancy greater numbers of EPF-negative sows were detected. Subsequently, a continuous increase of mean RITs occurred, which then declined gradually from Day 80 of pregnancy. Similar changes were observed for progesterone values in the dialysed serum samples. However, highest progesterone concentrations always followed elevated RITs with a delay of 1.5-4 days. A subthreshold cyclicity of ovarian and luteal function probably persists despite pregnancy.     

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.     

Detection of early pregnancy factor (EPF) using the rabbit ovary and oviduct perfused in vitro

Two peaks of rabbit serum EPF activity were seen over the course of pregnancy. Rabbit ovaries with or without attached oviducts were perfused in vitro for 5 h beginning 12, 16, 24, 48, 72 and 120 h after mating. Perfused isolated ovaries did not produce EPF in vitro, but significant EPF activity was detected in the perfusate of the ovary together with oviduct. Pseudopregnant animals and those rabbits that did not ovulate exhibited no perfusate EPF activity. Perfusate EPF activity was highest at the time embryos were at the pronuclear stage and continued through the morula stage. Although the location of embryos at 72 h after mating varied between oviduct and uterus, EPF activity was maintained over the perfusion period. The results suggest that EPF release occurs within 3 h of fertilization and that the presence of the preblastocyst embryo is crucial for EPF release.     

Early pregnancy affects the expression of toll-like receptor pathway in ovine thymus

Toll-like receptors (TLRs) participates in regulation of the maternal immune tolerance during pregnancy, and the thymus is critical for the adaptive immune system. This study hypothesized whether early pregnancy affected the expression of toll-like receptor pathway in the thymus of ewes. In this study, expression of TLRs, tumor necrosis factor receptor associated factor 6 (TRAF6), interleukin 1 receptor associated kinase 1 (IRAK1) and myeloid differentiation primary response gene 88 (MyD88) was detected in maternal thymus during early pregnancy in sheep. Ovine thymuses were collected on day 16 of the estrous cycle, and days 13, 16 and 25 of pregnancy, and expression of TLR members was analyzed by real-time quantitative PCR, western blot and immunohistochemistry analysis. The results revealed that there were decreases in the expression of the mRNA and proteins of TLR2, IRAK1, TRAF6 and MyD88, but increase in TLR5 mRNA and protein. Furthermore, expression of TLR3 and TLR4 proteins peaked at days 13 and 16 of gestation, and MyD88 protein was located in the epithelial reticular cells and thymic corpuscles. In summary, TLR signaling is implicated in regulation of maternal thymic immune, which may be via downregulation of TLR2, IRAK1, TRAF6 and MyD88 during early pregnancy in sheep.     

Expression of the chaperonin 10 gene during zebrafish development

We have isolated a cDNA encoding chaperonin 10 (cpn10) from the zebrafish. Using northern, western, and in situ hybridization analysis, we observed that the cpn10 gene is expressed uniformly and ubiquitously throughout embryonic development of the zebrafish. Upregulation of cpn10 expression was observed following exposure of zebrafish embryos to a heat shock of 1 hour at 37 degrees C compared to control embryos raised at 27 degrees C. The extracellular form of Cpn10 called early pregnancy factor (EPF), found in the serum of pregnant mammals, was not detected in the serum of either male or female zebrafish. These expression studies suggest that Cpn10 plays a general role in zebrafish development as well as being consistent with the hypothesis that EPF is involved in the embryo implantation process in mammals.     

Muscarinic Receptors in Amygdala Control Trace Fear Conditioning

Intelligent behavior requires transient memory, which entails the ability to retain information over short time periods. A newly-emerging hypothesis posits that endogenous persistent firing (EPF) is the neurophysiological foundation for aspects or types of transient memory. EPF is enabled by the activation of muscarinic acetylcholine receptors (mAChRs) and is triggered by suprathreshold stimulation. EPF occurs in several brain regions, including the lateral amygdala (LA). The present study examined the role of amygdalar mAChRs in trace fear conditioning, a paradigm that requires transient memory. If mAChR-dependent EPF selectively supports transient memory, then blocking amygdalar mAChRs should impair trace conditioning, while sparing delay and context conditioning, which presumably do not rely upon transient memory. To test the EPF hypothesis, LA was bilaterally infused, prior to trace or delay conditioning, with either a mAChR antagonist (scopolamine) or saline. Computerized video analysis quantified the amount of freezing elicited by the cue and by the training context. Scopolamine infusion profoundly reduced freezing in the trace conditioning group but had no significant effect on delay or context conditioning. This pattern of results was uniquely anticipated by the EPF hypothesis. The present findings are discussed in terms of a systems-level theory of how EPF in LA and several other brain regions might help support trace fear conditioning.