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.     

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.     

High molecular weight forms of adrenocorticotropic hormone in the mouse pituitary and in a mouse pituitary tumor cell line.

Denaturing solvents have been used to determine the molecular weight of the adrenocorticotropic hormone (ACTH) activity in mouse pituitary, in an ACTH secreting mouse pituitary tumor cell line (AtT-20/D-16v), and in the tissue culture medium from the pituitary tumor cells. ACTH activity was quantitated by radioimmunoassay and by bioassay. It is possible to utilize guanidine hydrochloride or sodium dodecyl sulfate in characterizing the multiple forms of ACTH because treatment of porcine ACTH (the 39 amino acid polypeptide form of ACTH, alpha(1-39)), pituitary extracts, tumor cell extracts, and tumor cell tissue culture medium with these denaturants does not diminish the immunological ACTH activity. Based on gel filtration in the presence of guanidine hydrocholoride, extracts of the pituitary tumor cells and the mouse pituitary contain three distinct molecular weight classes of ACTH activity. The major form of ACTH has a molecular weight similar to alpha(1-39) (molecular weight 4000-5500), but there are significant amounts of two higher molecular weight forms of ACTH: molecular weight 6500-9000 and molecular weight 20,000-30,000. The 6500-9000 molecular weight form of ACTH is the major form of ACTH in the tissue culture medium; there is no peak of alpha(1-39) size ACTH in the medium. In the radioimmunoasay all three forms of ACTH generate competitive binding curves parallel to that of porcine alpha(1-39); in the bioassay (stimulation of steroidogenesis in a mouse adrenal tumor cell line) the dose response curve for each of the molecular forms of ACTH is parallel to that for porcine alpha(1-39).     

Factors in the rat submaxillary gland that stimulate growth of cultured glioma cells: identification and partial characterization

The effect of rat submaxillary extract on the growth of rat C6 glioma cells in serum-free culture has been examined. Extracts (10-15 microgram/ml) of submaxillary glands from both male and female rats markedly enhanced the growth of serum-deprived C6 cells and, in combination with insulin, transferrin, and NIH-LH (a source of fibroblast growth factor), were able to stimulate C6 cell growth to an extent comparable to that achieved with an optimal amount of fetal calf serum. The mitogenic activity of rat submaxillary extracts was found to be heat-labile, acid-stable, and partially inactivated by protease and 2-mercaptoethanol. Under our assay conditions, biologically active preparations of purified mouse submaxillary gland epidermal growth factor (EGF) or nerve growth factor (NGF) were not mitogenic for C6 cells, nor was the mitogenic activity of rat submaxillary extracts inhibited by antiserum to these mouse submaxillary gland growth factors. These results suggest that the active component(s) of rat submaxillary extracts is unrelated to either EGF or NGF. The growth-enhancing effect also appears unrelated to esteropeptidase activity present in these extracts since the mitogenic activity was unaffected by several protease inhibitors. Moreover, two purified mouse submaxillary gland arginylesteropeptidases, EGF-binding protein and gamma-subunit of 7 S NGF, were unable to elicit a comparable growth response even when added to cell culture medium at unreasonably high concentrations. The C6 cell mitogenic activity of crude submaxillary extracts could be separated into two biologically similar components by either gel filtration on Sephadex G-100, preparative isoelectric focusing in a pH gradient of 3-10, or adsorption to DEAE-cellulose followed by elution with a sodium chloride gradient. One of the active components was acidic in nature and had an apparent molecular weight of 40,000, while the other was near neutral in charge and possessed a molecular weight of approximately 20,000. The relationship between these two C6 cell mitogenic components and the rat submaxillary gland component responsible for stimulating Balb/c-3T3 cell growth in serum-free, factor supplemented medium (McClure et al., 1979, J. Cell Biol. 83:96a) is also discussed.     

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.     

Release of nerve growth factor by human glial cells in culture

Non-transformed human glial cells obtained from brain biopsies (lines U-787 CG, U-1169 CG and U-1508 CG) release to their culture medium a factor which, in bioassay, induces neurite outgrowth in spinal and sympathetic embryonic chick ganglia. The neurite-stimulating activity, which was enhanced after pressure dialysis of glial-conditioned medium, is inhibited by specific antiserum prepared to mouse βnerve growth factor (NGF). The glial factor was partially purified by gel filtration on Sephadex G-200 of concentrated, serum-containing, conditioned medium. The activity eluted close to a molecular weight of 30000, as did mouse NGF run under identical conditions. Ion-exchange chromatography on DEAE-Sepharose CL-6B and flat-bed electrofocusing of conditioned medium showed the activity to be associated with a heat-labile entity having an isoelectric point of about 4.1. All purified preparations were blocked by anti(mouse)-βNGF. The results demonstrate the existence of a human glial NGF which in several respects resembles the mouse submandibular gland NGF.     

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.     

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.     

Impact of culture age on conidial germination,desiccation and UV tolerance of entomopathogenic fungi

The impact of culture age on conidial yields, germination and tolerance to UV exposure of freshly harvested and dry conidia produced by five entomopathogenic fungal (EPF) isolates was studied. Beauveria bassiana, Metarhizium anisopliae, Lecanicillium lecanii and Lecanicillium muscarium were grown on potato dextrose agar (PDA) medium for 7 or 14 days at 25°C. While the age of cultures had a significant impact on the germination rate of conidia produced by isolates L. lecanii CBS 122.175 and B. bassiana LMSA 1.01.093, other EPF isolates germinated at the same rate regardless of the culture age. When exposed to UV radiation, conidia produced by all isolates germinated at a lower rate compared to the non-irradiated conidia, although this decrease in germination (20–80% decrease) was unaffected by the culture age. Air-drying had only a slight impact on conidial germination (0–60% decrease). Under the conditions of this study, the stability of irradiated conidia produced by M. anisopliae LMSA 1.01.197 and B. bassiana CBS 110.25 was significantly increased when conidia were dried prior to UV exposure. This increase in tolerance to stress of dried conidia might be caused, at least partially, by the low metabolic activity associated with dehydration.     

Isolation and characterization of an apoptosis-inhibiting component from the hemolymph of Bombyx mori.

Silkworm (Bombyx mori) hemolymph showed an apoptosis-inhibiting activity in insect cells (Sf 9) infected with baculovirus (AcNPV). The addition of silkworm hemolymph into the culture medium increased the host cell longevity due to its apoptosis-inhibition activity. Components with an apoptosis-inhibiting effect were purified from the silkworm hemolymph by heat treatment, gel-filtration chromatography, and ion-exchange chromatography. The component with highest activity was characterized by periodic acid-Schiff staining, isoelectric focusing, MALDI-TOF-mass spectrometry, and N-terminal sequencing and was found to be a nonglycosylated monomeric protein with a molecular weight of ca. 28,000 Da.     

Characterization of a zinc finger DNA-binding protein expressed specifically in Petunia petals and seedlings.

In Petunia, the expression of the 5-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS) is tissue-specific and developmentally regulated. Nuclear extracts from Petunia petal contain a factor that interacts with the 5' upstream region of EPSPS. DNase I footprinting experiments revealed four strong binding sites (EP1-EP4) and several weaker sites that appear to bind the same factor. We have isolated a cDNA clone (EPF1) encoding a DNA-binding protein that has similar binding activity to that of the nuclear factor. The deduced amino acid sequence shows that the encoded protein, EPF1, contains two repeats of a Cys2/His2 zinc finger motif. EPF1 and the factor detected in nuclear extracts appear to differ in their molecular weight and Zn2+ requirements. Nevertheless, Northern blot analyses showed that the expression pattern of EPF1 is remarkably similar to that of EPSPS. In addition, as determined by translational fusion of the EPF1 upstream region to the beta-glucuronidase reporter gene, the cell specific expression pattern of EPF1 in flower and seedling is nearly identical to that of EPSPS. Taken together with the results of cis-element analyses, these observations suggest that EPF1 may be one of the factors involved in the activation of EPSPS.     

Synthesis and secretion of nerve growth factor by mouse astroglial cells in culture

Astroglial cells cultured from the mouse brain have been found to synthesize and secrete a material(s) with nerve growth factor-like immunoreactivity (NGF-LI) into their culture medium. A material(s) with NGF-LI showed identical properties to those of beta NGF purified from the mouse submaxillary gland in immunoreactivity, molecular weight, isoelectric point, and neurite outgrowth stimulatory activity. These results indicate that astroglial cells cultured from mouse brain are able to synthesize and secrete beta NGF in culture.     

Characterization of lysozyme synthesized by differentiated mouse myeloid leukemia cells.

Lysozyme was induced by dexamethasone during normal differentiation of cultured mouse myeloid leukemia cells (M1) to macrophages and granulocytes. A large amount of lysozyme was produced by macrophage-like line cells (Mm-1), established from spontaneously differentiated macrophage-like cells from a clonal line of M1 cells. Lysozyme purified from the culture medium of these Mm-1 cells (Mm-1 lysozyme) had a molecular weight of 15,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and showed maximal activity at pH 6.6 with an optimal NaCl concentration of 0.04 M. Its mobility on polyacrylamide gel electrophoresis at pH 4.5 was distinctly lower than those of lysozymes from hen egg white and human urine. Rabbit anti-Mm-1 lysozyme serum inhibited the activities of lysozyme preparations from peritoneal macrophages of normal mice and rats and dexamethasone-induced differentiated M1 cells, but not those of preparations from hen egg white and human urine. Lysozyme was also purified from normal mouse lung, which is rich in alveolar macrophages and was found to be similar to lysozyme purified from the culture medium of Mm-1 cells in size and electrophoretic mobility and in its pH optimum, trypsin peptide map, and antigenicity. Thus the molecular structure of the lysozyme induced in differentiated mouse myeloid leukemia cells is similar to that of lysozyme produced by normal cells.     

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.     

Effects of species and cellular activity of oviductal epithelial cells on their dialogue with co-cultured mouse embryos

An efficient co-culture system, especially with oviductal or uterine epithelial cells, is important not only for the production of high quality embryos, but also for the study of the molecular dialogue between embryos and their maternal environment. Although mouse embryos have been co-cultured successfully with oviductal epithelial cells (OECs) from several species, studies on the effects of species and functionality of OECs are few. Reports concerning the necessity of direct contact between the embryo and OECs and about the culture of mouse embryos in medium conditioned with heterologous OECs have been controversial. In this study, pronuclear embryos from Kunming mice, characterized by an obvious two-cell block in vitro, were co-cultured with mouse, goat, and chick OECs. The functionality of OECs was determined by analyzing the cell cycle, apoptosis, the numbers of mitochondria and cilia, and the ability both to support embryonic development and to remove hypoxanthine from the culture medium. The necessity of direct contact between OECs and embryos was studied by repeated renewal of culture medium with fresh conditioned medium, the culture of embryos in plastic wells connected by tunnels to wells with OEC monolayers, and the co-culture of embryos separated from OECs by a filter. Both goat and chick OECs supported mouse embryonic development, but their embryotrophic lifespan was shorter than that of the mouse OECs. Whereas media conditioned with mouse OECs supported mouse embryonic development satisfactorily, medium conditioned with goat OECs supported little development. Immediate dialogue between heterologous OECs and embryos was essential for efficient co-culture, whereas direct contact between the two cell types was not; neither dialogue nor contact was needed between isologous OECs and embryos. Embryotrophic activity and the ability to remove hypoxanthine from conditioned medium declined with time after confluence and number of passages of OECs, mainly because of apoptosis and dedifferentiation. Thus, the species and functionality of OECs have profound effects on their molecular dialogue with co-cultured embryos, and efficient co-culture depends upon both positive and negative conditioning.This study was supported by grants from the China National Natural Science Foundation (nos. 30430530 and 30571337) and the “973” Project of the Chinese Science and Technology Ministry (no. G200016108).     

A factor produced by feeder cells which inhibits embryonal carcinoma cell differentiation. Characterization and partial purification

Medium conditioned by STO mouse fibroblast cells inhibited both the spontaneous differentiation of NG2 embryonal carcinoma cells and the differentiation of F9 embryonal carcinoma cells induced by retinoic acid. This effect was due to a differentiation retarding factor (DRF). Reduction in DRF activity in conditioned medium by boiling and by pronase treatment suggested the involvement of a polypeptide, which had an apparent molecular weight of 57000 on gel filtration. A 28-fold purification of DRF was achieved. DRF delayed but did not prevent the extensive differentiation observed after prolonged culture of NG2 colonies. Conditioned medium could be successfully used to replace feeder cells in NG2 stock cultures. Media conditioned by a variety of other cell types also contained differentiation retarding activity.     

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.     

Partial purification and initial characterization of a novel differentiation factor for mouse myeloid leukemia cells

We found cells spontaneously differentiated from mouse myeloid leukemia M1 cells were producing a strong differentiation factor in culture medium and established a method to prepare a large quantity of conditioned medium containing the differentiation factor. The factor purified over 4,000-fold from the conditioned medium showed a single peak due to a peptide on a TSK 3000PW column which was coincident with differentiation activity. The molecular weight of the factor estimated by high-performance gel filtration chromatography was 1,300, which is remarkably lower than the values reported for protein differentiation factors reported thus far. M1 cells were induced to differentiate into macrophage-like cells by the factor.