Al2O3 and TiO2 atomic layer deposition on copper for water corrosion resistance.

Al(2)O(3) and TiO(2) atomic layer deposition (ALD) were employed to develop an ultrathin barrier film on copper to prevent water corrosion. The strategy was to utilize Al(2)O(3) ALD as a pinhole-free barrier and to protect the Al(2)O(3) ALD using TiO(2) ALD. An initial set of experiments was performed at 177 °C to establish that Al(2)O(3) ALD could nucleate on copper and produce a high-quality Al(2)O(3) film. In situ quartz crystal microbalance (QCM) measurements verified that Al(2)O(3) ALD nucleated and grew efficiently on copper-plated quartz crystals at 177 °C using trimethylaluminum (TMA) and water as the reactants. An electroplating technique also established that the Al(2)O(3) ALD films had a low defect density. A second set of experiments was performed for ALD at 120 °C to study the ability of ALD films to prevent copper corrosion. These experiments revealed that an Al(2)O(3) ALD film alone was insufficient to prevent copper corrosion because of the dissolution of the Al(2)O(3) film in water. Subsequently, TiO(2) ALD was explored on copper at 120 °C using TiCl(4) and water as the reactants. The resulting TiO(2) films also did not prevent the water corrosion of copper. Fortunately, Al(2)O(3) films with a TiO(2) capping layer were much more resilient to dissolution in water and prevented the water corrosion of copper. Optical microscopy images revealed that TiO(2) capping layers as thin as 200 Å on Al(2)O(3) adhesion layers could prevent copper corrosion in water at 90 °C for ~80 days. In contrast, the copper corroded almost immediately in water at 90 °C for Al(2)O(3) and ZnO films by themselves on copper. Ellipsometer measurements revealed that Al(2)O(3) films with a thickness of ~200 Å and ZnO films with a thickness of ~250 Å dissolved in water at 90 °C in ~10 days. In contrast, the ellipsometer measurements confirmed that the TiO(2) capping layers with thicknesses of ~200 Å on the Al(2)O(3) adhesion layers protected the copper for ~80 days in water at 90 °C. The TiO(2) ALD coatings were also hydrophilic and facilitated H(2)O wetting to copper wire mesh substrates.

Using trap crops for control of Acalymma vittatum (Coleoptera: Chrysomelidae) reduces insecticide use in butternut squash.

Striped cucumber beetle, Acalymma vittatum F., is the primary insect pest of cucurbit crops in the northeastern United States. Adult beetles colonize squash crops from field borders, causing feeding damage at the seedling stage and transmitting bacterial wilt Erwinia tracheiphila Hauben et al. 1999. Conventional control methods rely on insecticide applications to the entire field, but surrounding main crops with a more attractive perimeter could reduce reliance on insecticides. A. cittatum shows a marked preference for Blue Hubbard squash (Cucurbita maxima Duchesne) over butternut squash (C. moschata Poir). Given this preference, Blue Hubbard squash has the potential to be an effective perimeter trap crop. We evaluated this system in commercial butternut fields in 2003 and 2004, comparing fields using perimeter trap cropping with Blue Hubbard to conventionally managed fields. In 2003, we used a foliar insecticide to control beetles in the trap crop borders, and in 2004, we compared systemic and foliar insecticide treatments for the trap crop borders. We found that using a trap crop system reduced or eliminated the need to spray the main crop area, reducing insecticide use by up to 94% compared with conventional control methods, with no increase in herbivory or beetle numbers. We surveyed the growers who participated in these experiments and found a high level of satisfaction with the effectiveness and simplicity of the system. These results suggest that this method of pest control is both effective and simple enough in its implementation to have high potential for adoption among growers.

Significance of serum early pregnancy factor concentrations during pregnancy and embryonic development in Sminthopsis macroura (Spencer) (Marsupialia: Dasyuridae).

Marsupial pregnancy differs from that in eutherians in duration, placentation and hormonal profile so much so that maternal recognition of pregnancy may not occur in polyovular marsupials. However, a comparison of gravid and non-gravid uteri reveals differences indicative of histological and physiological adaptations to pregnancy. In the present study, the hypothesis that embryo-maternal signalling occurs in polyovular marsupials was tested by examining serum from non-pregnant and pregnant Sminthopsis macroura for the presence of early pregnancy factor (EPF), a serum protein secreted by the ovary in response to the presence of a newly fertilized egg in the oviduct. EPF is detectable in the serum of pregnant, but not in non-pregnant, females in all eutherians studied to date. In the present study, EPF was detected in S. macroura serum by the rosette inhibition test during the first 9 days of the 10.7 day gestation period in this marsupial. However, EPF was not detected on day 10, just before parturition, or in non-pregnant or preovulatory animals. Immunohistochemical analysis of ovaries from gravid and non-gravid animals demonstrates that EPF is found in the capillaries, interstitial spaces and secretory cells of the corpus luteum. It is concluded that the spatiotemporal pattern of EPF activity described strongly indicates that maternal recognition of pregnancy in marsupials is mediated, at least in part, by EPF. Because the endocrinological milieu is the same in pregnant and non-pregnant marsupials, the possibility of using marsupials as an experimental system for studying EPF function unconfounded by hormonal effects is presented.

The murine chaperonin 10 gene family contains an intronless, putative gene for early pregnancy factor, Cpn10-rs1.

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. Human platelet-derived EPF shares amino acid sequence identity with chaperonin 10 (Cpn10), a mitochondrial matrix protein which functions as a molecular chaperone. The striking differences in cellular localization and function of the two proteins suggest differential regulation of production reflecting either alternative transcription of the same gene or transcription from different genes. In mammals and more distantly related genera, there is a large gene family with homology to CPN10 cDNA, which includes intronless copies of the coding sequence. To determine whether this could represent the gene for EPF, we have screened a mouse genomic library and sequenced representative Cpn10 family members, looking for a functional gene distinct from that of Cpn10, which could encode EPF. Eight distinct genes were identified. Cpn10 contains introns, while other members are intronless. Six of these appear to be pseudogenes, and the remaining member, Cpn10-rs1, would encode a full-length protein. The 309-bp open reading frame (ORF) is identical to that of mouse Cpn10 cDNA with the exception of three single-base changes, two resulting in amino acid changes. Only one further single nucleotide difference between the Cpn10-rs1 and Cpn10 cDNAs is observed, located in the 3' UTR. Single nucleotide primer extension was applied to discriminate between Cpn10-rs1 and Cpn10 expression. Cpn10, which is ubiquitous, was detected in all tissue samples tested, whereas Cpn10-rs1 was expressed selectively. The pattern was completely coincident with known patterns of EPF activity, strongly suggesting that Cpn10-rs1 does encode EPF. The complete ORF of Cpn10-rs1 was expressed in E. coli. The purified recombinant protein was found to be equipotent with native human platelet-derived EPF in the bioassay for EPF, the rosette inhibition test.

Immunoelectron microscopy provides evidence for the presence of mitochondrial heat shock 10-kDa protein (chaperonin 10) in red blood cells and a variety of secretory granules.

Hsp10 (10-kDa heat shock protein, also known as chaperonin 10 or Cpn10) is a co-chaperone for Hsp60 in the protein folding process. This protein has also been shown to be identical to the early pregnancy factor, which is an immunosuppressive growth factor found in maternal serum. In this study we have used immunogold electron microscopy to study the subcellular localization of Hsp10 in rat tissues sections embedded in LR Gold resin employing polyclonal antibodies raised against different regions of human Hsp10. In all rat tissues examined including liver, heart, pancreas, kidney, anterior pituitary, salivary gland, thyroid, and adrenal gland, antibodies to Hsp10 showed strong labeling of mitochondria. However, in a number of tissues, in addition to the mitochondrial labeling, strong and highly specific labeling with the Hsp10 antibodies was also observed in several extramitochondrial compartments. These sites included zymogen granules in pancreatic acinar cells, growth hormone granules in anterior pituitary, and secretory granules in PP pancreatic islet cells. Additionally, the mature red blood cells which lack mitochondria, also showed strong reactivity with the Hsp10 antibodies. The observed labeling with the Hsp10 antibodies, both within mitochondria as well as in other compartments/cells, was abolished upon omission of the primary antibodies or upon preadsorption of the primary antibodies with the purified recombinant human Hsp10. These results provide evidence that similar to a number of other recently described mitochondrial proteins (viz., Hsp60, tumor necrosis factor receptor-associated protein-1, P32 (gC1q-R) protein, and cytochrome c), Hsp10 is also found at a variety of specific extramitochondrial sites in normal rat tissue. These results raise important questions as to how these mitochondrial proteins are translocated to other compartments and their possible function(s) at these sites. The presence of these proteins at extramitochondrial sites in normal tissues has important implications concerning the role of mitochondria in apoptosis and genetic diseases.

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.

Early pregnancy factor suppresses experimental autoimmune encephalomyelitis induced in Lewis rats with myelin basic protein and in SJL/J mice with myelin proteolipid protein peptide 139-151.

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. During pregnancy, it appears in maternal serum within 6-24 h of fertilization, is present for at least the first two-thirds of pregnancy in all species studied and is essential for embryonic survival. It is a homologue of chaperonin 10, a heat shock protein, but, unlike other members of this family, EPF has an extracellular role. As it has the ability to modulate CD4+ T cell-dependent immune responses, its role in treatment of experimental autoimmune encephalomyelitis (EAE) was investigated. EAE is a CD4+ T cell-mediated disease, the best available animal model of multiple sclerosis (MS). Two models of EAE were investigated, acute EAE induced in Lewis rats by inoculation with myelin basic protein (MBP-EAE) and chronic relapsing EAE induced in SJL/J mice by inoculation with myelin proteolipid protein peptide (residues 139-151) (PLP-EAE). EPF, delivered intraperitoneally or orally to rats or intraperitoneally to mice, suppressed clinical signs of disease. Mice with PLP-EAE were also treated with interferon-beta, with and without EPF. Both EPF and IFN-beta suppressed clinical signs of EAE and, when administered together, gave greater suppression than when given separately. These findings suggest that EPF may be a potential candidate for use in treatment of MS and may be of use in combined therapy with IFN-beta.

Isolation of antibodies which neutralise the activity of early pregnancy factor.

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It functions as an autocrine growth factor for tumour cells and as an autocrine or paracrine growth factor for regenerating normal cells. Anti-EPF antibodies have demonstrable anti-tumour activity and, as a result, hybridomas which produce such antibodies are unstable. In this study, the phage display antibody techniques have been investigated as a means of producing recombinant anti-EPF antibodies. Mice were immunised with synthetic peptides which correspond to the N or C terminal regions of EPF, and their splenic tissue was used to make combinatorial antibody libraries. The Fab repertoire was displayed on the surface of phage and panned over recombinant EPF. Reactive Fabs were identified by ELISA and their binding was characterised by BIAcore analysis and functional studies. Three libraries with a size of greater than 5x10(7)cfu were constructed and a total of 26 unique Fabs with specific reactivity against EPF were identified. Three Fabs were purified and of these one demonstrated strong EPF neutralising activity, one had intermediate activity and the other was not neutralising. Phage display has provided the means of circumventing the problems of anti-EPF hybridoma development and has resulted in the production of antibodies with potential applications in the diagnosis of pregnancy and the diagnosis and therapy of cancer.

Early pregnancy factor is required at two important stages of embryonic development in the mouse.

PROBLEM: The importance of early pregnancy factor (EPF) at the pre-implantation stage of development (days 1-3 post-coitum [p.c.]) has been previously established in this laboratory. However, the role of EPF at the implantation stage (days 4.5-5 p.c.) has not been determined. This present study therefore investigates the role of EPF at this important developmental stage, both in vivo and in vitro. METHOD OF STUDY: Mated mice were passively immunized with anti-EPF antibodies at the peri-implantation stage (days 3.5-4 p.c.) and embryo implantation recorded. Parallel studies were conducted in vitro, where the effect of anti-EPF antibodies on trophoblast outgrowth of blastocysts was determined. RESULTS: Administration of anti-EPF antibodies in vivo at the peri-implantation stage of development resulted in failure of embryos to implant. Similarly, trophoblastic outgrowth of blastocysts was adversely affected in the presence of anti-EPF antibodies. CONCLUSIONS: These results, together with previous findings that anti-EPF antibodies retard embryonic development when administered at the early pre-implantation stage, clearly demonstrate that EPF is required by the embryo at two important developmental stages- the one-two-cell stage and the peri-implantation stage.

Preparation and characterization of polyclonal antibodies against human chaperonin 10.

Early pregnancy factor (EPF) has been identified as an extracellular homologue of chaperonin 10 (Cpn10), a heat shock protein that functions within the cell as a molecular chaperone. Here, we report the production of polyclonal antibodies directed against several different regions of the human Cpn10 molecule and their application to specific protein quantitation and localization techniques. These antibodies will be valuable tools in further studies to elucidate the mechanisms underlying the differential spatial and temporal localization of EPF and Cpn10 and in studies to elucidate structure and function.

Mapping and characterization of the eukaryotic early pregnancy factor/chaperonin 10 gene family.

Early pregnancy factor and mitochondrial chaperonin 10 have very different functions within mammals but the mature peptides have identical amino acid sequences. In order to understand the mechanisms by which identical proteins can have different functions and sites of activity, we have examined genomic DNA which could encode the protein. In most species studied, there is a large gene family of at least ten members with homology to the DNA sequence for this protein. Using a monochromosomal somatic cell hybrid panel, we have mapped the gene for human chaperonin 10 to chromosome 2. Other members of the human gene family map to several chromosomes. Chromosomes 1, 2 and 9 contain pseudogenes with Alu insertions while chromosome 16 has a pseudogene containing a short direct repeat flanking an insert. Chromosomes 1 and 16 may also carry a functional intronless copy of the EPF/Cpn10 sequence.

The human early pregnancy factor/chaperonin 10 gene family.

cDNA clones corresponding to the sequence for human early pregnancy factor were isolated from a human melanoma library and hybridized to DNA digested with four restriction enzymes obtained from twelve different subjects. Up to 20 cross hybridizing bands were observed. When hybridized to metaphase spreads from four different humans, significant signals were present in nine locations, on eight different chromosome arms. These results suggest that the early pregnancy factor gene is a member of a large gene family. The coding sequence for early pregnancy factor has a high degree of homology with the sequence for human chaperonin 10, and the gene family described here should contain the genes for both of these proteins.

Identification of early pregnancy factor as chaperonin 10: implications for understanding its role.

Early pregnancy factor (EPF) is a secreted substance with growth regulatory and immunomodulatory properties. It is required for successful establishment of pregnancy and for proliferation of both normal and neoplastic cells, in vivo and in vitro. The rosette inhibition test was used as a bioassay, and the appearance of EPF in serum in the very early stages of pregnancy (in mice, within 4-6 h of mating) was first described two decades ago. However, because of the difficulty of this bioassay and the paucity of EPF in biological materials, the primary structure of the molecule has been identified only recently. Seventy per cent of the amino acid sequence of EPF derived from human platelets was determined. With the exception of a single residue, this was identical to the sequence of rat mitochondrial chaperonin 10 (cpn10). Cpn10 is a heat shock protein that functions as a molecular chaperone. It binds to and stabilizes cpn60 and, in concert, these molecules mediate protein folding in mitochondria, chloroplasts and bacteria. Characterizing EPF as an extracellular form of cpn10 raises unprecedented questions about the mechanism of action. It may be that, as a molecular chaperone in the extracellular compartment, EPF can functionally modify other proteins, serving as a regulator of regulators.

Purification and partial characterization of an early pregnancy factor-induced suppressor factor (EPF-S1).

PROBLEM: The immunomodulatory properties of early pregnancy factor (EPF) are mediated through induction of at least two lymphokines, designated EPF-S1 and EPF-S2 (previously estimated M(r) 15,000 and 55,000 respectively). The activity of the former is MHC-restricted while the latter is restricted to a locus (or loci) outside the MHC. The present study established further criteria by which EPF-S1 and EPF-S2 might be distinguished from each other and compared with other suppressor factors. In addition, techniques have been developed to purify EPF-S1 to homogeneity. METHOD: Congenic mouse strains were used to map the genetic restriction of EPF-S2 in the rosette inhibition test and high performance gel permeation chromatography was used to demonstrate that EPF-S1 induces EPF-S2 but not vice versa. Further studies then focused on isolation of this first component of the cascade, EPF-S1, from immune ascites (from growth in athymic mice of the anti-EPF-S1 producing rat-mouse hybridoma R2T gamma, in which EPF-S1 is complexed to antibody). Techniques used were acidification followed by application to Sep-pak C18 cartridges, high performance cation-exchange chromatography and two reversed-phased HPLC steps on a C3 column. Purified material was analyzed by SDS-PAGE and Edman degradation. RESULTS: Approximately 10 micrograms EPF-S1 were isolated fom 60 ml ascitic fluid. Homogeneity of the purified material was demonstrated by SDS-PAGE, where it ran as a single band of approximate M(r) 12,000 coincident with biological activity. Attempts at Edman degradation indicate that the molecule is N-blocked. CONCLUSION: Definitive primary characterization of EPF-S1 must await the preparation and isolation of proteolytic fragments of the molecule, but the present studies establish conditions which make such structural analysis possible.

A monoclonal antibody to an early pregnancy factor-induced suppressor factor (EPF-S1) disrupts implantation in mice.

PROBLEM: The importance of EPF during pregnancy has been established previously but the importance of the EPF-induced suppressor factor EPF-S1 in pregnancy has to date been unaddressed. Investigations were therefore conducted in order to study this. METHOD: Monoclonal antibodies to EPF-S1 were produced, and one antibody, designated R2T gamma, was characterized. Mated mice were passively immunized with R2T gamma and the effect on implantation determined. RESULTS: Characterization of anti-EPF-S1 R2T gamma revealed that it cross-reacted with EPF-S1 of different MHC restriction but not with EPF or EPF-S2. When injected into mated mice on days 1 to 4, R2T gamma had no effect on pregnancy but when injections continued to day 5, pregnancy was affected; the number of embryos implanted on day 7 were significantly less than the number of corpora lutea counted, signifying embryonic loss. CONCLUSION: These studies show that anti-EPF-S1 R2T gamma disrupts implantation in mice when injected on days 1 to 5 of pregnancy but not when injected on days 1 to 4, demonstrating that EPF-S1 exerts its effects around the time of implantation.

Early pregnancy factor in liver regeneration after partial hepatectomy in rats: relationship with chaperonin 10.

Early pregnancy factor is not only a product of dividing embryonic and neoplastic cells, as demonstrated previously, but also of normal proliferating cells. Eight hours after partial hepatectomy in rats, early pregnancy factor was detected in serum. It rose to a peak by 48 hr. Neutralization of early pregnancy factor in vivo by passive immunization with specific antibodies, 18 hr after partial hepatectomy, resulted in a significant decrease in the uptake of [3H]thymidine by the liver remnant, measured 4 to 6 hr later. These results suggest that during liver regeneration, early pregnancy factor is essential to the sequence of events that culminates in DNA synthesis and cell division. Recently we purified early pregnancy factor from human platelets and determined by mass spectrometry a precise molecular mass of 10,843 Da. Amino acid sequencing (approximately 72% of the molecule) demonstrated that early pregnancy factor is highly homologous with chaperonin 10, a stress-inducible mitochondrial protein, and that platelet-derived early pregnancy factor and rat chaperonin 10 share similar biochemical and immunological properties. In this study we show that early pregnancy factor, purified from regenerating rat liver and from serum taken 24 hr after hepatectomy, shares these properties. In addition, antibodies to early pregnancy factor, effective in passive immunization studies, recognize chaperonin 10, whereas chaperonin 10 antibodies bind to early pregnancy factor from regenerating liver and posthepatectomy serum. We propose that early pregnancy factor/chaperonin 10 is selectively released from proliferating cells and, in an autocrine or paracrine mode (or both) is involved in DNA synthesis.

The purification of early-pregnancy factor to homogeneity from human platelets and identification as chaperonin 10.

Early-pregnancy factor (EPF), first discovered in the early stages of gestation, is associated with and necessary for cell proliferation in a wide variety of biological situations. Like many other growth factors, EPF is present in platelets, and, by titration studies with a neutralising anti-EPF monoclonal antibody, platelets were identified as an extremely rich source of this growth factor. EPF has been purified from clinically outdated human platelets by heat extraction, ion-exchange and affinity chromatographies on SP-Sephadex and heparin-Sepharose respectively, high-performance hydrophobic interaction chromatography and three reverse-phase HPLC steps, with an average yield of 15 micrograms/100 platelet units (equivalent to approximately 50 1 blood). Using SDS/PAGE, EPF migrated as a single band with approximate M(r) 8500, coincident with biological activity. Mass spectrometry provided an accurate and precise determination of the molecular mass as M(r) 10843.5 +/- 2, along with definitive evidence of the homogeneity of the preparation. Attempts at Edman degradation indicated that the molecule was blocked at the N-terminus and sequencing of proteolytic fragments was undertaken. The amino acid sequence of approximately 70% of the molecule was determined which, with a single exception, is identical with rat chaperonin 10. This structural relationship was shown to extend to functional identity by studies using chaperonin 10 and its functional associate chaperonin 60. Investigations with the latter confirmed that chaperonin 10 is the moiety in pregnancy serum which initiates response in the EPF bioassay. Our studies identify EPF as a member of the highly conserved heat-shock family of molecules and demonstrate a molecular chaperone performing an extracellular role.

Early pregnancy factor has immunosuppressive and growth factor properties.

Early pregnancy factor (EPF) was first described as a pregnancy-associated substance, although recent studies suggest a more general link with cell development. It is a product of actively dividing cells and its apparent functional importance to them suggests its potential as a regulator of cell proliferation. The recent discovery of EPF in platelets has provided a comparatively rich and readily available source of EPF. The purification procedures employed to isolate EPF from this source have also been applied to pregnancy serum and urine, medium conditioned by oestrous mouse ovaries (stimulated with prolactin and embryo-conditioned medium), medium conditioned by tumour cells, and serum from rats 24 h after partial hepatectomy (PH). In all instances, biological activity followed the same pattern throughout. Furthermore, the final active reversed-phase high-performance liquid chromatography fraction from all sources was bound specifically by immobilized anti-EPF monoclonal antibodies (MAbs), indicating that the active fractions produced from these diverse sources are very closely related, if not identical. Some differences have been observed in the behaviour of EPF in various conditions. EPF is produced by proliferating tumour cells and by liver cells post-PH, and passive immunization studies with anti-EPF MAbs have shown that these cells need EPF for survival. In contrast, EPF has not been detected as a product of the pre-embryo, and addition of anti-EPF MAbs to embryo cultures does not adversely affect development from the 2-cell to the blastocyst stage. Although the pre-embryo is not dependent on EPF for its development in vitro, neutralization of EPF in vivo by anti-EPF MAbs retards its development. Thus, EPF appears to play an indirect role in maintaining the pre-embryo. By virtue of its ability to suppress the delayed-type hypersensitivity reaction, it has been suggested that EPF might act as an immunological response modifier of the maternal immune system. Alternatively, the effect of EPF on lymphocytes may be to reduce the expression of all or some cytokines and this could inhibit development. Whether or not EPF acts more directly as an autocrine growth factor from around the time of implantation, when the embryo first begins synthesis of EPF, is not known and remains to be investigated.

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.